Chemiluminescence immunodetection method based on double-enzyme amplification system
A chemiluminescence immunoassay and amplification system technology, applied in the chemiluminescence immunoassay, the detection of IP-10 in serum and plasma, can solve the problems of low sensitivity and low sensitivity, and achieve high sensitivity, wide detection range and stability. strong effect
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Embodiment 1
[0056] Include the following steps:
[0057] (1) Add 100 μL of 5 μg / mL rabbit anti-IP-10 polyclonal antibody to coat a white 96-well Immuno plate, and incubate at 37°C for 1 h; the coating solution is 50 mmol / L carbonate buffer, and the pH of the coating solution is 9.6;
[0058] (2) Wash three times with PBST buffer, 1 min each time; the formula of PBST buffer includes 8.1 mmol / L disodium hydrogen phosphate, 1.9 mmol / L sodium dihydrogen phosphate, 18 mmol / L sodium chloride and 0.05% (v / v)Tween-20;
[0059] (3) Add 200 μL of 5% nonfat milk blocking solution to each well and incubate at 37°C for 1 h;
[0060] (4) Add 100 μL of different concentrations of IP-10 fusion protein or 100 μL of the diluted sample to be tested to each well, and incubate at 37°C for 1 h; the preparation method of IP-10 fusion protein is as follows:
[0061] The IP-10CDS gene sequence with His tag was optimized and synthesized, and the sequence was subcloned into pET-28a vector using Nco I and Xho I ...
Embodiment 2
[0073] Include the following steps:
[0074] (1) Add 100 μL of 5 μg / mL rabbit anti-IP-10 polyclonal antibody to coat a white 96-well Immuno plate, and incubate at 37°C for 1 h; the coating solution is 75 mmol / L carbonate buffer, and the pH of the coating solution is 9.6;
[0075] (2) Wash three times with PBST buffer, 1 min each time; the formula of PBST buffer includes 8.1 mmol / L disodium hydrogen phosphate, 1.9 mmol / L sodium dihydrogen phosphate, 18 mmol / L sodium chloride and 0.05% (v / v)Tween-20;
[0076] (3) Add 200 μL of 5% nonfat milk blocking solution to each well and incubate at 37°C for 1 h;
[0077] (4) Add 100 μL of different concentrations of IP-10 fusion protein or 100 μL of the diluted sample to be tested to each well, and incubate at 37°C for 1 h; the preparation method of IP-10 fusion protein is as follows:
[0078] The IP-10CDS gene sequence with His tag was optimized and synthesized, and the sequence was subcloned into pET-28a vector using Nco I and Xho I ...
Embodiment 3
[0090] Include the following steps:
[0091] (1) Add 100 μL of 5 μg / mL rabbit anti-IP-10 polyclonal antibody to coat a white 96-well Immuno plate, and incubate at 37°C for 1 h; the coating solution is 100 mmol / L carbonate buffer, and the pH of the coating solution is 9.6;
[0092] (2) Wash three times with PBST buffer, 1 min each time; the formula of PBST buffer includes 8.1 mmol / L disodium hydrogen phosphate, 1.9 mmol / L sodium dihydrogen phosphate, 18 mmol / L sodium chloride and 0.05% (v / v)Tween-20;
[0093] (3) Add 200 μL of 5% nonfat milk blocking solution to each well and incubate at 37°C for 1 h;
[0094] (4) Add 100 μL of different concentrations of IP-10 fusion protein or 100 μL of the diluted sample to be tested to each well, and incubate at 37°C for 1 h; the preparation method of IP-10 fusion protein is as follows:
[0095] The IP-10CDS gene sequence with His tag was optimized and synthesized, and the sequence was subcloned into pET-28a vector using Nco I and Xho I...
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