| OBJECTIVE: To clon, express and purify the recombinant human globular domain of adiponectin (gAPN) with biological activity using genetic engineering technology , then to establish an enzyme-linked immunosorbent assay (ELISA) method for detecting adiponectin (APN) in human serum and explore its application in coronary heart disease.METHODS: 1. The expression plasmid pET22b(+)-gAPN was constructed, expressed in E.coli, and purified to obtain the recombinant human gAPN protein. Its biological activity was evaluated by Streptozocin(STZ) induced hyperglycemia model mice. 2. The microplate was precoated with anti-APN McAb, blocked with 10% calf serum, and detected by using another monoclonal antibody anti-APN McAb which was labeled by biotin. The recombinant gAPN protein was used as a standard with which the standard curve was established. Thus the sandwich ELISA method was developed for detecting APN in human serum quantitatively with biotin-streptavidin amplify system. 3. The method was then applied in detecting serum APN in 161 patients. In combination with results of coronary angiography(CAG), there were 103 patients with coronary heart disease (CHD) and 58 patients with non-CHD, then the relationship between serum APN levels and progression of CHD was evaluated.RESULTS : 1. Plasmid pET22b-gAPN was constructed and the 15kD recombinant human gAPN protein was obtained. The yield of recombinant gAPN protein was as high as 1.57mg, with its purity>90%. Western blot confirmed the recombinant protein had the antigen activity, and its biological activity was confirmed by significantly reducing the levels of blood glucose in STZ induced hyperglycemia model. 2. A quantitative ELISA method for detecting APN in human serum was successfully set up. The sensitivity of this method reached 150pg/ml, of which the accuracy and reproducibility was good, and the recovery rate was among 91.0%-108.0%. The result obtained from our ELISA method showed high correlation (r=0.935, P<0.001) with that from Shenzhen Innogent APN ELISA kit. 3. Our results showed the concentration of serum APN was significantly lower in CHD group than in non-CHD group [ 5.67(2.59-10.47)mg/L VS 7.14 (4.37-11.40)mg/L, P<0.01 ]. In non-CHD group, the concentration of serum APN was significantly higher in women than in men [ 8.92(5.84-15.53)mg/L VS 4.68(3.11-9.05)mg/L, P<0.01 ], and in CHD group, it was lower [ 4.20 (1.83-6.05)mg/L VS 5.96 (2.71-10.90)mg/L ], but there was no significant difference(P > 0.05). With the increasing Gensini's score, the extent of coronary artery stenosis was more serious and the level of APN became lower, but had no significant difference among the groups, serum APN levels were negatively correlated with hs-CRP and Gensini's score(r=-0.253, r=-0.162, P<0.05), and positively correlated with HDL-C (r=0.213, P<0.01).CONCLUSION: The human gAPN gene and recombinant gAPN protein with biological activity have been successfully obtained. Then a simple, rapid and reliable quantitative ELISA method for detecting APN in human serum has been established. According to the preliminary clinical trials,the levels of srume APN in CHD decrease significantly. Serum APN levels were negatively correlated with hs-CRP and Gensini's score and positively correlated with HDL-C. Hypoadiponectinemia could be used in clinical diagnosis for coronary heart disease and has the important clinical application.
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