| At present, the infectious diseases caused by a variety of pathogenic microorganisms continue to be hazardous to human health and lead to death of humans and animals. Outbreaks of avian influenza, tuberculosis and other infectious diseases have triggered major social and economic problems. Toxoplasma gondii infection and other zoonotic diseases have been a serious threat to the development of animal breeding and cause human health problems at birth. The development of new detection techniques and kits is important for infectious disease prevention and treatment. In this paper, rapid detection techniques and kits for the avian influenza virus H5 subtype (AIV-H5), Mycobacterium tuberculosis and Toxoplasma gondii infection were developed based on the monoclonal antibody technique, genetic engineering technique and colloidal gold labeling immunochromatography technique.BALB/c mice were immunized with purified AIV-H5 antigen and spleen cells were fused to myeloma cells SP2/0. The AIV-H5 monoclonal antibodies were screened and the Ig subclass and their antigen binding sites were analysed. Five AIV-H5 monoclonal antibodies with good specificity and stability were obtained. Except for one McAb which belongs to IgM subclass, the other four are IgG subclass with the highest ascites antibody titer of 1:2.56×105. Three of them have different antigen binding sites and are suitable for"double-antibody sandwich"immunoassay. AIV-H5 lateral flow assay was developed by colloidal gold labeling AIV-H5 McAb and AIV-H5McAb coated on nitrocellulose membrane. Except for AIV-H5 antigen, the developed lateral flow assay did not have cross reaction with the other AIV subtypes, NDV, IBV, EDSV or other avian viruses. The sensitivity reached 1: 22 HA (hemocyte agglutination) which was 10 times higher than that of agar diffusion test but 10 times lower than the fluorescence immunohistochemistry assay. The coincidence rate and reproducibility of the assay is 100% with precision coefficient of variation of zero (CV=0). The kit quality was stable after store at 37℃for 12 days. Except for the negative result from the heart, AIV-H5 was detected in the liver, spleen, lung and other organs of chicken artificially infected with AIV-H5 by this assay, which is 100% coincident to chicken embryo virus isolation assay. In field test the coincidence rate of the assay and fluorescent RT-PCR kit was 100%.The primers derived from TB16 and TB38 genes were used to amplify the target gene fragments from Mycobacterium tuberculosis H37Rv genomic DNA. After digested with restriction enzymes, the amplified TB16 and TB38 gene fragments were ligated to pET30a and transformed to E. coli DH5αcell. The transformants were identified by PCR, enzyme digestion and sequencing. The 0.4 kb fragment of TB16 and 1.1 kb fragment of TB38 were amplified. The pET30a-TB16 and pET30a-TB38 recombinant expression plasmids were successfully constructed and sequencing results indicated the 435 bp insert of TB16 and 1053 bp insert of TB38 were identical to the corresponding sequences in GenBank. The recombinant plasmid pET30a-TB16 and pET30a-TB38 were then transformed to E. coli BL21 (DE3), induced for expression and analysed with SDS-PAGE. After induction by IPTG, pET30a-TB16 transformant had an expression band at 21 kDa which reached 40% of bacterial total protein as soluble form. After induction by IPTG, pET30a-TB38 transformant had an expression band at 41 kDa which reached 30% of bacterial total protein as inclusion body form. Tuberculosis antibody colloidal gold labeling array assay was developed by colloidal gold labeling human IgG McAb with TB16 and TB38 recombinant antigens coated on nitrocellulose membrane. The detection coincidence rate and reproducibility of the assay is 100% with precision coefficient of variation of of zero (CV=0). The kit quality was stable after store at 37℃for 12 days. The sensitivity for detection active TB antibody was 78.3%, which was higher than acid-fast stain (27.96%) and Bacteria culture (57.99%) with very significant difference (P<0.01). The sensitivity was 6.1% higher than that of TB antibody lateral flow assay based on only TB38 recombinant antigen. TB antibody positive rate of the array assay was 77.66% in acid-fast stain negative patients and the detection rate was similar to positive patients (82.32%). TB antibody positive rate of the array assay was 65.50% in Bacteria culture negative patients but the detection rate was lower than positive patients (85.82%). Double-antigen sandwich T. gondii antibody colloidal gold labeling lateral flow assay was developed with T. gondii excretory and secretory antigen. The detection coincidence rate of the assay is 100% to T. gondii positive and negative control serum with the precision coefficient of variation of 0.3% (CV=0.3%). The kit quality was stable after storage at 37℃for 12 days or at room temperature for 18 months. The T. gondii antibody positive rate was 2.87%, 4.38% and 6.72% respectively in 852 normal physical examination serum samples, 547 pregnant or lying-in woman serum samples and 1206 tumor patients, which was 96% identical to that of ELISA. T. gondii infection in tumor patients indicated that T. gondii antibody positive rate in tumor patients (6.72%) was 2.38 times higher than that in normal medical population (2.82%) with very significant difference (P<0.01). There were some differences of T. gondii infection rates in different clinical forms tumor patients. T. gondii infection rate in the renal cell carcinoma patients was the highest (22.50%), followed by lung cancer (15.04%), skin cancer (12.05%) and breast cancer (9.03%) patients, which was 3 to 8 times higher than those in normal human serum (2.82%) with very significant difference (P<0.01). T. gondii infection rate in digestive tract cancer patients was 5.56% with significant difference to normal serum (P<0.05). T. gondii infection rate in uterine cancer patients was 3.70% and 3.68% in other malignant tumor patiens which was slightly higher than normal serum with no significant difference (P>0.05). While the T. gondii infection rate in blood lymphoid malignancies (2.27%), liver (1.79%) and thyroid cancer (0) was lower than those in normal serum. T. gondii infection rates were different among different clinical forms of tumor patients, the infection in renal cell carcinoma was 3.3 times higher than the average infection rate in other malignant tumors (6.72%) with very significant differences (P <0.01) .The developed AIV-H5 gold labeling detection kit, TB antibody gold labeling array assay kit and T. gondii antibody gold labeling lateral flow assay kit showed high specificity and sensitivity and could be used to rapidly detect these diseases.
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