Preparation Of The Anti-APs Monoclonal Antibody And Homology Modeling Of Its Fv Fragment

Alkylphenols(APs)is a kind of environmental endocrine disrupting chemicals.It has strong reproduction,immune and nervous biological toxicity,potentially harmful to human health.Because of its high biological toxicity and widely pollution range,some countries have issued relevant laws and regulations to limit the using of APs.So it is particularly important for the preparation of high affinity APs monoclone antibody on the content of APs testing.APs is a big family of structure similarity,have common phenol structure and different length of carbon chain.Taking amylphenol as the hapten,we prepared immunogen and coating antigen with BSA and OVA coupling by EDC method,and coupling ratio is 39:1 and 41:1 respectively.We measured serum titer of the vaccinated mice.It was higher than 128000 after three times immunization.Then,we adopt the method of electric fusion to fuse the spleen cells of immunized BALB/C mice and sp2/0 myeloma cells.And we selected out cell line F10 and Wu-13 which could secrete high affinity 4-AP monoclonal antibody by ELISA and flowcytometrty.By indirect competitive ELISA quantitative detection of APs,IC50 of mAb F10 and Wu-13 is 9.8 μg/mL and 185 ng/mL respectively.And it could recognize APs,best for APA and 4-AP.With mouse monoclonal antibody isotyping ELISA kit analyzed,it was found that heavy chains of mAb F10 and Wu-13 belonged to type IgGl and type IgM,respectively,Light chains belonged to type kappa.Through homology modeling,there are 22 and 12 amino acids different in the variable region of the heavy chains and light chains respectively.Most of them were located in the CDR region,and these variable amino acids made binding sites’ hydrophobic to enhance.Through molecular docking experiment,it was found that ser(99)in heavy chain CDR3 can form stable hydrogen bonds with 4-AP phenolic hydroxyl groups.In the CDR,a large number of hydrophobic amino acids form a hydrophobic cavity packing small molecule to realize the combination of antigen and antibody.The main reason for the increase of the affinity is that the increase of hydrophobic force of mAb Wu-13 binding sites.In this study,we prepared APs’s monoclonal antibodies with high affinity and good specificity,which provided a new idea for detection of APs.And the discovery of crucial amino acids and hydrophobic force by sequence alignment and homology modeling lay the foundation for further research of scFv and engineered antibody.