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