CN102072960A

CN102072960A - Method for detecting neutrophil gelatinase-associated lipocalin (NGAL) in sample

Info

Publication number: CN102072960A
Application number: CN 201010136227
Authority: CN
Inventors: 华权高; 沈鹤霄
Original assignee: WUHAN LIFE ORIGIN BIOTECH CO Ltd
Current assignee: WUHAN LIFE ORIGIN BIOTECH CO Ltd
Priority date: 2010-03-29
Filing date: 2010-03-29
Publication date: 2011-05-25

Abstract

The invention relates to a method for detecting neutrophil gelatinase-associated lipocalin (NGAL) in a sample. The method comprises the following steps of: reacting an antibody of the NGAL with human NGAL of a specimen to be detected to form a compound which causes turbidity change; characterizing the change by using light projection intensity or light scattering intensity; and finding out the content of the human NGAL in the specimen to be detected from the concentration of the NGAL standard product and a standard curve of corresponding light scattering intensity or light transmission intensity. The method has the advantages of high sensitivity, high recovery rate and good repeatability, and a detection measure having the advantages of simplicity, convenience, good repeatability, strong adaptability and high speed is determined for measuring the NGAL in the samples of urine or blood plasma, and the like.

Description

The detection method of neutrophil gelatinase-associated lipocalin in a kind of sample

Technical field

The present invention relates to the detection method of neutrophil gelatinase-associated lipocalin in a kind of sample.

Background technology

Neutrophil gelatinase-associated lipocalin (be called for short NGAL) be 1993 at first found in neutrophil leucocyte, relevant with processes such as the generation of inflammation, embryonic development, immune response, chemotaxis, signal transduction and kinds of tumors and development.Domestic and international research shows in recent years, and NGAL albumen is sent out the characteristics that have specific expressed variation in the process in multiple disease, makes NGAL become the biomarker that detects disease.

In ischemic and toxicity injury of kidney process take place, the NGAL in the renal cells will significantly increase, and in two hours of beginning, the NGAL level will significantly increase in urine and the blood, so NGAL is the responsive mark of early stage acute injury of kidney.

Acute renal damage prognosis will develop into acute renal failure.General diagnostic method is as measuring serum creatinine or cysteine proteinase inhibitor C (Cystatin C), can only damage one day after very the person detect in several days.There are some researches show, NGAL content detection distribution of results 0.7-9.6ng/ml in healthy volunteer's urine, mean value is 5.3ng/ml.And the content detection result in the blood plasma is 37-106ng/ml, and mean value is 63ng/ml.Behind injury of kidney, with the machine testing severe case, the concentration of urine NGAL is that 110ng/ml does not wait to 40000ng/ml.And their EDTA anticoagulate plasma testing result is that 25ng/ml is to 3491ng/ml.Judge that according to 90% positive predictive value that the acute renal failure patient detects the positive judgment value of NGAL is 350ng/ml in the urine, and the positive judgment value that blood plasma detects is 400ng/ml.

Homology analysis shows, (neu-related lipocalin NRL) has very high homology for NGAL and the 24p3 of mouse and the relevant lipocalin of neu of rat.The 24p3 of mouse is synthetic in liver, raises during inflammatory reaction, and 24p3 also sees macrophage and endometrium in addition, especially the endometrium in reproduction period.With steroid hormone the 24p3 level in the respective organization is raise, and some growth factors and tumour promote that the factor also can make its rising.Monkey poison SV40, polyoma poison or other malicious infection can stimulate the expression of the mouse kidney cell 24p3mRNA of cultivation to increase 7-10 doubly.And raise along with the synthetic increase of large T antigen.Therefore 24p3 is considered to oncoprotein.This shows that simultaneously NGAL may be human a kind of new oncogene.

The expression that experiment showed, the NGAL in ovarian cancer cell line, colon tumor and breast cancer etc. obviously strengthens.The NGAL strong positive of SABC proof in cancerous lung tissue, and BAC cancer and carcinoma muco-cellulare dyeing are the strongest, NGAL can be increased to strong positive from feminine gender in cancer of pancreas.

In sum, set up at the method for quick of NGAL in the sample and will provide a strong tool for the research of this albumen.

Summary of the invention

The invention provides the described method of method that detects NGAL in the sample and comprise following steps:

I) use related equipment to collect serum, blood plasma and urine that sample comprises the people,

Ii) sample and antibody contact reaction,

Iii) detect the concentration of NGAL in the sample.

Preferably measure the NGAL level by immuno-chemical method, the example of this method comprises but never is limited to immunoturbidimetry, sandwich method ELISA, competition law ELISA.

To biological specimen, can carry out with any method of satisfied analysis specificity, sensitivity and degree of accuracy that provides.In the test experience, use monoclonal, polyclonal antibody or any molecule that can combine of NGAL with the NGAL specificity.The binding molecule at first NGAL that catches in the sample of immobilization forms compound, and the detectable binding molecule of another one mark is again in conjunction with above-mentioned compound, thereby the value indirect reaction that demonstrates the compound amount by ad hoc approach at last goes out the content of NGAL in the sample.Can directly utilize antibody that free NGAL antibody or coupling latex particle exist with colloidal form and NGAL in the sample directly to combine formed compound in addition and cause that turbidity changes; Use up projection intensity or light scattering intensity and characterize this variation; Find the content of the people NGAL of sample to be checked from the typical curve of the concentration of people NGAL standard items and corresponding light scattering strength or transmittance intensity;

Description of drawings

Fig. 1 is that sandwich method ELISA detects recombined human NGAL canonical plotting;

Fig. 2 is that competition law ELISA detects recombined human NGAL canonical plotting.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

Embodiment 1 latex enhance immunity turbidimetry detects the NGAL content in the human urine

Realize the technical scheme of this example: the immunoturbidimetry of a kind of people of detection NGAL, at first crosslinked with latex with the polyclonal antibody of anti-NGAL, with the NGAL reaction of latex solution after crosslinked and sample to be checked, formed compound causes that turbidity changes then; Use up projection intensity or light scattering intensity and characterize this variation; Find the NGAL content of sample to be checked from the typical curve of the concentration of NGAL standard items and corresponding light scattering strength or light projection intensity; The polyclonal antibody of wherein used NGAL is tired and is at least 1: 500, does not contain assorted antibody, has many kinds of antigenic determinants of identification NGAL; Sample to be checked does 1 with thinning agent: 200-1: 1000 dilutions;

In the said method, described NGAL standard items are pure NGAL, and its concentration is 0.2mg/ml, do 1: 200 times of dilution with thinning agent earlier, do doubling dilution again, make the series standard product, are made for typical curve and use;

Embodiment 2 usefulness sandwich method ELISAs are determined human urine NGAL concentration

Concrete steps are: bag is by the NGAL antibody A; Prepare serial NGAL standard items; Prepare human urine; The human urine of series standard product or required value is added in the 96 micropore plate holes, make again mark, binding site is different from the antibody B combination that is coated on antibody A on 96 microwell plates, with matrix liquid colour developing color development stopping after 20-30 minute, measure the reacted absorbance of NGAL in above-mentioned series standard product and the human urine to be detected after 10 minutes; Set up the typical curve of the concentration of series standard product and absorbance and from curve, find the concentration of NGAL of the human urine of required value.

Embodiment 3 usefulness competition law ELISA determine human urine NGAL concentration

Concrete steps are: bag is by the NGAL polyclonal antibody; Prepare serial NGAL standard items; Prepare human urine; Make the human urine of series standard product or required value and the NGAL standard items mixing of mark; With matrix liquid colour developing color development stopping after 20-30 minute, measure the reacted absorbance of NGAL in above-mentioned series standard product and the human urine to be detected after 10 minutes; Set up the typical curve of the concentration of series standard product and absorbance and from curve, find the concentration of NGAL of the human urine of required value.

Claims

1. the detection method of neutrophil gelatinase-associated lipocalin in the sample.

2. method according to claim 1, it is characterized in that, be based upon on the antigen and antibody specific reaction principle basis, by the concentration of the relevant lipocalin protein (NGAL) of human neutrophil gelatinase in the immunological method test sample that includes but not limited to immunoturbidimetry and enzyme linked immunological (ELISA) method.

3. method according to claim 1 is characterized in that, comprises the steps:

I) use related equipment to collect sample and comprise serum, blood plasma and urine,

Ii) NGAL in the sample and antibody contact reaction,

Iii) detect the concentration of people NGAL in the sample.

4. sample according to claim 2 is characterized in that including but not limited to people's urine, blood plasma and serum.

5. antibody according to claim 3 is characterized in that this antibody is polyclonal antibody or the monoclonal antibody by the people NGAL immune animal acquisition of natural or reorganization.

6. immunoturbidimetry according to claim 2 is characterized in that: with the antibody of anti-people NGAL, with the people NGAL reaction of sample to be checked, formed compound causes that turbidity changes; Use up projection intensity or light scattering intensity and characterize this variation; Find the content of the people NGAL of sample to be checked from the typical curve of the concentration of people NGAL standard items and corresponding light scattering strength or transmittance intensity.

7. enzyme linked immunological according to claim 2 (ELISA) method is characterized in that: comprise double antibodies sandwich method ELISA, competition law ELISA.

8. enzyme linked immunological according to claim 2 (ELISA) method, it is characterized in that: people NGAL is immobilization in advance, or people NGAL antibody immobilization in advance.

9. enzyme linked immunological according to claim 2 (ELISA) method is characterized in that: the scope that detects NGAL content is: 7.8ng/ml-500ng/ml.

10. competition law according to claim 7 is if direct competition method is characterized in that: immobilization people NGAL antibody in advance.

11. competition law according to claim 7 is if the indirect competition method is characterized in that: immobilization people NGAL in advance.

12. immobilization according to claim 8, it is characterized in that: with people NGAL or people NGAL antibody sandwich to solid support, this holder can be, the polystyrene or the polyvinyl chloride surface that for example are used for enzyme linked immunosorbent assay, or latex (polystyrene) particle, or by the filter glass rod formed of polyethylene particle of compression, or porous digest cellulose matrix, or in fact be any suitable holder in the immunochemical analyses use.