Detection of HIV-1 p24 Antigen using New Generation ELISA Kit 2.0

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Human Immunodeficiency Virus Type 1 (HIV-1) is an enveloped retrovirus approximately 120 nm in diameter. It contains two copies of a single stranded, positive-sense RNA. HIV-1 (Strain: BaL)

HIV-1 p24 Antigen ELISA 2.0

The ELISA (enzyme-linked immunosorbent assay) is generally used to detect and quantify antigens and antibodies from human serum or plasma.

The HIV-1 p24 antigen is known as a capsid protein since it is the protein composing the capsid of the virus. It encloses into a two-domain structure forming a loop of about five to six members through self-association.

It is possible to detect the presence of p24 protein circulating in the blood before the presence of HIV antibodies during acute infection. As such, HIV-1 p24 is considered to be an early detection marker. This occurs early after infection because of the initial burst of virus replication. This early stage is associated with high levels of viremia during which the individual is highly infectious.

Antigen testing was implemented in the United States in 1995 to detect antibodies in donated blood components. It has been used to detect HIV-contaminated units, which had previously tested antibody non-reactive. The period from the time after infection and before seroconversion, during which markers of infection (p24 antigen and antibodies) are still absent or too scarce to be detectable, is referred to as the window period.

Analysis has shown that fourth-generation laboratory tests (which detect both antibodies and p24 antigen) detect HIV infections between one and three weeks earlier than antibody-only tests.

Intended Use

The HIV-1 p24 Antigen ELISA 2.0 is for research purposes only. It is not for screening, diagnosis, nor for use as a confirmatory test for reactive samples. The HIV-1 p24 Antigen ELISA 2.0 kit is used for the detection of Human Immunodeficiency Virus Type 1 (HIV-1) p24 antigen in cell culture media. It can also be used to monitor the purification and biochemical behaviour of HIV-1 or to determine the titer of HIV-1 based lentiviral samples.

This assay detects p24 from HIV-1 subtypes A.F.

There is no cross-reactivity to Human Immunodeficiency Virus Type.2, Human T-Cell Leukemia Virus Types 1 and 11 (HTLV 1 and 11) or Simian Immunodeficiency Virus (SIV).

Principle of the test

ELISA plates available for detecting HIV p24 antibodies or antigens are conventionally packaged to include microplates or pre-coated strips and related reagents.

A monoclonal antibody test specialized for the HIV-1 p24 gag gene product of HIV-1coats the microwells. While the specimen is incubated, a target antigen present in the sample is attached to a confined enzyme-labelled secondary antibody. Later, the captured antigen reacts with a human anti-HIV-1 antibody associated with biotin.

As a result of Streptavidin-Peroxidase formation, colour produces due to an enzyme-substrate reaction. The resultant optical density is in proportion with the HIV-1 p24 antigen composed in the specimen.

HIV-1 p24 Antigen ELISA 2.0 assay principle

HIV-1 p24 Antigen ELISA Kit 2.0 is convenient to analyze the quantitative in vitro of cell culture. It can also enable the detection of supernatant present in the recombinant and natural HIV-1 p24 Ab concentration.

The HIV-1 p24 Antigen ELISA Kit uses Double Antigen Sandwich Technique. The sandwich ELISA test is based on the characteristics of the antigen that is tested. It should have more than two valances to identify the detection of antigen and coated antigen at the same time. The process is as follows:

1. Develop unbound antibodies by associating the solid phase carriers and antigen. Later wash out the remaining impurities and antigens. Enclose the irrelevant proteins with balanced binding sites.

2. Perform under test for contact reaction with immobilized antigens. After some time, connect antibodies inside and outside of carriers into the captured antibody. Separate the impurities and antibodies that are not combined.

3. Combine antigens to the conjugated antibodies on immune complexes and wash out the antigens that are not combined. The amount of enzyme on the carrier is therefore matched to the number of substances tested in the specimen.

4. Include horseradish peroxidase for labelling the avidins and then bind to the antigens. Wash out the integrated markers. The amount of enzyme on the carrier is then positively matched to the number of substances tested in the specimen.

5. A substrate is added, and a blue colouring will develop in wells containing the viral antigen.

6. The reaction is stopped, providing a colour change from blue to yellow. The optical densities of each well are read at 450 nm using a microplate reader/photometer.

7. The absorbance values of a set of standards are then plotted, and the amount of p24 determined from a linear regression analysis of the standard curve or by interpolation from a point to point plot.

Features of HIV-1 ELISA assay kit

• Easy to follow procedures

• Refrigerator stable, ready to use reagents

• Increased sensitivity – quantification as low as 4 pg/ml (double the sensitivity of version 1 HIV-1 p24 Antigen ELISA kit, ref 0801111 / 0801200)

• Shorter assay time – 3 hour total incubation time

• Detachable 8 well strips

• Easy-to-read test results

The ELISA kit offers several advantages, besides high sensitivity and specificity, over other types of assays. ELISA kits are relatively simple, inexpensive and can be easily adapted to automated platforms.

The original Retro-TEK kits are also available for use with serum, plasma and cell cultures.

https://dev.h-h-c.com/product/hiv-1-p24-antigen-elisa-kit-3/

Contact our experts at Helvetica Health Care to get more information about the different types of ELISA kits.