Fibronectin detection kit

A fibronectin and detection kit technology, applied in the field of biomedicine, can solve the problems of complex operation, low sensitivity, large amount of antibody, etc., and achieve the effect of satisfying sensitivity, strong anti-interference ability, high automation and rapid batch detection

Inactive Publication Date: 2015-04-01
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA method has high sensitivity, but it has disadvantages such as complex operation and long detection time
The common immunoturbidimetric method uses polyclonal antibody to directly compare the turbidity, and can cooperate with the automatic biochemical instrument for detection. The detection speed is fast and the operation is convenient, but there are problems of large amount of antibody, high cost and low sensitivity.
However, most of the existing fibronectin assay kits are single reagents, which have problems such as low sensitivity, unstable reagents, and poor anti-rheumatoid factor interference ability.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Preparation of Fibronectin Detection Kit:

[0025] Reagent R1: 200mmol / L Tris-HCl buffer solution, 20g / L polyethylene glycol (PEG), 20g / L sodium chloride, 1g / L sodium azide, 0.5g / L anti-human rheumatism factor antibody.

[0026] Reagent R2: Preparation of latex particles labeled with anti-human fibronectin polyclonal antibody: Dilute latex particles with a particle size of 100nm to 3g / L with 50mmol / L MES, pH6.0 buffer solution, per ml of solution Add 0.5mg of EDAC to the solution, react at room temperature for 1 hour, centrifuge at 20000rpm for 30min, remove the supernatant, suspend the precipitate in 50mmol / L MES, pH6. , Suspend the precipitate in 50mmol / LMES, pH6.0 buffer solution, so that the final concentration of latex particles is 6g / L, ultrasonically disperse, add an equal volume of MES diluent containing 8mg / ml anti-human fibronectin antibody while stirring , mixed and stirred, reacted at room temperature for 2 hours, and the final concentration of latex partic...

Embodiment 2

[0028] Determination of Fibronectin in Human Serum Samples:

[0029] Measuring instrument: Hitachi 7060 automatic biochemical analyzer;

[0030] Analysis method: two-point endpoint method;

[0031] Analysis parameters: R1: 240ul, R2: 60ul, sample: 3ul; wavelength: 546nm;

[0032] Determination steps: first add reagent R1 and sample, incubate at 37°C for 5 minutes, then add reagent R2, immediately read the absorbance value of the first point, after timing the reaction for 5 minutes, read the absorbance value of the second point, and calculate the absorbance difference between the two points value.

[0033] Calibration curve creation: take the concentration of the calibration solution as the abscissa, and the absorbance difference corresponding to each concentration of the calibration solution as the ordinate, and make a logit-log(4p) function curve.

[0034] Calculation method of sample concentration: According to the difference of absorbance value of the sample, it is subst...

Embodiment 3

[0036] Kit performance test:

[0037] (1) Sensitivity test

[0038] Using water as a blank sample, select a low-value serum sample for doubling dilution to prepare samples with different concentrations (0, 5, 10, 20, 40mg / L), repeat the measurement 10 times for each sample, and calculate the average value and standard Poor, the results are shown in Table 1, as can be seen from Table 1, the detection sensitivity of the kit of the present invention to serum fibronectin is 5mg / L.

[0039] Table 1

[0040] Fibronectin (mg / L)

0

5

10

20

40

average

0.24

4.91

10.42

19.56

39.14

SD

0.22

0.46

0.41

0.39

0.57

cv

91.7％

9.4％

3.9％

2.0％

1.5％

[0041] (2) Linear range test

[0042] Prepare a high-value serum sample with a concentration of 400mg / L with pure fibronectin, and dilute it with physiological saline according to the dilution ratio of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3...

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Abstract

The invention discloses a fibronectin detection kit which comprises a reagent R1 and a reagent R2, wherein the reagent R1 adopts a buffer solution containing an anti-human rheumatoid factor antibody and a coagulant, and the reagent R2 adopts a buffer solution containing latex particles for marking an anti-human fibronectin antibody; the anti-human rheumatoid factor antibody is an intact antibody or an antibody fragment containing a functional part, and is a rabbit anti-human polyclonal antibody, a goat anti-human polyclonal antibody or a monoclonal antibody. The fibronectin detection kit can meet requirements for high sensitivity and automatic and fast batch detection simultaneously, the anti-interference capacity is high, the stability is high, the dosage of the antibody is small, and the cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a fibronectin detection kit. Background technique [0002] Fibronectin is also called fibronectin (abbreviated as fibronectin), fibronectin, and fibronectin. The English name is fibronectin, and the English abbreviation is Fn. Fibronectin is a high-molecular-weight glycoprotein, containing 4.5%-9.5% sugar, and its monomer relative molecular weight is 220,000-250,000. Each subunit forms a disulfide cross-link at the C-terminus. Plasma fibronectin is mostly a dimer, composed of two similar A chains and B chains, and the entire molecule is V-shaped. Fibronectin in cells is mainly multimer, especially in fibroblasts. Fibronectin is available in soluble and insoluble forms. Fibronectin is soluble in plasma and moves in the α2 or fast β region during electrophoresis. It is mainly synthesized by hepatocytes, endothelial cells and macrophages. Insoluble fibronectin widely exist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N21/82
CPCG01N33/68G01N21/82
Inventor 邹炳德邹继华方亮刘献文
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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