Production and Purification of Polyclonal Antibody against Cholera Toxin

• Main Authors: Shahram Nazarian, Mohammadali Arefpour, Mohammadjavad Bagheripour, gholamreza Olad
• Format: Article
• Language: English
• Published: Babol University of Medical Sciences 2015-02-01
• Series: Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Bābul
• Subjects: Vibrio Cholera; B Subunit of Cholera; Recombinant Protein; Polyclonal Antibody; GM1-ELISA
• Online Access: http://jbums.org/browse.php?a_code=A-10-1364-12&slc_lang=en&sid=1

BACKGROUND AND OBJECTIVE: Cholera is a debilitating enteric disease, caused by Vibrio cholerae. Cholera toxin is the most important virulence factor in the pathogenesis of Vibrio cholera. Cholera toxin B subunit (CTxB), which forms a bond between the toxin and eukaryotic cells, has immunogenic features. The purpose of this study was to produce and purify antibodies against CTxB recombinant protein. METHODS: The CTxB recombinant protein was expressed and purified by Ni-NTA affinity chromatography. In total, ten 5-week-old BALB/C mice were divided into control and test groups. The test group subcutaneously received 10 micrograms of the recombinant protein along with Freund's adjuvant. Antibody titers were measured by ELISA method. The serum of immunized mice, receiving phosphate-buffered saline, was used in ELISA as the control. Immunoglobulin G was purified by the use of affinity column of G protein. The inhibiting effect of antibody against CTxB on toxin was examined using GM1-ELISA method. FINDINGS: The results of ELISA method showed the binding of recombinant protein to cholera toxin antibody. The amount of purified protein for each liter of the medium was 9 milligrams. ELISA findings showed that after each injection, the amount of antibody in mice was increased. The absorption rate of serum with the dilution of 1:500 was higher than three. According to Bradford assay, the density of purified antibody was 1 mg/ml. In ELISA’S reaction, 156 ng of toxin-binding subunit was identified by the antibody. The binding of toxin to GM1 increased by 70%, using immunized animal serum. CONCLUSION: The results of this study showed the efficiency of CTxB recombinant protein as an effective immunogen for provoking humoral response against cholera toxin. The antibody against the recombinant B subunit was able to identify toxins and inhibit its binding to GM1 receiver