Double-antibody sandwich kit for detecting liver cancer and liver cirrhosis-related polypeptide-protein combinatorial markers
A technology for detecting antibodies and kits, which is applied in the field of double-antibody sandwich enzyme-linked immunosorbent assay or chemiluminescence kits
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Embodiment 1
[0074] Example 1: Optimization of Detection Conditions for Capture Antibodies and Detection Antibodies
[0075] 1) Dilute the capture antibody with coating buffer to 50ng / mL, 100ng / mL, 250ng / mL, 500ng / mL, 1000ng / mL and 2000ng / mL, respectively, according to 100μL / well, and place overnight at 4°C.
[0076] 2) Take the ELISA plate coated with the capture antibody in the kit, add 100 ng / mL HKa standard to each well, 100 μL per well. Incubate at 37°C for 1.5 hours;
[0077] 3) Discard the solution, wash each well 5 times with 300 μL PBST washing buffer, and pat dry;
[0078] 4) Dilute the HRP-labeled detection antibody with PBST at 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, add 100 μL to each well, and stand at 37°C Incubate for 40 minutes;
[0079] 5) Discard the solution, wash each well with 300 μL PBST washing buffer 5 times, and pat dry;
[0080] 6) Add 100 μL of TMB substrate chromogenic solution interacting with HRP to each well, and develop color at 37°C in...
Embodiment 2
[0083] Embodiment 2: the preparation of kit
[0084] 1. Preparation of reagents
[0085] Carbonate coating buffer: pH9.6, 0.1mol / L, weighed Na 2 CO 3 1.59g, NaHCO 3Add 2.93g of deionized water to 1000mL.
[0086] PBST washing buffer: pH value is 7.4, including 80mmol / L Na 2 HPO 4 , KH of 20mmol / L 2 PO 4 , KCl of 100mmol / L, NaCl of 1400mmol / L, Tween-20 whose volume fraction is 0.5%.
[0087] TMB Chromogenic Solution: Substrate Chromogenic Solution A: Sodium Acetate 13.6g, Citric Acid 1.6g, 30% Hydrogen Peroxide 0.3mL, Distilled Water to 500mL; Substrate Chromogenic Solution B: Disodium EDTA 0.2g, Citric acid 0.95g, glycerin 50mL, take 0.15g TMB and dissolve in 3mL DMSO, add distilled water to 500mL. Take substrate color development solution A and mix equal volumes of substrate color development solution B to obtain TMB color development solution.
[0088] Sulfuric acid stop solution: prepare a sulfuric acid aqueous solution with a concentration of 2mol / L.
[0089] 2...
Embodiment 3
[0095] Example 3: Identification of the minimum detection limit and detection range of the kit
[0096] Get the kit that embodiment 2 makes, prepare the HKa standard solution of gradient concentration as the sample to be tested, the concentration of HKa standard is: 0ng / mL, 30ng / mL, 60ng / mL, 90ng / mL, 120ng / mL, 150ng / mL.
[0097] Take the ELISA plate coated with the capture antibody in the kit, add different concentrations of HKa standard solution, 100 μL per well, and repeat 3 times for each concentration. Incubate at 37°C for 1.5 hours; discard the solution, wash each well 5 times with 300 μL PBST buffer in the kit, and pat dry;
[0098] Add 100 μL of HRP-labeled detection antibody to each well, and incubate at 37°C for 40 minutes; discard the solution, wash each well 5 times with 300 μL of PBST buffer in the kit, and pat dry;
[0099] Add 100 μL of TMB chromogenic solution to each well, and develop color at 37°C in the dark for 15 minutes; add 50 μL of 2mol / L sulfuric acid...
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