Anti-Gd-IgA1 monoclonal antibody and ELISA kit for auxiliary diagnosis of IgA nephropathy
A technology of monoclonal antibodies and kits, applied in disease diagnosis, biological testing, instruments, etc., can solve the problems of poor stability of polysaccharide recognition activity, unsuitability for large-scale research and clinical diagnosis, etc.
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Embodiment 1
[0030] Example 1. Preparation of anti-human Gd-IgA1 monoclonal antibody
[0031] This embodiment provides a method for preparing an anti-human Gd-IgA1 monoclonal antibody, and the specific method steps include:
[0032] 1) Entrust a third-party company to synthesize the antigenic epitope polypeptide in the hinge region of Gd-IgA1, the sequence of which is: VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT(GalNAc)PSPS-NH2, where GalNAc stands for N - Acetylgalactosamine;
[0033] 2) Weigh 20 mg of Gd-IgA1 epitope peptide and dissolve it in 5 mL of coupling buffer (0.1 M MES, pH 4.7) to prepare a 4 mg / mL peptide solution;
[0034] 3) Use Imject TM The EDC mcKLH Spin Kit (ThermoFisher Company) coupled the Gd-IgA1 epitope peptide with the carrier protein KLH and centrifuged and desalted, and the experimental steps were carried out according to the instructions;
[0035] 4) Use the conjugated Gd-IgA1 epitope peptide to immunize BALB / c mice. The co-immunization procedure is as follo...
Embodiment 2
[0042] The preparation of embodiment 2 people's Gd-IgA1 Elisa kit
[0043] The human Gd-IgA1 Elisa kit provided by the present invention is composed of anti-human Gd-IgA1 monoclonal antibody pre-coated ELISA plate, human Gd-IgA1 standard, sample diluent, concentrated washing solution, HRP-labeled anti-human IgA1 polyclonal Composition of antibody, TMB chromogenic solution and stop solution.
[0044] Wherein, the preparation method of anti-human Gd-IgA1 monoclonal antibody pre-coated microtiter plate comprises the following steps:
[0045] 1) Pre-coat the anti-human Gd-IgA1 monoclonal antibody onto a 96-well ELISA plate with ELISA coating buffer (0.05mol / L pH 9.6 carbonate buffer) at a coating concentration of 1-5 μg / mL, The coating volume was 50 μL / well, and coated overnight at 4°C.
[0046] 2) Block with 5% BSA (prepared in 0.01 mol / L, PBS with pH 7.2-7.4), 100 μL per well, incubate at 37° C. for 1 h, wash the plate 3 times, and pat dry.
[0047] 3) Add 200 μL ELISA Plate ...
Embodiment 3
[0066] The detection method of embodiment 3 people Gd-IgA1 Elisa kit
[0067] Utilize the human Gd-IgA1 Elisa kit of the present invention (embodiment 2) to detect the application of Gd-IgA1 level in human serum or blood plasma, its steps are:
[0068] 1) Prepare the standard product: ①When using for the first time, dissolve the standard product in 1mL sample diluent to 500ng / mL as the storage solution. . ②Take one EP tube, add 160 μL of sample diluent, and then add 40 μL of standard stock solution to make 100 ng / mL, marked as C1. ③Take another 6 EP tubes, add 100 μL of sample diluent to each tube, and mark them as C2-C7 in turn. ④ Take 100 μL standard substance from tube C1 and add it to C2, mix well; then take 100 μL standard substance from tube C2 and add it to C3, mix well; and so on. ⑤The concentrations of the obtained C1-C7 standard substances were as follows: 100, 50, 25, 12.5, 6.25, 3.13, 1.56 ng / mL.
[0069] 2) Sample preparation: Dilute the serum or other samples...
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