Our lab is involved in HIV studies and we routinely need to measure viral titer in our
in vitro cell cultures. To do this, we have been using the Beckman Coulter HIV-1 p24 Antigen EIA Kit, which measures expression of the p24 viral core protein. This kit provides an enzyme-linked immunoassay to compare the effects of experimental treatments on HIV-1 replication
in vitro and compare them to untreated HIV-1 infected cells. The p24 assay for HIV-1 replication is reliable and easy to use, requiring 4-5 hours (not all hands on time) depending on the numbers of wells used.
This kit contains all items necessary to perform the assay from start to finish. Plate wells are coated with a murine monoclonal antibody for HIV-1 p24. Following the addition of samples (i.e. supernatant from cell suspension or lysate) to the wells, the Lysis Buffer is added and the plate is incubated for 1 hour at 37 C, allowing binding to occur between the p24 antigen and the monoclonal antibody coated well. After washing with buffer (provided), biotinylated human anti-HIV-1 IgG is added to each well and again, the plate is incubated for 1 hour, allowing for binding of IgG to the HIV-1 antigen. The wells are then washed again and streptavidin-horseradish peroxidase is added to complex with the biotinylated antibodies. The wells are washed a final time and TMB reagent (tetramethylbenzidine and hydrogen peroxidase) is added, which reacts with the already complexed peroxidase generating a blue color. The enzyme reaction may subsequently be stopped using CSR-1 and the blue color intensity is directly proportional to the amount of uncomplexed HIV-1 p24. Using a spectrophotometer/plate reader, one can quantify the amount of free HIV-1 p24 antigen in the samples relative to the absorbance of the purified HIV-1 p24 calibration standards (provided in the kit).
One disadvantage of this kit is the number of washing steps. Following each incubation step, one has to use a pipettor to wash the wells with 300 microliters of buffer at least 5 to 6 times. If you have many wells, washing can become very tedious unless you have an automated system. Also, I noticed that the absorbance values of the calibration standards can exhibit as much as a 20% variability between experiments with no clear explanation. In addition, this kit is relatively expensive.
The advantages of this kit are that it is very easy to use, the protocol is self-explanatory, and we get fairly consistent data. Overall, the Beckman Coulter HIV-1 p24 Antigen EIA is a reliable and quick assay to test HIV-1 replication in vitro, providing a means to obtain results in as little as half a day, which is amazing. We recommend this product.
Hee Chul Lee, Ph.D.
Post-Doctoral Scientist
Dept. of Biochemistry
NYU School of Medicine