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Mouse TDAR experimental quantitative analysis detection kit and detection method
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A detection kit and quantitative analysis technology, applied in the direction of analyzing materials, biological testing, and analyzing materials through chemical reactions, can solve problems such as qualitative analysis only, and achieve good practicability, strong specificity, and detection sensitivity high effect
Inactive Publication Date: 2019-09-10
HAINAN MEDICAL COLLEGE
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A mouse TDAR experiment quantitative analysis detection kit proposed by the present invention relates to immune function quantitative analysis detection technology, includes immune protein and matching detection system, and can carry out effective T cell-dependent antibody response (TDAR) Quantitative analysis, which is mainly through collecting the serum of Balb / c mice immunized with the specific antigenic protein KLH, combined with the specific KLH antigen coating concentration, and the dilution of the goat anti-mouse enzyme-labeled secondary antibody and the corresponding serum to be tested magnification to meet the ratio conditions of quantitative analysis, so that the detection system can effectively quantitatively analyze the results of TDAR experiments through the OD value, and overcome the shortcomings of existing experiments that can only be qualitatively analyzed but not quantitatively analyzed, thus achieving the transformation of TDAR experiments from qualitative to quantitative It can directly carry out the quantitative analysis of mouse TDAR experiment, which has the characteristics of high detection sensitivity, strong specificity, convenient operation and good practicability
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Embodiment 1
[0035] Embodiment 1-a mouse TDAR experimental quantitative analysis detection kit, including kit body, antigenic protein KLH, enzyme-labeled plate coated with antigenic protein, biotin-labeled goat anti-mouse enzyme-labeled secondary antibody, standard product KLH Antibody (polyclonal IgG), sample diluent, washing buffer, substrate chromogenic solution A solution, substrate chromogenic solution B solution and stop solution; The coating concentration is 80-85 μg / mL; the dilution ratio of the biotin-labeled goat anti-mouse enzyme-labeled secondary antibody (Goat Anti-Mouse IgG / HRP) is 1:16000-1:20000.
[0036] The antigenic protein KLH (keyhole limpet hemocyanin) is commercially available, CAS number: 9013-72-3.
[0037] The sample diluent contains BSA 1g per 100ml, NaCl 0.8g, KCl 0.02g, KH 2 PO 4 0.024g, Na 2 HPO 4 0.144g, the rest is deionized water.
[0038] The washing buffer contains Tween-20 50 μL per 100 ml, NaCl 0.8 g, KCl 0.02 g, KH 2 PO 4 0.024g, Na 2 HPO 4 0....
Embodiment 2
[0042] Embodiment 2-a kind of detection method of mouse TDAR experimental quantitative analysis detection kit, comprises the steps:
[0043] 1. Experimental Preparation
[0044] A. Preparation of the microtiter plate coated with antigenic protein:
[0045] (1) The antigenic protein KLH was dissolved in the coating solution at a concentration of 80 μg / mL, and the coating solution was carbonatebuffer solution at pH 9.6;
[0046] (2) Add 100 μL of the solution prepared in step (1) to each well of the 96-well ELISA plate, coat at 4°C overnight, empty the liquid and remove the residual liquid, and clean the plate twice with washing buffer;
[0047] (3) 200 μL of 5% skimmed milkpowder was added to each well to seal at room temperature for 1 hour, and the liquid was emptied and vacuum-packed at 4°C in a dark place for future use.
[0048] B. Preparation of standard KLH antibody:
[0049] (1) SPF grade BALB / c mice were selected, and the antigenic protein KLH was injected intraper...
Embodiment 3
[0068] Embodiment 3-According to the detection method of the mouse TDAR experiment quantitative analysis detection kit of embodiment 2, the difference is that, in the preparation process of the microplate plate coated with the antigenic protein, the antigenic protein KLH is dissolved in 85 μ g / mL concentration In the coating solution, the rest of the experimental steps are the same as those in Example 2.
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Abstract
The invention provides a mouse TDAR experimental quantitative analysis detection kit and a detection method. The detection reagent comprises a kit body, an antigenprotein KLH, an enzymelabel plate coated with the antigenprotein, a biotin-labeled goat anti-mouse enzymelabel second antibody, a standard product KLH antibody, a sample diluent, a washing buffer, a substrate coloring solution A, a substrate coloring solution B and a stop solution, wherein in the enzymelabel plate coated with the antigenprotein, the coating concentration of the antigen protein KLH is 80-85[mu]g / mL, and the dilution ratio of the biotin-labeled goat anti-mouse enzyme label second antibody is 1:16000-1:20000. The mouse TDAR experimental quantitative analysis detection kit provided by the invention effectivelyrealizes the qualitative to quantitative conversion of TDAR experiments, can directly carry out quantitative analysis of mouse TDAR experiments, and has the characteristics of high detection sensitivity, strong specificity, convenient operation and good practicability.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a mouse TDAR experimental quantitative analysis detection kit and detection method. Background technique [0002] TDAR is an immune function response in which B cells are exposed to T cell-dependent antigens and produce specific antibodies with the participation of Th2 cells. In recent years, the TDAR test has been considered as a functional test with good predictive potential for detecting the potential immunotoxicity of drugs. By artificially introducing foreign antigen KLH and detecting the production level of KLH-specific IgG antibodies, it reflects the impact of the test substance on the immune system as a whole and predicts the immune system. Therefore, this method has gradually been widely used in the study of drug immunotoxicology. [0003] However, due to the problems of poor antigen specificity, complex operation and large variability in the traditional TDAR test, the spec...
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