Objective:To investigate the best preservation conditions of N-terminal pro-brain natriuretic peptide( NT-proBNP) and provide detection references with stable performance for detection kits.Methods: ELISA was used to quantitatively detect the changes in NT-proBNP contents in various preservation solutions.The effects of basic buffer system, preservative Proclin300 and antibiotics on the preservation of NT-proBNP were analyzed using univariate analysis.The combination of various factors was then optimized using orthogonal experiments, to identify the best preservation system for NT-proBNP.Results: The univariate analysis determined that the basic buffer system for NT-proBNP was 0.02 mol/L phosphate buffered saline(PBS) at pH7.2,the addition of pre-servative Proclin300 could extend the preservation time of NT-proBNP at 37℃ by one day, the combined addition of penicillin and streptomycin prolonged the preservation time of NT-proBNP by one day compared with individually adding penicillin or streptomycin.The orthogonal experiments identified a preservation solution for NT-proBNP as 20%calf serum,1/1 000 Proclin300,120 U/ml penicillin and 80 U/ml streptomycin in a basic buffer system of 0.02 mol/L PBS at pH7.2.This solution was used to preserve an NT-proBNP reference sample at 37℃.Seven days later,the calibrated fixed-value of the sample at 37℃was only 1.3%lower than that at 4℃.Conclusion:Optimized NT-proBNP serum preservation solution could preserve NT-proBNP standard sample at 37℃ for seven days.

Objective To construct and identify norepinephrine ( NE) complete antigen for the preparation of high sensitive and specific anti-NE monoclonal antibody .Methods Glutaraldehyde ( GA) and 1-Ethyl-3-(3-Dimethylaminopropyl ) carbodiimide ( EDC) were used to cross-link NE with carrier pro-teins (BSA, OVA) for NE complete antigen preparation under conditions of pH 4.5 or pH9.0.Three assays including UV scanning , SDS-PAGE and FeCl3 color reaction were performed for identification of NE com-plete antigen.Serum antibody titers were evaluated in mice model induced by intraperitoneal immunization with NE complete antigen .Results NE complete antigens were successfully prepared as indicated by the three identification assays .The coupling ratio was significantly increased in a time-depended manner under the condition of pH9.0 in comparison to that in the condition of pH 4.5.Indirect ELISA results showed that , when coating antigens and serum antibodies were prepared with the same cross -linking method , the serum antibody titers were significantly higher than those with different methods .Conclusion Anti-NE antibodies were successfully prepared by immunizing mice with NE complete antigens .

To prepare monoclonal antibody of carbohydrate antigen 19-9(CA19-9).Methods: Based on the titer test results of mouse ascites and its IC 50 values ,the mouse that prepare for fusion was identified.Positive monoclonal cell strains were established by cell fusing and screening.Monoclonal antibody from ascites was produced by peritoneal injection monoclonal cell , and then purified by octoic acid-ammonium sulfate precipitation method.After determine the protein concentrations by UV-spectrophotometry ,the monoclonal antibody against CA 19-9 was labelled with horseradish peroxidase.Based on antibody pairing test , DAS-ELISA method was established .To compared with abroad kit , analyzing performance of this method.Results: Three strains of monoclonal antibody were obtained.And the optimal working concentrations of mAb (ZJY3-1G9) ,as coated antibody,McAb(ZJY2-7F10),as HRP-IgG,were assured.Limit of detection was 26.4 U/ml.Linear range was 30-300 U/ml.By detecting patients with serum 33 , confirmed the correlation coefficient of r=0.950 4 , compared with abroad kit that measure simultaneously.Conclusion:Monoclonal antibody prepared for CA 19-9 can be used to develop a kit.

Objective:To establish quantitative a fluorescence immunochromatographic assay detection method for the heat shock protein 90α(HSP90α)in human serum.Methods: Indirect the technology of fluorescence microshers immunochromatographic assay was used to research fluorescent microsphere activation,ensure optimal testing time,determine linearity range,precision,recovery experiments,testing clinical samples and that sort of thing in the fluorescence immunochromatographicassay experiment.Results: In all the reaction conditions,it was determined that the optimal teaction time was 5 minutes,and in the best line range (0.39-100 ng/ml) precision quality control materials of high recovery (30 ng/ml) Intra-assay CV was 8.14%;quality control materials of low recovery (10 ng/ml) Intra-assay CV was 9.26%.With high,medium and low recovery of serum recovery was 100.56%,99.76%,94.1%,separately.Foreign kit(ELISA) for detection of 40 cancer patients clinical serum,the result showed that the correlation was 0.968 5.Conclusion: Establishing the method initially quantitative fluorescence immunochromatographic assay for the heat shock protein 90α(HSP90α)in human serum have high performance and linear,clinic accordance rate also can satisfy the demand of clinic quantitative detection,and will improve hopeful to applied to clinic after apprroved.

To establish a time-resolved fluorescence immunochromatographic assay for quantitative determination of carbohydrate antigen 19-9 (CA19-9) in serum, we prepared CA19-9 test strips by integrating double-antibody sandwich method and fluorescence immunochromatography technique. Carboxy fluorescent microspheres and nitrocellulose membrane were used as carriers for labeling and coating CA19-9 pairing antibodies. We optimized the process by adjusting the amount of labeling and coating antibody. According to the linear range, lowest detection limit and precision, We evaluated the time-resolved fluorescence immunochromatographic assay of CA19-9. When the amount of labeled antibody was 80 μg for 20 μL fluorescent microspheres, and the concentration of coated antibody on the test line was 1.5 mg/mL, the optimal reaction time was 15 minutes. Assay linear range was 12.5 to 800 U/mL and the minimum detection limit was 6.32 U/mL. The Within-run and between-run coefficient of variation were less than 15%. Average recovery rate was 101%. By detecting 50 clinical samples in parallel with Roche electrochemical luminescence detection kit, correlation coefficient was 0.980 6. The experiment, initially established a fluorescence immunochromatographic detection method to quantitative detection of serum CA19-9, which has a good clinical application prospect.

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.
Adjuvants, Immunologic ; Aluminum ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19/prevention & control* ; COVID-19 Vaccines/genetics* ; Humans ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/genetics* ; SARS-CoV-2/genetics* ; Spike Glycoprotein, Coronavirus/genetics* ; Tetanus Toxin ; Tuftsin ; Vaccines, Subunit ; Viral Vaccines

A rapid and simple method to detect tumor markers in liver cancer was established by combining immunochromatography technique with fluorescent microsphere labeling. According to the principle of double antibody sandwich, the cytoskeleton-associated protein 4 (CKAP4) paired antibody was used as the labeled and coated antibody, and the goat anti-rabbit polyclonal antibody was used as the quality control line coated antibody in the preparation of the CKAP4 fluorescent immunochromatographic test strips. After the preparation, the test strips were evaluated on various performance indicators, such as linearity, precision and stability. The CKAP4 immunochromatographic strip prepared by time-resolved fluorescent microspheres had high sensitivity, and good specificity. Its precision was within 15%, recovery between 85% and 115%, and linear range between 25 and 1 000 pg/mL. The test strip could be kept stable at 37 °C for 20 days, and it correlated well with commercial ELISA kits. The CKAP4 fluorescence immunochromatography method can quantitatively detect the content of CKAP4 in serum. Furthermore, it is rapid, sensitive, simple, economical and single-person operation. This method has the potential of becoming a new method for the diagnosis and treatment of liver cancer.
Animals ; Antibodies ; metabolism ; Chromatography, Affinity ; Fluorescence ; Humans ; Liver Neoplasms ; diagnosis ; Membrane Proteins ; isolation & purification ; Molecular Diagnostic Techniques ; instrumentation ; methods ; Sensitivity and Specificity

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.
Antibodies, Viral ; COVID-19 ; Chromatography, Affinity ; Fluorescent Antibody Technique ; Humans ; Microspheres ; SARS-CoV-2 ; Sensitivity and Specificity