HIV-1 P24 Antigen ELISA 2.0
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 the capsid protein since it is the protein that makes up the capsid of the virus. It is enclosed in a two-domain structure forming a loop of about five to six members through self-association.
It is possible to detect the presence of the circulating p24 protein in the blood before the presence of antibodies to HIV during acute infection. As such, HIV-1 p24 is considered an early detection marker. This occurs shortly after infection due to the initial burst of virus replication. This early stage is associated with high levels of viraemia during which the individual is highly infectious.
Antigen tests were implemented in the United States in 1995 to detect antibodies in donated blood components. It has been used to detect units contaminated with HIV, which had previously tested for non-reactive antibodies. The period from post-infection to before seroconversion, during which infection markers (p24 antigen and antibodies) are still absent or too few to be detectable, is called the window period. Tests have shown that fourth-generation laboratory tests (which detect both antibodies and p24 antigen) detect HIV infections one to three weeks earlier than antibody-only tests.
The HIV-1 p24 Antigen ELISA 2.0 is for research purposes only. It is not for screening, diagnosis, or use as a confirmatory test for reactive specimens. 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 of the A.F. of HIV-1. There is no cross-reactivity with human immunodeficiency virus type 2, human T-cell leukaemia virus types 1 and 11 (HTLV 1 and 11), or simian immunodeficiency virus (SIV).
Principle Of The Test
ELISA plates available for detecting HIV antibodies or p24 antigens are conventionally packaged to include pre-coated microplates or strips and related reagents. A specialized monoclonal antibody test for the HIV-1 p24 gag gene product of HIV-1 is coated on the microwells.
While the sample is incubating, a target antigen present in the sample binds to a confined enzyme-labelled secondary antibody. Later, the captured antigen reacts with a biotin-associated human anti-HIV-1 antibody. As a result of the formation of streptavidin-peroxidase, colour is produced due to an enzyme-substrate reaction. The resulting optical density is in proportion to the HIV-1 p24 antigen compound in the sample.
Principle Of The HIV-1 P24 Antigen ELISA 2.0
HIV-1 p24 Antigen ELISA Kit 2.0 is suitable for testing the in vitro quantity of cell cultures. It may also allow the detection of the supernatant present in the concentration of wild-type and recombinant HIV-1 p24 Ab. The HIV-1 p24 Antigen ELISA kit uses the double antigen sandwich technique. The sandwich ELISA test is based on the characteristics of the antigen being tested. You must have more than two borders to identify antigen detection and coated antigen at the same time. The process is the following:
- Develop unbound antibodies by associating the solid phase carriers and the antigen. Later, wash away any remaining impurities and antigens. Circle irrelevant proteins with balanced binding sites.
- Carry out under-contact reaction tests with immobilized antigens. After some time, connect the antibodies inside and outside the carriers on the captured antibody. Separate impurities and antibodies that do not combine.
- Combine the antigens with the conjugated antibodies in the immune complexes and wash the uncombined antigens. Therefore, the amount of enzyme on the support is adapted to the number of substances tested in the sample.
- Include horseradish peroxidase to label the avidins and then bind them to the antigens. Wash built-in markers. The amount of enzyme on the support is then positively compared to the number of test substances in the sample.
- A substrate is added and a blue stain will develop in the wells containing the viral antigen.
- The reaction stops, giving a colour change from blue to yellow. The optical densities of each well are read at 450 nm using a microplate reader/photometer.
- Absorbance values are then plotted from a set of standards and the amount of p24 is determined from a linear regression analysis of the standard curve or by interpolation from a dot-to-dot plot.
Characteristics Of The HIV-1 ELISA Test Kit
- Easy-to-follow procedures
- Refrigerator-stable, ready-to-use reagents
- Higher sensitivity: quantification as low as 4 pg/ml (twice the sensitivity of version 1 of the HIV-1 p24 antigen ELISA kit, ref. 0801111/0801200)
- Shortest test time: total incubation time of 3 hours
- Removable 8-well strips
- Easy-to-read test results
The ELISA kit offers several advantages, in addition to high sensitivity and specificity, over other types of essays. ELISA kits are relatively simple, inexpensive, and can be easily adapted to automated platforms.