Development Of Raw Materials For Immunological Detection Of HFABP,a Specific Biomarker In Myocardial Damage

Cardiovascular disease has become one of the main diseases threatening human health in twenty-first Century,one of the most dangerous cardiovascular disease is the acute coronary syndrome(ACS),which includes unstable angina and acute myocardial infarction(AMI).Nearly 1700 0000 people died from cardiovascular disease worldwide every year,more than half of them died of AMI.AMI may occur suddenly,and the acute mortality rate is about 30%.Electrocardiogram is the common detection method for AMI,but its role is very limited for AMI patients with normal ECG.Therefore,it is necessary to find a new biomarker for AMI,which has excellent specificity and short window period for AMI detection.Human heart-type fatty acid binding protein(HFABP)is the excellent myocardial damage biochemical marker with high sensitivity and specificity to early detection?risk stratification and prognosis for AMI.Moreover HFABP is also an excellent biomarker than cTnI?cTnT?CK-MB in early diagnosis of cardiovascular disease.In this study,total RNAs were extracted from human heart tissue,and then were reverse transcripted into cDNA.HFABP coding sequence was obtained by PCR amplification,and expressed in prokaryotic E.coli.Analysis results of mass spectrometry comfirmed that the recombinant protein was HFABP,and the relative molecular weight of recombinant protein is consistent with native HFABP.The recombinant HFABP had excellent immunoreactivity with anti-native HFABP monoclonal antiboby from Proteintech,and the recombinant HFABP can form dimer and oligomer.The recombinant protein may be simulate the conformation of the native HFABP,can be used as immune antigen for the preparation of anti-HFABP monoclonal antibody.The purity of recombinant protein reached 99%after purified by fractional precipitation of ammonium sulphate,his-tag column and molecular sieve chromatography.4 to 6 weeks BALB/c female mice were immunized four times with 100 ?g purified recombinant HFABP per mice using subcutaneous immunization,and then the antibody titers of immunized mice were investigated by indirect ELISA.The result indicated that antibody titers is mounting up in immunized mice.With the increase of immunization times,and the serum titers still had high OD value at the 107 times diluted concentration.Mouse spleen cells and myeloma cells SP2/0 were fused after booster immunization from spleen and tail vein,and the hybridoma cells were screened with 20%FBS-HAT-1640.Nine positive hybridoma cell strains were obtained after four times of cloning and they can stably secrete monoclonal antibody against HFABP with high titers,and the antibody types and subtypes were identified.Western blotting and indirect ELISA showed that all 9 monoclonal antibodies indicated good immunoreactivity with recombinant HFABP.A series of C-terminal amino acid deletion mutant HFABPs were expressed in E.coli,and linear and conformational antibody were identified by indirect ELISA and Western blotting used the purified mutant proteins as antigen,moreover,we have mapped the prepared monoclonal antibodies to their epitopes,respectively.In summary,the recombinant HFABP with high biological activity was expressed in E.coli,anti-HFABP monoclonal antibodies with high activity and high sensitivity were prepared and screened,which mapped one-for-one different antigen epitopes,the results also showed that there were both linear and conformational monoclonal antibody among these antibodies,which provided a basis for development of HFABP diagnostic kit with independent intellectual property rights.