Preparation and application of anti-human PD-L1 monoclonal antibodies / 中国免疫学杂志

Objective:To obtain a high specificity and high affinity anti-human PD-L1 monoclonal antibody which can be used for clinical diagnosis and block PD-L1 and PD-1 binding.Methods:BALB/c mice were immunized with recombinant PD-L1 protein.The positive cell clones stably secreting anti-human PD-L1 monoclonal antibody were obtained by classical hybridoma cell fusion technique.The specificity,affinity,subtype and other characteristics of the antibody were identified by ELISA.Immunofluorescence and indirect immunofluorescence were used to detect the tumor cells.Antibody blocking activity was confirmed by tumor killing test.Results:Two cell strains stably secreting monoclonal antibodies against human PD-LI were screened out.Abl and Ab2 had high titer and affinity.The antibody titers were 1:2.56×106 and 1:3×105,and the affinity was 1.5×109 L/mol and 2.5×10s L/mol respectively.There was no cross reaction between these two antibodies and PD-L2.Immunoblotting,indirect immunofluorescence confirmed that the antibody can be used to the diagnosis.Experiment showed that PD-L1 antibodies can increases tumor-killing activity of CIK cells.Conclusion:Two hybridoma cell lines capable of stably secreting highly specific and high affinity anti-human PD-L1 monoclonal antibody are obtained.They can specifically bind to PD-L1 molecules on tumor cells and can be used to the diagnosis of tumor phenotype and prognosis.Antibody blocking function can be applied to combined CIK cell immunotherapy.

Identification of a new shrimp allergen enolase from Litopenaeus vannamei / 中国免疫学杂志

Objective:To identify enolase,47 kD allergen,from Litopenaeus vannamei by Mass spectrometry. Methods: The proteins were extracted from Litopenaeus vannamei tissue with acetone precipitation method. The protein components were analyzed by SDS-PAGE and Western blot. By using Matrix-Assisted Laser Desorption/Ionization time of flight mass spectrometry ( MALDI-TOF/TOF-MS) ,the 47 kD allergen from Litopenaeus vannamei was identified as enolase. Results:By SDS-PAGE,we proved that the native protein components from Litopenaeus vannamei were completely. According to the Western blot result more than 14 components could react with the positive serum. MALDI-TOF/TOF analysis results showed that the suspected proteins were enolase. A sensitization frequency was 55%. Conclusion:Enolase was identified as a new allergen of Litopenaeus vannamei.

Expression of bFGF in malignant tumor and its clinical pathological sig-nificance / 中国病理生理杂志

[ ABSTRACT] AIM: To detect basic fibroblast growth factor ( bFGF ) expression in clinical common malignant tumor ( non-small-cell lung cancer,breast cancer, colon cancer and melanoma) , and to identify relationship between the expression and tumor clinicopathological characteristics.METHODS:Immunohistochemical SP method was used to detect the expression of bFGF at protein level in 208 cases of paraffin-embedded tissue of primary malignant tumor patients ( 68 cases of lung cancer, 80 cases of breast carcinoma, 41 cases of colon cancer and 19 cases of melanoma) .RESULTS:The bFGF protein expression levels were significantly higher in low differentiated non-small-cell lung cancer with lymph node metastasis, and were positively correlated with TNM.In addition, no significant influence of the bFGF protein expression on the patients with median survival period was observed.The protein expression of bFGF was higher in advanced breast cancer with lymph node metastasis and was commonly found in the middle/higher differentiated colon cancer with regional lymph node metastasis.Meanwhile, bFGF protein was highly expressed in advanced melanoma patients with lymph node metastasis.CONCLUSION:bFGF may participate in the process of occurrence and progression of malignant tumor.Ex-pression of bFGF protein may be an effective parameter for evaluating metastasis and prognosis of malignant tumor.

Analysis of allergen components in shrimps and crab corresponding to Chinese / 中国免疫学杂志

Objective:To identify the allergens which can react with Chinese allergic patients in shrimp and crab ,and analysis their reaction-rates.This results would provide foundation for further research on allergen-detection and desensitization therapy.Methods:Allergen components in Metapenaeus ensis ,Macrobrachium rosenbergii and Charybdis feriata by using 46 portions of shrimp(crab) allergic patients’ serum IgE in Western blot.Results:The reactions between shrimps and allergic patients ’ serum IgE were stronger than that between crab and serum.32-38 kD Tropomysin (TM),40 kD Arginine kinase (AK),60-80 kD Hemocyanin (Hc) and 21 kD arcoplasmic calcium-binding protein(SCP) were the major allergens in shrimps.TM,AK and Hc were common major allergens among shrimps and crab and TM shared the highest reaction-rate.Compare to the results of some from American researchers , AK,Hc and SCP have higher reaction-rate when react with Chinese patients serum ,and we also found a new allergen in shrimp.Con-clusion:For Chinese patients , shrimps have higher reaction-rate than crabs and the allergens among shrimps and crabs which are roughly same.There are some differences in allergens among different human races.A new allergen with 48 kD was found in this re-search.

Rapid detection of Listeria monocytogenes by immunomagnetic separation combined with selective medium / 生物工程学报

Listeria monocytogenes is a pathogenic bacterium, therefore, it is essential for food safety monitoring to establish a rapid and specific detecting method. In this study, immunomagnetic beads and selective medium were combined to detect Listeria monocytogenes at different concentrations (10(1)-10(5) CFU/mL). Other three types of Listeria spp., Staphylococcus aureus and Vibrio parahaemolyticus were also detected to conduct the cross-reaction analysis. Meanwhile, contaminated milk samples were prepared to explore the limit of detection of immunomagnetic beads combining with selective medium. Results showed that Listeria monocytogenes with the concentration of 10(3) CFU/mL and above was successfully detected. Milk samples were detected within 6 hours, with a detection limit of 0.7 CFU/mL. The method developed is capable of detecting milk samples within 30 h, which is 38 h faster compared with national standard method with the same sensitivity.

Screening, expression and characterization of single chain Fv (scFv) against basic fibroblast growth factor / 中华微生物学和免疫学杂志

Objective To obtain the specific human scFv basic fibroblast growth factor(bFGF)using phage antibody library technology. Methods The library was panned with human recombinant bFGF for 4 rounds. The antigen binding activities of random clones were tested by ELISA in order to select specific antibodies, which were then examined by DNA sequence analysis. Results The positive clone selected from the 104 random clones was able to bind bFGF specifically, while not able to bind other growth factors,such as aFGF, VEGF(vascular endothelial growth factor). By competition ELISA assay we found one clone 44 could inhibit bFGF binding to FGFR1. Conclusion Seven specific human phage antibody against bFGF was obtained by phage display technique, one clone could inhibit bFGF binding to its high affinity receptor FGFR1.

Screening of the antigen epitopes of basic fibroblast growth factor by phage display.

In order to investigate the epitope of basic fibroblast growth factor (bFGF) and its immunogenicity, the epitopes of bFGF were screened from the phage display library with monoclonal antibody GF22, which can neutralize the bio-activity of bFGF. By three rounds of screening, the positive phage clones with bFGF epitopes were selected, which can effectively block the bFGF to bind with GF22. Sequence analysis showed that the epitopes shared a highly conservative sequence (Leu-Pro-Pro/Leu-Gly-His-Phe/Ile-Lys). The sequence of PPGHFK was located at 22-27 of the bFGF. The specific immuno-response of mouse could be highly induced by phage clones with the epitopes. And the anti-bFGF activity induced by LPGHFK was 3 times higher than the original sequence, which showed that the mimetic peptide LPLGHIK might be used as a tumor vaccine in the prevention and treatment of tumor.

Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation.

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.

Expression, purification, and characterization of humanized anti-HBs Fab fragment.

Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.

Se-containing spirulina phycocyanin attenuated liver injury induced by carbon tetrachloride in mice / 中国病理生理杂志

AIM: To investigate the effect of Se-containing spirulina phycocyanin (Se-SPC) on liver injury of mice induced by carbon tetrachloride (CCl 4). METHODS: The mouse model was conducted by intragastric feeding with 2% CCl 4 oil for three times, meanwhile Se-SPC, spirulina phycocyanin (SPC) and Na 2SeO 3 were injected (ip) to various groups for 7 days. Then selenium (Se), glutathione peroxidase (GPx), superoxide dismutase (SOD), alanine aminotransferase (ALT), malondiaoldehyde (MDA) and nitric oxide (NO) levels in blood and liver were measured. RESULTS: The level of Se, GPx and SOD activities were obviously higher ( P

Involvement of bFGF in the lung response to silica in a mouse model / 中国病理生理杂志

AIM: To investigate the relationship between basic fibroblast growth factor (bFGF) and the development of silicosis in mice. METHODS: MTT test was utilized to examine the effects of bFGF-neutralizing antibody and the bronchoalveolar lavage fluid (BALF) of the mice exposed to silica on lung fibroblast cell growth. RESULTS: BALF from mice treated intrabronchially with silica promoted the growth of lung fibroblasts and anti-bFGF antibody inhibited the effect of BALF dramatically. CONCLUSION: These results indicates that bFGF secretion increases in lung in a mice silicosis model and participates in the development of silicosis.

Effects of antisense oligonucleotide on endogenous human bFGF, bFGF mRNA and FGFR1 in tumor cell SWO-38 / 中国免疫学杂志

Objective:To clarify the contribution of endogenous bFGF, bFGF mRNA and FGFR1 to the abnormal growth and phenotypic transformation of neoplastic tumors cells.Methods:The antisense oligonucleotide primers was used to evaluate the influence of endogenous bFGF on growth of human glioma malignant cell lines SWO-38 in vitro. MTT was used to examine the variety of cells growth treated with bFGF antisense oligonucleotide primers. The methods of ELISA, in situ hybridization, immuno-hischemistry and image analysis were used to detect the expression level of bFGF, bFGF mRNA and FGFR1. The colony formation of cells in soft agar was used to assess the cloning efficiency of the cells after exposed to bFGF antisense oligo-nucleotide primers.Results:The cells multiplication, expression of bFGF mRNA and FGFR1 was inhibited by bFGF antisense oligonucleotide primers,and the cells multiplication was dose-dependent. Treated with antisense oligo-nucleotide primers, the expression of FGFR1 and secretion of bFGF were distinctly reduced, and the inhibition efficiency of cells multiplication of WSO-38 was 48% and the inhibition efficiency of colonies of SWO-38 in soft agar was 35%. The inhibition of cells multiplication can be reversed completely by external bFGF, and the reverse efficiency was 8%.Conclusion:The synthesis of bFGF mRNA and expression of bFGF can be specifically inhibited by antisense oligonucleotide, but the inhibition can be cleared up with the addition of external bFGF. The study suggested that the bFGFantisense oligonucleotide could have good effect in inhibiting of tumor under special condition.