Aug 17, 2020
Immunoblot analysis for immunodetection of HIV proteins.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Immunoblot analysis for immunodetection of HIV proteins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjtpknmn
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Created: August 17, 2020
Last Modified: August 17, 2020
Protocol Integer ID: 40527
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Abstract
Enzyme-Linked Immunosorbent Assay (ELISA) was used for screening while the Western Blot was used for confirmation.
Aliquots of 3-5 µl of the serum are applied to the gel and run on a protein electrophoresis (SDS-PAGE).
Gels are transferred to nitrocellulose membranes (Immobilon-Nc, pore size 0.45 µm, during 75 min at 40 mAmps using a semi-dry electroblotter, HEP-1 Model, Owl Scientific Inc).
The running buffer contains 25mM Tris, 192mM glycine pH 8.3 and 20% methanol.
The nitrocellulose membranes are blocked overnight in 10% nonfat skim milk in PBS with 0.05% Tween-20 pH 7.4 and then washed 4 times for 10 minutes with PBS-Tween 20.
Peroxidase-labeled anti-HIV conjugate are added and incubated at 4ºC overnight.
Membranes are washed as above and then tetra-methyl-benzidine are added and the reaction is stopped with deionised water.
A positive test displays two or more HIV proteins.
Only patients with positive confirmatory tests are classified as HIV positive.