Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit
A technology for detection of outer membrane proteins and antibodies, applied in the biological field, can solve the problems of many false negative results, reduce test accuracy, false positives, etc., and achieve the effect of meeting the requirements of epidemic prevention
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Embodiment 1
[0038] The Brucella outer membrane protein OMP28 that embodiment 1 Escherichia coli expresses is prepared
[0039] 1. Materials
[0040] 1.1 Strain and serum
[0041] Brucella bovis A544 strain was provided by the China Veterinary Drug Administration, the prokaryotic expression vector pET-28a (+), and Escherichia coli BL21 (DE3) were preserved by the Immunology Laboratory of Capital Medical University.
[0042] 1.2 Reagents
[0043] DNA polymerase, various restriction enzymes and T4 DNA ligase were purchased from NEB Company; Sephadex G25 and Superdex75 prepacked columns were purchased from GE Company; DNA gel recovery kit and plasmid extraction kit were purchased from Dalian Bao Biological Engineering Co., Ltd. .
[0044] 2. Primer Design
[0045] Referring to the gene sequence of Brucella bovis OMP28 (CP003176) provided by NCBI, primers were designed with Primer5.0 software and synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The upstream prime...
Embodiment 2
[0057] The preparation of embodiment 2 antigen-coated microtiter plates
[0058] 1. Reagents
[0059] (1) Coating solution: 25mmol / L carbonate buffer solution, pH9.6. Na 2 CO 3 1.59g, NaHCO 3 2.93g,ddH 2 Add O to 1000mL, store at 4°C for later use;
[0060] (2) Washing solution storage solution (10*PBS): NaCl80g, KCl2g, NaCl 2 HPO 4 12H 2 O29g, KH 2 PO 4 2g,ddH 2 O was added to a constant volume of 1000mL;
[0061] (3) Washing solution (PBST): containing 0.05% Tween-20PBS, (pH7.4). Take 50mL10*PBS, 450mL ddH 2 O, 250uL Tween-20, stored at room temperature;
[0062] (4) Blocking solution: Weigh 5 g of skim milk, dissolve it in 100 mL of PBST washing solution, and store it at 4°C for later use.
[0063] 2. Coated
[0064] Under sterile conditions, dilute the purified recombinant protein OMP28 in the coating solution at 1 μg / ml, add 100 μL / well to a 96-well microtiter plate, and coat in a wet box at 37°C for 1 hour.
[0065] 3. Washing
[0066] Wash 4 times with...
Embodiment 3
[0071] Embodiment 3 Positive control serum, negative control serum preparation
[0072] 1. Reagents
[0073] Brucella standard positive serum and negative serum were purchased from China Veterinary Drug Administration, and thimerosal was purchased from Sinopharm Chemical Reagent Co., Ltd.
[0074] 2. Positive Control Serum Production
[0075] Under sterile conditions, dilute the Brucella standard positive serum (1ml dry powder package) with 2ml of high-pressure distilled water, add 0.01% thimerosal for preservation, then subpackage and label it.
[0076] 3. Negative Control Serum Production
[0077] Under sterile conditions, dilute the Brucella standard negative serum (1ml dry powder package) with 2ml of high-pressure distilled water, add 0.01% thimerosal for preservation, then subpackage and label it.
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