Protein biochip-based semiquantitative detection for plasma leptin.

PURPOSE: Plasma leptin is secreted from adipose tissues and plays pivotal roles in human physiological and pathological processes. Here, we aimed at conducting a protein biochip-based sandwich-like approach for detection of plasma leptin among healthy individuals, obesity, and diabetes patients. EXPERIMENTAL DESIGN: Totally, 96 plasma samples, including 45 healthy individuals with standard body mass index (BMI), 28 obesity and 23 diabetes patients, were recruited in the study. Plasma leptin was detected by a well-established protein biochip. Meanwhile an ELISA was also performed for assessment of the leptin detection by the protein biochip. RESULTS: We found that the plasma leptin level in the obesity and diabetes patients was significantly higher than that in healthy individuals with standard body mass index (p < 0.001). The limit detection concentration of leptin was as low as 0.006 µg/mL. The plasma leptin could be semiquantitatively detected by the protein biochip. The compatibility of the biochip-based detection approach seemed acceptable in comparison with the ELISA assay (R2 = 0.948). CONCLUSIONS: We provided a protein biochip-based approach for plasma detection. This approach would be a potential substitution for the ELISA assay.

[Preparation, identification and application of monoclonal antibody against matrix metalloproteinases-2].

AIM: To prepare and characterize monoclonal antibodies against matrix metalloproteinase-2 (MMP-2), check its expression in the tissues of human ovarian cancer and transplanted tumors in nude mice. METHODS: MMP-2 were linked to the carrier protein bovine serumalbumin (BSA) and keyhole limpet hemocyanin (KLH) using glutaraldehyde method to obtain MMP-2-BSA and MMP-2-KLH, respectively. The anti-MMP-2 monoclonal antibody was obtained through hybridoma technique. We established the cell strains secreting mAb by hybridoma technique and prepared the mAb by induction of ascites in vivo. The prepared mAb was purified by salting out with ammonium sulfate and identified by ELISA and Western blotting. We compared the mAb and commercial polyclonal antibody by immunohistochemistry and detected the expressions of MMP-2 and CA125 in ovarian cancer issues and transplanted tumor. RESULTS: The artificial antigen and 3 hybridoma cell lines secreting monoclonal antibodies (mAb) against MMP-2 were obtained. The subclasses of mAb were all IgG1. The titer of peritoneal exudates was 1:1×10(6);. The expressions of MMP-2 and CA125 in transplanted tumor and ovarian cancer tissues were all high. The positive expression rate of MMP-2 checked using generated antibody was 71.2%(57/80) in ovarian cancer tissues and 16.67% (5/30) in normal tissues, with significant difference between them (P<0.01). In early stage, the positive rate of MMP-2 and CA125 combined detection was higher than that of CA125 detection alone (P<0.01). The mAb was suitable for detecting the expression of MMP-2 in human tissues and gave results consistent with commercial polyclonal antibody. The mAb was more specific than commercial mAb (P<0.01). CONCLUSION: The anti-human MMP-2 mAb is successfully prepared, which may serve as a valuable tool in the functionaI studies of ovarian cancer.

[Preparation and characterization of human colon tumor-associated antigen and its clinical significance].

AIM: To purify the colon tumor-associated antigen from cultured colon tumor cells, and to investigate its expression in the sera of patients with colon cancer. METHODS: The monoclonal antibody (mAb) against human colon tumor-associated antigen 4D10 was employed as the ligand for the immunoaffinity chromatography to purify the colon tumor-associated antigen from the lysate of colorectal tumor cell LOVO. The purified antigen was identified by SDS-PAGE and Western blot. The expression of colon tumor-associated antigen in the sera of patients with colon cancer and in normal sera was detected by Sandwich ELISA. RESULTS: The purified colon tumor-associated antigen binding to mAb 4D10 was a heterodimer composed of two subunits with relative molecular mass M(r) of 30 x 10(3) and 35 x 10(3) respectively. The antigen was significantly higher expressed in sera from patients with colon cancer than that in normal sera (P<0.01). CONCLUSION: The tumor-associated antigen obtained from the colon tumor cells has been successfully purified through immunoaffinity chromatography with mAb 4D10, which may be useful for diagnosis on clinic.