Procalcitonin light-activated chemiluminescence immunoassay kit and preparation method thereof
A photo-excited chemiluminescence and procalcitonin technology, applied in the field of procalcitonin photo-excited chemiluminescence immunoassay kits, can solve the problems of EIA sensitivity limitation, easy inactivation of enzyme activity, low sensitivity, etc., and achieve a wide detection range. Range, high sensitivity, high sensitivity effect
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Embodiment 1
[0027] Embodiment 1 preparation kit
[0028] Preparation of receptor microspheres coated with anti-procalcitonin monoclonal antibody: Add 0.2 mg PCT monoclonal antibody to a centrifuge tube with a filter membrane, centrifuge at 8 000 r / min for 5-6 min, wash with labeling buffer solution (0.13mol / L, pH8.0PBS) was repeatedly washed 6 times, and 1mg of receptor microspheres, 10μL of 25mg / mL NaBH3CN (prepared with labeling buffer), 1.25μL of 10% Tween-20 were added to the antibody solution. The volume of the labeling buffer was added to 200 μL, and the reaction was shaken at 37°C for 48 hours in the dark. Add 10 μL of 65mg / mL CMO (prepared with 0.8M NaOH) to block the unbound sites, incubate at 37°C in the dark for 1 hour, then centrifuge and wash to obtain receptor microspheres linked to the antibody, which are diluted for use.
[0029] PCT calibrator: use standard buffer (50mmol / L Tris-HCl, 1.5% BSA, 0.9% NaCl, 0.05% Proclin-300, 0.01% Tween-20, pH7.8) to prepare PCT to 0ng / mL,...
Embodiment 2
[0041] Example 2 Evaluation Test
[0042] Reagents: PCT calibrator prepared by the method of Example 1, receptor microspheres coated with anti-procalcitonin monoclonal antibody, biotinylated anti-procalcitonin monoclonal antibody, streptavidin Donor microspheres.
[0043] Detection method: Add 25 μL each of the calibrator, receptor microspheres coated with anti-procalcitonin monoclonal antibody, and biotinylated anti-procalcitonin monoclonal antibody into the wells of a white opaque plate, and incubate with shaking at 37°C 15 min, then add 175 μL of streptavidin-coated donor microspheres, incubate with vibration at 37°C for 15 min, then detect the signal value on the AlphaScreen / Lisa detector, calculate the PCT content of the tested sample from the standard curve, and the unit is ng / ml, at the same time judge the negative or positive of the sample according to the S / CO value, and finally print the test report.
[0044] 1. Detection of linear range
[0045] The blood sample...
Embodiment 3
[0067] Embodiment 3 clinical comparison test
[0068] Reagents: PCT calibrator prepared by the method of Example 1, receptor microspheres coated with anti-procalcitonin monoclonal antibody, biotinylated anti-procalcitonin monoclonal antibody, streptavidin Donor microspheres.
[0069] Source of samples: 126 clinical serum samples came from patients with systemic bacterial, fungal and parasitic infections, systemic inflammatory response syndrome, sepsis, acute and chronic pneumonia, acute pancreatitis, active hepatitis, trauma, etc., and 405 were healthy individuals.
[0070] The serum samples were tested separately by PCT-TRFIA and the chemiluminescent immunoassay kit (ILMA) of the foreign BRAHMS company, and the correlation analysis was carried out by SPSS17.0 software: R=0.977, PAlphaLisa =1.155X ILMA -0.354; Comparing the detection performance of the two methods, it can be seen that the photochemiluminescence immunoassay kit developed by us has no obvious difference with fo...
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