Veterinary World

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Research (Published online: 26-04-2016)

13. Conjugation of ampicillin and enrofloxacin residues with bovine serum albumin and raising of polyclonal antibodies against them - B. Sampath Kumar, Vasili Ashok, P. Kalyani and G. Remya Nair

Veterinary World, 9(4): 410-416

 

 

   doi: 10.14202/vetworld.2016.410-416

 

 

B. Sampath Kumar: Department of Veterinary Biochemistry, College of Veterinary Science, Sri Venkateswara Veterinary University, Korutla, Karimnagar, Telangana, Andhra Pradesh, India; samkum85@gmail.com

Vasili Ashok: Department of Veterinary Biochemistry, College of Veterinary Science, Sri Venkateswara Veterinary University, Korutla, Karimnagar, Telangana, Andhra Pradesh, India; vasiliashok@gmail.com

P. Kalyani: Department of Veterinary Biochemistry, College of Veterinary Science, Sri Venkateswara Veterinary University, Korutla, Karimnagar, Telangana, Andhra Pradesh, India; parvathalakalyani@gmail.com

G. Remya Nair: Department of Veterinary Biochemistry, College of Veterinary Science, Sri Venkateswara Veterinary University, Korutla, Karimnagar, Telangana, Andhra Pradesh, India; remyagnair10@gmail.com

 

Received: 29-07-2015, Accepted: 14-03-2016, Published: 26-04-2016

 

Corresponding author: B. Sampath Kumar, e-mail: samkum85@gmail.com


Citation: Kumar BS, Ashok V, Kalyani P, Nair GR (2016) Conjugation of ampicillin and enrofloxacin residues with bovine serum albumin and raising of polyclonal antibodies against them, Veterinary World, 9(4): 410-416.



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 1 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.

Keywords: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, antibodies against antibiotics, conjugation, indirect competitive enzyme linked immunosorbent assay.



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