Conjugation of ampicillin and enrofloxacin residues with bovine serum albumin and raising of polyclonal antibodies against them

Abstract

Aim:The aim of this study is to test the potency of bovine serum albumin (BSA) conjugated ampicillin (AMP) and enrofloxacin (ENR) antigens in eliciting an immune response in rats using indirect competitive enzyme-linked immunosorbent assay (icELISA).Materials and Methods:AMP and ENR antibiotics were conjugated with BSA by carbodiimide reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linker. The successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sprague-Dawley rats were immunized with the conjugates and blood samples were collected serially at 15 days time interval after first immunization plus first booster, second booster, third booster, and the fourth sampling was done 1½ month after the third booster. The antibody titres in the antisera of each antibiotic in all the four immunization cycles (ICs) were determined by an icELISA at various serum dilutions ranging from 1/100 to 1/6400.Results:Analysis of antibiotic-BSA conjugates by sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining revealed high molecular weight bands of 85 kDa and 74 kDa for AMP-BSA and ENR-BSA respectively when compared to 68 kDa band of BSA. Both the antibiotic conjugates elicited a good immune response in rats but comparatively the response was more with AMP-BSA conjugate than ENR-BSA conjugate. Maximum optical density 450 value of 2.577 was recorded for AMP-BSA antisera, and 1.723 was recorded for ENR-BSA antisera at 1/100th antiserum dilution in third IC.Conclusion:AMP and ENR antibiotics proved to be good immunogens when conjugated to BSA by carbodiimide reaction with EDC as crosslinker. The polyclonal antibodies produced can be employed for detecting AMP and ENR residues in milk and urine samples.

Immunological properties of gold nanoparticles

“…Traditionally, this problem is solved by chemical attachment of a hapten to a protein matrix called a schlepper (from the German schleppen “to drag”), and by the use of adjuvants and intensive schemes of animal immunization with the obtained conjugate. 87 , 95 Bovine serum albumin (BSA), ovalbumin, thyreoglobulin, hemocyanin and diphtheria or tetanus toxoids (in the case of synthetic peptides) are generally used as schleppers. However, this method yields antibodies to both the hapten and the immunodominant sites of the carrier.…”

Study on immobilisation process on lateral flow assay test strip for detection of Enrofloxacin antibiotic by inkjet printing

“…Synthesis of the biological substance ink. In consideration of the small size with molecular weight less than 1 kDA [45], Enrofloxacin needs a protein carrier to achieve compatibility with the pore size of the nitrocellulose membrane. Therefore, BSA is chosen to be the carrier molecule that forms the conjugation with Enrofloxacin for the attachment at the test line.…”

Modifications of quinolones and fluoroquinolones: hybrid compounds and dual-action molecules

“…To obtain immunogens, the carbodiimide reaction was employed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as a crosslinker. Polyclonal antibodies were successfully produced in rats, which were confirmed by indirect ELISA [ 53 ].…”