MJAS

Manuscript Details

Production of polyclonal antibody against tetracycline using KLH as a carrier protein

Siti Noraini, B.* and Nur Azura, M.S.

Biotechnology Research Centre, Malaysian Agricultural Research and Development Institute, P.O. Box 12301, 50747 Kuala Lumpur, Malaysia

Malaysian J. Anim. Sci. 2011 14(1): 61-66

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The production of polyclonal antibody against tetracycline was described using tetracycline - KLH conjugate (Keyhole Limpet Hemocyanin). Tetracycline was conjugated with KLH as a carrier protein because it was small molecule and unable to stimulate an immune response by itself. The UV absorbance reading of the tetracycline-KLH conjugate and KLH alone slightly shifted the reading of UV absorbance. Ammonium sulphate precipitation and Protein A affinity column were used in the antibody purification. Two peaks were obtained from affinity Protein A column. Peak 1 indicated the unbound material from serum sample and peak 2 was bound antibody with protein A which was eluted with elution buffer. Peak 2 was collected for titer antibody determination using ELISA method. Antibody level was higher at the fourth bleed which reached 1.2 absorbance (UV/nm) and equivalent with 1 mgmL-1 concentration. The entire antibody level declined dramatically at dilutions greater than 0.0001 mgmL-1 protein. The optimum and significant antibody concentration was at 0.00001 mgmL-1 for use in ELISA or other assays