Role of Gonadotropin-releasing Hormone Stimulation Test in Diagnosing Gonadotropin Deficiency in Both Males and Females with Delayed Puberty / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Delayed puberty can result either from constitutional delay of growth and puberty (CDP) or idiopathic hypogonadotropic hypogonadism (IHH). Gonadotropin-releasing hormone (GnRH) stimulation test has been generally accepted as a current method for diagnosing delayed puberty. The objective of this research was to assess the cut-off values and the efficacy of GnRH stimulation test in the diagnosis of delayed puberty in both males and females.</p><p><b>METHODS</b>A study of 91 IHH, 27 CDP patients, 6 prepubertal children, and 20 pubertal adults was undertaken. Blood samples were obtained at 0, 30, 60, and 120 min after GnRH administration and the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured. For each parameter, the sensitivities and specificities were estimated, and the receiver operating characteristic (ROC) curves were constructed.</p><p><b>RESULTS</b>The ROC curves indicated that a serum basal LH <0.6 IU/L or peak LH <9.74 IU/L resulted in moderate sensitivity (73.8% or 80.0%) and specificity (90.9% or 86.4%) in the diagnosis of HH in males. Serum basal LH <0.85 IU/L or basal FSH <2.43 IU/L resulted in moderate sensitivity (80.0% or 100.0%) and specificity (75.0% or 50.0%) in the diagnosis of HH in females.</p><p><b>CONCLUSIONS</b>Our data suggest that isolated use of the gonadorelin stimulation test is almost sufficient to discriminate between HH and CDP in males, but unnecessary in females. The most useful predictor is serum basal or peak LH to differentiate these two disorders in males, but serum basal LH or FSH in females.</p>

Preparation and identification of monoclonal antibody against enoyl-CoA hydratase 1 / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (mAbs) against enoyl-CoA hydratase 1 (ECH1).</p><p><b>METHODS</b>Normal human liver tissues were homogenized, and the mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were characterized by ELISA, Western blotting and immunohistochemistry. The specificity of the antibody was identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening.</p><p><b>RESULTS</b>One clone of the hybridoma BGB095 secreting specific mAb against ECH1 was obtained. The mAb was identified to belong to Ig subclass IgG1 and could be used in ELISA, Western blotting, immunohistochemistry, and immunoprecipitation.</p><p><b>CONCLUSION</b>A hybridoma cell line stably secreting specific mAb against ECH1 has been established. The specific mAb against ECH1 can be of great value for functional and distribution studies of ECH1.</p>

Preparation and identification of monoclonal antibody against UGP2 / 中国实验血液学杂志

The study was aimed to generate monoclonal antibodies (mAbs) against homo sapiens UDP-glucose pyrophosphorylase 2 (UGP2). Normal human liver tissues homogenized, and cytosolic proteins isolated by centrifugation were used to immunize BALB/c mice to generate mAbs by hybridoma technique. The mAbs were identified by ELISA, Western blot, and immunohistochemistry assay. The antibody specificity was confirmed by Uni-ZAP expression library screening. The results indicated that one hybridoma BAD062 secreting specific mAb against UGP2 was established. The Ig subclass of this mAb was IgG(2b) (kappa), and it could be used in ELISA, Western blot, immunohistochemistry assay. The antigen recognized by BAD062 mAb was localized in the hepatocyte cytoplasm, with molecular weight of 56 kD in the cytosolic proteins of human liver tissue. The BAD062 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. It is concluded that a hybridoma cell line stably secretes specific mAb against UGP2. This mAb reacted with UGP2 in ELISA, Western blot, immunohistochemistry assay, and would be very useful for the UGP2 studies.

Preparation and identification of monoclonal antibody against Homo sapiens hemoglobin alpha 2 (HBA2) / 中国实验血液学杂志

This study was purposed to prepare and identify monoclonal antibodies (McAbs) against Homo sapiens hemoglobin alpha 2 (HBA2). Normal human fetal liver tissues were homogenized, and human liver nuclear proteins were isolated by centrifugation. The total human fetal liver nuclear proteins were used to immunize BALB/c mice for preparing McAbs by hybridoma technique. The McAbs specificity was identified by ELISA, Western blot, and immunohistochemistry. The antigen was identified by Uni-ZAP expression library screening. The results showed that one hybridoma cell line, AEE091, secreting specific McAb against HBA2 was established. The Ig subclass of this McAb was IgG1 (kappa). Data from immunohistochemistry assay showed that AEE091 could recognize human liver nuclear protein. Using AEE091 McAb, isolation of the protein antigen by IP revealed that AEE091 McAb could recognize 15 kD protein. Screening the Uni-ZAP XR pre-made liver cDNA library with AEE091 hybridoma cell supernatants demonstrated that AEE091 McAb specially reacted with HBA2. It is concluded that a hybridoma cell line stably secreting specific McAb against HBA2 is established. The specific McAb against HBA2 would be very useful for studying HBA2 function and screening thalassemia.

Recombinant human VEGF-D induces the angiogenesis of the chick embryo chorioallantoic membrane / 中国实验血液学杂志

Vascular endothelial growth factor-D (VEGF-D) and vascular endothelial growth factor receptor-2, -3 (VEGFR-2, -3) with their corresponding signaling pathway play significant roles in the development of the embryonic vascular system and pathological lymphangiogenesis. The study was aimed to express and purify the GST-VEGF-D fusion protein, and to explore the angiogenesis effect of VEGF-D. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGF-D was expanded by polymerase chain reaction (PCR), then the plasmid pGEX-5X-1/VEGF-D was reconstructed and the GST-VEGF-D fusion protein expressed in transformed E.coli BL21-DE3. The results showed that the molecular mass of this fusion protein was 38 kD and compassed more than 15% of the total bacteria proteins. The fusion protein was recognized by anti-GST and anti-VEGF-D antibodies. The soluble GST-VEGF-D fusion protein could interact with VEGFR-3/Fc and was able to stimulate the proliferation of human erythroleukemia cell line (HEL) cells. The data of chick embryo chorioallantoic membrane (CAM) experiments indicated that GST-VEGF-D could induce the CAM angiogenesis. It is concluded that the GST-VEGF-D fusion protein with biological activity was successfully expressed, and which may provide an experimental model for the investigation of the VEGF-D-induced angiogenesis and lymphangiogenesis.