CN103175971A

CN103175971A - IgA antibody detection kit

Info

Publication number: CN103175971A
Application number: CN2013100622397A
Authority: CN
Inventors: 陈菲; 武书; 霍如松; 朱凡
Original assignee: SUZHOU HERUI MEDICAL TECHNOLOGY Co Ltd
Current assignee: Suzhou sharp Biotechnology Co., Ltd.
Priority date: 2013-02-28
Filing date: 2013-02-28
Publication date: 2013-06-26
Anticipated expiration: 2033-02-28
Also published as: CN103175971B

Abstract

The invention relates to a human IgA antibody detection method and a kit and belongs to the field of immunoassay detection. The provided I2IgA antibody immunoassay kit comprises a porous plate coated with I2 polypeptides, an anti-human IgA antibody marked with horse radish peroxidase, a negative control product, a positive control product, a chemical luminous substrate solution, washing concentrate and antibody diluent, wherein the I2 polypeptides for coating the porous plate are obtained by an artificial amino acid synthesis method and comprise at least one SEQ1-4 sequence. The immunoassay kit has the characteristics of high accuracy, sensitivity and the like, can be used for detecting the anti-I2 IgA antibody in the human body and has the auxiliary diagnosis function on the Crohn's disease and other digestive system diseases.

Description

A kind of IgA antibody assay kit

Technical field

The present invention relates to the immunoassay detection field.Particularly, the present invention relates to a kind of immunoassay kits that detects anti-I2IgA antibody.

Background technology

Clone's disease (Crohn ' s disease, CD) be a kind of enteron aisle chronic nonspecific inflammation of cause of disease complexity.There have been various bacteria and virus to be proved to be relevant with the CD disease.In numerous CD pathogenesis, one of mechanism that body is extensively approved certain antigen or the composition radical response in normal diet of certain normal enteric bacteria.The certified antigen relevant with the CD pathogenesis comprises the mannan of Escherichia coli adventitia duct albumen (outer membrance protein C, OmpC), yeast cell wall etc.I2 is a kind of albumen of finding on activity CD patient pathology intestinal mucosa, by amino acid sequence analysis, finds that I2 is one section sequence of a kind of intestinal tract normal flora Pseudomonas fluorescens (pseudomonas fluorescens) pfiT gene.Studies show that, the IgA antibody positive of the anti-I2 of nearly 50%CD patients serum, and the IgA antibody of the anti-I2 in normal person and non-CD intestines problem patient seldom is detected.Therefore, the IgA antibody test of anti-I2 is one of efficiency index of CD diagnosis.

There is no at present I2 antibody immunoassay kit, reported once on document that the method with restructuring I2 albumen obtained I2 albumen, then exempt from or the detection of Salmonella method with putting.One aspect of the present invention replaces the I2 albumen of literature method coated with the coated microwell plate of synthetic I2 polypeptide, and coated efficient is higher, and quality is more controlled.Another aspect of the present invention when immunoassay, has replaced document TMB Color Appearance System used with the chemoluminescence method substrate, and chemoluminescence method is lower, sensitiveer than common ELISA method detectability.Therefore, the present invention compares with existing method, has advantages of sensitiveer, more stable.

Summary of the invention

The object of the present invention is to provide a kind of immunoassay kits of sensitiveer, more stable detection I2 antibody, is the immunoassay kits that detects the IgA antibody of the anti-I2 of people specifically.

The invention provides a kind of immunoassay kits of the I2IgA of detection antibody, this kit comprises: the microwell plate, the anti-human IgA antibody of horseradish peroxidase-labeled, negative control product, positive reference substance, Chemoluminescent substrate, concentrated cleaning solution, the antibody diluent that are coated with the I2 polypeptide.Wherein the I2 polypeptide of coated microwell plate is to obtain with the synthetic method of artificial amino acid, contains at least sequence of SEQ1-4.

The anti-human IgA antibody of described enzyme labeling adopts the chemical method coupling to obtain, and marker enzyme is horseradish peroxidase, the sodium iodate method of described coupling method for improveing.

Solid phase material for the preparation of described microwell plate is black lucifuge material, includes but not limited to, for example, polystyrene, tygon, polypropylene.The form of carrier is the micro-reaction plate shrinkage pool.

Described concentrated cleaning solution is the phosphate buffer of 0.05%tween20.Described Chemoluminescent substrate forms (for example, volume ratio is 1: 1) by A liquid and developer B liquid, and developer A liquid is hydrogen peroxide or urea peroxide, and developer B liquid is luminol and reinforcing agent thereof.Described sample diluting liquid is phosphate or the carbonate buffer solution that contains 0.5%BSA, and described antibody dilution is also for containing phosphate or the carbonate buffer solution of 0.5%BSA.

Another aspect of the present invention provides a kind of method that detects anti-I2IgA antibody in serum or blood plasma, comprises step:

(1) sample pre-treatments:

(2) detect with the arbitrary described kit of claim 1-9, add testing sample solution or reference substance in the coated micropore plate hole of I2 polypeptide, hatching rear washing pats dry, the anti-human IgA antibody that adds again horseradish peroxidase-labeled, hatching rear washing pats dry, add Chemoluminescent substrate, stop buffer, measure absorbance with biochemical instruments or Chemiluminescence Apparatus;

(3) analyzing and testing result.

The detection principle of kit of the present invention is:

the I2 polypeptide is coated on solid phase carrier, add detected sample or reference substance, anti-I2 antibody (all Ig in sample or reference substance, comprise IgG, IgA, IgE, IgM etc.) just with the I2 polypeptide combination on solid phase carrier, form antigen-antibody complex, remove irrelevant composition with cleansing solution, then the anti-human IgA antibody that adds horseradish peroxidase-labeled, the antigen of enzyme labelled antibody on microwell plate-IgA antibody is combined, form antigen-IgA antibody-hrp-antibody complex, go out unconjugated enzyme labeling thing by washing, stop after colour developing, the absorbance of working sample, the amount that contains the IgA antibody of anti-I2 in this value and sample is proportionate, the value that detects gained with negative control product and positive reference substance compares and can draw the IgA antibody content scope that whether contains anti-I2 in sample.

Description of drawings:

Accompanying drawing 1:SEQ:1

Accompanying drawing 2:SEQ:2

Accompanying drawing 3:SEQ:3

Accompanying drawing 4:SEQ:4

It is in order to understand better the present invention that following embodiment is provided, rather than content of the present invention and protection domain are consisted of any restriction.

The comparison that the different sequence I2 polypeptide of embodiment 1 are coated

SEQ:1, SEQ:2, SEQ:3, SEQ:4 polypeptide are synthesized by the safe medical sci-tech of Shanghai guest, and purity is more than 90%.

Press 100.0 μ g/mL, 50.0 μ g/mL, 25.0 μ g/mL, 12.5 μ g/mL with variable concentrations SEQ1-SEQ4 polypeptide, 6.25 μ g/mL, the serial dilution degree coated elisa plate of 3.13 μ g/mL, 100 μ L/ holes, 0～4 ℃ of placement is spent the night, and washes plate three times with cleansing solution, pats dry at every turn; 200 μ L/ hole lock solution sealings, room temperature was placed 3 hours, washed plate three times, patted dry at every turn; The positive negative antibody (1: 100) that adds the 100 a series of dilutions in μ L/ hole, room temperature was placed 2 hours, washed plate three times, patted dry at every turn; The anti-human IgA antibody of enzyme mark that adds the working concentration in 100 μ L/ holes, room temperature was placed 1 hour, washed plate three times, patted dry at every turn; Add the substrate nitrite ion in 100 μ L/ holes, measure luminous value after the acid adding cessation reaction.

Result shows as table 1.

The SEQ:1-SEQ:4 sequence all meets the demands.

Embodiment 2 immunoassay detection methods are set up

2.1 test method

The main I2 polypeptide coated elisa plate that adopts of this experiment, HRP enzyme mark goat-anti people I2 antibody (give birth to work bioengineering (Shanghai) incorporated company, article No. DAH005-0.1ml) is as detecting antibody.

1) detect antibody best effort concentration

(1) be coated with washing with 100ng/ml people IgA.

(2) the anti-human IgA antibody of enzyme mark is done to add respectively in coated hole after a series of dilutions insulation, washing with dilution.

(3) add the substrate colour developing.After the acid adding cessation reaction, in the Chemiluminescence Apparatus reading numerical values.The working concentration of getting the relative luminous intensity value and be the anti-human IgA antibody of enzyme mark of 15000 o'clock is optium concentration.Test figure is listed in table 2.

Determining of table 2 antibody working concentration

By can determine in table 2 data, the working concentration of the anti-human IgA antibody of best enzyme mark is: 1: 2000.

2) selection of washing lotion

This experiment has been chosen 2 kinds of lotion prescriptions and has been compared: the PBS of PBS and 0.05%Tween.By in example 2 1) described experimental technique, the relatively effect (the enzyme labelled antibody dilutability is 1: 2000) of two kinds of washing lotions.

Determining of the best washing lotion of table 3

2.2 conclusion

Pass through 2.1.1) experimental result (table 2) draw, when the working concentration of enzyme labelled antibody is 1: 2000 dilutability, determine its best effort concentration.

Pass through 2.1.2) experimental result (table 3), we find, use when containing the PBS washing lotion of 0.05%Tween20, background value can be reduced, and not affect the colour developing result, so selected its is the washing lotion composition in this experiment.

The antibody test of anti-I2 in embodiment 3 samples

3.1 detect with kit

1) add sample 100ul to be checked in antigen coated ELISA Plate micropore.Positive control and negative control respectively add 2 holes, every hole 100ul simultaneously.Shake up, put 37 ℃ of incubations 45 minutes.

2) discard that in the hole, solution adds cleansing solution 300ul/ hole, outwelled in standing 2 minutes, repeat this process and amount to 5 times, pat dry with thieving paper.

3) every hole adds enzyme labelled antibody 100ul, puts 37 ℃ of incubations repeating step 2 after 30 minutes.

4) every hole adds each 50ul of nitrite ion A nitrite ion B, mixing, and the room temperature lucifuge developed the color 5 minutes, measured the relative luminous intensity value in every hole.

3.2 interpretation of result

Result should satisfy following requirement:

1) positive quality control product luminous intensity values 〉=15000.

2) negative quality-control product luminous intensity values≤13000.

3) just can judge sample after satisfying above two requirements.Being patient higher than the positive control person, is the normal person lower than the negative control person.

The coated plate of table 4 sequence 1 is measured sample

The coated plate of table 5 sequence 2 is measured sample

The coated plate of table 6 sequence 3 is measured sample

The coated plate of table 7 sequence 4 is measured sample

By the result of showing 4-table 7 as can be known, SEQ:1-SEQ:4 all is suitable as coated polypeptide.

Claims

1. immunoassay kits that detects I2 antibody, comprising: the anti-human IgA antibody of microwell plate, enzyme labeling, negative control product, positive reference substance, Chemoluminescent substrate, cleansing solution, reaction terminating liquid, the sample diluting liquid that are coated with the I2 polypeptide.

2. enzyme linked immunological kit according to claim 1, is characterized in that, described I2 polypeptide is to obtain by the amino acid synthetic method.

3. I2 polypeptide according to claim 2, is characterized in that, contains SEQ:1; SEQ:2; SEQ:3; At least one sequence of SEQ:4.

4. immunoassay kits according to claim 1, is characterized in that, the anti-human IgA antibody of described enzyme labeling enzyme used is horseradish peroxidase.

5. immunoassay kits according to claim 1, is characterized in that, described negative control product are that the negative human serum of I2 is through deactivation, degerming gained.

6. immunoassay kits according to claim 1, is characterized in that, described positive reference substance is that I2 positive human serum is through deactivation, degerming gained.

7. immunoassay kits according to claim 1, is characterized in that, described Chemoluminescent substrate contains urea peroxide or analog, luminol or analog and 4-xenol.

8. enzyme linked immunological kit according to claim 1, is characterized in that, described cleansing solution is phosphate or the carbonate buffer solution that contains 0.05%Tween20.

9. enzyme linked immunological kit according to claim 1, is characterized in that, described sample diluting liquid is phosphate or the carbonate buffer solution that contains 0.05%Tween20.

10. the method for anti-I2-IgA antibody in a test sample comprises step:

(1) sample pre-treatments;

(2) detect with the arbitrary described kit of claim 1-9, add reference substance or testing sample solution in the coated microwell plate of I2 polypeptide, the anti-human IgA antibody that adds again enzyme labeling, hatching rear washing pats dry, add Chemoluminescent substrate, stop buffer, measure absorbance with biochemical instruments or Chemiluminescence Apparatus;

(3) analyzing and testing result.