Immune blotting in the diagnosis of HIV. Immunoblotting (detection of antibodies in the sera of patients to certain pathogen antigens) Hybridization of nucleic acids
Enzyme immunoassay (ELISA)
Enzyme immunoassay (ELISA) carried out in two stages: the first is the interaction of antibodies with the antigen, the second is the enzymatic indication of the antigen-antibody complex due to the appearance of staining of the reaction mixture and the registration of staining visually or by spectrophotometric method.
There are two variants of ELISA: solid-phase and liquid-phase, differing in the way the components of the immunochemical reaction are separated. Compared to the previously described methods for detecting antigens and antibodies, ELISA has significant advantages:
high sensitivity, allowing to determine up to 0.05 ng / ml of a substance;
the possibility of using minimal volumes of the test material (1--2 µl);
the possibility of instrumental or visual accounting of the reaction;
rapidity and the possibility of automating all stages of the reaction.
ELISA is currently widely used in practice for the diagnosis of many infectious diseases of bacterial, fungal etiology, protozoal infections and helminthiases, but especially viral infections, in particular hepatitis A, B, C, D, E, G, HIV infection, herpesvirus, rotavirus , adenovirus, astrovirus, parvovirus and other infections.
immune blotting
The principle of the immune blotting method is to detect antibodies to individual antigens of the pathogen. Using this method, antibodies to HIV antigens (virus envelope glycoproteins, core proteins and virus enzymes) are determined. The results of immune blotting are evaluated as positive, questionable and negative, depending on the quantitative and qualitative set of antibodies detected.
It should be noted that immune blotting is inferior in sensitivity to ELISA; in some cases, a negative result may be recorded in the presence of HIV infection in a patient. However, the possibility of registering false positive results in ELISA in HIV infection requires an integrated approach to the diagnosis of HIV infection, taking into account, in addition to the results of immunological reactions (ELISA, immune blotting), epidemiological and clinical data.
Polymerase chain reaction (PCR)
The PCR method was developed by the American biochemist Kary Mullis in 1983 based on the use of the thermostable DNA polymerase (Tag polymerase) discovered by him. The principle of the method is to increase by 10 6 -10 8 times the number of copies of a specific DNA region of the pathogen catalyzed in vitro by DNA polymerase in automatic mode.
Under artificial conditions, the reproduction of the process of replication of a genome region specific for a certain species or genus of pathogens is possible provided that its nucleotide sequence is known. The use of methods for detecting replication products of such regions (amplicons) makes it possible to ascertain the presence of a pathogen in the test sample.
The complementary chain completion described above begins only in certain starting blocks, which are short double-stranded sections. When such blocks are attached to specific regions of DNA, the process of synthesis of a new strand is directed only in the selected region, and not along the entire length of the DNA strand. Two oligonucleotide primers, which are called primers, are used to create starting blocks in given DNA regions. The primers are complementary to the DNA sequences on the left and right boundaries of a specific fragment and are oriented so that the completion of a new DNA strand occurs only between them.
To advantages of the PCR method should include:
high sensitivity, allowing to determine 10-1000 cells in a sample;
high specificity, since a DNA fragment unique for this pathogen is detected in the test material;
universality of the procedure for detecting various pathogens from one bioassay;
High analysis speed (4--4.5 h);
The possibility of diagnosing not only acute, but also latent infections.
The use of PCR is effective for diagnosing a wide range of bacterial and viral infections.
Recently, quantitative methods of PCR analysis have been quite successfully implemented, which make it possible to determine the concentration of the pathogen in the material (microbial or viral load), for example, to assess the replicative activity of the hepatitis B virus, HIV C.
However, it should be borne in mind that the PCR method also has its limitations, in particular, for the diagnosis of infections caused by opportunistic autoflora.
Hybridization of nucleic acids
hybridization of nucleic acids like PCR, allows you to identify the pathogen in the sample without prior isolation. For analysis, a single-stranded DNA or RNA probe is synthesized that is complementary to specific nucleotide sequences of the pathogen. The probe is labeled with a radionuclide, enzyme, or other easily recognizable label. The test material is subjected to processing for the purpose of lysis of microorganisms present in the bioassay, isolation and denaturation of DNA. Next, the probe is incubated with the test sample and the amount of labeled DNA that has entered into hybridization with the DNA in the test sample is measured. The reaction can occur both on solid-phase sorbents and in solution; however, a mandatory condition is the washing off of unbound amounts of the labeled probe. The sensitivity of the nucleic acid hybridization method is inferior to that of PCR and is 103 microbial cells per sample.
When I got tested for STDs, I decided to get tested for HIV. The next day, I received ready-made tests for ifa, except for HIV. As a result, I was diagnosed with chlamydia. Herpes and microplasmosis. But the vich never came. They call me in 3 days and say you need to come to the AIDS center to retake the blood. Can Imunablot Be False Positive in Chlamydia Diseases Mycraplasmosis HPV Herpes
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I don’t want to scare you, but when they call you to the AIDS center, it’s almost always positive, don’t retake it, good luck to you, the main thing is to take therapy and live long
One friend has been living with HIV for ten years, taking care of himself.
They say that in three years they will probably find a cure.
In any case, don't despair and don't spread it, get treated
No, IB cannot be false positive and no STDs affect this analysis, if it is positive, then alas, you have HIV, you need to monitor your immune cells and start therapy on time.
alas, no ((if they called to the center, then it’s definitely HIV
As far as I know, the first and even subsequent immunoblot results can be questionable. not false positives, but doubtful. and then a reanalysis is ordered. Here is the text from the site:
“Immune blotting is most often used to confirm the diagnosis of HIV infection. The WHO considers sera positive if antibodies to any two of the HIV envelope proteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (gp160, gp120, gp41) in combination or without reaction with other proteins, the result is considered doubtful and a second study is recommended using a kit from a different series or from another company. If after that the result remains doubtful, the studies are continued every 3 months.
you can google. if so, I hope you do not test positive for HIV. but if it is confirmed, know that today with this diagnosis they live and live as long as healthy people and give birth to children. the main condition is discipline in treatment. health to you!
Antiretroviral therapy online
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Deciphering the analysis - immunoblot
Please help me decipher the analysis
ENV(GP160)+
ENV(GP110/120)+
ENV(GP41)+
GAG(P55)+
GAG(P40)+
GAG(P25)+
POL(P68)+
POL(P52)+
POL(P34)+
Hello! 2.5 years ago I took an HIV test, there was such an immunoblot:
Gp-160+, Gp-110/120+, Gp-41+, p17+, p25+, p31+, p34+, p52+, p55+, p68+. And here is the immunoblot from 05/30/18: Gp-160+, Gp-41+, GAG 1 -, poll+, env2-. It is not clear what roll + means? Is he the only one there or hasn't been revealed? And where did the rest of the proteins go?
Pol and Env are genes that encode groups of proteins. Consider it just a different record. The question is different. And what are we waiting for? Why are we considering a blot? Everything is pretty clear. Something needs to be done.
Already got used to the fact that I have HIV. And ready for therapy.
Just curious to hear your opinion. Is this a fresh infection or am I missing something? Or sometimes it happens that the immunoblot slowly opens up a lot?
Today I looked at my analyzes in my personal file (done in the SC):
ELISA on the first test system - positive
ELISA on the second test system is negative (how is it?)
Further IB (done in invitro and SC a month ago):
immunoblot on the first test system: gp 160+, gp 41+
immunoblot on another test system: gp41+, p24+, p17+
immunoblot on the third test system: gp160+, gp120+, gp41+, p24+
According to my forecasts, I could have been infected a year ago (the acute stage was in April somewhere), or 9 months ago, but in general - xs when) I always used protection and had sex without condoms only once in the fall of 2017.
Today once again I passed on VN and IP. Teru will start in a month, when the order arrives.
Dates put to the tests mentioned.
First blot before ELISA? Doesn't beat. There is no moment here that would allow us to say that a fresh infection is highly likely, or vice versa.
It will remain a mystery if you do not accidentally find the source and compare.
To clarify, then
Handed over in Invitro.
The first IFA is May 11.
Then the same serum went to the blot and a positive analysis came in, where two proteins were identified.
gp 160+, gp 41+
Then I already handed over to the SC again.
Donated blood on May 29th.
And there he was run through several test systems, where the first showed negative, the second positive. Negative ELISA is funny though.
Then the same serum goes to the blot where the data came from:
First test system: gp41+, p24+, p17+
Second test system: gp160+, gp120+, gp41+, p24+
But not the point.
A new analysis of IP has come - 754. This pleases. VN is not ready yet. As soon as it arrives, I’ll probably start teru.
I am 80% sure that the infection was a year ago. And the fact that the blot is slowly opening and the ELISA is negative is strange. Or is it within the normal range?
Negative ELISA is funny though. I would also like to know the name of the system in order to write it down in a black notebook. Although, this moment, if you do not take the theory with marriage, is strongly for early infection.
I am 80% sure that the infection was a year ago. Poor blot for that period. Not impossible, but unlikely.
At some point, I even began to doubt the results of HIV tests - so I'm waiting for VN.
Here the last point in the question will already be put.
Is it also considered within the normal range that IP has grown by 200 copies without tera in a month? I am taking Ursosan now for a polyp in the gallbladder - the insert says that the drug improves immunity.
It is necessary to evaluate both with a relative content, and take into account the data of one laboratory with one calculation method. Because - God knows what's going on with CD4.
In general, CD4 count is 780 cells/µl. VN - 250 copies / ml.
This is without therapy. That is, CD4 out of 486 jumped to 780 in a month.
BH dropped from 550 to 250 copies.
Still, this is not strange, or are such jumps within the normal range? Is it time to start Tera?
Hello! I passed the ifa three times, each time +, an immunoblot p24+ p18+ came from the SC. The potential risk was more than six months ago. p24 indicates that it is still HIV?
Blot doubtful, repeat after 6-8 weeks. Most likely a false alarm.
Good day!
The test results came back, including a blot:
Dangerous contact: 04/24/2018
ELISA test for HIV 05/23/2018 (4 weeks from dangerous contact or 29 days) result "+"
ELISA test for HIV 05/28/2018 (5 weeks from dangerous contact or 34 days) “retake” result
ELISA test for HIV 06/01/2018 (5 weeks from dangerous contact or 38 days) result "+"
A blot came from the first analysis (05/23/18):
GP160 sl.
P24 sl.
New tests passed on 06.06.2018 ELISA and PCR HIV RNA at the local AIDS center. We are still waiting for the results.
Blot Questions:
1. If P24 is present, is it necessarily an HIV antigen or can such a protein be detected in other diseases?
2. What does the abbreviation "sl" mean next to the protein? Usually in the described blots there is + or -
3. What determines the deployment of a blot? From the term or from the individual characteristics of the body?
4. With such a blot at the 4th week from a dangerous contact, is there a chance that this is not HIV?
Have a nice day and thank you.
1. no, it may just be a similar protein of a different nature. 2. weak. those. doubtful. 3. Deadlines and realize individual features, but on average everything is very close. 4. the question is wrong, there is always a chance until the diagnosis is established.
Hello!
Please help me to navigate the results of the analyzes and understand how to behave correctly so that the repeated analyzes are correct.
Analyzes handed over 05/25/2018
HIV IB
NEW LAV BLOT 1 - undefined from 29.05.2018
IB HIV markers
gp 160+
gp 120 —
gp 41 -
p55+
p40-
p 24+
p18-
p 68 —
p 52 —
p 34 —
HIV ELISA
ADVIA Centaur HIV Ag-Ab Reactivity ELISA 12.00 positive (28.05.2018)
It is recommended to repeat the analysis after 2 weeks.
In November 2017, I had an NPA, after which I took tests on the HIV Ag\Ab Combo Abbott Architect test system, then the result was negative.
At the same time, I had a complete blood count. Lymphocytes are slightly increased, eosinophils are exactly 0 (but I had such indicators for eosinophils before.
The main question is:
What medicines can and cannot be taken during these two weeks? (I want nothing to “flash”)
I have occasional allergy attacks, usually I drink tavegil. Should it be abandoned?
Can antibiotics be taken before testing? (if prescribed by a doctor for the treatment of other infections and diseases)
Immunoblotting in HIV diagnostics
Immune blotting (immunoblot, Western Blot, Western blot)- combines enzyme immunoassay (ELISA) with preliminary electrophoretic transfer to a nitrocellulose strip (strip) of virus antigens.
In this beautiful scientific name, “blot” most likely translates as “blot”, and “western” as “western” reflects the direction of distribution of this “blot” on paper from left to right, that is, on a geographical map, this corresponds to the direction from west to east. ". The essence of the "immune blot" method is that the enzyme-linked immunosorbent reaction is carried out not with a mixture of antigens, but with HIV antigens, previously distributed by immunophoresis into fractions located according to molecular weight on the surface of a nitrocellulose membrane. As a result, the main proteins of HIV, carriers of antigenic determinants, are distributed over the surface in the form of separate bands, which appear during the enzyme immunoassay.
Immunoblot includes several stages:
Strip preparation. The immunodeficiency virus (HIV), which has been previously purified and destroyed to its constituent components, is subjected to electrophoresis, while the antigens that make up HIV are separated by molecular weight. Then, by blotting (analogous to squeezing out excess ink on a “blotter”), the antigens are transferred to a strip of nitrocellulose, which now contains a spectrum of antigenic bands characteristic of HIV that is invisible to the eye.
Sample study. The test material (serum, blood plasma of the patient, etc.) is applied to the nitrocellulose strip, and if there are specific antibodies in the sample, they bind to the strictly corresponding (complementary) antigenic bands. As a result of subsequent manipulations, the result of this interaction is visualized - made visible.
Interpretation of the result. The presence of bands in certain areas of the nitrocellulose plate confirms the presence in the studied serum of antibodies to strictly defined HIV antigens.
Currently, immune blotting (immunoblot) is the main method for confirming the presence of virus-specific antibodies in the test serum. In some cases of HIV infection, prior to seroconversion, specific antibodies are more effectively detected by immunoblotting than by ELISA. An immune blotting study revealed that antibodies to gp 41 are most often detected in the sera of AIDS patients, and the detection of p24 in persons examined for prophylactic purposes requires additional studies for the presence of HIV infection. Immune blot test systems based on genetically engineered recombinant proteins proved to be more specific than conventional systems based on purified virus lysate. When using a recombinant antigen, not a diffuse, but a clearly defined narrow band of antigen is formed, which is easily accessible for accounting and evaluation.
Serum of persons infected with HIV-1, detect antibodies to the following major proteins and glycoproteins - structural envelope proteins (env) - gp160, gp120, gp41; nuclei (gag) - p17, p24, p55, as well as virus enzymes (pol) - p31, p51, p66. For HIV-2, antibodies to env are typical - gp140, gp105, gp36; gag - p16, p25, p56; pol-p68.
Among the laboratory methods necessary to establish the specificity of the reaction, the detection of antibodies to HIV-1 envelope proteins - gp41, gp120, gp160, and HIV-2 - gp36, gp105, gp140 has the greatest recognition.
WHO considers sera positive if antibodies to any two HIV glycoproteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (rp 160, rp 120, rp 41) in combination or without reaction with other proteins, the result is considered doubtful and a second test is recommended using a kit from a different series or another company. If after this the result remains doubtful, observation for 6 months is recommended (research after 3 months).
The presence of a positive reaction with the p24 antigen may indicate a period of seroconversion, since antibodies to this protein sometimes appear first. In this case, it is recommended, depending on the clinical and epidemiological data, to repeat the study with a serum sample taken at least 2 weeks later, and this is exactly the case when paired sera are required in HIV infection.
Positive reactions with gag and pol proteins without the presence of a reaction with env proteins may reflect the stage of early seroconversion, and may also indicate HIV-2 infection or a non-specific reaction. Individuals with such results after testing for HIV-2 are re-examined after 3 months (within 6 months).
Question: Repeated analysis for HIV?
I was tested for sexually transmitted infections (no symptoms, I wanted confidence in a relationship with a woman). The HIV test was positive. Following the ultrasound showed a tumor on the kidney, removed (it turned out to be malignant).
I read the book by Sazonova I.M., it says that a malignant tumor can give a positive test result for HIV.
Could this be the case, or is there nothing to hope for?
You need to have a control test for HIV. If the first HIV test was determined by ELISA, then the result may be false positive. Its reliability can be checked by a more sensitive diagnostic method - PCR (polymerase chain reaction), which determines the DNA of the virus in the blood.
Help. 12/16/10 ELISA (+) IB (+) then from 03/23/11 to 05/19/11 nine negative ELISA (-) and quantitative PCR. are not determined. in 2002 during pregnancy ELISA then (+) then (-) but IB is always (-). from 2004 to 2008 I took ELISA (-) 2 times a year, but on 30.04.08 ifa (+) and IB-undefined. then again each 2 months handed over IFA always (-). and since December 2010, it has been written above. At the same time, I have never injected, my husband always has an ELISA (-). CD4 980 cells. and even blood for syphilis from 29.04 gave 3 +++. and then three times. negative every 10 days. hepatitis all (-). Has anyone had a similar one. thank.
Please specify whether you underwent RIBT (treponema pallidum immobilization reaction), if so, what are the results of this study.
no, no one offered me to do such an analysis. what will it show? I hope you understand that I was talking about HIV tests. thank. Have you had similar cases in your practice? by the way, about information security in 2008 was uncertain. was p24/25 protein. in 2010 IB(+) proteins gp160.41.120 p24.17.31. then when ifa was again 3 times (-) sent to IB on April 4th. the result came positive, but the proteins gp 120 and 41. the rest are crossed out with red paste and at the bottom with red IB REPEAT. but PCR from the same number will be denied. after April 4 I handed over IFA already 4 times deny. everything in the AIDS center, including antigen and antibody. Now I'm waiting for a second IB and a high-quality PCR. that's it. VERY TIRED TO THINK AND WAIT. Hoping for the best. THANK. I'm looking forward to a response.
If you ask any question, please try next time to formulate it more specifically, with a clarification of the diagnosis. RIBT is used to confirm the diagnosis of syphilis. For accurate diagnosis of HIV infection, antibodies to HIV in the blood are determined by ELISA and immunoblot. The diagnosis is confirmed only if both of these results are positive.
sorry for imprecisely formulating the question. I wrote that in December IFA and Imunoblot for HIV came positive. but since March ifa for HIV is negative 9 times. if I was registered in the AIDS center, then does this happen in principle. HIV is either always positive or negative. and how can an imunoblot be put on HIV if the result of ELISA is negative? then everyone will deny ifa needs to be checked for immunoblot, so what happens? in our speed center they can not answer me anything. Here is what I turned to you. thank.
Unfortunately, both ELISA and immunoblot can give false positive results. That is why the diagnosis of HIV is considered final, only with the simultaneous detection of HIV by ELISA and the immunoblot method.
hello. today I received the results of a PCR for HIV, a qualitative virus was not detected and a repeated immunoblot for HIV, the result is uncertain due to protein 41. in the AIDS CENTER they said that most likely there is no HIV, but in my body there are bodies similar in structure to HIV. and what do you think, given my questions of June 15 and 16 (see above), is there HIV or not. THANK.
In this case, the diagnosis of HIV infection is doubtful.
You write that only with the simultaneous detection of HIV with the help of IF and immunoblot, the diagnosis of HIV is considered final. But what about in my case? after all all ptsr will deny. and blot and ifa jump all the time. for 9 years. tell me, if the virus was in my blood, then its RNA and DNA could be accurately determined for so many years. and can the incubation period or "window" last that many years? Are there false negative results of PCR for HIV given such a period of time? Yes, I forgot to say that the rapid HIV tests that I take at the CPD are always negative. Or can I not rely on them either? thank.
In this case, PCR diagnostics is not the main method for identifying the dynamics of the process - serological methods are more informative. In this case, the probability of false negative results is high. rapid HIV tests have a high sensitivity threshold, therefore they can also give a false negative result.
sorry. I definitely wrote in the wrong place. please answer in the subject of HIV or not HIV. thank.
In the event that you have not received a notification of receipt of a response to your mail, you can view the answer to your question at this address http://tiensmed.ru/news/answers/vich-ili-ne-vich-.html
Hello! Please tell me, in order to register with the LCD (now 10 weeks pregnant), I passed tests for HIV, a couple of days ago the doctor called in and said that preliminary tests for HIV were positive (the first was done in Kirovograd, but there is still no official result from Kyiv ), on the same day, in our city laboratory, two express tests of the Pharmasco company CITO TEST HIV 1/2 were performed, both results are negative, the laboratory assistant said that these tests are reliable and I don’t have to worry, since this happens during pregnancy, And those analyzes could simply confuse. The doctor said to donate blood again and I donated my blood twice more for analysis in different hospitals (I still don’t have any of the three results). I'm very worried, I'm not a drug addict, there were no dubious sexual relationships, if I get sick very rarely, other tests are all normal. Can rapid tests be trusted? Does this really happen during pregnancy? Painfully strongly the doctor has frightened me. Thanks
First of all, you need to calm down and not think about the bad. Sometimes during pregnancy, there are false positive results. It is necessary to re-donate blood for HIV and wait for the results of the examination.
hello! The matter is that at me 2 months ago there was a sexual contact with the girl (till now we meet). after 1.5 weeks the temperature rose to 37.4. soon slept. to be sure, we passed the IF analysis after 2 weeks and again after 1.5 months. Both answers are negative. but I still have a fever and cough with a variable improvement. Can you please tell me if there is a risk? besides, I worked for a long time without days off and a week ago I was on sick leave (orvi). blood and lung tests are fine. thank.
This temperature may be associated with a viral disease, the body has not recovered yet, or chronic overwork. In the event that an organic pathology is excluded, a general blood and urine test is within the normal range, as well as the data of a fluorographic study are also within the normal range, then it is necessary to exclude sexually transmitted diseases: chlamydia, mycoplasmosis, ureaplasmosis, which can cause inflammation of the organs of the small pelvis and urethra and, as a result, an increase in body temperature. Read more about the reasons for the increase in body temperature by clicking on the link: High temperature.
Hello. There is such a thing - More than a year ago there was unprotected sexual contact with a walking girl. She assured me that she was not sick with anything, but I can’t believe her 100 percent. She also assured that she had undergone a medical examination before getting a job (she worked as a salesman) and everything was fine. 7 months after contact, I still passed an HIV test in the citylab laboratory - the result was negative. But recently I often began to get sick - for 3 weeks now, I have a red sore throat and I cannot cure it. Again he began to be afraid, but suddenly he caught it then? Tell me, is this possible, and is it worth trusting the analysis from citylab? I'm afraid to give up again, my nerves will not stand it ..
In the event that the result is negative, then most likely you are not sick and not infected with HIV / AIDS. However, to clarify the diagnosis, it is recommended to re-analyze in specialized laboratories at state institutions; this examination is carried out anonymously. In the event that self-treatment does not bring the desired result, it is recommended to consult with an otolaryngologist to conduct an adequate examination and prescribe appropriate treatment. Read more about HIV testing in a series of articles by clicking on the link: HIV.
Tell me, can you give any description of the citylab laboratory? All the same, it is not always possible to pass an analysis at a state institution. And what is the percentage chance for a man to get infected through unprotected contact?
Unfortunately, we do not give a comparative assessment of laboratories and private medical institutions. In the event that you doubt the reliability of the results, conduct an examination in another center and first ask for a license to provide these medical services, whether this center has the right to conduct this examination and whether everything complies with accepted standards. The risk of infection is the same for both sexes through unprotected intercourse. Read more about HIV testing in a series of articles by clicking on the link: HIV.
Good afternoon! An 8-month-old child was tested for HIV by ELISA, gp160 + and p25 + were found in the blood, the rest is all minus, the conclusion of IB is doubtful. Judging by these analyzes, it turns out that the child is +? gp160 + gp110/120 - p68 - p55 - p52 - gp41 - p34 - p25 + p18 -
Unfortunately, based on the data obtained, it is impossible to make a diagnosis with a 100 percent probability, since a false positive result is not excluded. To make an accurate diagnosis, you will need to undergo a series of examinations, including repeating this analysis by ELISA, as well as passing an analysis using the PCR method. After that, you should contact a specialized medical institution, where the infectious disease specialist will be able to evaluate the results in a complex. You can learn more about the manifestations of HIV infection in the thematic section of our website by clicking on the link: HIV
Can it show a false-positive result in "ARI" or more acute infectious diseases? Somewhere I read that with 58 diseases or even higher, it can show “+”, including vaccination against hepatitis B, if the kidneys, etc., suffer?
There is a possibility of a false positive result, so I recommend that you do the following: re-analyze - by ELISA and PCR, and then re-visit an infectious disease specialist. You can learn more about the diagnosis of HIV infection in the thematic section: HIV
Good afternoon! Immunoblot indeterminate due to p25 protein. What is the likelihood of HIV?
In this situation, it is necessary to carefully study the study protocols in combination with other indicators, since it is not possible to make an assumption based on these data. Presumably, the result can be considered doubtful and a re-examination is required after 3 months. Read more in the section of our website: HIV
Good afternoon.
Can you comment on ELISA for HIV
1 serum +3.559 k=13.3
+2.121 k=4.9
p 24 neg
Serum 2 +3.696 k=13.9
+2.477 k=5.7
In this case, a false positive result is not ruled out, given that the ELISA method is indirect, so I recommend that you take an analysis using a different, more sensitive method - immune blotting. You can find out more detailed information on this issue in the relevant section of our website by clicking on the following link: HIV
Good afternoon, tell me what to tune in? A year ago, when planning a child, my husband and I went through all the tests, including for HIV (they took it very seriously and correctly), I was examined in Kr. Rog, my husband in Kyiv, he had a negative answer, they told me that some kind of reagent did not work, I need to retake it at the Center AIDS in Kyiv. Having passed the analysis at the Center, the answer came negative for me too. Now I am in position at 14 weeks, i.e. I get registered, I go through all the tests and again the answer came back, the HIV test was indefinite, I re-passed it at the clinic and passed an express test to calm down at Dovir, but they didn’t calm me down, the express test showed a positive result (the second strip was less pronounced), immediately after all this procedure, without wasting time, I applied to the AIDS Center and also passed an analysis, I am waiting for the result. (I can’t calm down) Please tell me how much you can trust the express tests and why the first time there is no answer to the HIV test? (my husband and I lead a healthy lifestyle and love each other). Thank you.
Do not panic ahead of time - express diagnostics is not the basis for making an HIV diagnosis, it allows you to identify groups of patients who require a more in-depth study. In such situations, it is recommended to conduct immune blotting and personally consult with an infectious disease doctor. You can find out more detailed information on this issue in the thematic section of our website by clicking on the following link: HIV. You can also get additional information in the following section of our website: Laboratory diagnostics
Hello, I was in an infectious disease, only today the doctor called me out when I left and explained that I had a positive if, at first when I went to the hospital, it was negative, then when I retaken it became positive, they sent an immunoblot study to the falconers on the mountain they said it would be ready next week, I was in the hospital with a sore throat and parainfluenza viruses, I arrive in a state of shock, I still don’t understand how to regard it, an extract for my clinic was also drawn up indicating that ifa was found and lower that the immunoblot is in work, if tomorrow I’m discharged at your clinic, then everything will be indicated in this extract, how likely is HIV? Is it possible that due to the fact that I was treated for a sore throat of the parainfluenza virus, show positive results for ifa?
The probability of a false positive result is very high. The presence of one positive result does not yet give grounds for making a diagnosis of HIV, so we recommend that you wait for the result of immunoblotting, and then personally consult with an infectious disease specialist regarding further examination and observation. Angina, parainfluenza and other colds do not significantly affect the results of the analysis.
I want to believe it, but at the end of August I had a malaise, my temperature rose, 37.5-38 I had loose stools for about 4 days, it was on vacation where there were a lot of discos, I drank tap water like many others, because that it was very expensive a glass of water cost 300 rubles, I associated loose stools with such a temperature with some kind of intestinal infection caught in the water, I don’t remember exactly, but there was also a small rash in the upper body, when I arrived home with a temperature I called the doctor, she wrote a rotavirus infection, after 5 days of illness, I volunteered to leave him and go to work where I fell ill a few days later with sinusitis (during that period of time I had to be on the street due to work) I connected it that a large temperature drop from vacation and poisoning lowered my immunity and therefore I caught a cold once again with sinusitis, in total it is again a sick leave, at the direction of Laura I drank klacid cf 500 for 10 days, it passed, I went back to work after 3 weeks I was on a business trip to arch country for 3 days. the air conditioners in the transport and the hotel were merciless and upon returning home, on the plane I had a temperature already of 39.5. here I was at home with a temperature of 40, called the doctor to the house, wrote orvi and said my throat was very red, I had chronic tonsillitis and said this to Laura, she herself wrote to drink an antibiotic Levolet r. called an ambulance because she had a fever and the pace was 40 and did not decrease, hospitalization was not offered, the next day the same story - the ambulance gave an antipyretic injection and left. The third time I insisted on hospitalization, they barely took me away to the infectious diseases hospital, where a mixed infection of parainfluenza and adenovirus infection was detected, but upon discharge, the doctor - the head of the department said that I had a positive HIV ifa and that they did it twice, I'm in shock, I don't know what to do I can't eat and drink .she said that I have a pronounced acute HIV infection and for verification they sent an analysis of my blood for an immunoblot to the AIDS center,
now, drawing an analogy of the events that have happened to me lately, as well as 3 sick leave sheets in a row, I tried on all the symptoms and I am horrified by what could be, after being discharged on the same day, I went to take an analysis in an in vitro ananimally and the next day the result for ifa was the same +
I'm sorry for such detailed information, but I'm swept away and killed, I drink strong sedatives and I have no appetite and I practically don't eat, I've lost a lot of weight
I also have such a question that the doctor with an extract from the hospital indicated the result of HIV by ifa was found and below that the immunoblot is in work, but as soon as I close the bl in my clinic at the place, everything will be written there. what should I do? it will no longer be confidential. I asked the doctor not to write this analysis in the extract, to which she refused me, to what extent my rights on non-disclosure of information are respected here.
Unfortunately, the results of the studies carried out in the hospital fit into the extract, since the attending district doctor must have full information about your state of health. In this situation, we are not talking about the disclosure of information, since it is only transferred to another attending physician, who will continue to observe you.
Hello! I took tests for HIV because I needed a certificate for the FMS, they didn’t give the tests for a couple of weeks, then they invited me to the head and they gave me a positive result, they took a bunch of receipts and sent them to the regional AIDS center for further examination, as it says on the certificate. I want to pass in another clinic and then go to the regional one, or does it make sense to retake it? I just don’t understand why they didn’t give them away for so long, but the doctor said that they allegedly did some kind of analysis and I owe them another 4 thousand rubles, because if they did it, they would probably give detailed information about the disease in addition to the certificate?
In this situation, one should not panic ahead of time - obtaining one positive result does not yet allow one to judge reliably about a possible infection, since false positive results are not excluded. We recommend that you take the test again and if there is a positive result, you will need to undergo another examination - immunoblotting. As a rule, the laboratory does not give detailed information about the results, which is normal and common practice. All questions you may have can be answered by the attending physician after the examination at a personal consultation.
I forgot to add that from the beginning of June until mid-September I did myself a course of anabolic steroids, namely sustanon 250 is a mixture of testosterones and stanozolol with primabolan, I wanted to prepare myself for the summer and vacation, could they bring down my immunity and everything that happened to me.
Immune disorders, as well as the presence of autoimmune diseases, can give false positive results of an HIV test. That is why, in the case of obtaining 2 positive results by ELISA, immunoblotting is recommended, which will allow you to accurately answer the question of whether there is infection or not.
what does the presence of autoimmune diseases mean? what are they?
in general, I can say that I got sick quite often from early childhood and even a couple of three years ago I asked the attending physician to take care of my immunity, because I was constantly tired and often sick, mostly ear, throat, nose, but all the time there were negative results for HIV, I I handed them over with sufficient ease, without hesitation.
A false-positive result of an HIV test can be after a recent viral infection, hepatitis B vaccination, tuberculosis, hepatitis, herpes, as well as against the background of autoimmune diseases such as: rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, connective tissue diseases and etc.
I want to add to my question, my immunoblot came, it was negative, but the doctor said that since there were two ifs + when I was in the infectious diseases hospital, I still need to retake the analysis, but a little later
In this case, medical tactics are justified - we recommend that you take an immunoblot again in 1.5-2 months.
What is the probability: 2 ifa + difference between blood samplings of about 2 days, immunoblot - ; was in the infectious diseases hospital with adenovirus infection and parainfluenza, where the blood was taken, the immunoblot was sent to the AIDS center
Good afternoon! I got registered with the LCD, went through all the tests, the doctor says that I have herpes in my blood, then they call from the AIDS center and say that I need to retake it again, I applied and they tell me that I have HIV positive, in a panic I went with my husband to set up a second I also had an ifa and an immunoblot + my husband had an analysis, I gave it again a month later. I have + my husband - now I am 23 weeks pregnant!
In this situation, unfortunately, there is a possibility of HIV infection, but the final diagnosis cannot be made even with a positive immunoblot, given the state of pregnancy. In this situation, the exclusion of false positive results is required, therefore we recommend that you take the test again and personally consult with an infectious disease specialist.
if the immunoblot showed a positive result for HIV, and the screening is negative, which result should be trusted?
Immunoblot is a more accurate study, therefore, in this study, if a positive result is obtained, it is required to continue the study and personally visit an infectious disease specialist.
What is it - immunoblot? This is a common method for laboratory diagnosis of human viral infections. It is considered one of the most accurate and reliable ways to detect the presence of HIV. In its reliability, it surpasses even the results of the immunoblot are considered irrefutable and final.
general information
Immunoblot - what is it? In order to recognize an HIV infection in a person, it is necessary to undergo a laboratory test of blood serum for the presence of antibodies. The Western blot technique is also called Western blot. It is used to detect human viral infections as an additional expert method. It is necessary to confirm the ELISA - a laboratory test that allows you to determine the presence of HIV antibodies in the blood. A positive ELISA analysis is rechecked by immunoblotting. It is considered the most sensitive, complex and expensive.
purpose
What is it - immunoblot? This is a technique for laboratory testing of blood serum for the presence of antibodies to the virus. During the study, the specialist pre-separates the proteins of the virus in the gel and transfers them to a nitrocellulose membrane. The immunoblotting procedure is intended to determine HIV at different stages. At the first stage, the purified virus from its constituent parts is subjected to electrophoresis, and the antigens that make up its composition are separated by molecular weight.
It reproduces in a living cell, embedding its genetic information into it. At this stage, a person becomes a carrier of the HIV virus if he was infected. The specificity of the disease is that it may not manifest itself for a long time. The virus destroys lymphocytes, so the human immunity is reduced, and the body becomes unable to resist infections. If HIV is treated competently and on time, the patient will live to a ripe old age. Lack of therapy inevitably leads to death. From the moment of infection, but without treatment, the maximum life span is no more than ten years.
Peculiarities
Immunoblot analysis is a reliable method that allows you to determine the presence of antibodies to HIV antigens of the first and second types. If a person is infected, antibodies appear within two weeks, which can be detected much later. The peculiarity of HIV is that the number of antibodies increases rapidly and remains in the blood of the patient. Even if they are present, the disease may not manifest itself for two or more years. The ELISA method does not always accurately determine the presence of the disease, therefore, confirmation of the results using immunoblotting and PCR is required if the enzyme immunoassay is positive.
Indications for appointment
What is this "immunoblot" has already been found out, but to whom is this study prescribed? The reason to take tests for the method of immunoblotting is a positive ELISA result. It is necessary to go through enzyme immunoassay for patients who will be operated on. In addition, an analysis should be made for women planning a pregnancy, as well as for everyone who is sexually promiscuous. Assign immunoblotting to patients with HIV, if the results of ELISA are in doubt. The following alarming symptoms may be the reason for going to the doctor:
- sharp weight loss;
- weakness, loss of working capacity;
- bowel disorder (diarrhea) that lasts for three weeks;
- dehydration of the body;
- fever;
- swollen lymph nodes in the body;
- development of candidiasis, tuberculosis, pneumonia, toxoplasmosis, exacerbation of herpes.
The patient does not need to prepare before donating venous blood. 8-10 hours before the study, you can not eat. It is not recommended to drink alcoholic and coffee drinks a day before donating blood, to engage in heavy physical exercises, to experience excitement.
Where to do the analysis?
Where can I get tested for HIV? ELISA, immunoblot studies are carried out in urban private clinics, the results are issued within a day. Immediate diagnosis is also possible. In state medical institutions, ELISA tests and immunoblotting are carried out free of charge, in accordance with the legislation of the Russian Federation. Pregnant women, as well as patients who are to be hospitalized or undergo surgery, are required to be tested for infectious diseases.
How is the research done?
How is ELISA performed? Immunoblot positive/negative confirms or refutes the results of enzyme immunoassay. The research procedure is quite simple. The specialist conducts a venous blood sampling, in time it takes no more than five minutes. After sampling, the injection site should be disinfected and sealed with a plaster. The sampling is carried out on an empty stomach, so after the procedure it does not hurt to eat a bar of dark chocolate or drink a sweet hot drink.
In order to get a referral for a free analysis at a public medical institution, you need to visit a general practitioner. In general, the immunoblot does not differ from other blood tests in terms of the method of sampling. The research methodology is simple. If a virus is present in a person's blood, the body begins to produce antibodies to destroy it. Each virus has its own set of antigen proteins. The detection of these antibodies is the basis of the Western blotting technique.
Price
How much does the analysis cost? Immunoblot for HIV does not apply to cheap research. On average, a screening examination by enzyme immunoassay costs from 500 to 900 rubles. Immunoblotting is a verification study, the cost of which is from three to five thousand rubles. More complex methods are much more expensive. For example, you will need to pay about 12,000 rubles for it.
Interpretation of the result
The most common methods for diagnosing HIV infection are enzyme immunoassay and immunoblot. They are used to determine the serum antibodies to the immunodeficiency virus. The presence of infection is usually confirmed by two tests: screening and confirmatory. The interpretation of the results of the study should be carried out by a doctor, he also makes a diagnosis and prescribes treatment. If the immunoblot is positive, this means that a virus is present in the human body.
A positive result should not be a reason for self-treatment, since each patient may have his own picture of the disease. Qualitative analysis includes screening and verification. If the patient does not have a virus, then the result is indicated as “negative”. When detected by the screening method, an additional verification study is carried out. An immunoblot is an analysis that confirms or refutes a screening. If darkening appears on the test strip in certain areas (sites of protein localization), a diagnosis of HIV is made. If the results are in doubt, the tests are carried out within three months.
You can prevent infection with the immunodeficiency virus if you follow certain rules: avoid casual sex, use a condom during contacts, do not take drugs. If the disease is detected in a pregnant woman, it is important to strictly follow the recommendations of the attending physician, do not forget to undergo examinations for the presence of the virus.
IMMUNOBLOT(western blot) - a method of laboratory testing of blood serum for the presence of antibodies to HIV; it is a more accurate test than ELISA and is used to confirm ELISA results. ELISA - enzyme-linked immunosorbent assay (ELISA) - a laboratory test that allows you to determine the presence of HIV antibodies in the blood; HIV antibody test.
According to the WHO recommendation, immunoblotting (western blot) is used in the diagnosis of HIV infection as an additional expert method, which should confirm the results of ELISA. This method is usually used to double-check a positive ELISA result, as it is considered more sensitive and specific, although more complex and expensive.
Immunoblotting combines enzyme immunoassay (ELISA) with preliminary electrophoretic separation of virus proteins in a gel and their transfer to a nitrocellulose membrane. The immunoblot procedure consists of several stages (). First, pre-purified and destroyed to its constituent components, HIV is subjected to electrophoresis, while all the antigens that make up the virus are separated by molecular weight. Then, by blotting, the antigens are transferred from the gel to a strip of nitrocellulose or a nylon filter, which now contain a spectrum of proteins that is characteristic of HIV, invisible to the eye. Next, the test material (serum, blood plasma of the patient, etc.) is applied to the strip, and if there are specific antibodies in the sample, they bind to the strips of antigen proteins that strictly correspond to them. As a result of subsequent manipulations (like ELISA), the result of this interaction is visualized - made visible. The presence of stripes in certain areas of the strip confirms the presence in the studied serum of antibodies to strictly defined HIV antigens.
Immunoblotting is most commonly used to confirm the diagnosis of HIV infection. The WHO considers sera positive if antibodies to any two of the HIV envelope proteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (gp160, gp120, gp41) in combination or without reaction with other proteins, the result is considered doubtful and a second study is recommended using a kit from a different series or from another company. If after that the result remains doubtful, the studies continue every 3 months.
For immunoblotting, at the first stage, the proteins contained in the blood serum are separated in the gel according to their molecular weight and charge using an electric field (gel electrophoresis method). Then a nitrocellulose or nylon membrane is applied to the gel and "wetted" (this is blotting). This is carried out in a special chamber, which allows complete transfer of the material from the gel to the membrane. As a result, the pattern of protein arrangement that was on the gel is reproduced on the membrane (blot), which can then be easily manipulated. Initially, the membrane is treated with antibodies to the desired antigen, and after washing off the unbound material, a radioactively labeled conjugate is added that specifically binds to antibodies (as in ELISA). The location of the resulting antigen-antibody-labeled conjugate complex is determined by autoradiography using x-ray film. After its manifestation, everything becomes clear whether there are antigens in the blood or not.
Immunoblotting -(from the English "blot" - spot) - a method for identifying antigens (or antibodies) using known sera (or antigens). It is a combination of gel electrophoresis with ELISA. Initially, bacterial cells or virions are destroyed using ultrasound, and then all antigens of the virus or bacterial cells are separated by electrophoresis and a commercial reagent is obtained on a special nitrocellulose film. When setting up immunoblotting, the test serum of the patient is applied to the film with known antigens. After incubation and washing off of unbound antibodies, they proceed to ELISA - an antiserum to human immunoglobulins labeled with an enzyme and a chromogenic substrate that changes color when interacting with the enzyme are applied to the film. In the presence of antigen-antibody-antiserum to immunoglobulin-enzyme complexes, colored spots appear on the carrier. The method of immunoblotting allows you to separately detect antibodies to various antigens of the pathogen (for example, in HIV infection, immunoblotting detects antibodies to gp120, gp24 and other antigens of the virus).
Radioimmunoassay (RIA)
The method is based on the antigen-antibody reaction using an antigen or antibody label with a radionuclide. 125I, 14C, 3H, 51Cr and other radionuclides are used as a label. The resulting immune complexes are isolated from the system and their radioactivity is determined on counters (β-radiation). The radiation intensity is directly proportional to the number of bound antigen and antibody molecules.
The solid-phase version of RIA using labeled antibodies or antigens adsorbed in the wells of polystyrene panels is widely used.
RIA is used to detect antigens of microbes, viruses, various hormones, enzymes, medicinal substances, immunoglobulins, as well as other substances contained in the test material in minor concentrations of 10–12–10–15 g/l.
test questions
Immune bacteriolysis reaction: what kind of reaction is it; what is an antigen, what is an antibody, reaction mechanism, methods of setting, practical application. Immune hemolysis reaction: necessary ingredients, method of setting; controls, practical application. Reaction of local hemolysis in gel (Yerne reaction): principle of the reaction, method of formulation, practical application. Complement fixation reaction: reaction principle; what is formed when the immune serum interacts with a specific antigen; what happens to complement if it is present in this interaction? What is the fate of complement if there is no specific affinity between antigen and antibodies? What reaction can be used to determine what happened to the complement; why this reaction is used; what is the visible positive result of RSK? Why? What property of complement is used in the first phase of CSC? In the second phase? If the end result of RSK is hemolysis, does that mean a positive or negative result? Explain the results: RSK+ + + +, RSK+ + +, RSK+ +, RSK+. Name the ingredients of the first RSK system and the ingredients of the second RSK system. Why does the test serum need to be inactivated? How is complement titrated? Hemolytic serum: what does it contain, how is it obtained, what is a titer and how is it determined? What animals are used to obtain RSC components? The technique of setting RSC in the cold. When staging which of the following reactions, the participation of a complement is necessary: precipitation, flocculation, agglutination, detection of incomplete antibodies, immune bacteriolysis, immune hemolysis, Yerne, CSC? Immunofluorescence reaction (RIF) - indicate the sequence of events in the direct Coons reaction; the necessary ingredients. What is an antigen, what is an antibody, how are antibodies labeled, how is the result of the reaction taken into account, what does a positive result look like? Practical application - what can be determined using this reaction? Indirect immunofluorescence reaction - indicate the sequence of events in this reaction, the necessary ingredients, what is the antigen, what immune sera are used; practical use; advantage of indirect RIF over direct reaction. Enzyme immunoassay (ELISA) - the principle of the reaction; necessary ingredients; indicate the sequence of events when setting up a reaction in order to detect an antigen in the test material; necessary ingredients; what happens with a positive result, what does it look like? Specify the sequence of actions during ELISA in order to detect antibodies in the test serum; necessary ingredients; what happens with a positive result? Immunoblotting - the principle of the reaction; main stages; necessary ingredients; How is the result taken into account? reaction benefits. Radioimmunoassay (RIA) - what are the main stages of the reaction; what are antibodies or antigens labeled with, how is the result taken into account? Immunoelectron microscopy - the principle of the method; main stages; necessary ingredients; What are antibodies labeled with? how the result of the reaction is taken into account. Immobilization reactions - the principle of the method, the technique of setting, components, accounting for results.
Tasks to perform in the process of self-training.
Complete the Immunity Reactions table for the reactions covered in this topic.
Immune reactions
Student's work in a practical lesson
Start work immediately with the formulation of the 1st phase of the RSC, but write it down in a notebook later (see below).
1. The reaction of immune hemolysis. View a demonstration reaction of immune hemolysis, draw it as a diagram, explain the result in the experimental and control tubes.
2. Complement binding reaction
a) disassemble the RSC according to the table;
b) draw in a notebook a scheme for setting the RSC in the form of a table;
c) put the second phase of the RSK (the first phase is put at the beginning of the lesson);
d) disassemble the diagnostic preparations necessary for the RSK;
e) take into account the result. Formulate a conclusion about the presence of specific antibodies in the test serum.
3. Immunofluorescence reaction. Study the table, draw up a scheme for setting up the reaction in your notebook; see diagnostic sera; determine what the serum contains, how it is prepared, for which reaction (direct or indirect RIF) it is used. Look at the demonstration result of the RIF in a fluorescent microscope.
4. Enzyme immunoassay (ELISA). In your notebook, draw up a reaction scheme in two versions: for the detection of an antigen in the test material and for the detection of antibodies in the serum. Review the HIV and Hepatitis B Diagnosis Ingredient Kit. Determine what each ingredient contains and what it is used for.
5. Immunoblotting. Make a diagram of the reaction in your notebook; see the demo - the result of the reaction.
6. Radioimmunoassay (RIA). Draw the reaction scheme in your notebook.
7. Immune electron microscopy (IEM). Watch the demonstration - the result of the reaction, draw up a reaction scheme in your notebook, indicate the antigen (virus) and labeled antibodies with arrows.
Immunoblotting is a highly sensitive protein detection method based on a combination of electrophoresis and ELISA or RIA. Immunoblotting is used as a diagnostic method for HIV infection, etc.
In a general sense, immunoblotting is understood as the analysis of a mixture of proteins transferred to a solid support-membrane, with which they bind by covalent bonds, followed by immunodetection.
It is possible to analyze a mixture of proteins directly deposited on a substrate (dot blot analysis) or after its preliminary fractionation by electrofocusing, disk electrophoresis, or two-dimensional electrophoresis (Western blotting).
Pathogen antigens are separated by polyacrylamide gel electrophoresis, then transferred from the gel to activated paper or nitrocellulose membrane and developed by ELISA.
Firms produce such strips with "blots" of antigens. The patient's serum is applied to these strips. . Then, after incubation, the patient is washed from unbound antibodies of the patient and serum against human immunoglobulins labeled with the enzyme is applied. . The complex formed on the strip (antigen + antibody of the patient + antibody against human Ig) is detected by adding a chromogenic substrate that changes color under the action of the enzyme.
This methodology is also applied to the selection of clones of bacteria, phages or viruses expressing the products of the target cloned genes.
Transfer of proteins to the membrane is carried out either passively or using electrotransfer apparatus. The efficiency of protein transfer to the membrane is affected by many factors, such as the molecular weight of the proteins, the porosity of the gel, the transfer time, and the composition of the buffer solution (trans buffer) used.
Depending on the tasks and conditions of the experiment, transfer conditions are selected that provide the best results. The substrates commonly used are nitrocellulose, polyvinylidene difluoride (PVDF), or positively charged nylon membranes. Nitrocellulose can bind up to 80-100 micrograms of protein per 1 cm2.
Low molecular weight proteins (with a molecular weight of less than 20 kDa) can be lost as a result of washings, which makes it possible to preliminarily study the polymorphism of certain genetic loci by the lengths of the corresponding restriction DNA fragments.
In addition, using Southern hybridization, it is easy to find out whether the target gene has a site of hydrolysis by a certain restriction enzyme in its internal part, which allows you to choose the optimal strategy for cloning the region of the genome under study.
According to a similar scheme, RNA molecules can also be transferred from agarose gel to a nitrocellulose filter. This method has been called Northern blotting as opposed to Southern blotting, as the surname Southern means "southern" in English.
The transfer to filters from the protein gel was accordingly called Western blotting. Large proteins (greater than 100 kDa) denatured in sodium dodecyl sulfate (SDS) solution may be poorly transferred to the membrane if ethanol is present in the trans buffer. Alcohol significantly improves the transfer of proteins from the SDS-polyacrylamide gel, but narrows the pores in the gel, which leads to the retention of large proteins.
The PVDF membrane is optimized for immunodetection and is able to retain specifically bound proteins up to 160 µg/cm2 with a very low level of non-specific binding.
Immunoblotting
An important property of this membrane is the possibility of its repeated use. Zeta-Probe nylon membranes effectively bind SDS proteins in the absence of alcohol, and this binding is resistant to subsequent treatments. Low molecular weight proteins are also retained effectively. With a high binding capacity of approximately 480 μg of protein per 1 cm2, Zeta-Probe membranes allow the detection of trace amounts of protein in assay mixtures.
After the antigen is immobilized on the membrane, the remaining binding sites are blocked with solutions of gelatin, or bovine serum albumin, or skim milk.
Then the membrane is incubated in a solution of polyclonal or monoclonal antibodies to the tested antigen. After washing off unbound antibodies, the membrane is incubated in a solution of secondary antibodies, which are a conjugate of alkaline phosphatase enzymes (alkaline phosphatase, AP) or horseradish peroxidase (HRP) with anti-species antibodies (goat antibodies to rabbit, mouse or human immunoglobulins) or proteins A (Staphylococcus aureus protein) or G (Streptococcus sp. protein) having a high affinity for the Fc region of immunoglobulins.
Detection of the formed immune complexes is carried out by chemical or chemiluminescent method. Substrates for chemical reaction when using alkaline phosphatase conjugates are 5-bromo-4-chloro-3-indolyl phosphate (BCIP) or tetrazolium blue (NBT), and when using horseradish peroxidase conjugates - 4-chloro-1-naphthol and hydrogen peroxide.
As a result of enzymatic reactions, a colored band or spot is formed on the membrane at the site of the localization of the antigen-antibody complex.
The sensitivity of this method is 100 pg of protein when using AP conjugates and 100-500 pg when using HRP conjugates. Chemiluminescent detection of immune complexes can detect less than 5 pg of antigen. The principle of this method is that when HRP reacts with hydrogen peroxide and cyclic diacylhydrazineluminol, light is emitted at a wavelength of 428 nm, which can be recorded on a photosensitive film.
The immunoblotting reaction (RI) was developed on the basis of ELISA. It is the most specific and sensitive method of immunochemical analysis. Immunoblotting (from the English blot - to get wet, spot) combines ELISA with electrophoresis. It is used to detect not complex antibodies to HIV, but antibodies to its individual structural proteins (proteins-p24, glycoproteins-gp120, gp 41, etc.). Refer to expert (confirmatory) reactions for the diagnosis of HIV infection.
The reaction is carried out in several stages:
The virus is destroyed into components - antigens (p24, gp120, gp 41, etc.), which are subjected to electrophoresis in a polyacrylamide gel, that is, the separation of antigens into fractions by molecular weight.
2. The gel is covered with a nitrocellulose membrane and antigen fractions are transferred to it by means of electrophoresis. Nitrocellulose behaves like blotting paper. The membrane is cut into strips (strips). Firms produce such strips with "blots" of antigens.
Immunoblotting - an additional indirect method
Strips with HIV antigens applied to it are immersed in the serum of the subject and then washed from unbound material.
4. The strips are incubated with peroxidase-labeled antiglobulin serum and washed.
A substrate is added and the number of colored fractions (spots) is noted, which correspond to the localization zone of the AG-AT complex.
The presence of stripes in certain areas of the strip confirms the presence in the studied serum of antibodies to strictly defined HIV antigens. The result of immunoblotting is considered positive if bands corresponding to any two of the three HIV antigens - p24, gp41 and gp 120 are visible on the membrane (Fig. 37).
DRAWINGS
LIST OF USED LITERATURE
Main literature
Medical microbiology, virology and immunology: a textbook for medical students. universities 2nd ed., corrected. and additional — 702 p. Ed. A.A. Vorobyov. M. : MIA, 2012.
2. Microbiology, virology and immunology: a guide to laboratory studies: study guide / (V.B. Sboychakov et al.); ed. V.B.Sboychakova, M.M.Karapats. – M.: GEOTAR-Media, 2014.- 320 pp.: ill.
3. Medical microbiology, immunology and virology [Electronic resource]: a textbook for honey.
universities - 760 p. — Access mode: http://www.studmedlib.ru/book/ISBN9785299004250.html Korotyaev A.I., Babichev S.A. St. Petersburg: Spetslit, 2010.
4. Medical microbiology, virology and immunology [Electronic resource]: textbook: in 2 volumes / V. 1. - 448 p. — Access mode: http://www.studmedlib.ru/book/ISBN97859704142241.html Zverev V.V., Boychenko M.N.
M. : Geotar Media, 2010. .
5. Medical microbiology, virology and immunology [Electronic resource]: textbook: in 2 volumes. Vol. 2. - 480 p. Access mode: http://www.studmedlib.ru/book/ISBN97859704142242.html Zverev V.V., Boychenko M.N. M. : Geotar Media, 2010.
additional literature
1. Immunodiagnostic reactions: textbook / compiled by: G.K. Davletshina, Z.G. Gabidullin, A.A. Akhtarieva, M.M. .
Khusnarizanova, Yu.Z. Gabidullin, M.M. Alsynbaev - Ufa: Publishing House of GBOU VPO BSMU of the Ministry of Health of Russia, 2016. - 86p.
2. Features of some properties that determine the pathogenic potential of co-cultivated variations of Enterobacter, Сitrobacter, Serratia, E. coli, Proteus bacteria: scientific publication / Yu. Z. Gabidullin, R. S. Sufiyarov, I. I. Dolgushin - Ufa, 2015. - 250 s.
Home » Immunoblot - what is it? Immunoblot in the diagnosis of infectious diseases
Immunoblot - what is it? Immunoblot in the diagnosis of infectious diseases
What is an immunoblot? This is a common method for laboratory diagnosis of human viral infections. It is one of the most accurate and reliable ways to detect the presence of HIV.
For reliability, it is even larger than the enzyme-coupled immunosorbent (elisa) assay. Immunoblot results are considered conclusive and conclusive.General Information
Immunoblot - what is it? In order to recognize a person as HIV positive, you must undergo a laboratory test to test for the presence of antibodies in the blood serum.
The method of Western blot sections is also called Western blot (western blot). It is used to detect human viral infections, as an additional expert method. This is necessary to confirm the ELISA - a laboratory test that allows you to determine the presence of antibodies to HIV in the blood. Immunoblot recheck positive ELISA.
It is considered the most sensitive, complex and expensive.
Target
What is an immunoblot? This method of laboratory testing of blood serum for the presence of antibodies to the virus.
During special studies of the total viral proteins in the gel and on nitrocellulose membranes.
Immunoblotting (detection of antibodies in the sera of patients to certain pathogen antigens)
The Western blot section procedure is intended to determine HIV infection at different stages. In the first step, the purified virus from its constituent parts is subjected to electrophoresis and the antigens included in it, divided by the molecular weight.
The human immunodeficiency virus replicates in living cells embedded in its genetic information. At this stage, the person becomes a carrier of the HIV virus if you have been infected.
The specificity of this disease is that it does not manifest itself for a long time. The virus destroys lymphocytes, thus, a person's immunity decreases and the body becomes unable to fight infections.
If HIV is treated correctly and in a timely manner, the patient will live to a ripe old age. Lack of treatment inevitably leads to death. From the moment of infection, but without treatment, the maximum period is not more than ten years.
Peculiarities
Immunoblot analysis is a reliable method that allows you to determine the presence of antibodies to HIV antigens of the first and second type.
If a person is infected, after two weeks of antibodies, which can be detected much later. A feature of HIV is that the amount of antibodies increases rapidly and remains in the patient's blood. Even if they are present, the disease may not manifest itself for two or more years. The ELISA method does not always accurately indicate the presence of the disease, requiring confirmation of the results from PCR and Western blot sections if the enzyme immunoassay showed a positive result.
Indications for
What kind of “immunoblot” has already been found out, but which is being introduced in the study?
The reason for testing for the human immunodeficiency virus (HIV) immunoblot will be a positive ELISA result. It is necessary to go through an enzyme-linked immunosorbent assay in patients who are about to undergo surgery. In addition, you should make an analysis of women planning pregnancy, as well as those who are promiscuous. Western blot sections are given to patients with HIV infection if elisa results are questionable.
The following alarming symptoms may be the reason for visiting a doctor: rapid weight loss; weakness, loss of function; intestinal disorders (diarrhea) that lasts three weeks; dehydration; fever; swollen lymph nodes in the body; development of candidiasis, tuberculosis, pneumonia, toxoplasmosis, exacerbation of herpes.
The patient does not need to prepare before donating venous blood.
Do not eat for 8-10 hours before the test. It is not recommended to drink alcohol and coffee drinks, heavy physical exercises, to experience excitement the day before blood donation.
Where to do the analysis?
Where can I get tested for HIV?
ELISA, an immunoblot analysis carried out in urban private clinics, gives results within a day. Immediate diagnosis is also possible. In public institutions, elisa medical tests and Western blot sections are free of charge, in accordance with the legislation of the Russian Federation.
Mandatory screening for infectious diseases of pregnant women, and patients requiring hospitalization or surgery. How is this study carried out?
How to conduct an ELISA? Immunoblot positive/negative confirms or refutes elisa results. The procedure is quite simple. The specialist takes venous blood, in time it takes less than five minutes.
After sampling, the injection site should be disinfected and sealed with a plaster. The sampling is carried out on an empty stomach, so after the procedure it does not hurt to eat a bar of dark chocolate or a sweet hot drink.
To get a referral for a free analysis at a public medical institution, you must visit a therapist.
In general, the immunoblot does not differ from other blood tests by sampling. The research methodology is simple. If a virus is present in a person's blood, the body begins to produce antibodies to destroy it. There are many protein antigens for each virus. The detection of these antibodies is the basis of the Western blot sectioning method. Price
How much analysis? Immunoblot HIV refers to cheap research.
On average, screening immunoassay methods range from 500 to 900 rubles. Western blot sections is a study check, the cost of which ranges from three to five thousand rubles. More complex methods are much more expensive. For example, for the analysis of the polymerase chain reaction (PCR), you will need to pay about 12,000 rubles.
Interpretation of results
The most common methods for diagnosing HIV infection are enzyme immunoassay and immunoblot.
They are for the determination of serum antibodies to the human immunodeficiency virus. Infection is usually confirmed by two tests: screening and confirmatory. The interpretation of the results should be made by a doctor, he diagnoses and prescribes treatment. If the immunoblot is positive, it means that the human body has a virus.
A positive result should not be a reason for self-treatment, since each patient may have his own picture of the disease.
Qualitative analysis includes screening and certification. If the patient does not have the virus, the result is “negative”. When this certificate is found, additional screening tests are carried out. Immunoblot analysis that confirms or refutes screening. If the test strips appear in the darkening of certain areas (protein localization), the diagnosis is "HIV".
If the results are doubtful, then the tests are carried out within three months.
To prevent infection with the human immunodeficiency virus, it is possible if you follow certain rules: avoid casual sexual contact, use condoms during contact, do not use drugs.
If the disease is detected in a pregnant woman, it is important to follow the recommendations of the attending physician, do not forget about tests for the presence of the virus.
What is Western Blotting?
Protein identification in complex mixtures or extracts of various tissues is one of the frequently encountered problems. Using such a tool as specific antibodies, it is possible to determine the protein under study with a minimum of time and financial costs.
In the Western blotting method, at the first stage, a mixture of proteins is separated by electrophoresis in the presence of sodium dodecyl sulfate (SDS), then transferred to a nitrocellulose membrane by electroblotting.
The essence of this method lies in the fact that the gel after electrophoresis is placed on a nitrocellulose membrane between layers of filter paper. The "sandwich" assembled in this way is placed in an electric field so that the protein-SDS complexes move across the gel plate and are immobilized (as a result of nonspecific sorption) on the surface of the nitrocellulose membrane.
In the binding of the protein-SDS complex to the nitrocellulose membrane, mainly electrical forces are involved, and this interaction is multipoint and leads to the "spreading" of proteins on the membrane surface. Thus, after electrotransfer, we obtain a replica of the gel on nitrocellulose with proteins arranged in the same way as in the polyacrylamide gel.
After carrying out SDS - electrophoresis, electrotransfer and sorption of proteins from the gel onto a nitrocellulose membrane, the tertiary conformation of the protein is greatly changed, if it is generally correct to speak of the existence of a tertiary structure for the protein after such a harsh treatment. Therefore, for the immunochemical detection of the protein under study, only mono- or polyclonal antibodies specific to the linear regions of the protein molecule are usually used.
51. Enzyme immunoassay, immunoblotting. Mechanism, components, application.
Antibodies specific for conformational epitopes (or sites involving intersubunit contacts) are generally not suitable for use in the Western blot method.
After protein transfer, the membrane is incubated sequentially with antibodies specific to the protein under study, and then with secondary antibodies specific for the Fc fragments of the primary antibodies, conjugated with an enzyme (or some other) label (Fig.
1 A). In the case when primary antibodies specific to the studied antigen are directly conjugated with the label, secondary antibodies are not required (Fig. 1 B). The immune complexes formed at the site of the studied protein localization are “manifested” with the help of a chromogenic substrate (depending on the type of label).
The sensitivity and specificity of the method is highly dependent on which antibodies are used in the study.
The antibodies used must be specific to a unique amino acid sequence specific only to the protein under study. Otherwise, interaction (especially in the case of coarse protein extracts) of antibodies with several protein molecules is possible, which in turn will lead to the appearance of several colored bands on the membrane.
The identification of the protein under study in this case is often difficult or even impossible.
The second important factor to keep in mind when choosing antibodies is affinity. The higher the affinity of the antibodies used, the brighter and clearer the protein bands stain, the higher the sensitivity of the method. When using high affinity antibodies, sensitivity of 1 ng and even higher can be achieved.
To visualize the result of the interaction of the membrane-bound antigen and antibodies, secondary antibodies conjugated with agents capable of giving a certain signal under certain conditions are used.
Usually, an enzyme (peroxidase or phosphatase) is used as such an agent, the reaction product of which has a color and precipitates on the membrane in the form of an insoluble precipitate.
It is also possible to use fluorescent labels in this method.
Rice. 1. Scheme of immunochemical staining of the studied protein: A - using secondary antibodies conjugated with an enzyme label; B - the primary antibody is directly conjugated with an enzyme label.
Protocol:
I. Gel and membrane preparation and protein electrotransfer
The polyacrylamide gel after electrophoresis was placed in a bath with blotting buffer (25 mM Tris, pH 8.3, 192 mM glycine, 10% ethanol).
Two sheets of filter paper, cut to the shape of the blotting cassette and moistened with blotting buffer, are placed on the part of the cassette that will face the anode. Then, a nitrocellulose membrane previously moistened with the same buffer is placed on the filter paper, making sure that there are no air bubbles between the membrane and the paper.
After that, the gel should be carefully placed on the membrane, again paying special attention to the absence of air bubbles between the gel and the membrane. The sandwich is completed by two layers of wetted filter paper, which are placed on the surface of the gel (Fig. 2). The resulting sandwich is clamped in the cassette and placed between the electrodes so that the membrane faces the anode.
Rice. 2. Scheme of electrotransfer of proteins to the membrane.
II. electrotransfer
Electrotransfer of proteins to a nitrocellulose membrane is carried out in a buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, 10% ethanol for 30–50 min at a constant voltage of 100 V.
The electrotransfer time depends on the size of the transferred proteins, the larger the protein, the longer the electrotransfer takes. The quality of electrotransport and the arrangement of protein bands was assessed by staining the nitrocellulose membrane with 0.3% Ponceau S in 1% acetic acid. Before immunochemical staining, the membrane should be washed several times with a mildly alkaline aqueous Tris solution to remove protein-bound dye.
III. Immunochemical staining of proteins immobilized on a nitrocellulose membrane
To block non-specific antibody binding sites, the membrane is incubated with constant stirring at room temperature for 30 min in PBST (for better blocking, a PBST solution containing 10% skimmed milk powder can be used).
After blocking, the membrane is incubated for one hour at room temperature with constant stirring in PBST containing 1-10 μg/ml of specific antibodies.
The optimal concentration of antibodies is selected empirically and depends on the affinity of the interaction of antibodies with the antigen.
At the end of the incubation, the membrane was washed 5 times with PBST and transferred to a solution of secondary antibodies conjugated with horseradish peroxidase. The dilution of the conjugate is usually indicated by the manufacturer on the packaging, or is selected empirically by the researcher. Incubate the membrane in a solution of secondary antibodies for 1 hour with constant stirring.
After thorough washing (at least 5-6 buffer changes), the PBST membrane is transferred into a chromogenic substrate solution containing 3 mg diaminobenzidine (DAB) and 10 µl of 30% hydrogen peroxide in 10 ml of 0.1 M Tris-HCl, pH 7.6.
Incubation is carried out with stirring for 5 to 10 minutes. After the end of incubation with the substrate, the membrane should be washed with PBST, dried by blotting with filter paper, and immediately made an electronic copy by scanning in color. If the membrane dries out completely, the dyed protein stripes fade, and the image is less bright and contrasty.
Note: DAB is toxic and a potential carcinogen. Work only with rubber gloves!