Research On The GICA Dipsticks Of Brucella Strain Specificity Antigens And Antibodies And Application

This topic in view of at present in worldwide scale popular broad cow Brucella and sheep Brucella, developed the specificity antigen, the antibody immunity colloid gold dipsticks of B.abortus and B.melitensis, and carried on the preliminary application to test it.This research first recovers B.abortus from the liquid nitrogen pot hybridoma 4B8, 1C9, 1G9, 1E2 and B.melitensis hybridoma 3C6, 2D10, 1E10, after the expand-raising enables the cell quantity to achieve 5×106, the abdominal cavity injects the Balb/C mouse to produce the monoclonal antibody ascites.Passes through the caprylic acid-ammonium sulfate to purify the antibodies thickly first, then HiTrap Protein G HP affinity column further purifies antibodies IgG, and to the monoclonal antibody hypotype, the affinity, the IgG content, the titer and the specificity carry on the appraisal.After the purified antibodies have non-special belts, the titer is above 1×106,the Mcabs hypotypes are IgG1, IgG3, IgG2a and IgG2b, the affinity constants are between 3.75×106~8.99×108,antibodies IgG contents are between 1.6mg/mL~ 2.5mg/mL and purity in 95.4%~96.2%.After B.abortus 544A and B.melitensis the 16M purification, use the adjuvant to emulsify, the domestic rabbit back hypodermic multi-spot immunity prepare the multi-clone antibodies. Multi-clone antibodies purified method the same as Mcabs. After the purification the antibodies carry on the appraisal with SDS-PAGE, the ELISA examine antibodies titer, the ultraviolet dispersion of light luminosity instrument decides the protein contents, AlphaScreen Rabbit IgG Detection Kit examines the IgG contents, and calculates the purity.Finally the SDS-PAGE result show many anti-fragment size as the anticipated it, B.melitensis and B.abortus antibodies titer respectively are 1:25600, 1:51200, after purified antibodies, protein contents respectively are between 11.32 mg/mL and 14.03mg/mL, IgG contents respectively are 10.45mg/mL and 13.12mg/mL, also the purity achieve 92.3% and 93.5%.Using the citric acid reducing process preparation colloid gold particles, observes its dispersion degree and the uniformity result under the electron microscope.The electron microscope and ultraviolet scanning appraisal colloid gold particles mean diameters are 15nm, 25nm, 30nm and 40nm, the particle sizes are consistent, the distribution is even.To B.abortus 4B8, 1C9, 1G9, 1E2 and B.melitensis 3C6, 2D10, 1E10 carry on the inter-species pair, choosing 4B8, 2D10 as the golden sign antibodies and taking 1C9, 3C6 as the coating antibodies, useing the colloid gold immunity chromatographic assay to prepare colloid gold dipsticks,and to sensitivity, specificity, repeaTab.ility and sTab.ility test, the result indicate B.abortus 4B8, 1G9 best coating density are 25μg/mL, B.melitensis2D10, 3C6 best coating density are 20μg/mL, and the colloid gold best coating pH value is 8.4.The experiment show that the dipsticks lowest examination to limit are between 3×103~5×103CFU, Yersinia O9, coli O157, the salmonella bacillus and the peripneumonia line bacillus not to have the overlapping response.To prepared the water samples, the soil samples and the milk samples carry on the examination, the water samples detection line are 3×103CFU to pick out, the detection line of soil samples and the milk samples are 5×103 CFU, and the dipsticks'repeaTab.ility are very good, guarantee time is 12 months.In above the overseas correlation research report foundation, use the hot phenol method and cold phenol method to separately withdraw B.abortus 544A and B.melitensis 16M LPS, and anthrone color method determines LPS contents. Sephadex G-50 purifies LPS, then carries on the appraisal to its immunity and purity.Finally B.abortus 544A and B.melitensis 16M LPS contents are respectively 1.21mg/mL, 1.36mg/mL, after the SDS-PAGE molecular weights are respectively 46000Da and 59000Da, non-special belt vanishing.Using purification LPS as the coating antigen, with reorganizes golden yellow staphylococcus A protein (SPA) to take the golden sign protein, finally assembles the dipsticks.The best marking quantity of SPA protein is 25μg/mL, and the best marking pH is 6.2. And carries on appraisal to dipsticks, sensitivity,the specificity, the stability and actual samples, finally B.abortus and B.melitensis dipstick lowest examination protein quantity are respectively 1μg /mL, 2μg /mL, with no overlapping response for positive blood serum of coli O157, Yersinia O9, the salmonella bacillus, the peripneumonia bacterium, the keep time is 100d.The 20 pieces of B.abortus positive blood serum dilutes 1:1 with PBS, with this experimental dipsticks carries on the examination, the results show that 19 pieces are positive and the coincidence rate is 95%. After processing 18 pieces B.abortus blood serum samples, uses the plate agglutination test (PAT) and dipsticks separately to carry on the examination, the results of coincidence rates are 100%.