Time-resolved fluorescence immunochromatographic reagent for rapidly, quantitatively and simultaneously detecting cTnI and H-FABP, and preparation method thereof
A technology of time-resolved fluorescence and immunochromatography, which is applied in the field of clinical medical diagnosis, can solve the problems of reducing the false positive rate, achieve the effect of improving sensitivity, improving detection precision and accuracy, and eliminating the interference of samples
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Embodiment 1
[0053] (1) Preparation of cTnI, H-FABP time-resolved fluorescent immunochromatographic test strips:
[0054] A. Preparation of detection line (T1, T2) solution: dilute cTnI monoclonal antibody and polyclonal antibody, H-FABP monoclonal antibody to 1-2 mg / ml with 10 times, 50 mM citrate solution;
[0055] B. Preparation of the quality control line (C) solution: dilute the rabbit IgG antibody to 1-2 mg / ml with 10-fold, 50 mM citrate solution;
[0056] On the bottom plate (1) with adhesive backing, adopt the overlapping method, first paste the nitrocellulose membrane (3), and then paste the Fusion5 membrane (2) and the absorbent paper ( 4). On the nitrocellulose membrane (3) and close to the Fusion5 membrane (2), draw T1 (H-FABP capture line (5)), T2 (cTnI capture line (6)), respectively, at the end close to the absorbent paper (4) Draw the C (rabbit IgG) line, and the intervals between T1, T2, and C are all 4mm.
[0057] At the T line, draw T1 and T2 respectively, and at the ...
Embodiment 2
[0060] Detection of cTnI, H-FABP time-resolved fluorescent immunochromatographic test strips
[0061] (1) Preparation of cTnI, H-FABP goat anti-rabbit time-resolved fluorescent microspheres
[0062] Dissolve time-resolved fluorescent microspheres (particle size about 200nm) in 100mM MES buffer (pH 6.0), add activator (NHS, 20mg / ml; EDC, 20mg / ml) to activate for 15 minutes; wash, centrifuge, Reconstitute the time-resolved fluorescent microspheres with 60mM boric acid buffer (pH 8.5), then add troponin I monoclonal antibody 2 to react for 2 hours (the mass ratio of microspheres to antibodies is 10mg:0.5mg); the reaction is completed Then add blocking agent (BSA, 100mg / ml) to block for 2 hours; after blocking, wash, centrifuge, redissolve with 60mM boric acid buffer (pH 8.5) and blocking agent (BSA, 100mg / ml) again, sonicate, make The microspheres are evenly dispersed in the buffer, and stored at 2-8°C in the dark.
[0063] The preparation process of fluorescent microspheres la...
Embodiment 3
[0069] Precision testing
[0070] With the test method in embodiment 2, test the mixed standard substance of cTnI, H-FABP respectively (the concentration of cTnI is 1 and 25ng / ml, the concentration of H-FABP is 10 and 80ng / ml), each standard substance is repeatedly detected Ten times, the specific detection values are shown in Table 1 below.
[0071] Table 1 precision test result table
[0072]
[0073]
[0074] As can be seen from the data in Table 1, using the triple detection test strip of the present invention, the precision of cTnI and H-FABP are all less than 10%, fully meeting the requirement of POCT product precision less than 15%.
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