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Immune Latex Nephelometric Kit for Detection of Ngal Based on Single-Size Latex Particles

A latex particle and single particle size technology, applied in the biological field, can solve the problems of inability to detect normal human values, narrow linear range, and low analytical sensitivity, and achieve biotin-streptavidin immunolabeling and related tracer analysis sensitive effect

Active Publication Date: 2019-09-13
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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  • Immune Latex Nephelometric Kit for Detection of Ngal Based on Single-Size Latex Particles
  • Immune Latex Nephelometric Kit for Detection of Ngal Based on Single-Size Latex Particles
  • Immune Latex Nephelometric Kit for Detection of Ngal Based on Single-Size Latex Particles

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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 respectively with 50mM boric ...

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 immunolatex turbidimetric assay kit for detecting NGAL based on single-diameter latex particles. The kit includes reagent R1 and reagent R2, wherein the reagent R1 includes a buffer, a surfactant, Coagulants, chelating agents, preservatives, reducing agents, electrolytes and water, the reagent R2 includes buffers, stabilizers, electrolytes, latex particles coated with anti-human NGAL polyclonal antibodies, wherein the reducing agent is β ‑Mercaptoethanol, the anti-human NGAL polyclonal antibody is a biotin-labeled anti-human NGAL polyclonal antibody, and the latex particles are streptavidin-labeled single-diameter latex particles. The kit of the present invention can simultaneously detect NGAL in blood and urine samples, not only has improved detection sensitivity and linear range, but also can overcome the influence of interfering factors, and detect NGAL homodimers and heterodimers, Avoid misjudgment by doctors due to low NGAL test results.

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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Patent Type & Authority Patents(China)
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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