Selection And Identification Of Human Fab Fragement Antibody Against Amylin From Phage Antibody Library

Objective Amylin,also known as islet amyloid polypeptide,co-exists in the secretory granule of pancreaticβ-cells,regulating the balance of the serum glucose with glucagon and insulin.The recent research indicated the toxic form of amyloidogenic proteins appear not to be the extracellular amyloid fibrils,but rather smaller non-fibrillar oligomers.The later disrupts the pancreatic p-cell membrane, penetrates it and induce apoptosis.Since the amnio acid sequence of the unique constituent peptide of islet amyloid was successfully exracted and determined in 1988,people payes much more attention to the physical and pathological fuction of amylin. However there are so much difficulities in the study of the amylin changing methods in corpore,such as:the content of amylin in corpore is low,the molecular mass is small,the content of mRNA is only 0.2-0.3% of the insulin,the present methods of detecting the hormone is complex and the antibody is deficiency of stability and specificity.Therefore the key point to study the hormone is to search for a methods that can extract and purifity the antibody of amylin quickly. The objective of our research is to generate antibodies against amylin from a'naive'human Fab fragment antibodies phage diasplay library and to identify their specification and activities.Methods Amylin was coated on immunoplate. Panning from human Fab fragment antibodies phage diasplay library against amylin was conducted to select specific antibodies. And the eluted phage was collected and enriched for the next round of selection. The panning and propagating steps were repeated 5 times. The antigen binding activity was determined by ELISA. And the phage-display Fab fragments were detected by horseradish peroxidase (HRP)-conjugated anti-M13 antibodies. The phagemid of positive clones was extracted and were transformed into BL21(DE3)pLysS.And the bacteria was induced to give soluble expression of Fab antibody fragments by isopropyl-P-D-thiogalactoside (IPTG). During Western blot, soluble Fab fragments were detected by secondary HRP-conjugated sheep anti-human antibodies.Results After 5 rounds of panning and propagatin from a human native Fab fragment phage antibody library, Specific anti-amylin Fab antibodies were enriched. Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab, protein band of about 47KD was shown by SDS-PAGE. Western blot using the goat anti human IgG-HRP showed their binding activities. ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusions The appearance of phage antibody library develops a new pathway of preparating natural antibody.The technology is utility and can obtain specificity antibody directly.The high level expression and identification of the soluble human anti-amylin Fab fragment antibodies has been obtained successfully, which lays a solid foundation for further researching about the biological and pathological activities of amylin.