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2010 - 20122
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Subject: Biochemistry × End date: 2013 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    CONJUGATION OF ANTIBIOTIC RESIDUES PREVALENT IN MILK WITH CARRIER PROTEIN AND RAISING OF POLYCLONAL ANTIBODIES AGAINST THEM
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-08) SAMPATH KUMAR, B; ASHOK, VASILI; ESWAR PRASAD, P; Krishnaiah, N
    ABSTRACT : Antibiotics are the drugs used to kill or inhibit the growth of bacteria. Presence of antibiotic residues in milk has significant public health importance as they have adverse side effects on the health. The present study was undertaken to produce polyclonal antibodies against the most commonly used antibiotics in large animal practice so that a diagnostic test can be developed to screen milk samples for the presence of antibiotic residues. Ampicillin, ceftiofur, enrofloxacin and gentamycin were conjugated with carrier protein BSA by carbodiimide method in which EDC was used as a crosslinking agent. Successful conjugation was confirmed by SDS PAGE in which antibiotic conjugates revealed high molecular weight protein bands of 90kDa, 85kDa, 78kDa, 74kDa for gentamycin, ampicillin, ceftiofur and enrofloxacin respectively in contrast to 68kDa band of BSA. Sprague Dawley rats aged 7-8 weeks were immunized with the antibiotic-BSA conjugates by subcutaneous route according to the standard immunization schedule. A total of four blood samplings were done from each group (3 animals) as follows: first three samples serially at 15 days time interval after 1st immunization plus 1st booster, 2nd booster, 3rd booster and the 4th sampling one and half month after the third booster. The mean total protein content in the antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups were 12±1.15 g/dL, 30±1.20 g/dL, 24±1.73 g/dL and 8.25±0.20 g/dL respectively and in the control group serum it was 6.66±0.01 g/dL. The mean albumin concentrations were 3.44±0.020 g/dL, 3.36±0.173 g/dL, 3.21±0.003 g/dL & 2.93±0.014 g/dL for ceftiofur, ampicillin, enrofloxacin and gentamycin groups respectively compared to 3.36±0.173 g/dL in the control group. The mean globulin content in the antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups were 8.64±0.10 g/dL, 26.56±1.18 g/dL, 20.79±0.08 g/dL and 5.32±0.05 g/dL respectively and it was 3.30±0.04 g/dL in the control group. A/G ratios were less than the normal value (>1) in antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups. Indirect ELISA was standardized with antibiotic-casein conjugate as coating antigen to detect antibody titres. The optimum antigen concentration was chosen as 20ng/ml (which was in the range of Maximum residue limits) and optimum antiserum dilution was chosen as 1/100 by checker board titration. The antibody titres in the test samples of 4 immunization cycles for each antibiotic were determined by indirect ELISA at various serum dilutions ranging from 1/100 to 1/6400. Cut off value of 0.3 was calculated from PNT base line constructed with M+3SD values of control serum samples. All the four antibiotic-BSA conjugates elicited positive immune response in rats. The immune response in BSA-ampicillin conjugate immunized rats was maximum with the highest antibody titre of 2.577 (OD450) followed by BSA-ceftiofur conjugate immunized rats, BSA-enrofloxacin conjugate immunized rats and BSA-gentamycin immunized rats with OD450 values of 2.072, 1.723, and 0.928 respectively at the antiserum dilution of 1/00. These polyclonal antibodies are the efficient tools for developing immunoassays to detect specific antibiotic residues in biological fluids like milk and urine samples
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    ThesisItemOpen Access
    DIFFERENTIAL EXPRESSION OF CHICKEN BRAIN PROTEINS DURING DEVELOPMENT
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-06) NAVEEN SWAROOP, M; ASHOK, VASILI(MAJOR); NARSIMHA REDDY, Y; ESWAR PRASAD, P
    ABSTRACT: Understanding the proteins role and their spatiotemporal relationships in the development of brain will enrich the knowledge about structure – function relationships, control of productive traits, cognitive functions, behavior, artificial intelligence and neurological diseases. This study explores the different proteins involved in the development of the brain. Brains from Vanaraja breed embryos were collected from days 2 to 13continuously; from days 15,17,19,21 and from 6weeks birds. Brains were dissected under stereozoom microscope, collected in tris buffer pH 8.3, homogenized and centrifuged in refrigerated centrifuge at 10,000 rpm for 20 min. The supernatants were collected and analyzed by SDS PAGE (5% Stacking gel and 12% resolving gel). The proteins were differentially expressed during different developmental stages. 27 prominent differentially expressed proteins were identified. There was a progressive increase in the expression levels of higher molecular weights with advancing age. During early development of brain, may be low molecular weight proteins are playing a vital role in neural induction, anterio –posterior patterning, dorsovental patterning and cell differentiation. Based on molecular weights, in the light of earlier literature some of these proteins may be kinectin (160kDa), aggrecan (145kDa), PLC-β4 (130kDa), amyloid precursor proteins (120-123kDa), anosmin1 (100kDa), β catenin (92-102kDa), Brevican (97kDa), clusterin (80kDa), hyaluran receptor for motility (75kDa), heat shock protein (73kDa), GPI brevican (66kDa), occludin (60kDa), Glypican (58kDa), stanniocalcin (50kDa), synaptic vesicle protein (34kDa), claudin (22-25kDa), shhN protein (20kDa), FGF (16kDa), myelin basic protein (14kDa). Further research has to be done using gradient PAGE, 2D PAGE, immunohistochemistry and other molecular biological techniques like gene knockout and microarray to confirm the identity and to know the functional role and spatio-temporal relations of these proteins during brain development.