Objective  To establish and validate a double-antibody sandwich-ELISA for detection of pneumococcal capsular polysaccharide serotype 14 (PS14) concentration.  Methods  Rabbit anti-PS14 polyclonal antibody (pAb) was purified from rabbit serum by Protein G affinity chromatography. Double antibody sandwich-ELISA was established using mouse anti-PS14 monoclonal antibody (mAb) as capture antibody, rabbit anti-PS14 pAb as detection antibody, alkaline phosphatase-labeled goat anti-rabbit IgG as secondary antibody, and PS14 as reference. The working concentrations of mouse anti-PS14 mAb and rabbit anti-PS14 pAb were optimized to set up the standard curve. The accuracy, precision, and specificity of the method were verified. Influence of adjuvant on the method was observed. Results  The optimal working concentration of mouse anti-PS14 mAb was 5 μg/ml, and that of rabbit anti-PS14 pAb was 1 μg/ml. The standard curve ranged from 9.375 to 600.000 ng/ml with good linearity, and the coefficient of determination was 0.999. The recovery rate of polysaccharide samples was 110%~140%, and that of polysaccharide-protein conjugate samples was 90%~110%. The mean intra- and inter-assay coefficients of variation were 5.64% and 7.14%, respectively. The recovery rate of PS14 in 13-valent pneumococcal conjugated mixture sample was 85%~100%. The recovery rate of PS14 in the vaccine sample containing adjuvant was 85%~110%. Conclusion  The double antibody sandwich ELISA was successfully established. The method has good accuracy, precision, and specificity. The detection results are not affected by adjuvant, showing certain durability of the method.
 

Objective  To express recombinant human MHCⅠchain related protein A α3 domain (MICA-α3) and natural killer group 2D (NKG2D), prepare anti-MICA-α3 monoclonal antibodies and test antibody binding function. Methods  MICA-α3 and NKG2D expression vectors MICA-α3-pET11c and NKG2D-pET16b were constructed and transformed into BL21 for expression. Recombinant proteins were purified by polyhistidine-tag chromatography and gel chromatography. After immunizing mice with MICA-α3 protein and screening monoclonal antibody by hybridoma technology, the monoclonal antibody function was tested by cell-ELISA. Results  Recombinant proteins were expressed in the form of inclusion body in E.coli  BL21. Five monoclonal antibodys were screened, which could bind to MDA-MB-453 cells espressing MICA-α3，and this binding did not affect the binding of MDA-MB-453 cells to NKG2D. Conclusion  Anti MICA-α3 monoclonal antibody can bind to tumor cells without affecting the binding of tumor cells to NKG2D.

Objective To generate monoclonal antibodies (mAbs) against coxsackievirus A2(CV-A2)and to establish a quantitative antigen ELISA. Methods Mice were immunized by purifiedCV-A2 virions to obtain hybridomas secreting anti-CV-A2 mAbs. Linear range of established CV-A2ELISA was determined, and its accuracy, precision, stability and specificity were validated. Samples in all purification steps of CV-A2 particles were detected. Results High binding-efficiency mAbs againstCV-A2 were generated. A quantitative ELISA for antigen detection was established. The detection range was 5. 00-320. 00 ng/ml. The recovery rates for accuracy verification of samples with high, medium andlow concentrations were between 89. 58% and 104. 78%. Their respective coefficients of variation (CVs)of repeatability verification were 2. 10%, 2. 47% and 6. 18%, and CVs of intermediate precision verification were 2. 89%, 2. 69% and 1. 94%. The recovery rates of durability were 84. 26%-114.21%. Coated microtiter plate was placed at 37 ℃ for 3 d and samples recovery rates were90.31%-103.11%. Specificity verification showed that the method only recognized CV-A2 antigen, and did not cross react with other antigens. Conclusion A CV-A2 quantitative ELISA is established, which can be applied to antigen detection in samples at different purification stages of CV-A2, and in multivalent hand-foot-mouth disease vaccines containing CV-A2.

Objective  The prepare fluorescent monoclonal antibody (McAb) against Seoul virus (SEOV) and preliminarily apply to the inactivation verification in the stock solution of type Ⅱ hemorrhagic fever with renal syndrome inactivated vaccine. Methods Using the inactivated virus stock solution of SEOV L-99 strain as immunogen, mouse hybridoma cell fusion technology was used to screen specific anti-SEOV McAb hybridoma. McAb ascites was prepared by induction method in mice and purified by protein A affinity chromatography, and its specificity was identified by Western blot. The titer and relative affinity of McAb were determined by indirect ELISA. The purified McAb was labeled with fluorescein isothiocyanate (FITC) by stirring method, and the virus inactivation validation in the stock solution of type Ⅱ hemorrhagic fever with renal syndrome inactivated vaccine was detected by direct immunofluorescence method and mouse intracerebral inoculation method. Results  Four hybridomas 1D5, 2E3, 3A4 and 5B7 with specific anti-SEOV L-99 activity were screened. Western blot showed that the four McAbs reacted specifically with nucleocapsid protein of SEOV L-99 strain. The titer of ascites determined by indirect ELISA was 3.13×10⁸﹣1.25×10^-7. The relative affinity order of 4 McAbs was 1D5 > 3A4 > 2E3 > 5B7. Purities of purified antibodies were >95%. The binding ratio of FITC label 1D5 McAb was 3.4. The direct immunofluorescence method and mouse intracerebral inoculation method were used to detect inactivated vaccines of type Ⅱ hemorrhagic fever with renal syndrome for three generations blindly, and the results of virus inactivation validation were consistent. Conclusion A high-titer specific anti-SEOV McAb is Successfully screened , and fluorescently labeled, which can be applied to the verification of virus inactivation during the production process of the type Ⅱ hemorrhagic fever with renal syndrome inactivated vaccine.

Antibody drug attracts much attention in tumor treatment for its high specificity and low side effects. Bispecific antibody (BsAb) simultaneously bind two different antigens or two epitopes of one antigen. Compared with monospecific antibody, BsAb shows higher specificity as well as better therapeutic effect and safety, and reduces side effects. Several BsAb drugs have been approved to the market and increasing numbers of drugs enter clinical studies. BsAb formats are constantly updating, and application range of BsAbs is increasing, which shows a good application prospect in the future. In this paper, the types, mechanisms, and difficulties of BsAb preparation are summarized.

Objective　To structurally optimize and preliminarily identify the biological activity of anti-tumor necrosis factor receptor superfamily member 4 (OX40) monoclonal antibody Ab18.Methods　The species cross-binding activity of Ab18 to OX40 of different species was measured by ELISA. The binding activity of Ab18 to HEK293-hOX40 cells was measured by flow cytometry (FCM). The blocking activity and agonistic activity of Ab18 were measured by reporter gene methods. The Fc fragment of Ab18 was engineered by point mutagenesis technology to change the affinity of the Fc fragment with Fc receptors. ELISA and FCM were used to evaluate the binding activity to human Fcγ receptor ⅡB and human neonatal Fc receptor, and the reporter gene method was used to measure the agonistic activity and antibody-dependent cell-mediated cytotoxicity (ADCC) activity of Ab18 and its mutants.Results　The Ab18 antibody had species cross-binding activity with mouse, rat and rhesus monkey OX40s, and stronger binding activity and stronger blocking activity with HEK293-hOX40 than the control antibodies GBR830 and KHK4083. The median effect concentration (EC₅₀) value of binding activity of Ab18 with HEK293-hOX40 was 366.9 ng/ml, and the half maximal inhibitory concentration value of blocking activity was 657.3 ng/ml. In addition, Ab18, GBR830 and KHK4083 showed agonistic activity. The ADCC activity EC₅₀ value of Ab18 was 25.7 ng/ml, and the agonistic activity EC₅₀ value was 14.9 ng/ml. The mutated Ab18 had lower agonistic activity, better binding activity with human neonatal Fc receptor, consistent ADCC activity, and good biological activity.Conclusion　The Ab18 antibody is successfully optimized, showing better biological activity.

Objective To optimize the culture medium for Chinese hamster ovary (CHO) cells expressing anti-human IL-17A/F monoclonal antibody (anti-hIL-17A/F), and screen domestic culture media with high expression and comparable quality of antibody. Methods A CHO cell line expressing anti-hIL-17A/F was used to optimize the culture medium. The optimal basic medium and feed medium combination was selected, and the feed strategy optimization and 2 L bioreactor screening were carried out. Then the antibody expression and quality were measured in bioreactor final confirmation.Results The combination of basic medium BM-01, feed medium FM-01 and FM-02 was the optimal medium combination for the expression of anti-hIL-17A/F in recombinant CHO cells. Among them, the feed medium FM-01 was supplemented with a feed strategy of day3/6/8/10/12 (3%-7%), and the ratio of FM-01 to FM-02 was 10∶1. In 2 L bioreactor， the protein expression exceeded 6.5 g/L, more than twice of that of the original process, and the product quality was consistent.Conclusion High expression and comparable quality of anti-hIL-17A/F in CHO cells are achieved by using the combination of basic medium BM-01, feed medium FM-01 and FM-02, which lays a foundation for large-scale production of anti-hIL-17A/F using domestic culture media.

Objective To establish and compare 3 tetanus antibody detection methods in vitro. Methods The indirect method, double antigen assay and toxin binding inhibition (ToBI) test were established. The developed methods were validated for linearity and range, intermediate precision, accuracy and detection limit. Results The linear range of indirect method was 0.007 8-1.000 0 mili-international unit (mIU)/mL [coefficient of determination (R²) = 0.998 6], of double antigen assay was 0.078-10.000 mIU/mL (R²=0.998 7) and of ToBI test was 1.95-31.25 mIU/mL (correlation coefficient > 0.98). The validation results indicated that three methods meet the guiding principles of analytical method validation in terms of precision, accuracy, and specificity. Comparing the positive detection rates of these methods, there was no statistically significant difference (χ²=1.34，P=0.513). Conclusion Three detection methods for tetanus antibody in vitro, namely indirect method, double antigen assay and ToBI test, are successfully established, laying research foundation for in vitro method to replace in vivo neutralizing assay.