Enhancement of binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification

Nazamid Saari, . and Rashidah Sukor, . and Farina Mustaffa Kamal, . and Radhiahtul Raehan Mustafa, . and Siti Mariam Mohd Nor, . and Aliah Zannierah Mohsin, . Enhancement of binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. pp. 2451-2464. ISSN 2231-8526

Antibodies are glycoproteins found in peritoneal fluid serum and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized“ bovine serum albumin at methyl ester (C22-MG-cBSA) or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 10��M which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 10��´M. In addition the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

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Abstract

Antibodies are glycoproteins found in peritoneal fluid serum and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized“ bovine serum albumin at methyl ester (C22-MG-cBSA) or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 10��M which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 10��´M. In addition the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

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Item Type:	Article
AGROVOC Term:	Rubiaceae
AGROVOC Term:	Medicinal plants
AGROVOC Term:	Purification
AGROVOC Term:	ELISA
AGROVOC Term:	Chromatography
AGROVOC Term:	Antigens
AGROVOC Term:	Immunoassay
AGROVOC Term:	Immunoglobulins
AGROVOC Term:	Secondary metabolites
AGROVOC Term:	Polyclonal antibodies
Depositing User:	Mr. AFANDI ABDUL MALEK
Last Modified:	24 Apr 2025 00:55
URI:	http://webagris.upm.edu.my/id/eprint/10258

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