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Objective To prepare a Fc-silent chimeric antibody against human CD36 and analyse its bioactivity and phagocytic effect on CD36(+) platelets. Methods The genes encoding V H and V L fragments of the monoclonal antibody (mAb) 32-106 were obtained through RNA extraction from hybridoma cell lines, PCR amplification and sequence analysis. By using gene synthesis and recombination techniques, the vectors expressing light and heavy chains of the chimeric antibody against human CD36 were constructed. Stable cell lines secreting the chimeric antibody were established by selective culture using Zeocin and Blasticidin. Antibodies were purified by an affinity chromatography column. The purity and molecular weight of the chimeric antibody were detected by SDS-PAGE. The activity of the antibody binding to CD36 antigen was determined by ELISA and flow cytometry. The ability of the chimeric antibody to participate in CD36(+) platelet phagocytosis was analyzed by a platelet phagocytosis assay. Results The vectors expressing light and heavy chains of chimeric antibody were successfully constructed. After co-transfection into HEK293 cells, stable cell lines secreting chimeric antibody were obtained by screening and cloning. The high purity and correct molecular weight of the chimeric antibody were confirmed by protein silver staining. The results of Flow cytometry and ELISA showed that the chimeric antibody had the activity of binding to human CD36 antigen. Platelet phagocytosis assay showed that the Fc-silent chimeric antibody against human CD36 basically lost the ability of mediating monocyte phagocytosis of CD36(+) platelets. Conclusion In this study, the Fc-silent chimeric antibody against human CD36 was successfully prepared, and its loss of ability to mediate CD36(+) platelet clearance was confirmed in vitro, which provides a preliminary basis for the study of modified antibodies for the therapy of CD36-antibody-mediated fetal and neonatal alloimmune thrombocytopenia(FNAIT).

To achieve rapid detection of enramycin in feed, we employed the competitive inhibition method to develop a colloidal gold immunochromatographic test strip based on the anti-enramycin A monoclonal antibody (anti-Er.A-mAb). Colloidal gold probes were prepared with a laboratory-prepared high-purity anti-Er.A-mAb. The effects of pH, antibody titer, and antigen concentration (test line) on the test strip performance were investigated. The colloidal gold test strip prepared with 8 μL potassium carbonate addition, 4 µg/mL antibody, 1.0 mg/mL antigen (test line), and 3 μL gold-labeled antibody showed acceptable specificity and a low limit of detection. The test strip showed the detection limit of 25 ng/mL for enramycin A, with a linear range of 25-300 ng/mL. The experiments on the feed with positive sample addition proved that the test strip had good repeatability and was more sensitive than high-performance liquid chromatography, being applicable for the rapid detection of enramycin in large batches of feed samples.

Objective To screen a monoclonal antibody (mAb) of anti-human Claudin-18 splice variant 2 (Claudin18.2) and construct chimeric antigen receptor T (CAR-T) cells targeting Claudin18.2 based on this antibody sequence for the development of CAR-T cell therapy. Methods Mice were immunized with human Claudin18.2 antigen, and then mice spleen cells were isolated and fused with SP2/0 cells to generate hybridoma cells. By hybridoma screening, we obtained the mouse against human Claudin18.2 mAb. The heavy chain variable region (VH) and light chain variable region (VL) sequences were amplified by PCR with the antibody sequence serving as the template. The linker peptide was used to link VL and VH into a single chain antibody (scFv) for CAR construction. The CAR was cloned into a lentiviral expression vector, and T cells were infected with the packaged lentivirus to prepare targeting Claudin18.2 CAR-T cells. Results The screened mouse anti-human Claudin18.2 mAb exhibited binding ability to both human and mouse Claudin18.2 antigens, with higher affinity than the control antibody. The constructed CAR-T cells showed a killing rate between 50% to 70% against Claudin18.2-overexpressing positive target cells at an effector-to-target ratio of 1:9. Conclusion The prepared mouse anti-human Claudin18.2 mAb exhibites cross-species specificity to humans and mice antigens, with good tissue specificity and high affinity. The constructed anti-Claudin18.2 CAR-T cells show effective killing of target cells.

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli . The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

In recent years, there has been a frequent occurrence of various epidemics worldwide such as COVID-19, monkeypox, influenza, and others additionally, there has been an increase in the number of new patients diagnosed with various types of tumors. Traditional drugs have limited effectiveness against emerging infectious diseases, tumors, and autoimmune diseases. However, with the emergence of hybridoma technology, monoclonal antibodies have achieved extensive applications and antibody drugs are playing an important role in modern medicine. Monoclonal antibodies have undergone various development stages, starting from mouse-derived antibodies to human-mouse chimeric antibodies, humanized antibodies, and ultimately human antibodies. Throughout this process, their immunogenicity has gradually decreased, while their safety for human use steadily increased. Fully human antibodies are currently the safest form of antibody, because their sequences all come from human sources and they do not induce human anti-murine antibody reactions. With the advance of genetic engineering technology, flow cytometry coupled to single B cell gene amplification technology has made it easier to construct and screen for fully human monoclonal antibodies. The development of antibody drugs has provided new opportunities, and the market for monoclonal antibody drugs will further expand. This article reviews the research progress of monoclonal antibodies and presents information on the 163 monoclonal antibody drugs approved by the United States Food and Drug Administration (FDA) as of Oct 1 st , 2023. The aim is to offer new insights for the development and production of monoclonal antibodies in China.

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P <0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P <0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P <0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P <0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P <0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl β-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:10 8 . Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.

Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group ( P <0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group ( P <0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group ( P <0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group ( P <0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group ( P <0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

Objective To prepare monoclonal antibodies (mAbs) against GII.4 norovirus P domain by multiple antigens in an immunization program. Methods BALB/c mice were immunized with the multiple GII.4 NoV P domain, namely 1996cluster (VA387), 2004cluster, 2006b cluster and 2010 cluster. The spleen cells from the immunized mice were fused with SP2/0 cells and the hybridoma cells were screened by ELISA. The supernatant of the mAbs was collected and purified by the limiting dilution assay. Its subtype was identified, and the specificity and neutralization were analyzed by indirect ELISA and HBGA blocking, respectively. Results We obtained thirteen hybridoma cell lines that stably secreted mAbs against GII.4 NoV P domain. Their titers reached above 10 -4 after purification. The subtypes of the mAbs were identified as IgG1. Indirect ELISA showed that all the mAbs specifically bound to all GII.4 norovirus variants. Five mAbs specifically bound to GII.17, GII.3 and GII.6 variants. Three mAbs specifically bound to GII.2 variants and strongly blocked NoV P particle from binding to the histo-blood group antigen (HBGA) receptors. Conclusion The mAbs against GII.4 norovirus P domain have been obtained by combined antigens immunization program. Multi-antigen immunization can enhance immune response significantly and cross-react with other GII.4 norovirus variants. The findings provide a basis for further development of novel GII.4 norovirus vaccines and for the optimization of the immunization programs of combined multi-antigen vaccine candidates.

Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.

Objective To prepare the monoclonal antibodies (mAb) against hemagglutinin of H4 subtype avian influenza virus (AIV), and develop a sandwich ELISA for the detection of H4 subtype AIV. Methods The BALB/c mice were immunized with inactive H4 subtype AIV. A mAb against H4 subtype AIV, designated as 6G4, was obtained by cell fusion, hemagglutination inhibition (HI) screening and subcloing. Immuofluorescence cytochemistry and Western blotting were used to detect the reactivity of 6G4 with H4 subtype AIV, and the specificity, broad spectrum and stability of 6G4 were characterized by HI assay. Subclass of 6G4 was determined by kit. With chicken polyclonal antibody against H4 subtype AIV as coated antibody, 6G4 mAb as capture antibody and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody, a sandwich ELISA for the detection of H4 subtype AIV was established by optimization of the reaction conditions and serial verification. Results 6G4 belonged to IgG1 subclass, and the light chain belonged to κ. It could secrete antibody stably and had good reactivity, specificity, broad spectrum and stability. ELISA based on 6G4 was specific, sensitive, accurate and suitable for the detection of a large number of samples. Conclusion We successfully achieved the anti-H4 subtype AIV mAb, and developed the sandwich ELISA for the detection of H4 subtype AIV.

Objective To study B cell epitopes of the influenza virus hemagglutinin (HA) by computer simulation and ELISA blocking experiment, and to establish a new method for pathogen microbial epitope detection. Methods The 2009 H1N1 influenza virus inactivated vaccine was used as the immunogen, and the monoclonal antibodies (mAbs) were prepared by conventional hybridoma fusion and screening techniques. The characteristics of mAbs were identified by ELISA, hemagglutination inhibition (HI) and Western blot analysis. The obtained mAbs were used as a tool to predict the epitope of H1N1 influenza virus by ELISA blocking experiment combined with computer simulation method. Results Four mAbs against the HA antigen on H1N1 influenza virus were obtained, and HA epitopes were classified into two types by ELISA blocking experiment. The computer simulation revealed that the four antibodies could bind to two epitopes on HA. Conclusion The results of computer simulation and ELISA blocking experiment are consistent, and a new method for predicting the epitopes of other pathogenic microorganisms had been established.

Objective To prepare the monoclonal antibodies specifically against P protein of human respiratory syncytial virus (HRSV) and identify it. Methods HRSV P protein prepared by prokaryotic expression in the form of overlapping peptides was used as the immunogen, and monoclonal antibodies (mAbs) were screened by hybridoma technology. Western blot analysis was used to verify the binding activity of the screened mAbs and P protein, and immunofluorescence cytochemical staining was used to determine whether the obtained mAbs could be used to detect the expression of P protein in HEp-2 cells infected with HRSV. Results P181-15A3 and P211-16D8 with great reactivity and specific recognition of HRSV P protein were screened. Both mAbs could bind to P protein by Western blot analysis and could be used for immunocytochemical detection of P protein in HEp-2 cells after HRSV infection. Conclusion We have successfully prepared the mAbs against HRSV P protein.

Objective: To observe the effect of tumor necrosis factor-α (TNF-α) monoclonal antibody on autophagy in allergic rhinitis (AR) mice. Methods: Thirty six weeks old BALB/c mice were randomly divided by random number table method into five groups: control group, model group (AR group), TNF-α antibody intervention group (AR+TNF-α group), autophagy inhibitor (3-methylindole, 3-NA) intervention group (AR+3-MA group), TNF-α antibody combined with autophagy inducer rapamycin (RAP) intervention group (AR+TNF-α+RAP group), with 6 mice in each group. AR model was established by conventional method, the corresponding reagent was administered before nasal cavity stimulation sensitization and during the whole experiment. Behavioral scores of mice were obtained, blood was collected from the eye socket, and mice in each group were sacrificed to collect nasal mucosa tissue samples. Pathological changes of nasal mucosa were observed by hematoxylin-eosin staining. Expression levels of inflammatory factor and IgE in serum were detected by enzyme-linked immunosorbent assay (ELISA). Expressions of autophagy related indicators microtubule-associated protein-1 light chain-3B (LC3B), Beclin-1, sequestosome1 (p62), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7) were measured by Real-time PCR and Western blot. The aggregation of LC3B protein was observed by immunofluorescence. SPSS 19.0 software was used for statistical analysis. Results: Compared with the AR model group, symptoms of AR in AR+TNF-α group and AR+3-MA group were mild; the pathological changes of nasal mucosa were weak; the expression of IgE, TNF-α, interleukin 4 (IL-4), interferon-γ (IFN-γ) in serum significantly reduced (IgE: 666.19±78.35 ( x ± s ) vs. 692.38±64.29 vs. 1 059.05±146.44, TNF-α: 112.06±12.95 vs. 113.17±15.43 vs. 161.22±17.96, IL-4: 54.05±7.14 vs. 58.26±5.67 vs. 79.95±6.33, IFN-γ: 28.58±4.51 vs. 30.67±2.60 vs. 39.83±3.31, all P< 0.05), and the expression of LC3B Ⅱ/Ⅰ, Beclin-1, ATG5, ATG7 in nasal mucosa significantly decreased, the expression of p62 significantly elevated. After intervention with autophagy inducer RAP, the therapeutic effect of TNF-α monoclonal antibodies on AR was antagonized. Conclusion: TNF-α monoclonal antibody significantly improves nasal symptoms in AR mice by inhibiting autophagy levels.

Objective To prepare monoclonal antibodies (mAbs) against bovine tumor necrosis factor-alpha (BoTNF-α). Methods Recombinant BoTNF-α with His tag (rHis-BoTNF-α) was expressed in a prokaryotic system as immunogen, and recombinant BoTNF-α with GST tag (rGST-BoTNF-α) was expressed as detection antigen. The mAbs were developed by hybridoma cell technology. The reactivity of mAbs with rHis-BoTNF-α and rGST-BoTNF-α was detected by Western blot analysis. Indirect ELISA was used to detect the titer and specificity of mAbs, and the reactivity to commercialized recombinant BoTNF-α (rBoTNF-α) was also evaluated. The reactivity of mAbs against natural antigens was identified by flow cytometry. Results Seven hybridomas stably secreting anti-rBoTNF-α mAbs were successfully obtained. The results showed that all seven mAbs had good reactivity and specificity. Flow cytometry analysis also showed that mAb 4G4 had good reactivity with natural BoTNF-α antigens. Conclusion The anti-rBoTNF-α mAbs are successfully achieved.

Monoclonal antibodies have become the main type of antibody drug because of their high specificity and strong affinity to antigen. However, with the intensive study of the natural monoclonal antibody, many defects have faced, such as the limit times of binding to antigen, the unanticipated antibody clearance and antigen accumulation. Therefore, studies are no longer limited to the natural antibody screening, but rather to improve the efficiency of antibody drugs by engineering. In recent years, the bottlenecks in the development of conventional antibody have been solved effectively since the discovery of a novel recycling antibody. Recycling antibody binds to an antigen in plasma and dissociates from the antigen in endosome, thus maximizing the use of antibody and reducing antigen-mediated antibody clearance and antibody-mediated antigen accumulation. In addition, recycling antibodies can enhance the affinity with Fc receptors through further Fc modification. This paper reviews the research progress of circulating antibodies, including its characteristics, transformation methods and prospects.

Objective To prepare a mouse anti-human B7-1 monoclonal antibody (mAb) and study its effect on growth and migration of tumor cells naturally expressing human B7-1 (CD80) molecular in vitro. Methods BALB/c mice were immunized with L929-B7-1 cells transfected with human B7-1 gene and mAbs were prepared by B lymphocyte hybridoma technology. Then, the recognition ability of mAb to tumor cells expressing membrane B7-1 was detected by flow cytometry. Different concentrations of mAbs (5, 10, 20, 40 μg/mL) were added into Daudi, Raji, and 8266 cells to investigate the anti-proliferation effect by MTT assay. Transwell TM assay was used to analyze the effect of mAb on tumor cell migration in vitro, and flow cytometry was used to analyze the induction effect of mAb on apoptosis of tumor cells. Results A hybridoma cell stably secreting mouse anti-human B7-1 mAb was successfully obtained and named 5G10. The binding rates to tumor cells Daudi, Raji, 8266, U266, Bel-7402 and MCF-7 were (96.3±2.12)%, (95.7±1.79)%, (96.8±2.48)%, (23.2±2.35)%, (1.68±0.35)%, (0.55±0.04)%, respectively. Compared with control group, 20 μg/mL 5G10 mAb significantly inhibited the proliferation of Daudi, Raji and 8266 cells, reduced their migration, and increased cell apoptosis rates remarkably. Conclusion The 5G10 mAb can specifically recognize and bind to membrane B7-1, inhibit the proliferation, migration and promotes apoptosis of tumor cells in vitro.

Objective To prepare monoclonal antibodies (mAb) against RecQ helicase, characterize their biological properties, and then investigate their activity against RecQ helicase in tumor cells. Methods BALB/c mice were immunized with purified recombinant RecQ helicase from E.coli. Hybridoma technique was used to generate the mAb against RecQ helicase. The chromosome karyotype analysis of the hybridoma cells was performed by colchicine blocking. The Ig subtype and titer of mAb in ascitic fluid were determined by ELISA. The biological properties of mAb were detected via Western blotting and fluorescence polarization. Immunofluorescence was used to detect the interactions of mAb with BLM, RecQ4 and RecQ5 helicases in human breast cancer MDA-MB-231 cells. Immunohistochemistry was applied to detect the binding of mAb and RecQ helicase in K562 tumor cells and the corresponding stem cells. Results One stable hybridoma cell line expressing anti-RecQ mAb was obtained and named 6H5. The number of chromosomes was from 94 to 104, and Ig subtype was IgG1. The titer of 6H5 in ascitic fluid was 1×10 -7 . The mAb could specifically recognize E. coli RecQ helicase and thus inhibit its DNA binding activity. Besides, the mAb could also recognize BLM, RecQ4 and RecQ5 in MDA-MB-231 cells, and sensitively detect the expression of the RecQ helicase in K562 tumor cells and the corresponding stem cells. Conclusion The mAb against RecQ helicase was successfully prepared with a high titer and specificity.

Objective To express prokaryotically the non-structural protein 1 (NS1) of duck Tembusu virus (DTMUV) and prepare NS1-specific polyclonal antibodies. Methods The NS1 gene of DTMUV strain AH-F10 was amplified by PCR, followed by subcloning and expression in the prokaryotic vector pET-32a. The recombinant NS1 protein was successfully expressed in Escherichia coli BL21 (DE3), and purified with hydroxymethyl urea and renatured by gradient centrifugation. The BALB/c mice were immunized with the purified recombinant NS1 protein to prepare polyclonal antibodies against the NS1 protein. Furthermore, the titer of the polyclonal antibodies was determined by agar diffusion test (AGP), and the specificity of the polyclonal antibodies was verified by Western blotting and indirect immunofluorescence assay (IFA). Results Polyclonal antibodies against NS1 protein in serum was successfully obtained with an AGP titer of 1:8. Western blotting and IFA demonstrated that the serum with polyclonal antibodies had a high-level specificity and reactivity to the NS1 protein of DTMUV. Conclusion Polyclonal antibodies against DTMUV NS1 protein were successfully prepared and validated in this study.

Objective To prepare recombinant protein of human neuropilin 1 b1b2 domain (HuNRP1 b ) and monoclonal antibodies (mAbs) against the recombinant HuNRP1 b (rHuNRP1 b ). Methods The coding sequence of HuNRP1 b was amplified and cloned into vector pET22b to construct recombinant plasmid pET-HuNRP1 b . After analyzed by restriction enzyme digestion and DNA sequencing, pET-HuNRP1 b was transformed into Escherichia coli and induced to express rHuNRP1 b with histidine tag (His-HuNRP1 b ), which was identified by SDS-PAGE analysis. Then His-HuNRP1 b was used as immunogen to immune BALB/c mice. After the fusion of spleen cells and Sp2/0 cells, the positive hybridoma cells were screened by indirect ELISA that was established with recombinant HuNRP1 b with a trigger factor tag (TF-HuNRP1 b ). The specificity of mAbs against HuNRP1 b was identified by ELISA and Western blotting. The ability of mAbs to bind native HuNRP1 was revealed by indirect fluorescence assay (IFA). Results His-HuNRP1 b was expressed and obtained. Western blotting showed that mAbs 1A10, 6A2 against rHuNRP1 b could bind His-HuNRP1 b with the band of 34 kDa and TF-HuNRP1 b with 89-kDa band, respectively, but not the proteins from bacteria with empty vectors. IFA revealed that the mAbs could bind native HuNRP1 expressed on breast cancer cell line MDA-MB-231. Conclusion The mAbs specific to rHuNRP1 b were generated successfully.

To develop a clinical diagnosis technique for bluetongue virus infection, we established serotype-specific methods to detect serotype 4 of bluetongue virus (BTV-4). Two monoclonal antibodies (mAbs) against VP2 protein of BTV-4, named 4A-1G7 and 4B-1B6, were used as competitive antibodies in the competitive enzyme-linked immunosorbent assays (C-ELISA). We detected 50 negative serum samples from sheep, goats and cattle by C-ELISA. The cut-off values of 4A-1G7 and 4B-1B6 mAbs were 49% and 40%, respectively. The results of the sensitivity, specificity and repeatability by detecting standard positive serum, were consistent with the general standard of Office International Des Epizooties. Furthermore, serum samples of BTV-4, BTV-18 and BTV-20 infection could be screened out through the combined C-ELISAs by 4A-1G7 and 4B-1B6 mAbs. Thus, this technique may diagnose BTV-4, BTV-18 and BTV-20 infections.

To confirm the B cell epitope recognized by monoclonal antibody (MAb) 3G11 of bluetongue virus type 8 (BTV-8) VP2 protein prepared in our laboratory, antigen epitopes recognized by 3G11 were screened and identified by phage display technology. KLLAT sequence was found by sequencing of blue spot after four rounds panning and 283LL284 of common short peptide sequence was obtained after comparison to amino acid sequence of BTV-8 VP2 protein. The peptide sequences KLLAA, KALAT, KLAAT and KLLAT were synthesized and identified by indirect ELISA. KLLAA and KLLAT bound strongly with supernatant and as cites of 3G11 cells and reacted specifically with BTV-8 positive standard sera. Further sequence analysis showed that amino acid sequence 283LL284 was conserved among different serotypes of BTV-8 strains, and283LL284 was the key amino acids of antigen epitopes recognized by 3G11. This study laid the foundation to establish type 8 BTV specific immunological detection methods.

Objective To prepare and characterize the monoclonal antibodies (mAbs) against chicken interleuikin-4 (ChIL-4). Methods Using lymphocyte hybridoma technique, 8-week-old BALB/c mice were immunized with purified recombinant ChIL-4 (rChIL-4) with GST tag (GST-ChIL-4), and rChIL-4 with histidine tag (His-ChIL-4) was utilized to set up an indirect ELISA to screen positive hybridomas. Limited dilution methods were used to generate hybridoma cell lines stably secreting mAbs against ChIL-4. The specificity of the mAbs was identified by indirect ELISA, immunofluorescent assay (IFA) and Western blot. Results Four mAbs against rChIL-4 were obtained and named 6A8, 18F12, 19F1 and 20G3. ELISA titers of the 4 mAbs were (1.6~6.4)×10 4 . ELISA, IFA and Western blot showed that the 4 mAbs could only react with rChIL-4, but not with the other proteins like bovine IL-4 (BovIL-4), chicken IFN-γ (ChIFN-γ). Conclusion The mAbs against rChIL-4 were successfully obtained.

Objective: To prepare human cystatin C( CysC) recombinant protein and produce monoclonal antibodies with high affinity and specificity. Develop a competitive ELISA detection system to detect of CysC in human serum., Methods: The CysC gene sequence was found on NCBI. The optimized gene fragments were synthesized and the recombinant CysC protein was expressed in Escherichia coli then used to immunize Balb/c mice. The positive hybridoma cell lines were obtained by hybridoma cell fusion techniques and ascites monoclonal antibody was prepared and purified. Affinity of the antibody was measured by indirect ELISA. Then competitive ELISA detection system was established, and 52 cases of human serum samples were detected by the detection system., Results: Four stable cell lines secreting CysC monoclonal antibodies were obtained. Antibody Ab3 was used as a detection antibody and HRP labeling was performed. Its affinity constant was 4. 26 × 10~6L/mol. The linear range of detection was 0. 011-1. 924 μg/mL. The detection limit was 4. 598 ng/mL and IC&#95;(50) was 0. 145 μg/mL. The established competitiveELISA serum detection system could accurately detect those 52 serum samples., Conclusion: The monoclonal antibody against CysC with high affinity and specificity has been successfully obtained. A reliable competitive ELISA serum detection system is established. The method provides a basis for the development of CysC rapid immunoassay kit.

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS). An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid (NGNA). The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid (NANA) which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.

Objective To prepare the monoclonal antibody (mAb) against influenza A virus (H1N1) using purified viral particles as antigen and investigate the characterization of host cells infected with influenza virus utilizing the mAb. Methods A/PR/8 (H1N1) virus was cultured in embryonated chicken eggs and further purified by differential and density gradient centrifugation. The structure of viral particles was identified by transmission electron microcopy (TEM). Immunogenicity of purified virus was evaluated by Balb/c mice immunized with formalin-inactivated virus. Hybridomas secreting mAbs were established through a fusion of Sp2/0 myeloma cells and splenocytes from the mice immunized with the virus. The characteristics of mAb were identified by ELISA, immunofluorescence assay (IFA), Western blotting, hemagglutinin inhibition assay (HI) and microneutralization assay. The outside hemagglutinin (HA) on the plasma membrane of the host MDCK cells in which the viruses were propagated and the apoptosis of MDCK cells infected with the viruses were measured using flow cytometry. Cell-based ELISA was established using mAb specific to HA. Subsequently, the growth of virus was analyzed by cell-based ELISA. Results Transmission electron microscopy revealed that the physical structures of the purified virus were spherical, elliptical and extended threadlike. Serum IgG titer to influenza virus showed a progressive increase, and the IgG titer reached 10 6 after the immunization for 6 weeks. Six hybridoma clones secreting mAb specific to A/PR/8 were developed by hybridoma technology. The HI and neutralization activities of PR8-10 mAb were significantly higher than those of the other mAbs. HI and neutralization titers of PR8-10 mAb were 1:2048 and 1:640, respectively. IFA and Western blotting confirmed that PR8-10 mAb could recognize HA. Flow cytometry showed that PR8-10 mAb also recognized HA on the membrane of MDCK in which the viruses were replicated and virus infection induced the apoptosis of MDCK cells. Based on the previous test results that PR8-10 mAb was able to recognize HA on the membrane of the host cells in which the viruses were replicated, cell-based ELISA we established was good at analyzing the growth of virus in MDCK cells. Conclusion We obtained whole viral particles that were demonstrated to be able to stimulate the production of a high IgG titer in a mouse model with formalin-inactivated viral particles, and successfully prepared the mAb against H1N1 of high binding affinity and neutralization potency. HA-specific mAb can be used to analyze the characteristics of virus infection process and the effect of virus infection on the host cells as well.

Objective To express the V1/V2 domain of HIV-1 subtype CRF01&#95;AE gp120 in eukaryotic cells, and then prepare its monoclonal antibody (mAb) and identify its antigen reactivity. Methods Eukaryotic expression vector of pTriEx-3-V1/V2CNE55 was constructed and transiently transfected into HEK293F cell line. The V1/V2-His chimera protein was purified and injected into BALB/c mice. After the fusion between spleen cells of immunized BALB/c mice and myeloma cells SP 2/0, ELISA was used for screening the positive hybridoma cell clones against the V1/V2 recombinant protein. The specificity, titer and type of its mAb were characterized. Results We obtained a stable hybridoma cell line which secreted anti-HIV-1 AE subtype gp120 V1/V2 domain mAb. The ascite titer of the mAb was 1:81 000, and the type of the mAb was IgG1/κ. Western blotting showed that the mAb could recognize recombinant HIV-1 gp120 of different HIV-1 subtypes. Conclusion The study prepared successfully the anti-HIV-1 V1/V2 domain mAb.

Objective: To establish the method of colloidal gold immunochrom atographic assay for detecting cadmium ions rapidly. Methods: The anti - cadmium ion monoclonal antibody - gold conjugate was labeled on the binding pad, cadmium ion hapten and goat anti - mouse IgG were coated on nitrocellulose membrane as the detection line (T line) and quality control line (C line) respectively. The sample pad, colloidal gold bonding pad, nitrocellulose membrane and absorption pad were orderly assembled on the PVC board to cut into a test paper strip. The qualitative results of the assay were visualized in color. Results: When detecting the human urine cadmium ions, the results were tested qualitativly within 15 minutes. The detection limit was 30 μg/L. No cross - reactivity with other heavy metal ions. The test paper strip could be stored at 4 ℃ for 3 months. Conclusion: The method has the advantages of low cost, strong specificity, good stability and reliable results, and is suitable for rapid screening of cadmium poisoning of enterprise and occupational health.

Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10⁻⁹, 1.61×10⁻⁹, and 2.36×10⁻⁹, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1-40, 80-112, and 40-80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

Immunoglobulin (Ig) is considered a part of the innate immune system and cooperates with the complementary system as the first line of defense. In this study, a monoclonal antibody (MAb) direct against the light chain of goose Ig (GoIgCL) was generated, characterized and identified in various immunoassays to detect goose Ig. An immunoaffinity chromatography column prepared with this MAb was used to separate the goose Ig from sera. After being conjugated with horseradish peroxidase (HRP), this MAb was used as the secondary antibody to evaluate the goose-specific antibody. In addition, this MAb distinguished and localized the SIg+ lymphocytes from peripheral blood lymphocytes. MAb against GoIgCL may be good candidate to detect or purify goose Ig under various conditions and as a powerful tool for humoral immunity research on goose.

To establish a method for measurement of the neutralizing activity of a monoclonal antibody against the rabies virus. Twenty-four rabies street virus-positive samples were isolated by the mouse inoculation test. Isolated rabies street viruses were cultured and the virus titer tested in N2A cells. We established a rabies street virus bank with viruses that could adapt to the growth of N2A cells with a high titer. Then repeatability was evaluated after three detections of the TRN006 monoclonal antibody. Of the 24 positive samples,15 strains of the virus could adapt to N2A cells and form fluorescent foci in cells.Finally,10 strains with a high titer were selected for the rabies street virus bank, which covered nine Provinces of China and four Chinese lineages. Three lineages were used for the neutralization test for the monoclonal antibody, and the Student’s t-test showed that it had good repeatability.

Objective To generate mouse anti-human monoclonal antibodies against the molecules expressed on immune cells from malignant pleural effusions, and identify the antigen recognized by the monoclonal antibody named Y4F11. Methods The cells separated from pleural effusions of lung cancer patients were used to immunize BALB/c mice. Hybridoma technology was used for cell fusion and screening; the monoclonal antibody named Y4F11 was chosen as the object for the following experiment. Flow cytometry was performed to analyze the molecules expressed on naive and activated T, B, NK cells and monocytes; Co-immunoprecipitation was conducted to get the antigen pulled down by Y4F11. Mass spectrometry sequencing and BLASTP were used to identify the antigen molecules. Western blotting, dot-blotting and flow cytometry were utilized to identify Y4F11. Results The study obtained 78 hybridoma clones secreting antibodies stably which could recognize surface molecules on cells from pleural effusions. The hybridoma named Y4F11 was chosen as the following experiment object for further identification. The molecule which Y4F11 recognized was about 50 Kd, and further was confirmed as B7-H3 molecule. Furthermore, Y4F11 could recognize B7-H3 expressed on B7-H3 gene-transferred cells as well as B7-H3 fusion protein. Besides, the expression pattern of B7-H3 on immune cells was confirmed by Y4F11 antibody. All these results indicated that the antigen molecule recognized by Y4F11 antibody was B7-H3. Conclusion A hybridoma cell line with stable expression of monoclonal antibody B7-H3 has been successfully obtained and could be used for biological function study on B7-H3.

Objective To prepare monoclonal antibodies (mAbs) against chicken cell cycle checkpoint kinase 2 (cChk2). Methods The cChk2 gene was amplified by reverse transcription PCR (RT-PCR) and subcloned into the prokaryotic expression vector pGEX-4T-3. After induced by IPTG, cChk2 was expressed in BL21 (DE3) E.coli cells and analyzed by SDS-PAGE to determine its soluability. BALB/c mice were immunized with cChk2 protein peritoneally. Indirect immunofluorescence assay (IFA) and Western blotting were used to detect anti-serum; if the detection result was positive, IFA and limited dilution was performed to screen hybridoma clones that produced antibodies against cChk2. Results cChk2 was mainly expressed in inclusion bodies. The anti-sera were able to recognize Chk2. Nine positive hybridoma clones were obtained and identified as 1F4, 2D9, 2G1, 3D9, 3E3, 4B5, 4E2, 5C9 and 5F7. Conclusion The study has prepared mAbs against cChk2 with a good specificity and a high titer.

Objective To develop the monoclonal antibodies (mAbs) against enterovirus type 71 (EV71). Methods Two neutralization epitopes, SP70 and SP55, from EV71 were cloned into the hexon gene of adenovirus type 3 to generate a recombinant adenovirus type 3 (R1R2A3) presenting SP70 and SP55 antigens. BALB/c mice were immunized with the R1R2A3. The mAbs were developed with hybridoma technology and were analyzed with microneutralizing assay, indirect ELISA, Western blotting and direct immunofluorescence assay (DFA). Results The study obtained four hybridoma cell clones, 2C4, D2C9, I2G2 and I12C3. ELISA showed that the titer of D2C9 against EV71 was 1:8 000 000 and the titers of 2C4, I2G2, and I12C3 all were 1:500 000. ELISA and Western blotting demonstrated that all mAbs could specifically recognize the VP1 of EV71. In addition, D2C9 recognized the SP70 epitope, and 2C4, I12C3 and I2G2 all recognized the SP55 epitope. DFA revealed that all mAbs could react with EV71, but not with Coxsackie virus A16 (CoxA16). Conclusion Four mAbs against EV71 have been developed successfully, and all of them could react with EV71 rather than CoxA16.

Objective: To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function., Methods: BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed., Results: A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration., Conclusion: McAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.

To evaluate the neutralizing potency and spectrum of three recombinant human mAbs CR57(Ⅰ), RV08(Ⅱ), RV3A5 (Ⅲ) and the triple combination cocktail against antigenic site I, II and III on rabies virus glycoprotein, a standard fluorescent antibody virus neutralization test(FAVN)on several RV vaccine strains, fixed strains, and street strains of total 11 trains was performed by incubation of RV with varying concentrations of antibody followed by incubation with BHK-21 cells. To investigate whether the antibodies display neutralizing activity against a lethal RV infection in vivo, we performed a Syrian hamster study by infecting with 50LD(50)/100μl of CVS-11 strain intramuscularly (i. m.) in the gastrocnemius muscle. Three recombinant human mAbs CR57 (I), RV08 (II), RV3A5 (III) and the compatibility triple cocktail showed broad cross-neutralizing reactivity to all 11 RV strains. The cocktail composed of three mAbs CR57 (Ⅰ) RV08 (Ⅱ), RV3A5 (Ⅲ) by neutralizing titers of 1 : 1 : 1 has no less in neutralizing ability against these strains, indicating that no mutual interference between the three antibodies. The cocktail exhibited neutralizing synergistic activity against individual strains(JX08-45,Flury,SRV9).The treatment with CR57,RV08,RV3A5 or the triple combination cocktail alone respectively provided better protection with a survival range of 100%against the lethal RV infection compared HRIG immunized alone. Combined immunization with the vaccine, recombinant mAbs protected hamsters with a survival rate of 100%equally as well as HRIG after exposure to a lethal RV infection. Our results provide more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

Objective: To explore the effect and mechanism of CD4 + CD25 + Tregs (Tregs) on the protective efficacy of gluthatione-S-transferase (GST) against Schistosoma japonicum in mice., Methods: Female BALB/c mice were divided randomly into five groups：a normal control group, an infected control group, an anti-CD25mAb group, a GST immunization group and a combination group with GST immunization and anti-CD25 mAb. The GST group and combination group were injected percutaneously with GST 50 μg each mouse, the other two groups were injected with equal volume PBS. The immunization was performed for 3 times for two-week interval, and 2 weeks after the last immunization, each mouse was challenged with 40 S. japonicum cercaria. Two weeks post-infection, the combination group and anti-CD25 mAb group were injected intraperitoneally with 300 μg antiCD25 mAb each mouse. The mice were succumbed 2 weeks, 3 weeks, 4 weeks and 5 weeks post-infection respectively. The percentages of CD4 + CD25 + Tregs in splenocytes of mice were measured with flow cytometer. The levels of IFN-γ, IL-2, IL-4, IL-5 and TGF-β in cell cultural supernatants were determined by sandwich-ELISA after stimulation with Con A. The liver sections were stained with hematoxylin and eosin., Results: The worm burden in the combination group (15.80±2.74) was significantly lower than those of the infected control group (27.78±3.15), anti-CD25 mAb group (21.50±4.21), and GST group (20.84± 6.46). Compared to those of the infected control group, the percentages of CD4 + CD25 + Tregs were significantly higher in the GST group, while the percentages of CD4 + CD25 + Tregs were significantly lower post-anti-CD25 mAb-administration. Regardless of GST administration, the levels of IFN-γ, IL-2, IL-4 and IL-5 after anti-CD25 mAb were significantly higher than those of the infected control groups. There were no significant differences of egg granuloma and the level of TGF-β between each group., Conclusions: CD4 + CD25 + Tregs could be partially blocked by anti-CD25 mAb while Th1 and Th2 type immunization response could be enhanced, which plays a role in improving the protective efficacy of GST against of S. japonicum .

This study was designed to prepare a monoclonal antibody against Orf virus 118 protein and explore the biological properties of ORFV118 using this antibody. We constructed a recombinant plasmid pET33b-ORFV118 that contained a full-length ORFV118 gene. The plasmid was transformed into E.coli BL21,and the expression of ORFV118 was induced by isopropyl-β-d-thiogalactoside (IPTG).Prokaryotic ORFV118 was purified via Ni-NTA affinity chromatography and was subsequently used as an antigen to immunize mice. An anti-ORFV118 antibody was prepared using hybridoma technology. The titer and specificity of this antibody were tested by an indirect ELISA and Western blot/immunohistochemistry, respectively. We successfully obtained three antibody-secreting hybridomas,1A2,3B5,and 5D10.The titers of the three hybridomas were 1:10000,1:6400,and 1:8000.The monoclonal antibody (mAb),1A2 and the highest titer was selected for further research. The mAb 1A2,an IgG1 type antibody was bonded to its immunizing antigen, both the eukaryotic and natural ORFV118 with high specificity. The immunohistochemical analysis showed that the focal specific staining was restricted to the epidermal layer and subcutaneous tissue, which conformed to the characteristics of an ORFV infection. The mAb 1A2 recognized ORFV118with high specificity. Further study of mAb 1A2 will facilitate our understanding of ORFV118 and provide potentially novel methods for the diagnosis, prevention, and treatment of Orf.

Objective: To explore the influence of His-tag on recombinant proteins in vaccination, immunization and pathogenesis., Methods: Multiple mouse monoclonal antibodies (mAb) against His-tag were prepared. The biological and immunoreactive characteristics of these mAbs and their cross-reactivity with the normal human tissues were investigated by ELISA, Western blotting and immunohistochemistry (IHC), respectively., Results: The binding activity of these anti-His mAbs was associated with the steric configuration of the his-tagged antigen. In addition, most of these mAbs reacted with human hemoglobin and some normal human tissues., Conclusion: Anti-His antibodies could be elicited by His-tagged recombinant proteins in vivo experiments. Moreover, the functional studies of the His-tagged recombinant proteins might be affected by the reactions of anti-His6 antibodies with human hemoglobin and normal human tissues.

Objective: To prepare and characterize a monoclonal antibody (mAb) against human tissue factor (hTF) with anticoagulation activity., Methods: BALB/c mice were immunized with truncated recombinant protein (rhTF243). Hybridoma cell lines were generated from cell fusion, and screened using indirect ELISA and prothrombin time (PT). After ascites was developed in BALB/c mice, antibody titers were determined using indirect ELISA. Western blotting was performed to study the antibody specificity. Anticoagulant activity of the antibody was detected by PT assay., Results: A mAb to hTF with excellent anticoagulation activity was identified. Its immunoglobulin subclass belonged to IgG1. Titer of ascites fluid was 1:200 000. Western blotting and PT analysis confirmed the specificity and anticoagulant activity of the antibody. The mAb reacted specifically to both recombinant hTF243 and natural TF on SW620 colon cancer cell surface., Conclusion: A hTF mAb with anticoagulation activity and high specificity has been successfully prepared.

Objective: To prepare 5-fluorouracil (5-FU) immunogen and develop polyclonal antibodies against 5-FU., Methods: The derivant of 5-FU (5-fluorouracil-1-yl-aceto amino acid, 5-FUAA) was synthesized, and then conjugated with bovine serum albumin (BSA) or ovalbumin (OVA) by carbodiimide (CDI) method. 5-FUAA conjugating BSA (5-FUAA-BSA) was used to immunize BALB/c mice to produce antiserum, and 5-FUAA conjugating OVA (5-FUAA-OVA) was used as coating antigen to detect the titer of the antiserum by indirect ELISA. Furthermore, specificity of the polyclonal antiserum was identified by ELISA and Western blotting., Results: 5-FU derivant 5-FUAA was successfully synthesized and conjugated with BSA or OVA. Indirect ELISA showed that the titer of the antiserum from BALB/c mice immunized with 5-FUAA-BAS reached 1:1 280 000. Moreover, ELISA and Western blotting proved that the anti-serum could combine 5-FU specifically., Conclusion: The experiment has prepared high-specific and high-titer polyclonal antibody against 5-FU.

Objective: To obtain recombinant mouse collagenase ADAMTS2 (ADAM metallopeptidase with thrombospondin type 1 motif, 2) C terminal (1109-1213), and prepare the corresponding rabbit anti-ADAMTS2 polyclonal antibodies., Methods: The recombinant expression plasmid pGEX-6p-1-ADAMTS2 (1109-1213) was transformed into E.coli. The target protein was induced by IPTG and identified by mass spectrometry following affinity purification. The expressed and purified ADAMTS2 (1109-1213) protein was used to immunize New Zealand rabbits to prepare anti-ADAMTS2 polyclonal antibodies. The antibody titers were detected by ELISA and the antibody specificity by Western blotting., Results: The protein ADAMTS2 (1109-1213) was expressed in E.coli after IPTG induction, and with the purified protein, we prepared antiserum in the immunized rabbits. The titer of the antiserum reached over 1:160 000. The antiserum showed a good specificity., Conclusion: The high titer and specific rabbit anti-ADAMTS2 antibody has been prepared successfully.

Objective: To prepare the rabbit polyclonal antibody against canine bocavirus (CBV) structural protein VP2 and identify its specificity., Methods: The target sequence of gene fragment encoding VP2 C-terminal region (300 AA) was amplified from CBV infection clone. After the restriction enzyme digestion and nucleotide sequencing, the target gene was successfully inserted into pET32a(+) prokaryotic expression vector to form recombinant plasmid pET-32a(+)-VP2. Then pET-32a(+)-VP2 was transferred into E.coli (BL21), and VP2-his fusion protein was induced under the optimized induction of isopropyl β-D-thiogalactopyranoside (IPTG). The products were identified and analyzed by SDS-PAGE. The purified fusion protein was inoculated into adult rabbits to develop polyclonal antibody. After the titer of the antiserum was detected by ELISA, Western blotting and immunofluorescence staining were performed to evaluate the features of the prepared antiserum., Results: Restriction enzyme digestion and sequencing showed that prokaryotic expression vector pET-32a(+)-VP2 was successfully constructed. The soluble recombinant protein was highly expressed in E.coli BL21 as expected. After purification and inoculation into adult rabbits, the high-titer polyclonal antibody was prepared and the titer was 1:6 400 000. Western blotting and immunohistochemistry demonstrated that the specificity of the prepared polyclonal antibody was perfect., Conclusion: The polyclonal antibody against CBV structural protein VP2 has been successfully prepared.

Objective: To prepare and identify monoclonal antibody (mAb) against human vascular endothelial cadherin 5 (VECDH5)., Methods: BALB/c mice were immunized with recombinant VECDH5 protein for preparing mAb using hybridoma technique. The positive clones were confirmed and selected by indirect ELISA for titer determination. Western blotting, flow cytometry, immunofluorescence staining, immunohistochemical staining were performed to identify the specificity and epitope., Results: One hybridoma cell strain secreting specific mAb against VECDH5 was obtained (2C11). ELISA showed that the titer of the ascites was 1:10 000. Western blotting, flow cytometry, immunofluorescence, immunohistochemistry demonstrated that the mAb could specifically recognize and bind VECDH5. Epitope identification showed that the amino acid sequence recognized by 2C11 was LDREVVPWYNLTVEA., Conclusion: We have prepared mAb against human VECDH5, which has good binding ability and specificity.

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

Objective: To prepare and apply monoclonal antibody (mAb) against human LOC339524 protein., Methods: The non-glycosylated antigenic gene sequence of the LOC339524 protein was expressed in triplicate to enhance immunogenicity. Then this synthetic gene was connected to pET28a plasmid. Recombinant LOC339524 protein was obtained by E.coli expression system and was administered intraperitoneally as an immunogen to BALB/c mice to obtain mAb. The specificity and titer of the mAb were characterized by ELISA. Recombinant LOC339524 protein was identified through Western blotting. The expression of the LOC339524 protein in human myocardial tissues and H9C2 cells were detected by immunohistochemistry, and its level in the sera of patients with different heart diseases was detected with the antibody., Results: We obtained two hybridoma cell lines, 5-D3 and 4-F8, secreting specific mAbs against LOC339524 protein. The titer of 5-D3 was up to 2×10(6), higher than the titer of 4-F8. Western blotting and immunohistochemistry demonstrated that 5-D3 could specifically recognize LOC339524 protein of Homo sapiens and Rattus norvegicus. With the antibody we obtained, we successfully detected the serum level of LOC339524 protein in patients with different heart diseases., Conclusion: The mAb against human LOC339524 protein with good specificity and high titer has been successfully prepared.