ELISA, Western Blot, and Other Tests for HIV (2022)

HIV is a virus that attacks the immune system. If HIV infection isn’t treated, a person can develop AIDS, which is a prolonged and often fatal condition. HIV is spread through vaginal, oral, or anal sexual contact. It’s also spread through blood, blood factor products, injection drug use, and breast milk.

To test for HIV, a series of blood screenings may be done, including one called the ELISA test. Read on to learn how these tests are done, what to expect during the tests, and what the results can mean.

The enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), detects HIV antibodies and antigens in the blood.

Antibodies are proteins produced by the immune system, which helps your body fight disease. The immune system produces the antibodies in response to the presence of foreign substances, such as viruses. By contrast, antigens are any foreign substance in the body that causes the immune system to respond.

The ELISA test is typically the first test ordered by a healthcare provider. In case of a positive result from this test, the ELISA test was previously followed by a test called a Western blot to confirm the diagnosis. However, the Western blot is no longer used, and today the ELISA test is followed by an HIV differentiation assay to confirm HIV infection. The provider may also order an HIV genetic material detection test.

The ELISA test is recommended if a person has been exposed to HIV or is at risk for contracting HIV. Those at risk for contracting HIV include:

  • people who use intravenous (IV) drugs
  • people who have sex without a condom, especially with someone who has HIV or an unknown HIV status
  • people who have had sexually transmitted diseases (STDs)
  • people who had blood transfusions or blood clotting factor injections before 1985

People may opt to have the test done if they’re uncertain about their HIV status, even if they’re not in a high-risk group. For people who participate in high-risk behaviors, such as IV drug use or sex without a condom, it’s a good idea to get tested on a regular basis. And the Centers for Disease Control and Prevention (CDC) recommends that all adults get tested at least once for HIV.

(Video) Diagnosis and Testing of HIV Infection

There’s no need to prepare for an ELISA test or a differentiation assay. These tests are done using a blood sample, and it takes very little time to give a blood sample. However, to get the test results, it may take several days, and in some cases weeks.

People with a fear of needles or who faint at the sight of blood should be sure to tell the healthcare provider as well as the laboratory technician. These clinicians can take precautions to help ensure safety in case the person faints.

(Video) HIV Testing - ELISA & Western Blot Test

Before the test, a healthcare provider will explain the procedure. The person having the test will probably need to sign a consent form.

To help prevent any problems during the test, the person should be sure to tell the healthcare provider if:

  • they’ve had trouble giving blood in the past
  • they bruise easily
  • they have a bleeding disorder, such as hemophilia
  • they’re taking anticoagulant medications (blood thinners)

During the test

The procedure for getting a sample of blood is the same for both tests. A medical professional will:

  • clean the skin site where they plan to draw blood
  • apply a tourniquet, or elastic band, around the arm to make the veins swell with blood
  • place a needle into one of the veins and draw a small sample of blood into a tube
  • remove the needle and apply a bandage

To decrease further bleeding, after the test the person may be asked to elevate or flex their arm to reduce blood flow.

Giving a blood sample isn’t painful, though the person may feel a sting or a pricking sensation as the needle goes into their vein. Their arm may throb slightly after the procedure.

Testing the blood

For the ELISA test, the blood sample will be sent to a laboratory for analysis. A lab technician will add the sample to a device that contains HIV antigen and anti-HIV antibodies.

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An automated process will add an enzyme to the device. The enzyme helps speed up chemical reactions. Afterward, the reaction of the blood and the antigen will be monitored. If the blood contains antibodies to HIV or antigens of HIV, it will bind with the antigen or antibody in the device. If this binding is detected, the person may have HIV.

The differentiation assay is very similar, but instead of an automated machine, the device can be handled by a lab technician. The specific antibodies and antigens in the blood are separated and identified in a different immunoassay device.

These tests are very safe, but rare complications can occur. For example, the person may:

  • feel lightheaded or faint, especially if they have a fear of needles or blood
  • get an infection at the site of the needle insertion
  • develop a bruise at the puncture site
  • have trouble stopping the bleeding

The person should contact their healthcare provider right away if they experience any of these complications.

What do the test results mean?

(Video) Western blot | Western blotting protocol | Application of Western Blot | Limitations of Western blot

If a person tests positive for HIV on the ELISA test, they might have HIV. However, there can be false positives with the ELISA test. This means that test results indicate that the person has HIV when they actually do not. For example, having certain conditions such as Lyme disease, syphilis, or lupus may produce a false positive for HIV in an ELISA test.

For this reason, after a positive ELISA test, more sophisticated tests are done to confirm whether the person has HIV. These tests include the differentiation assay and a test called the nucleic acid test (NAT). If the person tests positive for HIV with either one of these tests, they probably have HIV.

Sometimes, HIV doesn’t show up on the ELISA test even though a person has an HIV infection. This can happen if someone is in the early stages of the infection, and their body hasn’t produced enough antibodies (in response to the virus) for the tests to detect. This early stage of HIV infection, in which a person has HIV but tests negative for it, is known as the “window period.”

According to the CDC, a person’s window period is usually between three and 12 weeks. However, in rare cases, some people can take as long as six months to develop antibodies.

Though both the ELISA test and the differentiation test are simple and straightforward, waiting for the results can create anxiety. In many cases, a person will have to talk to someone either in person or over the phone to receive their results, regardless of whether they’re positive or negative. A positive test result can trigger strong emotions. If needed, the person’s healthcare provider can refer them to counseling or HIV support groups.

(Video) Diagnosing HIV - Concepts and tests | Infectious diseases | NCLEX-RN | Khan Academy

Although HIV is very serious, it’s important to keep in mind that today there is medication available that can help prevent HIV infection from developing into AIDS. It’s possible for someone with HIV to live a long and full life. And the earlier a person learns their HIV status, the earlier they can begin treatment to prevent health complications or transmission of the infection to others.


What are the ELISA and Western blot tests used to detect HIV? ›

ELISA Test ELISA, which stands for enzyme-linked immunosorbent assay, is used to detect HIV infection. If an ELISA test is positive, the Western blot test is usually administered to confirm the diagnosis. If an ELISA test is negative, but you think you may have HIV, you should be tested again in one to three months.

Which is more accurate ELISA or Western blot? ›

The Western blot test separates the blood proteins and detects the specific proteins (called HIV antibodies) that indicate an HIV infection. The Western blot is used to confirm a positive ELISA, and the combined tests are 99.9% accurate.

What is the most accurate way to test for HIV? ›

Antigen/antibody tests are recommended for testing done in labs and are common in the United States. An antigen/antibody test performed by a lab on blood from a vein can usually detect HIV 18 to 45 days after exposure. There is also a rapid antigen/antibody test available that is done with a finger stick.

How accurate is ELISA test for HIV? ›

Although false negative or false positive results are extremely rare, they may occur if the patient has not yet developed antibodies to HIV or if a mistake was made at the laboratory. When used in combination with the confirmatory Western blot test, ELISA tests are 99.9% accurate.

Can ELISA test give false positive? ›

False positive results are not uncommon in ELISA for HIV [4], specially in studies of low-risk individuals. In one such study only 13 per cent of ELISA-positive individuals among voluntary blood donors actually had HIV infection [5].

What is the difference between ELISA and Western blot? ›

ELISA vs Western Blot

ELISA is an immunosorbent assay that is used to detect antibodies or antigen in a sample. Western blotting is an analytical technique that is used to separate and identify proteins from a mixture.

Can Western blot test be wrong? ›

Since the Western blot test checks for antibodies, it may give false negatives for both conditions if it's administered too soon. In some instances, certain conditions such as lupus may also cause false positives to occur.

What if ELISA test is negative? ›

If you get an HIV test after a potential HIV exposure and the result is negative, get tested again after the window period for the test you took. If you test again after the window period, have no possible HIV exposure during that time, and the result is negative, you do not have HIV.

How accurate is ELISA test after 6 weeks? ›

Yes. Modern HIV tests are very accurate.

For example, 4th generation tests will pick up 99% of infections at six week. Testing earlier, for example after four week, only detects 95% of infections. Testing earlier needs a confirmatory test three months after the risk.

Can I test negative if my viral load is undetectable? ›

The bottom line is that if you're living with HIV and have an undetectable viral load, you will still test positive for HIV if you get tested. But, this is expected, and doesn't mean that your treatment is not working or that you aren't undetectable.

What does a positive ELISA test indicate? ›

An ELISA test is a blood test that looks for antibodies in your bloodstream. When certain antibodies are present, it's a sign your immune system is trying to fight off a disease.

What are the advantages of ELISA test? ›

ELISA exhibits the following advantages: (i) Simple procedure. (ii) High specificity and sensitivity, because of an antigen–antibody reaction. (iii) High efficiency, as simultaneous analyses can be performed without complicated sample pre-treatment.

When is ELISA used? ›

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum.

Why Western blot is not recommended? ›

The HIV-1 Western blot (WB), the historic gold standard for laboratory diagnosis of HIV-1 infection, is no longer part of the recommended algorithm. The two main reasons for this are the inability of the WB to detect acute infection and the potential to misclassify HIV-2 infection as an HIV-1 infection.

What is the ELISA test used for? ›

This test is often used to see if you have been exposed to viruses or other substances that cause infection. It is also used to screen for current or past infections.

What does Western blot detect? ›

Western blotting is a laboratory technique used to detect a specific protein in a blood or tissue sample. The method involves using gel electrophoresis to separate the sample's proteins. The separated proteins are transferred out of the gel to the surface of a membrane.

What type of test is Western blot? ›

The Western blot test is an antibody test that's done on a blood sample. It's used to confirm or disprove the results of an earlier test for HIV or Lyme disease. Technological advances have produced more accurate tests that give faster results than the Western blot test.

Which disease is diagnosed by ELISA test? ›

The assay used most widely to detect or diagnose virus infection, especially infection of blood borne viruses e.g. HBV, HCV, HIV and HTLV, is the enzyme linked immunosorbent assay (ELISA), whose sensitivity and practicability have rendered it the most common primary screening assay.

How accurate is ELISA test after 6 weeks? ›

Yes. Modern HIV tests are very accurate.

For example, 4th generation tests will pick up 99% of infections at six week. Testing earlier, for example after four week, only detects 95% of infections. Testing earlier needs a confirmatory test three months after the risk.

What are the 4 steps of ELISA? ›

The Direct ELISA Procedure can be summarised into 4 steps: Plate Coating, Plate Blocking, Antibody Incubation, and Detection.

Is ELISA qualitative or quantitative? ›

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical.

Why is it called a western blot? ›

Burnette definitely gave the technique the name "Western blotting" as a nod to Southern blotting and because their lab was on the west coast. He developed his technique independently, including the electrophoretic transfer step, but became aware of Stark's and Towbin's publications before he submitted his in 1979.

What are the limitations of western blot? ›

The main limitation of western blotting is that it can only be carried out if a primary antibody against the protein of interest is available. To detect post-translational modifications such as phosphorylation of target proteins, specific antibodies against the phosphorylated residues are needed.

How do you present western blot results? ›

When presenting a western blot in a Starr lab meeting or presentation, include the following information: Title: Date, protein(s) and cell lysates including conditions being analyzed. Subtitle: Your initials, and date where details can be found in your lab book (see Lab Book Details).

Can a Western blot be false positive? ›

The rate of false-positive Western blot results documented in this study was 0.00041% of all donations tested (95% CI, 0.00026%-0.00058%) and 4.8% of donations with results classified as Western blot positive.

Can ELISA detect virus? ›

Whereas virus neutralization, haemagglutination inhibition and enzyme-linked immunosorbent assay (ELISA) have been used for the detection of viral specific antibodies or viral proteins [5].

How can a viral infection be detected by ELISA? ›

The bound antibodies are then detected by using a second antibody that binds to the first antibody. ELISA is used in both experimental and diagnostic virology. It is a highly sensitive assay that can detect proteins at the picomolar to nanomolar range (10-12 to 10-9 moles per liter).

What are three limitations of ELISA? ›

Three most common ELISA Challenges
  • Weak or Low Signal Intensity. Are you repeatedly getting readings below the lower limit of absorbance? ...
  • High Background. ...
  • High Well-to-Well Variation.
18 Dec 2018


1. How to run HIV BLOT 2.2 (CE) immunoassay using AutoBlot System 48 [MP Biomedicals Asia Pacific]
(MP Biomedicals)
2. AIDS:HIV : Diagnosis
(USMLE exam gym)
3. mr i explains: The ELISA test for HIV (Enzyme-linked Immunosorbant Assay)
(mr i explains)
4. Is rapid HIV testing more accurate than ELISA? - Dr. Ramakrishna Prasad
(Doctors' Circle World's Largest Health Platform)
5. Negative results of 4th generation HIV & ELISA tests after few months - Dr. Ramakrishna Prasad
(Doctors' Circle World's Largest Health Platform)
6. Accuracy of HIV antibody ELISA test after 5 6 months of exposure - Dr. Ramakrishna Prasad
(Doctors' Circle World's Largest Health Platform)

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