Homogenous phase fluorescence immunoassay reagent group for fast and quantitatively detecting myeloperoxidase and preparation method thereof
A myeloperoxidase and immunoreagent technology, which is applied in the field of homogeneous fluorescent immunoreagent for rapid quantitative detection of myeloperoxidase and its preparation, can solve the problems of low non-specific fluorescence and the like, and achieves simple operation and good specificity. , the effect of low cost
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Embodiment 1
[0043] 1. Preparation of anti-MPO for labeling:
[0044] Purified anti-myeloperoxidase monoclonal antibody expressed by genetic engineering was selected. Eu 3+ The product code of anti-myeloperoxidase monoclonal antibody for labeling is 16E3; the product code of anti-myeloperoxidase monoclonal antibody for fluorescein labeling is 18B7 and 19G8.
[0045] 2. Preparation of rare earth element chelate labeled anti-MPO:
[0046] The mouse anti-human MPO monoclonal antibody 16E3 solution (3 mg / ml) was dialyzed twice at 4° C. with 3 L of 0.9% NaCl, 24 hr each time. Add water to adjust the concentration to 1.5mg / ml. Take 0.6ml of the antibody solution, add 1ml NaHCO 3 (0.2mol / L), and adjust the pH to 9.1 with 1mol / L NaOH. 20 μl of BHHCT methanol solution (30 μg / ml) was added dropwise to the antibody solution under stirring, and the stirring reaction was continued for 1 hr. After centrifugation (10000rpm, 10min) to remove insoluble matter, put on the SephadexG-50 column, use 0.05...
Embodiment 2
[0052] The preparation method of the present embodiment is basically the same as that of Example 1, the difference is:
[0053] In step 2, the preparation method of rare earth element chelate-labeled anti-MPO is as follows: dialyze the mouse anti-human MPO solution (3 mg / ml) twice at 4° C. with 3 L of 0.9% NaCl, 24 hours each time. Add water to adjust the concentration to 1.5mg / ml. Take 0.6ml of the antibody solution, add 1ml NaHCO 3 (0.2mol / L), and adjust the pH to 9.1 with 1 mol / L NaOH. 20 μl of BHHBCB methanol solution (30 μg / ml) was added dropwise to the antibody solution under stirring, and the stirring reaction was continued for 1 hr. After centrifugation (10000rpm, 10min) to remove insoluble matter, put on SephadexG-25 column, use 0.05mol / L NH 4 HCO 3 (pH8.0) to separate the labeled protein and free label. UV / visible spectrophotometer detects the A of each collection liquid 330 value, pool the solutions containing the labeled antibodies. Add final concentrations ...
Embodiment 3
[0055] The preparation method of the present embodiment is basically the same as that of Example 1, the difference is:
[0056] In step 3, dilute anti-MPO monoclonal antibodies 18B7 and 19G8 with 0.1M sodium bicarbonate solution to 1mg / ml respectively, take 5ml of antibody solution, add 40mg of fluorescein DyLight-DY647 solution, stir well, and incubate at room temperature 1.5 hours, mixing every 15 minutes. Finally, use a G25 gel column to separate and purify, collect the labeled fluorescein-labeled antibody, dilute it with 0.02M phosphate buffer containing 0.015% PEG600, 4.5% BSA, 12.5% glycerol, and 0.025% surfactant. The bottle is sealed and stored at 4°C.
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