Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample

A detection kit and protein-binding technology, applied in the field of biomedicine, can solve the problems of good sensitivity, low sensitivity, and reduce the amount of antibodies, and achieve the effects of wide linear range, high sensitivity, and cost reduction.

Active Publication Date: 2013-06-05
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the use of a single polyclonal antibody, the common immunoturbidimetric method has the characteristics of good specificity but poor sensitivity or good sensitivity but poor specificity, which cannot meet the requirements of specificity and sensitivity at the same time.
Among them, the website of the State Intellectual Property Office of China discloses a double-antibody composite retinol-binding protein detection kit, which uses a mixture of monoclonal antibodies and polyclonal antibodies for common immunoturbidimetric methods, which can meet the requirements of specificity and sensitivity at the same time. However, the general immunoturbidimetric method requires a large amount of antibodies, and the cost of monoclonal antibodies is high, which limits its clinical ap

Method used

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  • Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample
  • Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample
  • Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation method of RBP detection kit:

[0024] Reagent R1: containing 200mM tris-HCl buffer, 2% polyethylene glycol (PEG), 2% sodium chloride.

[0025] Reagent R2:

[0026] (1) Preparation of small latex particles labeled with anti-human retinol binding protein monoclonal antibody: Dilute small latex particles with a particle size of 90nm to a concentration of 1% with 50mM pH=6.0MES buffer, add EDAC1. 917mg / 10ml and 115mg / 10ml, react at room temperature for 1 hour, centrifuge at 20000rpm for 15min, remove the supernatant, suspend the precipitate in 50mM MES pH6. 50mM MES pH6.0, so that the final concentration is 2%, ultrasonic dispersion. To prevent foaming, add an equal volume of 50mM MES pH6.0+0.72mg monoclonal antibody coating solution while stirring, mix and stir, react at room temperature for 1 hour, and the final concentration is 1%. Centrifuge at 20,000rpm for 15min, remove the supernatant, suspend the pellet in 50mM MES pH6.0, with a final concentratio...

Embodiment 2

[0030] Determination of serum RBP:

[0031] Measuring instrument: Hitachi 7060 automatic biochemical analyzer;

[0032] Analysis method: two-point endpoint method;

[0033] Analysis parameters: R1: 225ul, R2: 75ul, sample: 3ul; wavelength: 600nm;

[0034] Measurement steps: first add R1 and sample, incubate at 37°C for 5 minutes, then add 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 difference between the two points of absorbance.

[0035] 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.

[0036] Calculation method of sample concentration: According to the difference of absorbance value of the sample, it is substituted into the calibration curve to calculate...

Embodiment 3

[0038] Determination of urinary RBP:

[0039] Measuring instrument: Hitachi 7060 automatic biochemical analyzer;

[0040] Analysis method: two-point endpoint method;

[0041] Analysis parameters: R1: 225ul, R2: 75ul, sample: 20ul; wavelength: 600nm;

[0042] Measurement steps: first add R1 and sample, incubate at 37°C for 5 minutes, then add 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 difference between the two points of absorbance.

[0043] 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.

[0044] Calculation method of sample concentration: According to the difference of absorbance value of the sample, it is substituted into the calibration curve to calcul...

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Abstract

The invention provides a kit for simultaneously detecting RBP in a urine sample and a serum sample. The kit comprises a reagent R1 and a reagent R2, the reagent R1 is a polymer buffer solution having an agglomeration promotion effect, the reagent R2 is a buffer solution containing a latex-antibody crosslink, and the latex-antibody crosslink comprises anti-RBP polyclonal antibody marked large latex particles and anti-RBP monoclonal antibody marked small latex particles. The kit which adopts a composite monoclonal and polyclonal antibody sensitized latex enhanced immune detection method has the advantages of simultaneous satisfying of the requirements comprising good specificity, high sensitivity and wide linear range, small antibody application amount, and cost reduction, and can be simultaneously used for the clinic detection of the RBP in human blood and urine.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a kit capable of simultaneously detecting retinol-binding protein in urine and serum samples. Background technique [0002] Retinol-Binding protein (RBP) is a transporter of retinol (vitamin A) in the blood, involved in the transport of retinol / acid in serum and cells, it is mainly synthesized in the liver, and It is released into the blood and enters various tissues. By interacting with retinol, prealbumin and cell surface receptors, it plays an important role in the storage, metabolism and transport of vitamin A to surrounding target organs. In the blood, RBP mainly exists in the form of complexes bound by retinol and prealbumin. When retinol in the complex binds to target cells, RBP is separated from prealbumin and filtered out from the glomerulus. End renal tubular epithelial cells absorb and degrade. RBP dysfunction will lead to abnormal storage, transport, distributio...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/31
Inventor 邹炳德邹继华方亮刘献文周海滨
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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