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Latex enhanced immunonephelometry detection kit and preparing and detecting method thereof

A technology for detection kits and latex enhancement, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problems of time-consuming, complicated operation, and high cost

Active Publication Date: 2018-10-26
SHENZHEN AMTECH BIOENGINEERING LTD INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The main purpose of the present invention is to provide a latex-enhanced immunoturbidimetric detection reagent for detecting small organic molecules such as vitamin D, aiming at the problems of low sensitivity, time-consuming, complicated operation and high cost in clinical detection of small organic molecules such as vitamin D The kit has the characteristics of high detection sensitivity, rapid detection, and simple operation, and expands the application of latex-enhanced immunoturbidimetric method in organic small molecule detection reagents, which has good practical value

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  • Latex enhanced immunonephelometry detection kit and preparing and detecting method thereof
  • Latex enhanced immunonephelometry detection kit and preparing and detecting method thereof
  • Latex enhanced immunonephelometry detection kit and preparing and detecting method thereof

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Embodiment 1

[0054] The preparation method of the latex-enhanced immune turbidimetric detection kit of the present invention, taking vitamin D as an example, comprises the following steps:

[0055](1) Preparation of biomacromolecules covalently coupled with vitamin D markers:

[0056] Add 0.1 mmol bovine serum albumin to 100 mM, pH=7.4 phosphate buffer solution, stir well to obtain solution A; add solution A dropwise to 2 mmol vitamin D markers containing maleimide groups ; After stirring at room temperature for 2 hours, remove the excess vitamin D marker with a desalting column to obtain a purified biomacromolecule covalently coupled with the vitamin D marker;

[0057] (2) Reagent R 1 Preparation of:

[0058] NaCl, Tris, mass percent are 0.05% sodium azide, 0.2% bovine serum albumin, 0.05% Triton100 and 0.8% polyethylene glycol 8000 are added in the container that 800mL ultrapure water is housed, make the NaCl concentration in the solution be 100mM, Tris concentration is 50mM, stir at ...

Embodiment 2

[0064] The preparation method of the latex-enhanced immune turbidimetric detection kit of the present invention, taking vitamin D as an example, comprises the following steps:

[0065] (1) Preparation of synthetic polymers covalently coupled with vitamin D markers:

[0066] Add 0.1 mmol synthetic polymer, 2 mmol N-hydroxymaleimide and 1 mL dimethylformamide to 2 mmol 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, After stirring at room temperature for 1 hour, a vitamin D marker with a free amino group was added, reacted at room temperature for 3 hours, and washed 3 times with ether to obtain a purified synthetic macromolecule covalently coupled with vitamin D; wherein, the synthetic macromolecule was A polymer containing free amino groups or a free carboxyl group, and the molecular weight of the synthetic polymer is 2-300KD, and the molecular weight of the small organic molecule α is below 2KD;

[0067] (2) Reagent R 1 Preparation of:

[0068] Add NaCl, Tris,...

Embodiment 3

[0074] 25-Hydroxyvitamin D 3 detection method and calibration curve.

[0075] The present invention uses a two-point endpoint method to detect 25-hydroxyvitamin D 3 , the detection wavelength is the main wavelength 600nm, the secondary wavelength is 800nm, the reagent R 1 The dosage is 180uL, reagent R 2 The dosage is 40uL, and the calibrator dosage is 8uL. Reagent R 1 Mix well with the calibrator, incubate at 37°C for 3 minutes, then add reagent R 2 React for 30s, read the first absorbance A 1 After that, continue to react for 5 minutes and read the second absorbance A 2 , calculate △OD600 value, the formula is △OD600=A 2 -A 1 , the results are shown in Table 1. Take the concentration of the calibrator as the X-axis, and the corresponding △OD600 value as the Y-axis to obtain the calibration curve, refer to the attached image 3 .

[0076] Table 1 25-Hydroxyvitamin D 3 △OD600 value

[0077] Concentration (ng / mL)

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Abstract

The invention discloses a latex enhanced immunonephelometry immunoassay detection kit and a preparing and detecting method thereof. The kit comprises a reagent R1 and a reagent R1; the reagent R1 comprises an electrolyte, a buffering solution, an accelerant, a preservative, a stabilizer, a surfactant, and bio-macromolecules or synthetic macromolecules which are in covalent coupling with organic micromolecules alpha containing maleimide perssad, and the reagent R2 comprises an electrolyte, a buffering solution, a preservative, a stabilizer, a surfactant and latex microspheres which are in antibody coupling with organic micromolecules alpha containing free amino groups. By means of the latex enhanced immunonephelometry immunoassay detection kit, through combination of the organic micromolecules alpha with the bio-macromolecules or the synthetic macromolecules, the organic micromolecules in a sample and antigens combined on the bio-macromolecules or synthetic macromolecules are competitively combined with antibodies on the surfaces of the microspheres in the reagent R2, a net-shaped combined substance is formed, so that the turbidity of a reaction system is lowered, thus the concentration of a detected marker can be measured by measuring the change of light absorbancy of permeated turbid liquid, and detection of the organic micromolecules is more convenient.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic reagents, in particular to a latex-enhanced immune turbidity detection kit and a preparation and detection method thereof. Background technique [0002] At present, the clinical methods for detecting vitamin D and other small organic molecules mainly include chemiluminescence, liquid chromatography-mass spectrometry, radioimmunoassay, enzyme-linked immunoassay and cloned enzyme donor immunoassay. Chemiluminescence method has high sensitivity, but requires a specific chemiluminescence instrument, and the detection cost is high; enzyme-linked immunoassay is time-consuming and expensive; mass spectrometry takes a long time and is complicated to operate; radioimmunoassay has environmental problems; cloning enzyme donor immunoassay The reagents of the method are less stable. [0003] The latex particle-enhanced immunoturbidimetric method is a method for determining the concentration of the detected ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/558
CPCG01N33/54313G01N33/558
Inventor 陈小茹吴向东
Owner SHENZHEN AMTECH BIOENGINEERING LTD INC
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