Aug 12, 2020
Protocol of preparation of horseradish peroxidase (HRP) conjugated to anti-human IgG to be used as secondary antibody in immunoassays.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Protocol of preparation of horseradish peroxidase (HRP) conjugated to anti-human IgG to be used as secondary antibody in immunoassays. . protocols.io https://dx.doi.org/10.17504/protocols.io.bjmxkk7n
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 12, 2020
Last Modified: August 12, 2020
Protocol Integer ID: 40343
Abstract
Peroxidase labeled anti-human IgG is a conjugated secondary antibody that can be used to detect specific antibodies against millions of antigens in humans. This reagent is usually used in enzyme-linked immunosorbent assay, immunoblot analysis and dot blot [1,2].
Reference
1. Stubenrauch K, Wessels U, Essig U, Kowalewsky F, Vogel R, Heinrich J. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.J Pharm Biomed Anal. 2013;72:208-215. doi:10.1016/j.jpba.2012.08.023
2.Justiz Vaillant AA, McFarlane-Anderson N, Akpaka PE, Smikle MP, Ramirez N, et al. (2013) Use of Dot Blots Analysis in the Separation of Anti-HIV Antibodies in Animals. J Chromat Separation Techniq 4: 181. doi:10.4172/2157- 7064.1000181
Guidelines
All reagents but specially the enzyme and sodium periodate solution has to be prepared freshly before mixing it with the enzyme. Anti-human IgG can be prepared by immunizing an animal species with purified human IgG. Then, it can be isolated by a protein purification method and refined by affinity chromatography.
Materials
MATERIALS
Ammonium SulfateP212121
Sodium periodateBio Basic Inc.Catalog #SB0875.SIZE.100g
sodium borohydrideSigma AldrichCatalog #452882
Horseradish Peroxidase (HRP) type IVSigma AldrichCatalog #P8375-25KU
ZyMAX™ Goat Anti-Human IgG (H+L) - BTThermo FisherCatalog #817140
Human IgG can be isolated from human serum or plasma by Protein-A agarose affinity chromatography. To develop the anti-human IgG a laboratory or farm animal is usually immunized and then, the anti-IgG is isolated. The Mancini test can estimate the anti-human IgG concentration.
Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
Mix 500µg/ml of anti-human IgG with 500 µg of the mix of horseradish peroxidase-sodium periodate. The mixture is incubated for 3 hours at 4°C with gentle agitation.
Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
The preparation is incubated for 90 min at 4°C in the dark with gentle agitation.
Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
The pellets is re-suspended in 200 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
An equal volume of glycerol is added to the dialysate followed by 100 µl of bovine serum albumin, BSA (20 mg/ ml).
The peroxidase conjugated to anti-human IgG is then stored at -20°C until further used.