Assessing Lentiviral Titer Using an Enzyme-Linked Immunosorbent Assay

Take lentiviral vectors containing HIV-1 p24 antigen, a protein marker used for detection.

Add a detergent to lyse and release p24, then serially dilute the sample.

Take a microplate coated with anti-p24 monoclonal antibodies and a blocking agent to prevent non-specific binding.

Add the sample dilutions and p24 standards as references.

Incubate to allow p24 interaction with the antibodies via a specific epitope, then remove unbound components.

Incubate with anti-p24 polyclonal antibodies that bind to different epitopes on p24. Remove any unbound antibodies.

Add peroxidase-linked antibodies that bind to the polyclonal antibodies. Remove unbound antibodies.

Add a peroxidase substrate that converts into a colored product.

Add an acid to inactivate peroxidase and stabilize the product.

Measure the absorbance corresponding to the color intensity, which is proportional to the p24 concentration.

Generate the standard graph and estimate the sample concentrations to determine the viral titer.

Coat a high-binding 96-well plate with 100 microliters of monoclonal anti-p24 antibody at a dilution of one to 1,500 according to NIH AIDS vaccine program for HIV-1 p24 antigen capture assay kit. Incubate the plate overnight at four degrees Celsius. The next day, wash the plate with 200 microliters of 0.05% TWEEN-20 in cold PBS three times.

To avoid non-specific binding, block the plate with 200 microliters of 1%bovine serum albumin for one hour at room temperature. Then, wash the plate with 200 microliters of 0.05% TWEEN-20 in cold PBS three times. Then, prepare the concentrated and non-concentrated vector samples to a final concentration of 10% by diluting the vector with double-distilled water and Triton X-100.

Next, prepare the HIV-1 standards using two-fold serial dilution starting at five nanograms per milliliter. After diluting the concentrated and non-concentrated vector samples, pipette it on the plate in triplicates and incubate at four degrees Celsius overnight. The next day, wash the plate, then add 100 microliters of polyclonal rabbit anti-p24 antibody on each plate and incubate at 37 degrees Celsius for four hours.

After six washes, incubate the plate with goat anti-rabbit horseradish peroxidase at 37 degrees Celsius for one hour. After multiple washing, incubate the plate with TMB peroxidase substrate at room temperature for 15 minutes. Add one normal hydrochloric acid to stop the reaction. And read the absorbance at 450 nanometers with an absorbance plate reader.