A homogeneous fluorescent immunological reagent set for rapid quantitative detection of heart-type fatty acid binding protein and its preparation method
A technology for binding and quantitative detection of fatty acids, applied in the field of medical testing, can solve the problems of low non-specific fluorescence, low fluorescence value, and inability to excite, and achieve the effects of simple operation, good specificity and low cost.
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Embodiment 1
[0042] 1. Preparation of anti-FABP for marking:
[0043] Purified genetically engineered monoclonal antibodies against heart fatty acid binding protein were selected. Eu 3+ The product code of anti-cardiac fatty acid binding protein monoclonal antibody for labeling is 10E1; the product code of anti-cardiac fatty acid binding protein monoclonal antibody for fluorescein labeling is 9F3 and 5B5.
[0044] 2. Preparation of rare earth element chelate labeled anti-FABP:
[0045] The mouse anti-human H-FABP monoclonal antibody 10E1 solution (3mg / ml) was dialyzed twice with 3L 0.9% NaCl at 4°C for 24hr each time. Add water to adjust the concentration to 1.5mg / ml. Take 0.6ml of the antibody solution and 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 was continued for 1 hr. After centrifugation (10000rpm, 10min) to remove insoluble matter, apply t...
Embodiment 2
[0051] The preparation method of this embodiment is basically the same as that of embodiment 1, except that:
[0052] In step 2, the preparation method of the rare earth element chelate labeled anti-FABP is: dialyze the mouse anti-human H-FABP solution (3mg / ml) twice with 3L 0.9% NaCl at 4°C for 24hr each time. Add water to adjust the concentration to 1.5mg / ml. Take 0.6ml of the antibody solution and add 1ml NaHCO 3 (0.2mol / L), and adjust the pH to 9.1 with 1mol / L NaOH. 20μl of BHHBCB methanol solution (30μg / ml) was added dropwise to the antibody solution under stirring, and the stirring was continued for 1hr. After centrifugation (10000rpm, 10min) to remove the insoluble matter, apply to SephadexG-25 column, use 0.05mol / L NH 4 HCO 3 (pH8.0) elution, separation of labeled protein and free label. UV / Visible spectrophotometer detects A in each collection liquid 330 Value, combine the solution containing the labeled antibody. Add the final concentration of 0.1% BSA and 0.05% NaN ...
Embodiment 3
[0054] The preparation method of this embodiment is basically the same as that of embodiment 1, except that:
[0055] In step 3, dilute the anti-H-FABP monoclonal antibodies 9F3 and 5B5 with 0.1M sodium bicarbonate solution to 1mg / ml, take 5ml of the antibody solution, add 40mg of fluorescein DyLight-DY647 solution, and stir well. Incubate at room temperature for 1.5 hours, mixing every 15 minutes. Finally, use G25 gel column to separate and purify, collect the labeled fluorescein-labeled antibody, dilute with 0.01M phosphate buffer containing 0.025% PEG, 2.5% BSA, 15% glycerol, 0.03% surfactant, and use plastic The bottle is sealed and packaged and stored at 4°C.
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