Aug 12, 2020
Enzyme-linked immunosorbent assay (ELISA) for detection of anti-HIV antibodies (ELISA) developed in various animal species.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Enzyme-linked immunosorbent assay (ELISA) for detection of anti-HIV antibodies (ELISA) developed in various animal species. . protocols.io https://dx.doi.org/10.17504/protocols.io.bjmzkk76
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Created: August 12, 2020
Last Modified: August 12, 2020
Protocol Integer ID: 40345
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Abstract
Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-HIV antibodies in layer hens, rats and cats was a reproducible and feseable test. The presence of the anti-HIV antibodies was further confirmed by dot blot analysis.
Materials
MATERIALS
3,3′,5,5′-TetramethylbenzidineSigma AldrichCatalog #54827-17-7
ELISA Diluent 2 x 120 mL
Stemcell TechnologiesCatalog #1916
Nunc™ 96-Well Polystyrene Round Bottom Microwell Plates, V 96 well plate, Non-Treated, clear, without lid, SterileThermo FisherCatalog #260210
ELISA Blocker Blocking BufferThermo FisherCatalog #N502
Coating BufferBioLegendCatalog #421701
Protein LAG-anti-IgY-peroxidase (homemade)
Safety warnings
The risk associated with a particular microbe can be consulted in this website of the CDC (http://www.cdc.gov/biosafety/publications/BiologicalRiskAssessmentWorksheet.pdf)
The 96 well polystyrene microplates (U-shaped bottom) were coated with 50 ng of the synthetic gp-120 peptide in coating buffer overnight at 4°C.
The microplate is washed 4X (PBS-Tween-20) and blocked with 3% non-fat milk in PBS, 25 µl/well, 1h, RT.
The microplate is washed 4X (PBS-Tween-20) and triplicates of 25 µl of IgY (1 mg/ml), 25 µl of sera from cats or rats diluted 1:16 in PBS-non fat milk is added.
After incubation for 2 h at RT the microplate is washed 4X and 25 µl of a universal conjugate SpLAG-anti-IgY-HRP (comprised of peroxidase labelled-IgY chemically coupled to protein A, protein G and protein L) diluted 1:1000 was added and the plate incubated for a further 1 h at RT.
Then, the microplate is washed 4X and after that 3,3’,5,5’-tetramethylbenzidine (Sigma Chemical Co., St Louis, MO, USA) solution (50 µl) is added to each well.
After a further incubation of 15 min in the dark, the reaction is stopped when positive controls are coloured and blanks and negative controls do not.
Positive controls are from a positive human serum for anti-HIV antibodies, negative controls are from a turtle serum (Sigma Chemical Co., St Louis, MO, USA) and blanks 0.9% normal saline solution.
Cut-off point is calculated as the mean of negative controls multiplied by four.