Study On Expression And Monoclone Antibody Preparation Of H-FABP, The Concerned Establishment Of Detection Method And The Clinical Application

Fatty acid-binding proteins (FABPs) are one of the members of the multi-gene family which consists of a kind of conservative in-cell lipid-binding proteins (iLBPs). They are expressed in all the issues of the vertebrate, and each of the FABPs has the different mode of expression. The proteins have multi functions, including promoting cell-intake and transportation of fatty acid, looking for and participating the special way of fatty acid metabolism, participating gene expression and the modulation of the cell growing. In 1972, Ockner, for the first time, found a group of homology small proteins in cells on intestinal mucous when he was engaged in the research of the modulation of absorbing the fatty acid in small intestine of the rat. They widespread in multi cells of the small intestine, liver, fat, heart, brain and skeletal muscle of the mammal.( MW: 12~16KD). The FABPs (fatty acid-binding proteins) we have known have 9 types , which include: H-FABP (heart type fatty acid-binding proteins), I-FABP ( intestinal fatty acid binding protein), L-FABP (liver fatty acid binding protein), A-FABP (adipocyte fatty acid binding protein), B-FABP (brain fatty acid binding protein), K-FABP (kidney fatty acid binding protein), S-FABP (skeletal muscle fatty acid binding protein), PA-FABP (psoriasis fatty acid binding protein) andE-FABP (epidermal fatty acid binding protein) . FABPs was named by their distribution in the tissues and multi FABPs could be distributed in the same cell. For instance, there are two different FABPs, L-FABP and I-FABP on the intestine endothelial cells. Both of them have 29% homologyo FABPs was also found in plants. The sequences of different types of FABPs have more homology.H-FABP is a kind of dissolvable cytoplasm protein and its MW is 14~15KD, which is distinctive and exists in the issues of cardiac muscle in large amount, about 4%-8% of the dissolvable proteins of the whole heart. For the health adult, each gram of cardiac muscle contains H-FABP for 0.52 ±0.06 mg, or so. H-FABP is a kind of acidic protein, its isoelectric point (pi) is 5.1. The primary structure of human's H- FABP is composed of 132 amino acids, in which there are threonines and lysines, deficiency of cysteine, and with a residue of acetyl valine on its N terminal.H-FABP has the specific bindings with hydrophobic ligand molecules, such as LCFA ( long-chain fatty acid), vitamin(vitamin A1), RA (retinoic acid) and some organic negative ions. H-FABP is the key carrier protein of fatty acid. It can transport the fatty acid from cytomembrane to the parts of esterization and oxydation and thereby enter the energy metabolism system of mitochondria where the fatty acid are oxided and broken down, producing ATP to provide energy. H-FABP exists specifically in the tissues of the cardiac muscle. The blood plasma and urine of health adult don't contain H-FABP or just very few.Recently the attack rate of heart infarction keeps rising, so the early diagnosis and early treatment have become the focal point doctors show solicitude for. The first 6 hours during the attack of heart infarction are the best time for the thrombolysis. After the 6 hours, the cardiacmuscle cells will appear irreversible damage because of oxygen deficiency. H-FABP will appear in the blood during the 1.5 hours after the AMI (acute myocardial infarction), and the content in the blood will reach the peak during the first 4-6 hours, then return to the baseline after 24 hours. Thereby, it could be the marker of the early diagnosis for the patients of AMI. Besides, it could be the judgment for the efficacy of the treatment and the prognosis. H-FABP could also detect the recurrence of AMI and evaluate the infarct size.The assay method of H-FABP has made a big progress. Ockner was the man who reported for the first time about quantitative detection of the FABP and put forward the method of radio-immunity in 1974. The method was used to detect the corresponding concentration of FABP by determining the specific activity of the radioactivity in immune-oprecipitation.The method was not effectual because of its elaborate operation, high cost and the radioactive pollution. Afterwards, a competitive EIA{ enzyme immunoassay} was used to determine H-FABP in blood plasma. However, EIA was less employed due to the limit of the time for the determination and the amount of the sample. A sandwich ELISA ( enzyme-linked immunosorbent assay ) appeared in 1997. This method is used to determine the concentration of H-FABP in human plasma and urine by making use of the complex of two different mice anti-human H-FABP monocloneal antibody, and the total determining time is just less than 2 hours so that the efficiency is greatly improved.In our research, for the first time we made use of RT-PCR technique to amplify the gene segments of H-FABP (396bp) from the cardiac muscle issues in human fetus, then we made the target gene construct respectively onto the prokaryotic expression vector pQE-30,pBV220 and Pichia yeast pPICZ eukaryotic expression vector so that we got recombination expressplasmid pQE-H-FABP^ pBV-H-FABP and pPICZ-H-FABP. We transformed the recombination plasmid pQE-H-FABP into E.coli M15[pREP4] to express the fusion protein with 6 terminals of histidine The recombination plasmid pBV-H-FABP was transformed into E.coli DH5a, to express human fatty acid-binding protein by temperature inducement. The recombination plasmid pPICZ-H-FABP was transformed into the yeast strain X-33 to express glycosylated human fatty acid-binding protein by methanol inducement. The content of the purified recombination of H-FABP is respectively about 480mg ? L1 (pQE-H-FABPX 850mg ? L1 (pBV-H-FABP) and 700mg ? L1 (pPICZ-H-FABP) .Monoclonal antibodies with 7 strains of anti-human H-FABP were obtained when the purified recombination of human H-FABP was applied into the immunized mice. We got 2 strains of monoclonal antibodies of anti-human H-FABP, 4D7 and 9H10, with high affinity and could resist different species-specific epitope, after detecting its valence of antibody, affinity, specificity by means of SPA-Sepharose CL-4B, gel affinity chromatograph of purified antibody. The immunoglobulin was respectively lgG2b and lgG2a > the content of the antibody was respectively 1.43g ? L"1, and 1.56g ? L"1, the dilution of ascitic fluid was 1 : 2*107 1 : 2*107, the relative index of affinity is 4.8 and 5.15. The cross reactivity between H-FABP 4D7, 9H10 monoclonal antibody and pig, mouse, cow serum was respectively 100, 6.15, 1.22, 0.76% and 100,59.3, 1.74,0.63.%.The ELISA method of double antibody sandwich determination for H-FABP was established by means of the purified monoclonal antibody. First we prepared and purified the tagged antibody 4D7 to make theHRP molecule and antibody form the stable conjugate of enzyme.Then the HRP tagged antibody was obtain by the conjugate going through Sepharose G-100 column chromatography. The working optimization concentration of enzyme labelled antibody was selected by the experiment, it was 1: 2000, and the yield of the conjugate was 29.8%. The betweenrun CV were respectively 7.41%, 4.3%, 3.0% and the withinrun CV were respectively 7.0%, 3.4%, 2.3%, when the determination was conducted by the concentration of 8.5, 51, 223 v-g ? L'1 H-FABP. The linear range of standard curve was from 0 to 250 u g *L'1, when H-FABP was under the circumstances of 0, 0.3, 0.15, 0.625 u g ? L'1. When H-FABP was 1.25u g ? L"1, the obtained optical density didn't overlap the background of the topmost value (blank), so the lowest limit of the ELISA detecting was 1.25 u g ? L'1. The result of the experiment showed that the sensitive range of ELISA method applied in the human H-FABP was from 1.25 to 250 v- g ? L"1. The stability of the recombinated human H-FABP of 10, 52, 160 tig ? L"1 was invariable within 7 days, at the temperature of -20°C> 4°C or 25°C. We observed the effects of the repeated freeze-thawing on the 3 different concentration of H-FABP in the blood plasma, and after 7 cycles of the freeze-thawing, the amount of H-FABP respectively kept 96.0%, 97.8 %, and 97.6 % of the origin. The experiment showed that the 7 cycles of freez-thawing didn't affect the immune assay for H-FABP in the human blood plasma. The average recovery of the two blood plasmas in the experiment was respectively 97.9% and 98.4. In the experiment to dilute the sample of the blood plasma, if the samples with the H-FABP concentration of 84.6 and 214.4 u g ? L"1 were diluted by 1/2, 1/4, 1/8, and the H-FABP concentration of the samples ranged from 0 to 214.4 ng ? L"1,the linear relationship was good. Under the same circumstances and with the same equipment, the determined results were almost the same with the results which determined by the imported ELISA agent for the common determinations of different concentration. The correlation coefficient r = 0.99.We detected the serum H-FABP of 110 healthy people by medical examination and 85 AIM (acute myeloblastosis patients) by means of ELISA method , meanwhile we determined MYO (myoglobin ), cTnl (troponin I), CK-MB (Creatine kinase isoenzyme MB), and had a dynamic observation for AMI. The sensitivity, specificity and chronergy of early diagnosis were analyzed. The result was the serum concentration of H-FABP began to rise during 1.87+0.56 hours after AMI , earlier than cTnl and CK-MB (P <0.01);the dynamic curve of time concentration was similar to MYO, and antelocated more than the curve of cTnl and CK-MB . For two hours after AMI, the sensitivity and specificity were respectively 72.9%, 91.8%;for 6 hours, the sensitivity and specificity were respectively 94.1%, 91.8%. The conclusion is: the detecting for H-FABP after AMI had higher sensitivity and specificity for the early diagnosis and hopefully will become the important marker of the serum cardiac muscle for the early diagnosis.With the ELISA method to detect the serum H-FABP from 110 healthy people of the medical examination and 67 UA (unstable angina ) patients, we determined cTnl, CK-MB and CRP (c- reactive protein) , making the dynamic observation for the prognosis in the near future for the UA patients. The sensitivity, the specificity and the chronergy of early diagnosis were analyzed. The result was the serum concentration of H-FABP began to rise during 1.87±0.56 hours after UA, earlier than cTnl and CK-MBCP <0.01);the dynamic curve of timeconcentration antelocated more than the curve of cTnl, CK-MB and CRP. For 2 hours after UA, the sensitivity and specificity were respectively 94.0%, and 94.7%. The peak concentration was 85.79±63.24|jg ? L"1, the peak time was 4 hours and the recovery time was between 24-48 hours. The positive predictive value of H-FABP for the UA patients, of the duration of hospital stay was 22.6%, who suffered from the non-fatal Ml (myocardial infarction) or died from other heart diseases. The negative predictive value concerned was 97%. The cardiac accident of UA patients took place within 1 hour to 2 weeks, and at least one item of the detection was abnormal when H-FABP, cTnl, CK-MB and CRP were detected in association. The diagnostic positive proportion was 100%. After UA, the detection for H-FABP had higher sensitivity and specificity to the early diagnosis. If the H-FABP was detected in association with other cardiac muscle markers, which would have significant value for the early diagnosis of UA, thrombolysis treatment, the recovery of metabolic effect, the recovery of injured muscle and the prognosis improvement.in the experiment, we employed the RT-PCR technique to amplify gene segment (396bp) of H-FABP from the cardiac muscle tissue of human foetus. We successfully constructed the prokaryotic expression vectors and eukaryotic expression vectors and expressed H-FABP efficiently. By use of the monoclonal antibody technique, we obtained anti-human H-FABP and established the ELISA method of human serum H-FABP for determination. The clinical detection of the serum H-FABP, from 110 healthy people in medical examination, 85 AMI patients and 67 UA patients, showed that after AMI and UA, the detection of H-FABP had higher sensitivity and specificity for the early diagnosis of AMI and UA. If it was detected in association with othercardiac muscle markers, which would have significant value for the early diagnosis of AMI and UA, thrombolysis treatment, the recovery of metabolic effect or the recovery of injured muscle , and the prognosis improvement. So hopefully H-FABP will become the important marker of the serum cardiac muscle for the early diagnosis of AMI and UA.