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

Siti Noraini B., . and Nur Azura M.S., . Production of polyclonal antibody against tetracycline using KLH as a carrier protein. pp. 61-66.

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

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Abstract

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

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Item Type:	Article
AGROVOC Term:	Tetracyclines
AGROVOC Term:	Antibiotics
AGROVOC Term:	Antibodies
AGROVOC Term:	ELISA
AGROVOC Term:	Protein content
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	24 Apr 2025 06:27
URI:	http://webagris.upm.edu.my/id/eprint/21567

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