Immunity latex turbidimetry kit for detecting NGAL (neutrophil gelatinase associated lipocalin) based on single-grain-size latex particles

A technology of latex particles and kits, which is applied in the field of latex-enhanced immunoturbidimetric detection kits, can solve the problems of inability to detect normal human values, false negative test results, narrow linear range, etc., and achieve biotin-streptavidin affinity Sensitive effects of immunolabeling and related tracer assays

Active Publication Date: 2018-11-23
宁波海尔施智造有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Various NGAL latex-enhanced immunoturbidimetric detection kits currently available in the market have many shortcomings: the use of single-size latex particles and antibody coupling often has the disadvantages of low analytical sensitivity and narrow linear range, and the detection sensitivity can only reach 10ng / ml, which is far from detecting the value of normal people; in addition, few kits can avoid the interference of NGAL homodimer and heterodimer, resulting in false negative test results

Method used

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  • Immunity latex turbidimetry kit for detecting NGAL (neutrophil gelatinase associated lipocalin) based on single-grain-size latex particles
  • Immunity latex turbidimetry kit for detecting NGAL (neutrophil gelatinase associated lipocalin) based on single-grain-size latex particles
  • Immunity latex turbidimetry kit for detecting NGAL (neutrophil gelatinase associated lipocalin) based on single-grain-size latex particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The preparation of embodiment 1 reagent

[0058] 1.1 Preparation of reagent R1

[0059] During the reagent preparation process, prepare according to the concentration in the table below, pass through a 0.22 μm filter membrane after preparation, and store at 4°C for later use.

[0060]

[0061] 1.2 Preparation of reagent R2

[0062] During the reagent preparation process, prepare according to the concentration in the table below, pass through a 0.22 μm filter membrane after preparation, and store at 4°C for later use.

[0063] components

concentration

2-Morpholineethanesulfonic acid buffer

150mM

Sodium chloride

100mM

Proclin 950

3mM

bovine serum albumin

0.1g / L

purified water

make up

pH

7.0

[0064] 1.3 Streptavidin-labeled latex

[0065] 1) Dilute latex particles (purchased from JSR Life Science, article numbers P0014 / P0113 / P0220) with particle diameters of 80nm, 198nm, and 240nm res...

Embodiment 2

[0078] Embodiment 2 kit detection process

[0079] 2.1 Test conditions:

[0080] dominant wavelength

546nm

Reaction direction

up

temperature reflex

37℃

Response method

two-point endpoint

[0081] 2.2 Operation process:

[0082]

[0083] The reading points in AU680 are 11~27.

Embodiment 3

[0084] The determination of embodiment 3 standard curve

[0085] 3.1 Comparison of different ratios of reagent R1

[0086] Use reagent R1 (formulation in 1.1, but without β-mercaptoethanol) to dilute NGAL protein so that the concentrations are 0, 150, 600, 1800, 3600, 7200 ng / ml, respectively. Prepare reagent R1 according to the formula of ratio 1, ratio 2, and ratio 3 in 1.1 and the optimal conditions of 1.2-1.5 to prepare reagent R2, and perform different concentrations of NGAL protein on AU680 according to the detection method of Example 2 detection, the result is as figure 1 shown. It can be seen from the figure that the linear relationship between the NGAL protein concentration and the △A value of the second ratio is better, the slope of the fitted standard curve will be larger, and the accuracy and sensitivity will be greatly improved.

[0087] 3.2 Comparison of different latex particle sizes

[0088] Use reagent R1 (formulation in 1.1, but without β-mercaptoethanol)...

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Abstract

The invention discloses an immunity latex turbidimetry kit for detecting NGAL (neutrophil gelatinase associated lipocalin) based on single-grain-size latex particles. The kit comprises a reagent R1 and a reagent R2; the reagent R1 is prepared from buffer solutions, surfactants, coagulants, chelating agents, preservatives, reductants, electrolytes and water; the reagent R2 is prepared from the buffer solutions, stabilizers, electrolytes and latex particles coated with antihuman NGAL polyclonal antibodies; the reductants are beta-mercaptoethanol; the antihuman NGAL polyclonal antibodies are biotin-labeled antihuman NGAL polyclonal antibodies; the latex particles are streptavidin-labeled single-grain-size latex particles. The kit has the advantages that NGAL in blood and urine samples can bedetected at the same time, so that the detection sensitivity is improved, the linear range is widened, and influences of interference factors can be avoided; NGAL homodimers and heterodimers are detected, and accordingly, misjudgment of doctors due to low NGAL detection results is avoided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a latex-enhanced immunoturbidimetric detection kit based on single-diameter latex particles and capable of simultaneously detecting neutrophil gelatinase-associated lipocalin in serum and urine. Background technique [0002] Neutrophil gelatinase-associated lipocalin (NGAL), also known as human lipocalin 2 (Ln2) or siderocalin, is a member of the human lipocalin family new member. In recent years, NGAL has attracted much attention as a new marker of renal injury. [0003] NGAL was discovered by Kjeldsen et al. in 1993 and is closely related to gelatinase B, matrix metalloproteinase 9 (matrixmetalloproteinase-9, MMP-9). The full length of the human NGAL gene is 25000bp, and its protein exists in three forms: a monomer with a molecular weight of 25kDa, a 46kDa homodimer of self-polymerization or a 135kDa heterodimer formed by polymerization with MMP-9. NGAL may be involv...

Claims

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Application Information

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IPC IPC(8): G01N33/92G01N33/577
Inventor 柳建敏林威彦裘艳群
Owner 宁波海尔施智造有限公司
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