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NGAL latex immunoturbidimetric detection kit and preparation method thereof

A detection kit and latex immunization technology, applied in the field of biochemistry, can solve the problems of inability to accurately measure the health status of patients, insufficient anti-interference performance, etc., and achieve the effects of good clinical application prospects, great application value, and strong specificity.

Active Publication Date: 2019-05-10
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reagents produced by this method have insufficient performance in anti-interference, and cannot accurately measure the health status of patients with special constitutions.

Method used

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  • NGAL latex immunoturbidimetric detection kit and preparation method thereof
  • NGAL latex immunoturbidimetric detection kit and preparation method thereof
  • NGAL latex immunoturbidimetric detection kit and preparation method thereof

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preparation example Construction

[0061] The preparation method of neutrophil gelatinase-related lipocalin latex immunoturbidimetric assay kit adopts the following steps:

[0062] I. Preparation of reagent R1:

[0063] Take purified water, add buffer, anti-interference agent, sensitizer, electrolyte, preservative in sequence, each time adding materials need to be stirred until completely dissolved, adjust pH to 6.5-9.0, and filter with 0.22μm filter membrane;

[0064] II. Preparation of reagent R2:

[0065] (1) Activated polystyrene latex microspheres: add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride to the latex microspheres, mix and activate at 20℃-25℃ for 30 minutes ;

[0066] (2) Conjugation antibody: Add the neutrophil gelatinase-related lipocalin antibody to the activated polystyrene latex microspheres and react for 2 hours at 20°C-25°C for covalent coupling;

[0067] (3) Washing to remove uncoupled antibodies: the obtained latex microsphere reaction solution is centrifuged to remove the supernatan...

Embodiment 1

[0075] Preparation of reagent R1: Take 995g of water, then add 4.8g sodium dihydrogen phosphate, 22.8g sodium hydrogen phosphate, 10.0g polyethylene glycol 8000, 30.0g NaCl, 2g polyoxyethylene polyoxypropylene block copolymer L61 , 1.5g Triton X-45 (w / w) and 0.5g Pr ocl in300, each addition of materials need to be stirred until completely dissolved, adjust the pH to 7.4: filter with a 0.22μm filter membrane to make 1L reagent R1.

[0076] To prepare reagent R2, the steps are as follows:

[0077] (1) Activation of polystyrene latex microspheres: Take 30g 200nm latex microspheres (100mg / mL), add 10mL 500mM HEPES solution with pH 7.4, 4mL freshly prepared EDC solution with a concentration of 20mg / mL, and make up with purified water To the final volume of 100mL, mix well, mix at room temperature (20~25℃) for 30min;

[0078] (2) Coupling: Add NGAL antibody to the reaction solution in the previous step at a ratio of microsphere: antibody mass ratio = 12:1, mix and couple for 2 hours at ro...

Embodiment 2

[0086] Preparation of reagent R1: Take 995g of water, then add 4.8g sodium dihydrogen phosphate, 22.8g sodium hydrogen phosphate, 10.0g polyethylene glycol 8000, 30.0g NaCl, 8g polyoxyethylene polyoxypropylene block copolymer L61 , 6g Triton X-45 (w / w) and 0.5g Pr ocl in300, each time the materials are added need to be stirred to completely dissolve, adjust the pH to 7.4: filter with 0.22μm membrane to make 1L reagent R1.

[0087] To prepare reagent R2, the steps are as follows:

[0088] (1) Activation of polystyrene latex microspheres: Take 30g 200nm latex microspheres (100mg / mL), add 10mL 500mM HEPES solution with pH 7.4, 4mL freshly prepared EDC solution with a concentration of 20mg / mL, and make up with purified water To the final volume of 100mL, mix well, mix at room temperature (20~25℃) for 30min;

[0089] (2) Coupling: Add NGAL antibody to the reaction solution in the previous step at a ratio of microsphere: antibody mass ratio = 12:1, mix and couple for 2 hours at room tempe...

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Abstract

The present invention relates to an NGAL latex immunoturbidimetric detection kit and a preparation method thereof. The kit comprises a reagent R1, a reagent R2, a calibration product, and a quality control product. The reagent R1 comprises a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative, and the reagent R2 comprises latex microspheres with an NGAL antibody, a buffer solution, a stabilizer and a preservative. Compared with the prior art, according to the technical scheme of the present invention, by using the anti-interference agent in the R1 reagent, the anti-interference ability can be significantly improved, and the applicable population of the neutrophil gelatinase-associated lipocalin assay kit is expanded; and the kit detection provided by the present invention has high sensitivity, high specificity, and good stability of the kit, the NGAL content in urine, plasma and serum can be efficiently detected, and the detection results can bewell correlated with imported reagents.

Description

Technical field [0001] The invention relates to the field of biochemical technology, in particular to a method for preparing a latex immunoturbidimetric neutrophil gelatinase-related lipocalin detection kit with high sensitivity and strong specificity. Background technique [0002] Human neutrophil gelatinase-associated lipocalin (NGAL) has a molecular weight of about 25kDa and is covalently bound to neutrophil gelatinase. NGAL is a growth factor under physiological conditions, mainly involved in the occurrence and growth of early renal epithelium. The expression level is low in normal tissues including kidney, lung, stomach and colon epithelial tissues. The high level of NGAL in the process of acute kidney injury can be directly detected in urine and blood, and the prognosis will develop into acute renal failure. Acute kidney injury (AKI) is a disease with high morbidity and mortality. The pathogenesis of this disease is unknown and there is a lack of accurate and reliable ear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/531G01N33/543
Inventor 陈国锋景晟
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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