CN104833804A

CN104833804A - Helicobacter pylori IgG antibody ELISA semi-quantitative detection kit and application thereof

Info

Publication number: CN104833804A
Application number: CN201510219965.4A
Authority: CN
Inventors: 刘峰
Original assignee: Will Europe Han Biotechnology (hefei) Co Ltd
Current assignee: Will Europe Han Biotechnology (hefei) Co Ltd
Priority date: 2015-04-30
Filing date: 2015-04-30
Publication date: 2015-08-12

Abstract

The invention provides a helicobacter pylori IgG antibody ELISA semi-quantitative detection kit. The kit is formed by a helicobacter pylori antigen coated enzyme labeled reaction plate, a sample diluting solution, a washing liquid, an enzyme labeled second antibody, a substrate solution, a stopping solution, a positive control solution, a negative control solution and a standard solution. A method for rapid and sensitive ELISA semi-quantitative detection of a helicobacter pylori specific IgG antibody in serum is established by using the kit. The helicobacter pylori IgG antibody standard solution is prepared, the helicobacter pylori antigen coated enzyme labeled reaction plate is prepared based on the standard solution, an indirect ELISA method is adopted to detect the specific helicobacter pylori IgG antibody in the serum, and the specific helicobacter pylori IgG antibody is quantified through calculation of an enzyme immunity unit (EIU). The kit has the advantages of high specificity and high sensitivity, and is mainly used for laboratory researches and clinic auxiliary diagnosis.

Description

A kind of Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit and application thereof

Technical field

The present invention relates to a kind of biological detection reagent kit, a specifically Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit, can be used for detecting Helicobacter pylori IgG Antibodies in serum fast and the ELISA detection kit of IgG antibody content being carried out to sxemiquantitative calculating.

Background technology

Helicobacter pylori (Helicobacter pylori, H.p), by Barry Marshall (Barry J.Marshall) and guest sieve Warren (J.Robin Warren) two people's Late Cambrian, therefore this two people obtains the Nobel prize's soul of 2005.Helicobacter pylori is the bacterium of a kind of one pole, many flagellums, the blunt circle of end, helically bent, long 2.5 ~ 4.0 μm, wide 0.5 ~ 1.0 μm.Normal in typical spiral fashion or arc on gastric epithelial cell surface.

Helicobacter pylori infections is chronic active gastritis, peptic gastric ulcer and gastric mucosa-associated lymphoid tissue (MALT) lymphadenomatous main pathogenic, and with cancer of the stomach there is Close relation.Within 1994, helicobacter pylori is decided to be I class procarcinogen by the World Health Organization (WHO)/international cancer research institution (WHO/IARC).In Asia, the infection rate difference 60%, 40%, 70% of the juvenile helicobacter pyloris such as China's Mainland, Vietnam, India, in the Gastric Biopsy of Patients with Chronic Gastritis, helicobacter pylori recall rate can reach 80% ~ 90%, and peptic gastric ulcer patient Geng Gao, can more than 95% be reached, even close to 100%.There is alienation due to topical epithelial cell in cancer of the stomach, therefore its recall rate height report differs.The stomach infections of helicobacter pylori has been a worldwide medical problem, carries out to it monitoring that Accurate Diagnosis is conducive to controls H.p spread and epidemic and elimination HP treatment of infection.

Nineteen eighty-three by gastroscope get biopsy specimen be separated cultivate successfully since, many methods have been developed to the diagnosis of helicobacter pylori infections, have included bacteriology, pathology, serology, tagging, molecular biology etc.But total says, from collection of specimens angle, invasive and the large class of Noninvasive two can be divided into.

Invasive method mainly refers to must pass through the method that biopsy specimen inspection got by gastroscope, is the conventional method of current disease for digest subject.It comprises separation cultivation and direct smear, the rapid urease test of bacterium, drug sensitive test.

Noninvasive method mainly refers to the method for not got the infection of biopsy specimen diagnosing helicobacter pylori sample by gastroscope.These class methods comprise antibody test, antigen detection, urea 13C/14C breath test etc.The current existing method of antibody test comprises euzymelinked immunosorbent assay (ELISA), Western blot, colloidal gold method and latex enhancing immune turbidimetry etc.

Serologic detection is a conventional Noninvasive detection method, is particularly useful for the area that Hp infection rate is higher.Hp causes chronic inflammation, and the immune response meeting sustainable existence of system, produces IgG antibody.Conventional serological test comprises ELISA method, rapid whole blood/method such as serum test, Western blot, and wherein ELISA method is the most frequently used.ELlSA method accuracy rate is up to 90% ~ 95%, and ELISA kit can provide the quantitative testing result of antibody, and this method is not by the impact of medicine.But regrettably at present various Helicobacter pylori IgG Antibodies ELISA detection kit is qualitative detection on domestic and international market, and IgG antibody content all has important suggesting effect for the residence density of helicobacter pylori and helicobacter pylori eradication Outcome measure.The present invention prepares Helicobacter pylori IgG Antibodies titer, adopts indirect elisa method on this basis, can carry out half-quantitative detection quickly and easily to specificity Helicobacter pylori IgG Antibodies in serum.

Summary of the invention

The invention provides a kind of helicobacter pylori IgG type antibody ELISA half-quantitative detection kit.Kit of the present invention is mainly used in the chronic gastritis instructing Helicobacter pylori infection to cause, the adjuvant clinical diagnosis of the diseases such as peptic ulcer and clinical monitoring index.

Technical solution of the present invention is as follows:

A kind of Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit, it is characterized in that: be made up of by enzyme reaction plate, sample diluent, cleansing solution, ELIAS secondary antibody, substrate solution, stop buffer, positive control solution, negative controls and titer Heliobacter pylori antigen bag

The antigen coated enzyme reaction plate of described helicobacter pylorus is prepared from through the following steps:

(1) 96 hole ELISA Plate one piece is got;

(2) multiple of Heliobacter pylori antigen coating buffer by 1:1000 is diluted, the antigen liquid diluted is added 110 μ l in each ELISA Plate hole, ELISA Plate is inserted in wet box, spent the night at 2-8 DEG C of bag;

(3) clean with cleansing solution: with cleansing solution cleaning bag by the ELISA Plate of spending the night, every hole adds 350ul cleansing solution, cleans 2-4 time;

(4) stabilizing buffer is added: every hole adds 150ul stabilizing buffer, hatches 2-4 hour, then drain under 18-25 DEG C of condition;

(5) get the antigen coated microplate drained and add 10% bovine serum albumin of 200 μ l PBS dilutions at 37 DEG C of closed 1-2 hour, obtain antigen coated enzyme reaction plate;

(6) pack: with packaging of aluminium foil bag, and put into drying agent in bag, be stored in the freezer of 2-8 DEG C after packaging;

Described coating buffer: 0.05mol/L carbonate buffer solution (pH9.6) 0.75g sodium carbonate, 1.46g sodium bicarbonate, adds deionized water and is settled to 500ml;

Described sample diluent: the phosphate buffer (PBS) of 10% bovine serum albumin+0.1%ProClin300+0.01M pH7.4;

Described cleansing solution: the phosphate buffer (PBST) of 0.05%Tween 20+0.01mol/L pH7.4;

Described ELIAS secondary antibody: the rabbit anti-Hb poly-IgG of horseradish peroxidase-labeled;

Described substrate solution: TMB-H ₂o ₂urea liquid;

Described stop buffer: 2mol/L H ₂sO ₄solution;

Described negative controls: containing human serum base helicobacter pylori IgG negative controls;

Described positive control solution: containing human serum base helicobacter pylori IgG positive control solution;

Described titer: containing human serum base helicobacter pylori IgG titer.

Described a kind of Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit detects the application in serum in Helicobacter pylori IgG Antibodies in ELISA method.

Beneficial effect

The present invention obtains a kind of kit, the present invention prepares Helicobacter pylori IgG Antibodies titer, on this basis by Heliobacter pylori antigen bag by enzyme reaction plate, adopt indirect elisa method, detect specificity Helicobacter pylori IgG Antibodies in serum, and by the calculating of enzyme immunizing unit (EIU), it is carried out quantitatively.Kit of the present invention, has high specific and susceptibility, and this kit is mainly for laboratory study and clinical assistant diagnosis.

Kit of the present invention is utilized to adopt ELISA method to detect Helicobacter pylori IgG Antibodies in serum, the testing result of kit of the present invention and the result consistance of INOVA company Helicobacter pylori IgG Antibodies ELISA detection kit high, sensitivity is 95%, and specificity is 99.5%.

Embodiment

1, reagent

(1) coating buffer: the sodium carbonate-bicarbonate damping fluid of 0.05mol/L, pH9.6,

Na ₂cO ₃1.5g, NaHCO ₃2.9g, Na ₂n ₃0.2g, adds distilled water and is settled to 1000ml, be adjusted to pH9.45-9.75;

(2) bag is by stabilizing solution: the aqueous solution of 1% casein+5% sucrose,

Sucrose 5g, casein 1g, add distilled water and be settled to 1000ml dissolving, regulate pH value to 7.15-7.25;

(3) confining liquid: 10% calf serum+0.02mol/L PBS (pH7.4)

100ml calf serum joins the 0.02mol/L phosphate buffer prepared and dissolves quantitatively to 1000ml;

(4) sample diluent: 10% bovine serum albumin+0.01mol/L phosphate buffer PBS (pH7.4), 100g calf serum, 8.0gNaCl, 0.2gKH ₂pO ₄, 2.9gNa ₂hPO ₄12H ₂o, 0.2gKCl, 0.1g thimerosal, 1g antiseptic ProClin300, adds distilled water and is settled to 1000ml, be adjusted to pH7.4, to obtain final product;

(5) cleansing solution: 0.01mol/L PBS (pH7.4)+0.05%Tween-20PBST:

NaCl 8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o 2.9g, KCl 0.2g, Tween 20 0.5ml, thimerosal 0.1g, add distilled water and be settled to 1000ml, be adjusted to pH7.4, obtain final product;

(6) ELIAS secondary antibody: the rabbit anti-Hb poly-IgG of (rabbit anti-Hb poly-IgG of HRP mark) horseradish peroxidase-labeled,

Composition: 0.2mL horseradish peroxidase-labeled helicobacter pylori anti-human igg damping fluid, use after adding sample diluent by 1:100, containing 0.02% methyl-isothiazol ketone and 0.02% bromine nitro dioxan and 0.02% other washing isothiazole ketones as antiseptic;

(7) substrate solution: TMB-H ₂o ₂urea liquid,

Preparation: 1. substrate solution A:3, absolute ethyl alcohol 100ml, added distilled water and was settled to 1000ml for-tetramethyl benzidine, TMB, TMB200mg 3 ', 5,5 ',

2. substrate solution B damping fluid (0.1mol/L citric acid-0.2mol/L sodium hydrogen phosphate damping fluid, pH5.0 ~ 5.4): Na ₂hPO ₄14.60g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml, adds distilled water and is settled to 1000ml, be adjusted to pH5.0 ~ 5.4;

3. substrate solution A and B is pressed 1:1 mixing, TMB-H ₂o ₂urea liquid;

(8) stop buffer: (2mol/L H ₂sO ₄solution): preparation: the 100ml98% concentrated sulphuric acid adds in 600ml distilled water, is settled to 1000ml, room temperature preservation;

(9) titer

Composition: 1.5mL is containing human serum anti-helicobacter pylori IgG titer+0.1% antiseptic ProClin300;

(10) negative controls

Composition: 1.5mL is containing human serum anti-helicobacter pylori IgG negative controls+0.1% antiseptic ProClin300;

(11) positive control solution

Composition: 1.5mL is containing human serum anti-helicobacter pylori IgG positive control solution+0.1% antiseptic ProClin300;

(12) sealing of hole film: hatch for 3 and use plastic seal pore membrane.

2, key instrument

(1) microplate reader: BIO-RAD Model 550

(2) ELISA reaction plate: Corning Incorporated Costar R 96Well EIA/RIA plate

3, method

The selection of the suitableeest titre of 3.1 ELIAS secondary antibody

A) get 96 hole ELISA Plate one piece, use 100ng/ml human IgG, 110 4 DEG C, μ l/ hole bags are spent the night, and cleansing solution washes plate 3 times, 350 μ l/ holes, dry;

B) by how anti-for enzyme mark anti-human igg do a series of dilution with dilution after add respectively and wrapped in the hole of quilt, 100 μ l/ holes, hatch 40min for 37 DEG C, and rear cleansing solution washes plate 3 times, 350 μ l/ holes, dry;

C) add substrate colour developing, every hole adds substrate solution 100 μ l, and 37 DEG C or room temperature lucifuge develop the color 15 minutes; After add stop buffer 50 μ l cessation reaction, microplate reader reads 450nm wavelength place absorbance, i.e. A value, get the enzyme labelled antibody dilutability of A value 1.0 time, the suitableeest titre as ELIAS secondary antibody is 1:1000.

3.2 Checkerboard titration methods select the suitableeest titre of envelope antigen

(1) 96 hole ELISA Plate one piece is got; Get after antigen is done a series of dilution by Heliobacter pylori antigen coating buffer and carry out bag quilt, every hole 110 μ l, 37 DEG C of incubators spend the night oven dry; 10% calf serum 37 DEG C that the antigen coated microplate that getting spends the night dries adds 200 μ l PBS dilutions closes 1.5 hours, obtains the antigen coated enzyme reaction plate of gradient dilution;

(2) strong positive reference serum, weak positive reference serum and negative reference serum sample diluent are doubly diluted by 1:100, application of sample, insulation, washing.

(3) add the enzyme mark anti-human igg of diluting by the suitableeest titre to resist, insulation, washing more.

(4) add substrate colour developing, in microplate reader, read 450nm wavelength place absorbance after acid adding cessation reaction, i.e. A value,

(5) select that the A value of strong positive reference serum is between 1.8 ~ 2.0, the A value of negative reference serum is less than the dilutability of the envelope antigen of 0.2 as the suitableeest titre, the suitableeest bag of this kit is 1:1000 dilution by the dilutability of Heliobacter pylori antigen, 100 μ l/ holes.

The preparation of 3.3 titers

After collecting 100 parts of positive serum mixing, concentrated and purified through Protein A tubing string affinity chromatography, ultimate density is that 2.0mg/ml is decided to be 100EIU unit.

3.4 Heliobacter pylori antigen bags are by the preparation method of reaction plate

Get 96 hole ELISA Plate one piece, Heliobacter pylori antigen coating buffer is diluted in 1:1000 ratio, then wrapper sheet, every hole 110 μ l; 37 DEG C of incubators spend the night oven dry; 10% calf serum 37 DEG C that the antigen coated microplate that getting spends the night dries adds 200 μ l PBS dilutions closes 1.5 hours, obtains antigen coated enzyme reaction plate..

3.5 kit assemblings

Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit, is made up of by enzyme reaction plate, sample diluent, cleansing solution, ELIAS secondary antibody, substrate solution, stop buffer, positive control solution, negative controls and titer above-mentioned Heliobacter pylori antigen bag.

Specifically be divided into:

(1) sample diluent: containing the phosphate buffer (PBS) of the 0.01M pH7.4 of 10% bovine serum albumin and 0.1%ProClin300;

(2) cleansing solution: the phosphate buffer (PBST) of the 0.01mol/L pH7.4 containing 0.05%Tween 20;

(3) ELIAS secondary antibody: the rabbit anti-Hb poly-IgG of horseradish peroxidase-labeled;

(4) substrate solution: TMB-H ₂o ₂urea liquid;

(5) stop buffer: 2mol/L H ₂sO ₄solution;

(6) negative controls: containing human serum base helicobacter pylori IgG negative controls;

(7) positive control solution: containing human serum base helicobacter pylori IgG positive control solution;

(8) titer: containing human serum base helicobacter pylori IgG titer.

3.5 utilize mentioned reagent box to adopt ELISA method to detect Helicobacter pylori IgG Antibodies in serum

3.5.1 prepare before test

All reagent and micro reaction plate are returned to room temperature (20 ~ 25 DEG C).Concentrated cleaning solution (i.e. 100ml cleansing solution+900ml distilled water or deionized water) is diluted with 1:10.Quick-thawing sample in water-bath (room temperature) also mixes.When almost dissolving completely, be placed in trash ice bath.Instructions should be read over before test.All blank solutions, titer, contrast liquid and sample is recommended all to do the test of multiple hole.Each experiment all should carry out the test of blank solution, titer and contrast liquid.

3.5.2 Sample Dilution application of sample

With dilution, blood plasma or serum sample are diluted 200 times (5 μ l sample+995 μ l dilutions), fully mix.

Abundant mixing reagent and the sample diluted, sample (the S1 adding 100 μ l dilutions (blank), titer (standard), negative controls (feminine gender), positive control solution (positive) respectively and dilute in reacting hole, S2, S3 etc.), do multiple hole test (with reference to table 1).After sealing of hole film shrouding, at 37 DEG C, hatch 30 minutes.

Table 1 application of sample example

	1	2	3	4	5
						A	Blank	Blank
B	Standard	Standard
						C	Negative	Negative
D	Positive	Positive
						E	S1	S1
F	S2	S2
						G	S3	S4
H	Other sample	Other sample

3.5.3 wash: after the cleansing solution 350 μ l that every hole is diluted rinses 3 times, be inverted microwell plate knocking several on clean thieving paper.Suggestion second step uses 8 channel pipettor to the 6th step.

3.5.4 ELIAS secondary antibody is added: diluted 100 times (such as: 40 μ l enzyme mark liquid+3960 μ l dilutions) by enzyme mark liquid with dilution, fully mix, each hole adds the enzyme mark liquid that 100 μ l have mixed respectively.After sealing of hole film shrouding, at 37 DEG C, hatch 30 minutes.

3.5.5 wash: with reference to 3.4.2.2.

3.5.6 substrate solution is added: each hole adds the substrate solution that 100 μ l have mixed.After substrate solution adds the first lath, start timing, under room temperature (20 ~ 25 DEG C), hatch 30 minutes, avoid illumination.

3.5.7 stop buffer is added: each hole adds the stop buffer that 100 μ l have mixed.

3.5.8 microplate reader measures: in 30 minutes, at 450nm wavelength place, microplate reader measures absorbance.

3.5.9 result calculates: the mean light absorbency (A) calculating each multiple hole test of blank (dilution), titer, contrast liquid and sample, and the average absorbance value of each test deducts the average absorbance value of blank solution.Enzyme immunizing unit (EIU) is calculated as follows:

Sample=sample; Blank=is blank; Calibrator=titer

[reference value (term of reference)]

Negative < 30EIU

The positive >=30EIU

3.6 specificity experiments

The antigen coated enzyme reaction plate ELISA kit of the full bacterium of helicobacter pylori is adopted to detect with helicobacter pylori standard I gG positive serum, japanese encephalitis virus IgG positive serum, measles virus IgG positive serum, rubella virus IgG positive serum, human cytomegalovirus IgG positive serum and helicobacter pylori standard I gG negative serum, P/N value is respectively 6.23,1.22,1.15,1.27 and 1.06, illustrates that this kit specificity is good.

3.7 clinical samples detect

INOVA Inc Bioisystech Co., Ltd Helicobacter pylori IgG Antibodies ELISA detection kit is adopted to detect 700 routine normal human serums, wherein positive 400 examples of IgG antibody, negative 300 examples, adopt the full bacterium of helicobacter pylori antigen coated enzyme reaction plate method testing result to be 98.5% with INOVA company helicobacter pylori IgG ELISA detection kit phase specific sensitivity, specificity is 98.7%.

Table 1 two kinds of kit IgG testing results compare

Meter sensitivity=98.5%, specificity=98.7%.

3.8 kit assay Performance Evaluations

With reference to the content of national regulations and standard, formulate the quality standard of this product.Based on this standard, we assess the analytical performance of product, and result of study shows that every analytical performance is good, and concrete outcome is as follows:

3.8.1 accuracy: join in normal serum with the Helicobacter pylori IgG Antibodies of concentration known, its recovery should between 90%-110%.

3.8.2 difference between batch; With the sample 10 times that the concentration of the helicobacter pylori IgG detection kit duplicate detection helicobacter pylori IgG of three different lot numbers is 20-30EIU, calculate mean value M and the standard deviation SD of 10 measurement results, coefficient of variation CV is drawn according to formula CV=SD/M × 100%, the difference between batch CV scope of serum sample is 4.2-7.0%, and the difference between batch CV scope of the plasma sample of EDTA anti-freezing is 4.2-8.1%.

3.8.3 criticize interpolation: the sample duplicate detection 10 times by the concentration of helicobacter pylori IgG being 20-30EIU, calculate mean value M and the standard deviation SD of 10 measurement results, draw coefficient of variation CV according to formula CV=SD/M × 100%.Batch interpolation CV scope of serum sample is 3.8-4.6%, and batch interpolation CV scope of the plasma sample of EDTA anti-freezing is 3.5-4.9%.

3.8.4 susceptibility is analyzed: the value being determined minimum quantitative limit by the dilution series (diluting by dilution buffer) of examination criteria liquid (definite concentration is marked on label, is about 100EIU).Dilution series is stoste, 1:2,1:4,1:8,1:16, l:32 and 1:64.The sample (namely each dilutability 10 parts repeats 10 times) of each dilution series is added respectively in the reacting hole of microwell plate.Calculate mean absorbance and each dilution concentration value.

Blank detectability (LoB): 1.1EIU.

Lowest detectable limit (LoD): 1.8EIU.

3.8.5 stability study

The accelerated test behavior pattern of 8 days is studied after placing 14 months to continuous 3 batches of products under 2-8 DEG C of condition and under 37 DEG C of conditions, measures reagent end properties, observes sample with or without mass change.Test result shows, this product is steady quality with this understanding, meets the requirements.

Claims

1. a Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit, it is characterized in that: be made up of by enzyme reaction plate, sample diluent, cleansing solution, ELIAS secondary antibody, substrate solution, stop buffer, positive control solution, negative controls and titer Heliobacter pylori antigen bag

(1) 96 hole ELISA Plate one piece is got;

(2) multiple of Heliobacter pylori antigen coating buffer by 1:1000 is diluted, the antigen liquid diluted is added 110 μ l in each ELISA Plate hole, ELISA Plate is inserted in wet box, spent the night at 2-8 DEG C of bag

Described substrate solution: TMB-H ₂o ₂urea liquid;

Described stop buffer: 2mol/L H ₂sO ₄solution;

Described titer: containing human serum base helicobacter pylori IgG titer.

2. the application of a kind of Helicobacter pylori IgG Antibodies ELISA half-quantitative detection kit according to claim 1 in ELISA method detection serum in Helicobacter pylori IgG Antibodies.