Aug 13, 2020
Inhibition immunoassay of the HIVgp120_anti-HIVgp120 (Ab-1) reaction by anti-anti-idiotypic-HIVgp120 antibody (Ab-3).
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Inhibition immunoassay of the HIVgp120_anti-HIVgp120 (Ab-1) reaction by anti-anti-idiotypic-HIVgp120 antibody (Ab-3). . protocols.io https://dx.doi.org/10.17504/protocols.io.bjnqkmdw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2020
Last Modified: August 13, 2020
Protocol Integer ID: 40368
Materials
MATERIALS
Carbonate Coating BufferBioLegendCatalog #421701
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
PBS Blocking buffer with non fat milkBio Basic Inc.Catalog #SB0625.SIZE.1pk
96-Well Microtiter™ Microplates, Polystyrene, 400µL, Nonsterile, U-bottomThermo FisherCatalog #2205
TMB Substrate SetBioLegendCatalog #421101
Fragment 254-274 of Gp120 of HIV (peptide)
Ninety six well polystyrene microplate (U-shaped bottom, Sigma-Aldrich Co, St Louis Missouri) is coated with 50 µl/well of 1 ng/µl solution of gp-120 peptide, fragment 254-274 (Sigma) in carbonate-bicarbonate buffer pH 9.6 (Sigma) for 4 hours at 37°C.
The microplate is then washed 4 times with PBS Tween-20.
The microplate is blocked with 3% non-fat milk in PBS, 25 µl/well, 1h, RT.
Triplicates of serial doubling dilutions of cat serum diluted in PBS-3% non-fat milk, pH 7.4 are added to the microplate which is then incubated for 90 min at 37°C.
The microplate is washed 4 times with 150 µl/well of PBS-Tween 20, and 25 µl of a water soluble fraction (WSF) containing anti-HIV-gp120 antibodies (titer =1:1024) is added to each well.
After adding WSF and it follows an incubation period (90 min, 37°C).
The microplate is washed 4 times and 25 µl of a 1:30,000 dilution of rabbit anti-chicken IgY-HRP (Sigma-Aldrich Co) is added to each well.
The microplate is then incubated at 37°C for 1 h.
The microplate is washed with PBS-Tween 20, 4 times.
Add 25 µl of TMB to the each well of the microplate and incubate the plate in the dark for 15 min.
The reaction was stopped with 3M H2SO4.
The microplate is read at 450 nm in a microplate reader.