Role of Gonadotropin-releasing Hormone Stimulation Test in Diagnosing Gonadotropin Deficiency in Both Males and Females with Delayed Puberty.

Background: 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.

[Prepared the monoclonal antibody associated with hepatocellular carcinoma and analyzed its epitope].

Aim: To prepare the monoclonal antibodies associated with hepatocellular carcinoma (HCC) for diagnosis.

Methods: 3 BALB/c mice were immunized with high metastasis characteristic HCC cells (HCCLM-6). The splenocytes from the immunized mice were fused with murine myeloma cells (Sp2/0).

Antigenically dominant proteins within the human liver mitochondrial proteome identified by monoclonal antibodies.

Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning.

[Preparation and identification of PON2 monoclonal antibodies.].

Aim: To prepare and characterize the mouse monoclonal antibodies against human PON2 (paraoxonase 2).

Methods: A fragment of human PON2 gene of low homology with mice but of strong hydrophilicity and immunogenicity was selected for recombinant expression. Mice were immunized with the purified HIS fusion protein 3 times.

[Preparation and identification of monoclonal antibody against enoyl-CoA hydratase 1].

Objective: To prepare monoclonal antibodies (mAbs) against enoyl-CoA hydratase 1 (ECH1).

Methods: 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.

[Expression of procalcitonin and characterization of antibodies against PCT].

Aim: To construct the expression vectors of procalcitonin (PCT), prepare polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) against PCT and identify their specific biological activity.

Methods: The recombinant expression plasmids of pGEX-4T-1-PCT and PET-32a-PCT were constructed using thyroid carcinoma cell line (TT cell) cDNA as template. The fusion protein of His-PCT was expressed in E.

[Preparation and preliminary application of rabbit anti-human PON2 antibodies(paraoxonase-2)].

Aim: To preparation and characterize the rabbit polyclonal antibodies against human PON2 (paraoxonase-2).

Methods: A fragment of human PON2 gene which was of low homology with rabbits but of higher hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times.

[Preparation, characterization and application of monoclonal antibody against CPS1].

Aim: To prepare and characterize the monoclonal antibody (mAb) against human carbamyl phosphate synthetase I (CPSI) and make a study of its application.

Methods: Normal human liver tissues were homogenized, and their mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAb using the routine hybridoma technique.

[Method of antibody analysis based on visual protein array].

Aim: To establish a novel method for antibody analysis based on visual protein microarray.

Methods: The antibodies spotted on the aldehyde-modified slides were incubated with corresponding antigens labeled by biotin, followed by silver enhancement detection method, and the chromogenic images were scanned by CCD camera.

Results: The affinity and specificity of each antibody was assayed by this method and six antibodies were found to have higher specificity than the others.

[Preparation and characterization of antibodies against clusterin].

Aim: To prepare rabbit polyclonal antibodies (pAb) and mouse monoclonal antibodies (mAbs) against human clusterin(CLU) and characterize these antibodies' properties.

Methods: CLU fragment was amplified from human liver cDNA library, and recombinant expression vectors pGEX-4T-1-CLU and PET-32a-CLU were constructed. GST-CLU fusion protein was expressed in E.

[Generation and characterization of monoclonal antibody against HSD11B1].

Aim: To generate and identify monoclonal antibodies (mAb) against homo sapiens hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1).

Methods: Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to generate mAb by hybridoma technique.

[Identification of protein antigen for antibody DBD02 by biopanning of T7 phage display library].

Aim: To identify the specificity of mAb DBD02, we developed and optimized a new method by biopanning of T7 Select human liver cDNA phage display library.

Methods: After two rounds of biopanning, eluted phages were subjected to Dot blot using mAb DBD02 as primary antibody. The positive PFUs (plaque forming unit) which recognized by DBD02 were further confirmed by Western blot, PCR and sequencing.

[Generation and characterization of monoclonal antibody against GRHPR].

Aim: To generate monoclonal antibodies (mAb) against glyoxylate reductase/hydroxypyruvate reductase (GRHPR).

Methods: Normal human liver tissues were homogenized, and human liver cytosolic proteins were isolated by centrifugation. The total human liver cytosolic proteins were used to immunize BALB/c mice to prepare mAb by hybridoma technique.

[Preparation and identification of monoclonal antibody against Homo sapiens hemoglobin alpha 2 (HBA2)].

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.

[Recombinant human VEGF-D induces the angiogenesis of the chick embryo chorioallantoic membrane].

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.

Heat shock protein 70 inhibits alpha-synuclein fibril formation via interactions with diverse intermediates.

alpha-Synuclein (AS) is a main component of Lewy bodies in midbrain dopamine neurons pathologically characteristic of Parkinson's disease. We show that heat shock protein (Hsp) 70 inhibits AS fibril formation via preventing the formation of prefibrillar AS (PreAS), binding with PreAS to impede nuclei formation, and binding with nuclei to retard fibril elongation. Also, Hsp70 suppresses the PreAS-induced permeabilization of vesicular membrane through interactions with PreAS.

[Preparation and identification of the rabbit antibody against human sialin].

Aim: To prepare the rabbit antibody against human sialin and identify its properties.

Methods: Recombinant expression vector pGEX-5X-1-sialin was constructed, in which the sialin cDNA encoding the 1-38 aa was fused to the C-terminal of the gene encoding the GST protein. The GST-sialin (N1-38) fusion protein was expressed in E.

[Preparation and characterization of monoclonal antibody against human LSECtin].

Aim: To prepare and characterize monoclonal antibody (mAb) against human LSECtin (liver and lymph node sinusoidal endothelial cell C-type lectin) protein.

Methods: BALB/c mice were immunized with prokaryotically expressed human LSECtin protein. The splenocytes from the immunized mice were fused with murine myeloma cells (Sp2/0) and then the mAb-positive hybridoma cells were screened by indirect ELISA.

[Preparation and characterization of monoclonal antibody against DCXR].

Aim: To prepare monoclonal antibodies (mAbs) against Dicarbonyl/L-xylulose reductase (DCXR).

Methods: Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique.

[Generation and preliminary application of rabbit anti-human polyclonal antibodies against NH2-terminal peptides of CXCR3-B].

Aim: To generate and identify the rabbit polyclonal antibodies against NH(2)-terminal peptides of CXCR3-B.

Methods: Three peptides (aa. 1 to 19, aa.

An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens.

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies.

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs.

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration.

Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins.

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation.