Elisa cross-reactivity method to confirm recombinant flic from salmonella typhimurium and evaluate impurities in purified recombinant protein

Mohammad-hosein Khani kangarshahi,1,* Masoumeh bagheri,2 Fattah sotoodehnejad nematalahi,3

1. Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
2. Department of genomics and engineering genetics, Razi Vaccine and Serum Research Institute, Agricultural Research, Educa
3. Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran

Abstract

Introduction

Recombinant proteins as important materials for biomedicinal research, need high level of purity in order to be used in downstream applications. in previous studies some methods have been introduced to confirm recombinant proteins such as western blotting and protein sequencing. other methods were developed to confirm protein purity like sds-page. in this study we tested the purity and validity of flic, a tlr5 agonist, by elisa cross-reactivity experiment.

Methods

Purified recombinant flic from salmonella typhimurium was expressed in e.coli bl21 and used to immunize new zealand white rabbits. subsequently, elisa was performed using an indirect method with anti-rabbit hrp conjugated antibody. immunized serum was used as source of polyclonal anti-flic antibodies. flic, salmonella typhimurium and e.coli bl21 lysate was coated on micro titer plate to investigate affinity of antibody.

Results

Both interested antigen (flic protein) and salmonella typhimurium showed reactivity with polyclonal anti-flic antibodies. nevertheless, bl21 lysate did not show any reactivity with polyclonal anti-flic antibodies.

Conclusion

Results showed that using elisa cross-reactivity method could be promising application in order to confirm recombinant protein. moreover, it helped to evaluate protein purity. this method may be more convenient in those experiments which require determination of humeral immunity. in small percentage, investigation of impurities is costly and time consuming. for instant, the protein had to expressed in e.coli bl21, thus any impurities should be traced back to mentioned bacteria. in conclusion, lack of reactivity of antibodies and bl21 lysate showed high purity of recombinant protein.

Keywords

Elisa cross-reactivity, flic, polyclonal antibody , recombinant protein