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Use of an ELISA for detection of antibody responses in Argentine boa constrictors (Boa constrictor occidentalis)

Brad A. LockDepartment of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.
Present address is Disney's Animal Programs, Veterinary Services, 1200 E Savannah Cir, Bay Lake, FL 32830.

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Linda G. GreenHybridoma Core Laboratory, University of Florida, Gainesville, FL 32610.

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Elliott R. JacobsonDepartment of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Paul A. KleinDepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610.

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Abstract

Objective—To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis.

Animals—Two 3-year-old Argentine boa constrictors.

Procedure—Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 µg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity.

Results—A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas.

Conclusions and Clinical Relevance—Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens. (Am J Vet Res 2003;64:388–395)

Abstract

Objective—To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis.

Animals—Two 3-year-old Argentine boa constrictors.

Procedure—Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 µg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity.

Results—A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas.

Conclusions and Clinical Relevance—Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens. (Am J Vet Res 2003;64:388–395)