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Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm

A detection kit and amino acid technology, which is applied in the field of immunoassay, can solve the problems of unreported reagent detection performance evaluation, antibody can not be correctly oriented coupling, detection sensitivity and linearity impact, etc., to improve sensitivity and linear range, reduce binding Effect of steric hindrance and high sensitivity

Inactive Publication Date: 2014-12-10
NANJING PERLONG MEDICAL EQUIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Wuhan Shengzhiyuan Biotechnology Co., Ltd. (Chinese patents CN102253217A, CN103048464) and Nanjing Nuoerman Biotechnology Co., Ltd. (Chinese patent CN102680698) have developed a reagent for the determination of NGAL by a biochemical analyzer using the latex-enhanced turbidimetric method. The coupling method, that is, directly coupling the antibody to the carboxylated latex microspheres, will inevitably cause a part of the antibody to be coupled incorrectly, resulting in increased steric hindrance when the Fab end of the antibody binds to the antigen, which will affect the detection sensitivity and linearity to a certain extent. ; and it did not report the detection performance evaluation of reagents for rapid detection

Method used

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  • Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm
  • Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm
  • Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Preparation of NGAL rapid detection kit based on amino acid interarm:

[0042] 1. Preparation of NGAL reagent 1 (R1)

[0043] Add Tween-20, BSA, PEG-6000, NaCl, NaN to 50mM pH7.4 phosphate buffer 3 , so that the final concentration is Tween-20 0.1%, BSA 0.1%, PEG-6000 2.5%, NaCl 0.8%, NaN 3 0.1%, sterilized by suction filtration, that is, reagent 1 (R1).

[0044] 2. Preparation of NGAL reagent 2 (R2)

[0045] (1) Activation of carboxylated polystyrene microspheres:

[0046] Take 1ml of carboxylated polystyrene microspheres (Bangs Laboratories, Inc.) with a particle size of 110nm and a solid content of 10% and add it to a centrifuge tube, add 9ml of activation buffer (50mM pH5.5 MES buffer), centrifuge, pour Remove the supernatant and wash twice. After washing, add 5ml of activation buffer to resuspend the microspheres, then add 2.5ml of 9.6mg / ml Sulfo-NHS and 2.5ml of 12mg / ml of EDC (both dissolved in activation buffer), shake and mix immediately, and...

Embodiment 2

[0053] Embodiment 2 Preparation of NGAL rapid detection kit based on amino acid interarm:

[0054] 1. Preparation of NGAL reagent 1 (R1)

[0055] Add Tween-20, BSA, PEG-4000, NaCl, NaN to 50mM pH7.4 phosphate buffer 3 , so that the final concentration is TritonX-100 0.01%, BSA 0.01%, PEG-4000 1%, NaCl 0.8%, thimerosal 0.01%, and filter to sterilize, which is reagent 1 (R1).

[0056] 2. Preparation of NGAL reagent 2 (R2)

[0057] (1) Activation of carboxylated polystyrene microspheres:

[0058] Take 1ml of carboxylated polystyrene microspheres (Bangs Laboratories, Inc.) with a particle size of 120nm and a solid content of 10% into a centrifuge tube, add 9ml of activation buffer (50mM pH5.5 MES buffer), centrifuge, pour Remove the supernatant and wash twice. After washing, add 5ml of activation buffer to resuspend the microspheres, then add 2.5ml of 9.6mg / ml Sulfo-NHS and 2.5ml of 12mg / ml of EDC (both dissolved in activation buffer), shake and mix immediately, and set Mix a...

Embodiment 3

[0065] Embodiment 3 Preparation of NGAL rapid detection kit based on amino acid interarm:

[0066] 1. Preparation of NGAL reagent 1 (R1)

[0067] Add BL-9, BSA, PEG-4000, NaCl, NaN to 50mM pH7.4 phosphate buffer 3 , so that the final concentration is BL-9 5%, BSA 10%, PEG-8000 1%, NaCl 0.8%, proclin300 1%, filter and sterilize, that is, reagent 1 (R1).

[0068] 2. Preparation of NGAL reagent 2 (R2)

[0069] (1) Activation of carboxylated polystyrene microspheres:

[0070] Take 1ml of carboxylated polystyrene microspheres (Bangs Laboratories, Inc.) with a particle size of 140nm and a solid content of 10% into a centrifuge tube, add 9ml of activation buffer (50mM pH5.5 MES buffer), centrifuge, pour Remove the supernatant and wash twice. After washing, add 5ml of activation buffer to resuspend the microspheres, then add 2.5ml of 9.6mg / ml Sulfo-NHS and 2.5ml of 12mg / ml of EDC (both dissolved in activation buffer), shake and mix immediately, and set Mix and react in a blood mi...

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Abstract

The invention discloses a rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on an amino acid spacer arm and used for quantitatively and rapidly determining NGAL in human serum (plasma) or urine. The kit comprises a reagent 1 (R1) and a reagent 2 (R2), wherein the R1 comprises a biological buffer solution, a surfactant, a stabilizer, a coagulation accelerator and a preservative; the R2 comprises latex particles for enveloping an NGAL antibody, a biological buffer solution, a protective agent, a surfactant and a preservative; in R2, an antibody against human NGAL is connected with a polystyrene latex microsphere through the amino acid spacer arm. The invention also discloses a preparation method of the latex particles for enveloping the antibody against the human NGAL. The kit has high sensitivity and wide linear range; when being used in cooperation with a POCT (Point Of Care Testing) scattering nephelometry analyzer, the kit can achieve the aim of accurately and rapidly determining the NGAL in human serum (plasma) or urine.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to a rapid detection kit for NGAL based on amino acid interarms for measuring neutrophil gelatinase-associated lipocalin (NGAL) in human blood and urine by latex-enhanced immune turbidimetry It also relates to a method for using the kit to detect NGAL and a preparation method thereof. Background technique [0002] Acute kidney injury (AKI) is a clinical syndrome caused by a sudden decline in renal function in a short period of time (hours to days) caused by various reasons. Due to the lack of specific biological indicators in the early stage, it brings obstacles to the early treatment of the disease. Neutrophil gelatinase-associated lipocalin (NGAL) is a new member of the lipocalin family, and its structure and function research progress has attracted people's attention. Numerous studies have shown that NGAL gene may be an important oncogene and an important human cancer gene. Bact...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/531G01N33/68
Inventor 姚长进韩东成黄宝福黄丹程睿淳林
Owner NANJING PERLONG MEDICAL EQUIP
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