Qian Feng
Department of Rheumatology and Immunology, Changzheng Hospital, The Second Military Medical University, Shanghai 200003, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Dec 30;30(6):442-5.
To introduce a procedure of peptide-protein conjugation and prepare anti-malaria antibodies using a peptide-protein conjugate.
The recombinant atoxic form of Pseudomonas aeruginosa exotoxin A (rEPA) was used as carrier protein and modified by maleimide groups using the chemical linker of Sulfo-EMCS. The number of the maleimide group added onto the protein was measured by indirect Ellman's reaction. The maleimide modified protein was used to titrate the Pfs48/45-158, a synthetic peptide including a short amino acid sequence of Plasmodium falciparum Pfs48/45 with a cysteine residue at its N-terminus, and the curve fitting of the titration data was performed using linear regression. Based on the titration curve, the theoretic titration end point was determined and the conjugation ratio of peptide to carrier protein (mole peptide per mole carrier protein) was calculated. To scale up the preparation of the peptide-protein conjugate of Pfs48/45-158-rEPA, the peptide was used in excess to react with the modified carrier protein, and the resulting product was examined by SDS-PAGE. BALB/c mice were immunized with the peptide-protein conjugates. The mouse immune sera were examined by ELISA and immunofluorescence assay (IFA), two assays used respectively to determine the antibody titers against the peptide and the ability of the sera to recognize malaria parasites.
About 6.94 moles of maleimide groups were added onto one mole of rEPA via the chemical linker used in the reaction. The peptide-protein conjugate of Pfs48/45-158-rEPA was prepared with a conjugation ratio being about 7.33. High antibody titers against the Pfs48/45-158 peptide (12 500 ELISA units) were induced in mice immunized with the conjugates. The mouse immune sera were able to recognize not only the peptide but also the malaria parasites.
Peptide-protein conjugation is a convenient way that can be used to prepare anti-malaria antibodies. The indirect Ellman's reaction, titration and SDS-PAGE used in the study form a set of quality control methods, which ensures the quality and reproducibility of the conjugate.
