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The different generations of ELISA

When HIV was identified as the virus that causes AIDS way back in 1986, there was a flurry of research looking into ways of identifying individuals who have been infected with the HIV virus.

At the time, there was already a technique developed to detect various antibodies in the blood. This technique was called ELISA which stood for Enzyme Linked Immuno Sorbent Assay.

This technique is based on the lock and key theory of antibodies. Basically, antibodies and antigens work like locks and keys. One key for one lock. One antibody fits one antigen. Having the antibody means the antigen is also present.

So the ELISA technique basically involves getting a little cup (aka microwell) and sticking HIV antigens (locks) all over the bottom. The cup is then filled with the serum to be tested. If the appropriate anti-HIV antibodies are present (keys), they will stick to the antigens (locks). So far so good?

Now this is the clever bit. Since these antigens and antibodies are microscopic, the scientists had to figure out a way to be able to see if the locks have captured any keys. Microscopes do not work because these antigens and antibodies are just too small. They figured out that since antibodies are proteins too, they themselves are also antigens! In other words, the other end of the key is also a lock. So the scientist developed an anti-HIV antibody antibody. So this new antibody sticks to the back of the first antibody. This second antibody is also special in the fact that an enzyme is attached to it. When a special substrate is added to the mix, the enzyme will turn the substrate a different color (usually red). So there you have it. If the serum to be tested contains anti-HIV antibodies, after all these steps, the liquid in the microwell will turn red. So elegant and so simple. This came to be known as the 1st generation HIV ELISA test.

Basically, antibodies and antigens work like locks and keys.

However there were several problems associated with this test. First of all, it only detected antibodies to the HIV 1 virus, not HIV 2. Secondly, it only detected the IgG antibodies which can take some time to be produced in the body up to levels which where detectable and hence the testing window period. Thirdly, the antigen purification was not very good which led to a high level of false positives and hence the need for confirmation by Western Blot.

There wasn’t a big leap of technology going into the 2nd generation test. Scientist just basically figured out a way to incorporate HIV 2 antigens into the test. There were also better antigen production techniques that improved the false positive rate. However the science behind the test went largely unchanged.

There was however a huge leap of technology from the 2nd to 3rd generation ELISAs. The main change was the 3rd generation ELISAs could detect IgM as well as IgG antibodies. Because the body produces IgM way before it produces IgG, this significantly reduced the testing window period.

How the scientist did this was to develop what is now known as a ‘Sandwich ELISA’ technique. Similar to the previous ELISA tests, HIV 1 and 2 antigens were placed in the microwell. The anti-HIV IgM antibody would attach to the antigens. Now the thing about the IgM antibody is that it is like many keys attached to each other. In other words there are multiple antigen binding sites. This is very different from IgG that has just 1 antigen binding site. So what the scientist figured out how to do was to stick the enzyme and a HIV antigen together. So this antigen-enzyme conjugate will stick onto the other binding sites of the IgM antibody that has stuck onto the antigens in the microwell. The enzyme on the antigen then converts the substrate color thus enabling detection. Since the IgM antibody is ‘sandwiched’ between 2 antigens, the name ‘sandwich ELISA’ was coined.

3rd generation ELISAs could detect IgM as well as IgG antibodies

Lastly, we come to the so called 4th generation ELISA test. Not many people agree with this nomenclature because technically, it is identical to the 3rd generation ELISA test. Basically the 4th generation ELISA test is putting 2 different tests (i.e 3rd generation ELISA and P24 antigen test) onto the same test strip. Because the P24 antigen is produced even earlier than the IgM antibody, this reduces the testing window period even further. So you can see that there isn’t really a ‘4th generation ELISA’.

So now you know everything you need to know about the different generation ELISA tests. Hopefully that gives you more confidence in discussing tests and test results with your doctor.

Feel free to post any questions you have in the comments section below.
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About Dr. Tan
Dr. Tan graduated from the National University of Singapore in 2001. His residency was in the two largest public hospitals in Singapore; Tan Tock Seng Hospital and Singapore General Hospital.

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65 Comments

  1. Worried guy

    Dear Dr Justin, I had protected sex with fingering and kissing and have hiv negative test after 47th days with cmia duo test. Some people are getting variations in index value in increasing order in more than 1 test with negative cutoff . Its a cause of concern for me and is my test conclusive or hv to retest again after 3 months. Regards

  2. Robert

    Hello sir,

    I encountered a possible exposure to HIV. Had a 4th gen combo test at 6th week. The result came out to be negative. Is it reasonable to conclude that I am HIV negative or do I absolutely need to do the 3 month test as well.
    Also, could you clarify if the P24 antigen is reduced to undetectable levels because of the antibody production. Or, is there other reason for that at around 6 week. And, does it also mean that one of the two (either P24 or antibodies) will be detected by the test at 6th week ?

    • Yes it is reasonable to assume a 4th Gen test is conclusive at 6 weeks. Whether or not you need a 3 months test is something you should verify with your doctor.

  3. John abraham

    I got a tattoo around three years ago. I recently got elisa 2nd generation test done and it turned out to be non-reactive. What are the chances that it is a false negative. Can technical or human error give false negative. Am I confirmed negative or do you recommend getting the test done again at a different health centre. I also wanted to know if 3rd generation is more accurate than 2nd generation.

    • I am not able to comment on the chances of false negative because every test in the world has a chance of giving false negative results. If you are still concerned, you can get another test done for the peace of mind.

  4. Dr tan
    i protected sex with a unknow girl after 2 days i have noticed a rash on my stomach and i feel like tried so i am go to hiv elisa test after 4 week that was negative.. Now ver worried about hiv symPtoms..

    • OK. So what is your question?

      • Dixon Pj

        My elisa test after 39 days negative but i dont know which generation test am take(cmia vitros eci this technology used) ……. what is elisa test window period?

        • That is something you really should ask the lab/doctor who did the test for you. ELISA simply refers to a test methodology and does not tell us what test was run for you.

  5. charles

    Hi Dr. Tan this is my situation. I had unprotected sex with a stranger. At 2 weeks I take a antigen p24 chemiluminescence lab test with no reactive results. At 9 weeks I take a 4th gen hiv test ( hiv 1 & 2 ab & ag p24) by chemiluminescence. Can I take this results as conclusive? Or should retest at 12 weeks?

  6. charles

    Hi dr. I had unprotected sex about 9 weeks before with a stranger. At 15 days i had a p24 lab test by chemiluninescence and At 9 weeks after my exposure i take a hiv 4th generation ab & ag p24 lab test by chemiluminescence. Both test were negative. Should i consider my results conclusive? or should test at 12 weeks?

  7. Hi doctor, i had test 4th gen combo sd bioline on 53 days (7.5 weeks) and 76 days (10 weeks 6 days) both results are NEGATIVE. Can i take these as conclusive as 12 weeks? I didn test at 6 weeks at all. Thank you.

    • drtan

      In my opinion your tests are conclusive. Please verify this with your doctor.

      • Yeah my doctor told me this is conclusive and no further test needed. But m still worry since it is not full 84 days yet

      • So it is included hiv-2 as well doctor? Since i heard hiv 2 can only b detected after 3 months. Thank you doc

  8. Hi,

    Much useful information.

  9. Dear Dr
    I had unprotected vaginal sex with a woman while touring a different country. On return to my country I got a 3rd gen ELISA test after approx 3-4 weeks of intercourse. The result was non reactive negative. Should I consider the test conclusive? Also I am going in for a P24 antigen check tomorrow. Will a non reactive test prove conclusive now or should I be concerned?

    • 3rd generation ELISA test is not conclusive on week 3-4. It is only conclusive after 12 weeks. Please go ahead with p24 antigen check. If it is negative, it is indicative that you have not been infected with HIV.

      • Hello Dr,
        The test I carried out mentions its name as “HIV 4th Generation Sandwich ELISA HIV1 P24 Antigen test”. The OD value result given was 0.42 which as per the result parameters said that it was negative(0.9 being the limit). Is my test conclusive today because I last had unprotected sex 3 weeks or more back?

        • 4th generation Hiv test is conclusive on day 28. A negative result on week 3 is a good indicator that you have not been infected. If you are worried, please repeat the test on day 28.

  10. hello, please how accurate is alere determine hiv 1 / 2 antibody after four months of possible exposure. broken condom to a lady of unknown status. I have symptoms like oral thrush and slight rash on my arms and tingling hands. thanks

  11. rohit sharma

    hello sir,
    is Hiv-1 Rna pcr test negative is conclusive after 20 days and what about Hiv-2 rna test, is there any
    is ortho-Vitros Eci a 3 gen antibody test or 4 gen

  12. Hello Doctor.

    I had my risky exposure 26 days before my negative result from a antigen/antibody hiv test. Would you consider that negative to be conclusive?

    God bless

  13. Hello Dr tan
    I am bahu from India.I had unprotected vaginal sex on 24/06/2014 with aunt who is HIV 1/2 tridot NEGATIVE after 6 months from my sex I.e first and last in my life.I suffer with chronic pharyngitis from 7 months,red bumps on my chest,weight loss 6 kg/70kcokcold from 4 months.These symptoms appears from 3 months of my sex contact and its doesn’t go away to now. My hiv test results are
    (1) 1.5 ,3 month hiv1/2 tridot j.mitra antibody NEGATIVE .
    (2) 4th,5th,6th month sd bioline hiv 1/2 3.0 finger prick antibody NEGATIVE.
    (3). At 6.5 months hiv1rna pcr qualitative antigen NOT DETECTED
    50 copies/ml.
    (4) at 8th month sd bioline antibody NEGATIVE
    (5) at 9th ,10 th month comb aids 3 rd gen antibody NEGATIVE.
    (6) at 11 the month hiv1/2 ELISA 3rd gen antibody NEGATIVE
    (7) at 12 th month,13 month sd bioline hiv1/2 antibody NEGATIVE.

    Please clarify my questions sir
    1 is the above said symptoms are
    belongs to hiv symptoms 100% or not
    2.my test results are gives false negative or not
    3. Are my HIV status 100 % NEGATIVE or not
    4 some one says its show positive results after 2,3,4 years later, is it truth or not
    Please tell me answer to all

    • Dr Justin Sim

      Your test results are conclusive and show you are negative for HIV. This means your symptoms are caused by something else entirely. It is almost zero chance that this many tests will be all false negatives. It will not take 2-4 years for the antibody to show up. It only takes 3 months.

  14. worryandworry

    dear doctor…i intend to have a 3rd gen test at your clinic when exactly 12 weeks after exposure~so.the result comes out should be conclusive, right?

  15. Dr. I had a 3rd Generation negative at 8 weeks (Technically 54 Days not 56) – and I tested negative..

    HIV 1/O/2 Abs-Index Value Result <1.00

    Is this fairly conclusive or do I need to re-test?

  16. Sridevi Rayasam

    Dr. Tan

    The description and the way you went about the history and evolution of HIV ELISAs is extremely simple and very very comprehensive. I found it easy to follow as ‘a lay man’s inspite of being a PG in Medical Microbiology and knowing all the technical terminology.
    I look forward to more such clear & crisp responses in the future.
    Have a good day Sir.
    Sridevi Rayasam

    • Thank you very much for the positive feedback. It is even more meaningful coming from a fellow scientist. We will strive to continue to put out more of such information.

  17. Please what type of test are we using in Africa especially Nigeria. Becouse am concern about our type of test, I have just finished PEP 1month now.

    • I’m afraid I do not know that. Also, different hospitals and labs in any one country can also use different tests. The best thing to do is to ask the doctor doing the test for you exactly what HIV test he is using.

  18. i tested negative 5 months after exposure upon PEP,i have had fever,headache and now joint paint in the legs and hands (elbow & knee and thighs),this started days after i went for my last hiv test on 08th dec,2014,since the result was negative,could it now be an infection of the virus like AIDS?
    should i go again this January as is the 6th month? please help me

    • Some guidelines say testing 3 months post exposure for people on PEP is enough. Some say 6 months. Personally I think 3 months is enough.

  19. i Had a protecteed sex but I noticed blood stain on the condom after have ejauclated inside the condom. My concern is can hiv penetrate condom through blood because I found out that my partner is positive but lied to be negative before the sex. The condom didn’t break and never expired. I did test at 20days,27days and 35days after exposures and all came back negative but am still scared(anxiety). Can my hiv negative result change or is it a good indicator of what to expect in 3months. The laboratory said they used determine hiv 1/2kits. Thank you.

    • Well, condoms are not 100% protective against HIV even if they do not break. The presence of blood does increase the HIV risk. So all in all I must say that you are at risk of HIV but I think the risk is low. Also, the Determine HIV test at 35 days is already very predictive. Although I cannot say you are 100% free of HIV, I think your chances are pretty good.

      • Have done another hiv test at 46days and 70days or 10weeks all negative using the same determine hiv 1/2kits. Is my test scientifically conclusive. Thank you Sir

        • No you have to do it again at 3 months post exposure.

          • But is my 70days negative test reliable and is it a sign of what to expect in 84days. Thank you.

          • According to your analysis of window period as analysed below

            Between day 28 and 48, the IgG Antibody starts to become detectable in the blood.
            This is detectable by 1st, 2nd, 3rd and 4th Generation HIV tests.

            Since have passed the stated window period of all hiv generation kits even with additional 22days (70daysminus48days). Thank you Sir

  20. shasha

    Hi Dr Tan,

    my friend had rapid test few weeks ago. we are surprised when the result is ‘reactive’. and now, he’s going to do the confirmation test. He said, he never have unprotected sex/practice/expose with any related sexual activity or other risky life style. It is possible the result is ‘false positive’? What do you suggest?

    • Hi shasha,

      I understand this must be quite a worrying experience for your friend. There are cases where you may get a false positive. For this reason, all tests must be confirmed with a follow-up test, usually a Western Blot or a HIV RNA/DNA PCR. I’d advise your friend to follow- through with the testing to know for sure if this was just a false positive or something else. DO speak to your doctor for more advice.

  21. Dear Dr.Tan

    i have done a rapid HIV test (oralquick based on fingerprick) about 5 month aft the exposure(i am not sure if the man got HIV), the result showed negative.
    But i am still worrying abt if i need to do another ELISA test in your clinic, more lab-based, instead of the rapid oralquick test, in order to confirm that i am 100% negative..i doubt the accuracy of the rapid HIV test..thanks

    • Most people will accept the OraQuick at 5 months to be conclusive. You should too. Test again if you need the peace of mind.

  22. hello dr tan,i have a question;
    1) i have been expose for about 6 weeks and 5 days before my hiv test
    as the result were negative,as written on my medical report the test was done by cmia principle
    ans i saw ag/ab there..and shown undetected..was that mean they test both p24 antigen
    and my antibody since i saw in my report that effective from january 2013 all test in Malaysia
    will use cmia principle..so can consider that as a fourth generation test?

    2) im planning to do another test after 3 months post exposure but for the time being pls
    tell me that i can rely on my 6 weeks result..im too depress now…thank you dr tan.

  23. Dear doctor,
    What is the difference in doing dna pcr and rna pcr for hiv

    • RNA – virus. DNA – pro-virus. There is more data for using the RNA as a diagnostic test. Also, RNA is detectable earlier than DNA.

  24. Hello Dr. Tan,

    I’d like to ask you a question, is a 3th Generation ELISA at 6 weeks after exposure conclusive? or 8 weeks? And how about the length of the window period for the 4th?

    And also, is ICMA conclusive at 6 weeks?

    thank you so much!

    • 3rd Gen is as good as conclusive at 6 weeks much less 8 weeks. But officially the window period is 12 weeks. Window period for 4th Gen is 4 weeks but some countries say 8 weeks. ICMA is like 3rd Gen.

  25. patrick

    Quick question, what generation is the ICMA test comparable too and what type of window of detection are we looking at. I had a PCR RNA 10 days after possible exposure with <20 copies and one done at 29 days. Both came back negative. With the RNA test done at 29 days I also took this ICMA antibody test which was also negative. I just cant seem to find what this test is comparable in terms of the 4th gen duo, 3rd gen elisa, etc..

    Thanks for your time!!

    • There is no direct comparison. A dependable window period for the ICMA is 3 months. I do not see why you are concerned about the ICMA when you already have 2 negative RNA PCRs. This is already conclusive. Please post follow up questions on http://www.askdrtan.com

  26. hi dr. tan,

    i had unprotected sex last august 10, 2012. when i got here in singapre i was tested for hiv test assuming that it is elisa since it is needed by my employer for my spass approval. that test was on oct 8 and retaken on oct. 23 of the same year.. the result test is both negative. do u think i still need to be retest again? do u think its conclusive enough? thank u so much..

    • I think it is conclusive enough. However, the official HIV testing window period as recommended by MOH here in Singapore is still 3 months. Please post any follow up questions on http://www.askdrtan.com

  27. Hi Dr Tan

    I recently did a HIV testing at your medical centre, I was just wondering if the results are extremely accurate after 11 months plus to a year after exposure? And are Rapid Hiv tests more accurate then others ?

    Thanks

  28. Hi Dr greetings from malaysia.

    Could I ask whether negative on Abott 4th generation combo test on 35 days after vaginal sex conclusive ?

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