TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method

A time-resolved fluorescence and quantitative detection technology, applied in the field of clinical medical diagnosis, can solve problems such as background signal interference, unsatisfactory sensitivity, long detection time, etc., to improve detection precision and accuracy, reduce background signal value, and exclude samples the effect of interference

Inactive Publication Date: 2016-08-24
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-linked immunosorbent assay is complex in operation, long in detection time, and narrow in detection range; latex-enhanced immunoturbidimetry needs to be combined with large-scale instruments, and the cost is high; colloidal gold immunochromatography for qualitative or quantitative detection has low sensitivity and narrow linear range, which cannot meet quantitative , Accurate detection requirements; Most of the fluorescent immunochromatography methods use fluorescein or other substances as light sources. Although they have a high linear range, they cannot guarantee low-end sensitivity due to bac

Method used

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  • TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method
  • TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method
  • TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Preparation of H-FABP time-resolved fluorescence immunochromatographic test strips:

[0065] (1) Preparation of detection line (T) solution: Dilute H-FABP monoclonal antibody 1 to 1 to 2 mg / ml with 10 times, 50 mM citrate solution;

[0066] (2) Preparation of quality control line (C) solution: dilute rabbit IgG antibody to 1~2mg / ml with 10-fold, 50mM citrate solution;

[0067] On the bottom plate (1) with adhesive backing, the method of overlapping is adopted. First, paste the nitrocellulose membrane (3), and then paste the Fusion5 membrane (2) and absorbent paper (2) on both ends of the nitrocellulose membrane (3). 4). On the nitrocellulose membrane (3) and near the Fusion5 membrane (2), mark T (H-FABP capture line (5)), and mark C (rabbit IgG) at the end near the absorbent paper (4), T, The spacing of C is 5mm.

[0068] Draw a T line at the T line and a C line at the C line. The final concentration of T line antibody is 1 mg / ml, the final concentration of C line rabbit IgG...

Embodiment 2

[0071] Detection of H-FABP time-resolved fluorescence immunochromatographic test strip

[0072] (1) Preparation of H-FABP, goat anti-rabbit time-resolved fluorescent microspheres

[0073] Dissolve the time-resolved fluorescent microspheres (with a particle diameter of about 200nm) in 100mM MES buffer (pH 6.0), add an 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 H-FABP monoclonal antibody 2 to react for 2 hours (the mass ratio of microspheres to antibody is 10mg:0.5mg); after the reaction is complete Add blocking agent (BSA, 100mg / ml) for blocking for 2 hours; after blocking, wash, centrifuge, reconstitute with 60mM boric acid buffer (pH 8.5) and blocking agent (BSA, 100mg / ml), and sonicate The balls are evenly dispersed in the buffer and stored at 2-8°C in the dark.

[0074] The preparation process of goat anti-rabbit antibody-labeled fluore...

Embodiment 3

[0085] Precision testing

[0086] The test method in Example 2 was used to test H-FABP standard products (concentrations of 10 and 80 ng / ml), and each standard product was tested ten times. The specific test values ​​are shown in Table 2 below.

[0087] Table 2 Precision test result table

[0088]

[0089]

[0090] As can be seen from the data in Table 2, using the H-FABP detection test strip of the present invention, the precision of the H-FABP is less than 10%, which fully meets the requirement that the precision of the POCT product is less than 15%.

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Abstract

The invention discloses a TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and a preparation method and belongs to the field of clinical medical diagnosis. The reagent comprises two parts including a test strip and a fluorescent liquid, wherein the test strip comprises a bottom plate, Fusion5, a nitrocellulose membrane and a water absorbent pad; the Fusion5, the nitrocellulose membrane and the water absorbent pad are horizontally and sequentially connected and fixed onto the bottom plate; the nitrocellulose membrane is coated with a detection line for H-FABP monoclonal antibodies 1 and a quality control line comprising rabbit IgG (immunoglobulin G) antibodies; the fluorescent liquid contains TRF microspheres labeled by H-FABP monoclonal antibodies 2 and TRF microspheres labeled by goat anti-rabbit antibodies. According to the reagent, the fluorescence intensity is improved by the aid of the TRF microspheres, background signals are reduced, meanwhile, the content of the H-FABP in whole blood, serum or plasma is quantitatively detected, and only 10-20 microliters of samples are required. The test strip is convenient, rapid, simple to operate, short in detection time, high in specialty, high in sensitivity, more accurate in detection result and applicable to rapid diagnosis for clinical POCT (point-of-care testing).

Description

Technical field [0001] The invention belongs to the field of clinical medical diagnosis, and specifically relates to a time-resolved fluorescence immunochromatographic reagent for rapid quantitative detection of H-FABP and a preparation method. Background technique [0002] Fatty acid binding proteins (FABPs) are abundant in active fatty acid metabolism tissues, such as the heart and liver, and their main function is to promote the transport of long-chain fatty acids in cells. Nine different types of FABP have been identified, of which H-FABP (heart-type fatty acid binding protein) is the most widespread because it is abundantly present in cardiomyocytes. The combination of its low molecular weight and cytoplasmic location makes H-FABP a highly sensitive early marker of acute coronary syndrome (especially within 6 hours of onset of chest pain), which can be detected 30 minutes after an ischemic attack. This may be because after myocardial ischemia and myocardial necrosis, it qui...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/32G01N2800/324
Inventor 朱传增石晓强朱奇朗安永君张蕾
Owner SHANGHAI UPPER BIO TECH PHARMA
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