CN102305857A - Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof - Google Patents
Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof Download PDFInfo
- Publication number
- CN102305857A CN102305857A CN201110219417A CN201110219417A CN102305857A CN 102305857 A CN102305857 A CN 102305857A CN 201110219417 A CN201110219417 A CN 201110219417A CN 201110219417 A CN201110219417 A CN 201110219417A CN 102305857 A CN102305857 A CN 102305857A
- Authority
- CN
- China
- Prior art keywords
- helicobacter pylori
- antigen
- quality control
- latex
- damping fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention provides a latex immunoreagent for determining a helicobacter pylori antibody and a method for performing latex immunoturbidimetry detection by utilizing the immunoreagent. The immunoreagent comprises buffer solution with the pH value of 8.0+/-0.3, a helicobacter pylori specific antigen latex reagent, a calibrator, a low-value quality control substance and a high-value quality control substance, wherein a helicobacter pylori specific antigen can be a helicobacter pylori whole-cell protein antigen, a helicobacter pylori urease antigen, a helicobacter pylori cytotoxin-associated protein A antigen or a helicobacter pylori vacuolating toxin A antigen; and the calibrator, the low-value quality control substance and the high-value quality control substance are helicobacter pylori antibody solutions used for comparing with a sample and performing result calculation and quality control. The immunoreagent is a liquid double-reagent which is suitable for various types of full-automatic biochemical analyzers and semi-automatic biochemical analyzers and has the advantages of high sensitivity, good repeatability, good detection accuracy and good precision.
Description
Technical field
Application of the present invention relates to technical field of biological, is specifically related to the latex immunoreagent and the detection method thereof of helicobacter pylori antibody in a kind of human body serum.
Background technology
Helicobacter pylori (Helicobacter pylori is called for short HP) is the bacterium of a kind of one pole, many flagellums, terminal blunt circle, helically bent, is the Grain stain feminine gender, often is typical spiral fashion or arc on the gastric epithelial cell surface.
Helicobacter pylori infections is the lymphadenomatous main paathogenic factor of chronic active gastritis, peptic gastric ulcer and gastric mucosa-associated lymphoid tissue (MALT); And closely related with the generation of cancer of the stomach, the World Health Organization (WHO)/international cancer research institution (WHO/IARC) was decided to be I class procarcinogen with helicobacter pylori in 1994.The helicobacter pylori recall rate can reach 80%～90% in chronic gastritis patient's the gastric mucosa biopsy specimen, and peptic gastric ulcer patient Geng Gao can reach more than 95%, even near 100%.Cancer of the stomach is because alienation has taken place in local epithelial cell, so its recall rate is just reported and differed.The infection of helicobacter pylori has been a worldwide problem, it is accurately diagnosed help controlling HP propagation and monitoring popular and that eradicate the HP treatment of infection.
At present, the HP detection method of having set up both at home and abroad is divided into invasive and Noninvasive two big classes from detection means.
One, invasive detection method
The characteristics of these class methods detect after being to use endoscope to obtain gastric biopsy, and test method mainly contains fast furosemide enzyme test method, pathology detection method and bacterial cultivation.
1, fast furosemide enzyme detection method: produce ammonia but have abundant urease decomposing urea, develop the color by the pH indicator according to HP.This method is easy, rapid, sensitive, produces false positive but also might exist because of other bacteriums that contain urease.In addition, used the sample that reduces the stomach amount of bacteria or directly suppress the medicine of urease activity can produce false negative in the recent period.
2, pathology detection method: through mucosa tissue being carried out section statining inspection, can directly show the HP thalline and confirm that wherein the argentation verification and measurement ratio is high, but the otherness that the different pathological scholar observes has certain difficulty property for the diagnosis of lipogastry sample.
3, bacterial cultivation: get the gastric mucosa living tissue and make HP and cultivate, less because of culture of bacteria, operating process is complicated, the time cycle is long, the suitable widespread usage of expensive.
Two, Noninvasive detection method
These class methods characteristics are to use endoscope to take gastric biopsy, thereby avoid patient painful or the infection of other type takes place because of the infringement that threshes OGD and bring.Test method mainly contains urea breath test, PCR detection method and immunoserology method.
1, urea breath test:, use because HP is rich in urease
13C or
14After the urea of C mark is taken by the experimenter, detect the isotope-labeled carbon dioxide sample of being with that decomposes generation, judge the HP gradient of infection according to the gas nuclide mass spectrometer.This method is highly sensitive, can be quantitative, but certain Radio Active Hazard is arranged, and be subjected to device-restrictive to be difficult to generally promote.
2, PCR detection method: mainly detect HP urease (UReA) gene and toxin associated protein A (CagA) gene in gastric juice, the mucous membrane; But complicated operation; Be prone to pollute; To reviewer's professional knowledge and having relatively high expectations of operative technique; And increase patient's economy spending greatly, therefore application clinically is also comparatively limited.
3, immunoserology method: existing at present method comprises euzymelinked immunosorbent assay (ELISA), Western blot and colloidal gold method; For the epidemiology survey of HP provides favourable convenient means; But detection speed is slow; Except that euzymelinked immunosorbent assay (ELISA) can be used full-automatic enzyme non-analysis meter (and need 2～3 hours, step many); All the other all need manual operations; It is qualitative analysis that the patient obtains testing result, is inappropriate for treatment monitoring and curative effect assessment.
At present,, also do not have a kind of suitable Biochemical Analyzer though it is various to detect the method for Helicobacter pylori infection, can be easy, quick, in enormous quantities and quantitative detect.If can set up this kind method; Then can make greatly easy of testing process; Operating personnel only need set set factors; After reagent and sample placed the relevant position; Biochemical Analyzer can automatically be accomplished detection; And can obtain the result rapidly, and be easy to apply in medical institutions at different levels, have great clinical meaning for the control of helicobacter pylori and be worth with popularizing.
Summary of the invention
The purpose of application of the present invention is to overcome the defective of existing method and technology, a kind of latex immune reagent kit of measuring helicobacter pylori antibody and preparation method thereof is provided and uses this reagent to carry out the method that clinical sample detects.This reagent is applicable to all types of Biochemical Analyzers, have sample need not to dilute, simple to operate, quick, quantitatively accurately, the characteristics of high, the high specificity of good reproducibility, good stability, susceptibility.
For realizing the object of the invention, adopt following technical scheme:
A kind of latex immunoreagent of measuring helicobacter pylori antibody comprises that the pH value is 8.0 ± 0.3 damping fluid, helicobacter pylori specific antigen emulsion reagent, calibration object, low value Quality Control thing and high value Quality Control thing.
Wherein, the pH value is that 8.0 ± 0.3 damping fluid comprises one or more in Tris/HCl damping fluid, phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or the HEPPS damping fluid.
As preferably; Described pH value is that 8.0 ± 0.3 damping fluid is a Tri s/HCl damping fluid, and contains NaCl, PEG 6000 and Tween 80, and inorganic salts NaCl can regulate ionic strength; PEG 6000 can accelerate the immune response speed of antigen-antibody, shortens detection time.
Described helicobacter pylori specific proteins comprises helicobacter pylori whole cell albumen, helicobacter Pylori urease, cytotoxin-associated protein A (Cytotoxin associated gene A; Cag A) or vacuolate cytotoxin A (Vacuolating cytotoxin A, Vac A).
Helicobacter pylori can produce a large amount of ureases, and this urease reaches to cause a disease to bacterium field planting is in vivo bringing into play important effect.Urease mainly is made up of A (UreA) and two subunits of B (UreB), and molecular weight is about 30kD and 63kD respectively, has confirmed that already urease B subunit (ureB) is the important protective antigens of helicobacter pylori.
Existing research further shows; The helicobacter pylori that accounts for 50-60% greatly can be at external generation cytotoxic activity; Wherein cytotoxic expression is closely related with the cytotoxin-associated gene A that is exposed to the helicobacter pylori surface; The albumen of this gene expression exists only in and produces in the cytotoxic helicobacter pylori; And has a good immunogenicity; Think that now this albumen can provide foundation for the diagnosis of clinical stomach illness as antigen.
Vacuolate cytotoxin A (VacA) also is the specific expressed albumen of helicobacter pylori, has important value for the detection of clinical helicobacter pylori.
Helicobacter pylori specific antigen emulsion reagent of the present invention, preparation process comprises:
Step 1: the preparation of helicobacter pylori specific antigen;
Step 2: at latex particle size is to add step 1 gained antigen in the latex solution of 50-150nm, and stirring at room 8-15 hour, supernatant was removed in centrifugal (10,000rpm, 10-20 minute);
Step 3: in step 2 gained deposition, add confining liquid, mix, stirring at room 0.5 hour, supernatant is removed in centrifugal (10,000rpm, 10-20 minute);
Step 4: in step 3 gained deposition, add the damping fluid washing that contains stabilizing agent and antiseptic and disperse.
Preferably, the concentration after washing disperses is 1%.
As preferably; The antigen that step 1 obtained is adjusted to neutrality with 0.05M glycine buffer (pH=7.0); After being added in the said latex solution of step 2; Within 10 minutes with latex particle solution chemical crosslinking sensitization; Form covalency antigen-latex compound, otherwise the space three-dimensional structure of antigen is returned to the natural conformation that does not have activity easily.
The said latex solution of step 2 is with polyvinyl benzyl chloride latex particle mixing, centrifugal, cleaning back acquisition with PBS.Polyvinyl benzyl chloride latex particle is a kind of latex particle of nucleocapsid form, and its latex nuclear is the vinyltoluene polymkeric substance, and the latex shell is the polymkeric substance of vinyl chloride, is a kind of hydrophobicity latex.Have highdensity chloro-methyl group on the surface of shell, can in the aqueous solution of gentleness, react by amino groups direct and antibody, antigen or other parts, produce a kind of stable covalent compound through single step reaction.Through adding suitable surfactant, the surface of latex particle forms mechanicalness or electrical diaphragm, can suppress the flocculation of latex particle; According to the proportion of latex particle, regulate damping fluid proportion and viscosity through suitable suspending agent, can make the latex particle stable suspersion and can sedimentation.
Step 3 is the covalency antigen-latex compound solution that forms, and seals the not surface active groups of conjugated antigen---methyl chloride of latex particle with the phosphate buffer of 0.05%BSA.
The said stabilizing agent of step 4 is selected from one or more in protein, amino acid, inorganic salts, surfactant, suspending agent, the antioxidant.Be preferably the phosphate buffer that contains 0.05%BSA, the solution after the sealing is disperseed with the damping fluid washing that contains stabilizing agent and antiseptic, promptly obtain stable helicobacter pylori specific antigen emulsion reagent.
Said antiseptic is selected from suitable antiseptic well known by persons skilled in the art, comprises Sodium azide, Proclin-300, the gentamicin of 0.1% (g/ml).
The preparation process of helicobacter pylori specific antigen according to the invention comprises:
Step 1: microbe growth comprises solid-state cultivation, the liquid cultivation in a small amount and liquid a large amount of the cultivation;
Step 2: Bacteria Identification, fragmentation, centrifugal, extraction supernatant, purifying and determination of protein concentration.
In specific embodiment, a kind of method for preparing helicobacter pylori whole cell antigen is disclosed.
Calibration object according to the invention, low value Quality Control thing and high value Quality Control thing are that a kind of being used for compared with sample; Carry out the helicobacter pylori antibody solution of result's calculating and quality control, comprise PBS damping fluid, FBS, an amount of stabilizing agent, an amount of antiseptic and certain density helicobacter pylori specific antibody.
The concentration of calibration object can be high concentration single-point reference calibrations article; Be diluted to the reference calibrations article of a plurality of variable concentrations in use with physiological saline; Also can directly be prepared into the reference calibrations article of a plurality of variable concentrations; Can also be the single-point reference calibrations article of fixed concentration, draw calibration curve jointly with physiological saline.The single-point reference calibrations article of preferred immobilization concentration of the present invention, the concentration of calibration object, low value Quality Control thing and high value Quality Control thing is respectively 40AU/mL, 10AU/mL and 30AU/mL.In specific embodiment, a kind of method for preparing calibration object, low value Quality Control thing and high value Quality Control thing is disclosed.
The principle that the present invention measures sample is the latex immunoturbidimetry; Selected sample is a human serum; Sample and reagent R1(pH value are 8.0 ± 0.3 buffer solution) behind the preincubate 3-5 minute (antibody combining site in the sample is fully exposed); Add reagent R2(helicobacter pylori specific antigen emulsion reagent); Continued to hatch 3-5 minute; Helicobacter pylori specific antibody in the human serum combines with helicobacter pylori specific antigen among the reagent R2; Form insoluble antigen-antibody complex; Produce certain turbidity, its turbidity height is directly proportional with specific anti bulk concentration in detecting sample.Under provision wavelengths, measure the absorbance of this insoluble antigen-antibody complex, compare, then can calculate the concentration of helicobacter pylori antibody in the sample with the helicobacter pylori specific antibody calibration object of concentration known.
Reagent provided by the invention is liquid double reagent, is applicable to all types of automatic clinical chemistry analyzers and semi-automatic biochemical analyzer, compared with prior art, has following characteristics:
1, detection by quantitative, highly sensitive, can reach 2.0AU/mL;
2, relevant at (5～180) AU/mL sensing range inner height, good reproducibility;
3, accuracy in detection and precision are good;
4, high specificity, and be not subject to disturb;
5, good stability, but 2～8 ℃ of lucifuges stored at least 12 months, can preserve at least 14 days after each reagent uncork;
6, sample need not dilute in advance, simple to operate, quick, only need 10 minutes from detecting out the result, be specially adapted to examination in enormous quantities, detection speed can improve with the raising of Biochemical Analyzer detection speed.
Description of drawings
Accompanying drawing is a kit range of linearity correlativity synoptic diagram according to the invention.
Embodiment
The invention discloses a kind of latex immunoreagent and preparation and application of measuring helicobacter pylori antibody, those skilled in the art can use for reference this paper content, realize through suitable improvement technological parameter.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included within the present invention's technical scheme required for protection.Product of the present invention and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
For making the present invention be more prone to understand, below in conjunction with specific embodiment, further set forth the present invention, these embodiment only are used to the present invention is described and are not used in restriction scope of the present invention.
Embodiment one
The preparation of helicobacter pylori antibody latex immune reagent kit
1, the preparation of helicobacter pylori specific antigen (is example with the whole cell proteantigen)
(1) microbe growth
(a) solid-state cultivation
The HP bacterial classification inoculation in chocolate culture-medium, is placed 5%O
2, 10%CO
2And 85%N
2Environmental baseline under, cultivated 3～4 days for 37 ℃, it is transparent and like gluey bacterium colony, this is helicobacter pylori, provokes several bacterium colonies with oese promptly to produce flat flatband point, and bacterium after scratching with three zoning collimation methods on the chocolate culture-medium, is placed 5%O
2, 10%CO
2And 85%N
2Environmental baseline under, cultivated 3～4 days for 37 ℃, every bacterium that propagated at a distance from four days is once with the activity of maintenance bacterium.
(b) liquid in a small amount the cultivation
With solid-state helicobacter pylori bacterium colony of turning out, hang several bacterium colonies down with oese, and shake off in the 125ml conical flask; Fill BHI nutrient solution 50ml in the bottle; Wherein contain 1ml newborn calf serum and 0.1ml helicobacter pylori screening liquid, the conical flask bottleneck is covered tightly with tampon, place 5%O
2, 10%CO
2And 85%N
2Environmental baseline under, cultivated 3～4 days for 37 ℃.
(c) a large amount of liquid cultivations
Draw 0.5ml culture bacteria liquid in a small amount, carry out preliminary urea evaluation and outward appearance and observe, confirm not receive other living contaminantses.Draw 15ml bacterium liquid and add in the 500ml conical flask, fill BHI nutrient solution 250ml in the bottle, wherein contain 5ml newborn calf serum and 0.5ml helicobacter pylori screening liquid, the conical flask bottleneck is tight with the tampon covering, place 5%O
2, 10%CO
2And 85%N
2Environmental baseline under, cultivated 3～4 days for 37 ℃.
(2) Bacteria Identification, fragmentation, centrifugal and extraction supernatant
(a) Bacteria Identification
The outward appearance kenel is observed: observe the growth kenel of bacterium colony on chocolate culture-medium, be the transparent seemingly gluey bacterium colony of flat flatband point.
The urea method of inspection: the urease of helicobacter pylori is very abundant, 15% of about mycetome albumen, and the urease catalyzes hydrolysis of urea can produce ammonia.Get 0.5ml urea test solution and place test tube, add 0.5ml bacterium liquid, react half an hour approximately in 37 ℃.If contain helicobacter pylori, then test solution can change pink into by yellow.
(b) bacteria breaking, centrifugal and extraction supernatant
Get 250ml helicobacter pylori bacterium liquid; In 8000rpm centrifugal 20 minutes; Abandon supernatant, deposition adds 10ml0.05M glycine buffer (pH=7.0) cleans, in 8000rpm centrifugal 20 minutes; Abandon supernatant; Deposition adds 10ml 0.05M glycine buffer (pH=7.0) and mixes, and carrying out ultrasonic bacteria breaking is in 18; Centrifugal 20 minutes of 000rpm takes out supernatant and its placement-80 is ℃ subsequent use.
(c) antigen protein concentration determination
With the quantitative Heliobacter pylori antigen protein concentration of Bio-Rad protein determination kit; At first hyclone albumen (BSA) is dissolved in the standard items of following each concentration of preparation among the PBS: 0mg/ml; 0.1mg/ml; 0.2mg/ml; 0.3mg/ml; 0.4mg/ml; 0.5mg/ml; Then with 5 times of dilution analysis damping fluids of secondary water (containing Coomassie brilliant blue R-250); Then in 96 micropore dishes, add the analysis buffer after 100 μ l dilute; Add the Heliobacter pylori antigen protein solution that obtains in each concentration standard article of 10 μ l and (2) at last respectively; In room temperature reaction 5 minutes; Under the 595nm wavelength, read the OD value with visible spectrophotometer or microplate reader; The drawing standard curve, and calculate the Heliobacter pylori antigen protein concentration.
2, the preparation of reagent R1
Earlier with a little dissolving NaCl of redistilled water (3.5～9.0g), add the Tris-HCl damping fluid again, add redistilled water at last to 1000ml, fully shaking up and making the Tris final concentration is 0.05mol/L, adds a small amount of PEG 6000 and Tween80 again, mixes to get final product.
Those skilled in the art also can select other conventional damping fluid, like in phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid one or more.
3, the preparation of reagent R2
Be 1% (g/V) with the 1XPBS diluted latex; Mix back 10; Centrifugal 10 minutes of 000rpm; Remove supernatant; And with 1XPBS with the latex cleaning many times after; Adding 1XPBS breaks up latex; Add the helicobacter pylori whole cell antigen that extracts in 1 or purified after urease antigen; Cytotoxin-associated protein A or vacuolate cytotoxin A; Stirring at room 12 hours; 10, centrifugal 10 minutes of 000rpm removes supernatant; Adding sealer (1XPBS that contains 0.05%BSA) in the deposition mixes; Stirring at room 0.5 hour, 10, centrifugal 10 minutes of 000r pm; Remove supernatant, it is 1% (g/V) that deposition is dispersed into the antigen latex particle concentration that the milky suspension makes sensitization with the 1XPBS that contains 0.05%BSA.
4, the preparation of calibration object, low value Quality Control thing and high value Quality Control thing
With the repeatedly immune New Zealand of helicobacter pylori specific antigen rabbit, extract blood, left standstill 4 hours in room temperature, treat that blood clotting is complete, with 3, centrifugal 10 minutes of 000rpm just contains lot of antibodies in the serum.
Rabbit anteserum is carried out purifying through steps such as ammonium sulfate extracting, PBS dialysis, Protein A (Zymed) tubing string chromatographies, measure and collect the absorbance of liquid, confirm the existence of antibody at 280nm.
Measure helicobacter pylori antibody concentration with (3) in 1 said method; With 1XPBS hyclone is diluted 10 times again; Add helicobacter pylori antibody solution; Make its ultimate density be respectively 0.5mg/ml, 0.375mg/ml, 0.125mg/ml (convert active unit to and be respectively 40AU/ml, 30AU/ml, 10AU/ml); In 4 ℃ mix 30 minutes after, place 4 ℃ subsequent use.
Embodiment two
The assay method of helicobacter pylori antibody latex immunoreagent
This kit is applicable to Beckman, Hitachi, Olympus, Toshiba, Luo Shi, Abbott Laboratories, Siemens, step the full-automatic or semi-automatic biochemical analyzer of brand such as auspicious, and assay method is following:
1, condition determination (table 1)
The condition determination of table 1 kit according to the invention
| Temperature | 37℃ |
| Predominant wavelength | 546nm |
| Commplementary wave length | 800nm |
| Sample size | 4μL |
| The R1 consumption | 160μL |
| The R2 consumption | 40μL |
| Analysis type | 2 end-point methods |
2, assay method
Add sample: 4 μ L add R2:40 μ L
R1：160μL
Sample size, R1 consumption and R2 consumption can be adjusted in the sample of regulation and the ratio of reagent dosage in " condition determination " by the requirement of different model Biochemical Analyzer.Long like aphalangia standing wave in the instrument, can select and the immediate numerical value input of specified wavelength.
(3) calibration, quality control and sample are measured
Use the calibration object in the kit to calibrate by the calibration procedure requirement in institute's operational analysis instrument instructions, pattern is 2 points (being that physiological saline and concentration are the 40AU/mL calibration object) calibrations, and instrument generates calibration curve automatically.
After the calibration, measure low value Quality Control thing and high value Quality Control thing in the kit respectively, testing result should the sign value ± 15% scope in.
Quality Control can be by measure clinical serum sample with quadrat method after control.
Embodiment three
The analytical performance assessment of helicobacter pylori antibody latex immunoreagent
1, sensitivity for analysis
With the zero standard article is sample; Press embodiment two said method replications 20 times, result of calculation mean value is 0.8AU/mL, and standard deviation S D is 0.16; Mean value and 3 times of standard deviation sums are 1.2AU/mL, so the sensitivity for analysis of kit of the present invention is 2.0AU/mL.
2, accuracy and withinrun precision
With low value and high value quality control material substance as a sample, the method described in Example two repeated measurements 20 times, respectively, were calculated and measured results indicated that the relative deviation, mean
standard deviation (SD) and coefficient of variation CV, results as shown in Table 2.
The accuracy of table 2 kit according to the invention and withinrun precision are investigated
Investigate accuracy of measurement with relative deviation, relative deviation all in ± 15% scope, is investigated withinrun precision with the coefficient of variation, and low value Quality Control thing is respectively 5.9% and 5.3% with the coefficient of variation of high value Quality Control thing.
3, betweenrun precision
Measure same serum sample 10 times by embodiment two said methods respectively with the kit according to the invention of 3 lot numbers, the coefficient of variation (CV) of calculating 30 mensuration results is to investigate betweenrun precision, and the result shows that the coefficient of variation is 7.3%.
4, the range of linearity
With physiological saline with high value sample (concentration is 181.6AU/mL) by 1: 1,1: 2,1: 4,1: 8,1: 16,1: 32 totally 6 gradients dilute; Press each concentration determination of embodiment two said methods 3 times; Actual measurement mean value and corresponding theory value are done regretional analysis; The calculating regression equation is y=1.0843x-2.9756; Correlation coefficient r=0.9916; Show kit of the present invention good relationship in (5～180) AU/mL range of linearity, see accompanying drawing.
5, the influence of interfering material
To indicate concentration respectively and be the high value Quality Control thing of 30AU/mL and bilirubin solution that physiological saline, concentration are 0.5mg/mL, hemoglobin solutions that concentration is 5mg/mL, chyle that turbidity is 3000FTU is even by 9: 1 mixed; Press embodiment two said each sample of method test 3 times, get average.Observe the relative deviation of measuring the result after adding interfering material and adding physiological saline.The result shows: add above-mentioned concentration interfering material sample and be no more than 7% with adding the relative error of measuring the result with the sample of volume physiological saline, can think that the testing result of this assay method is interference-free basically when mild or moderate haemolysis, jaundice or chyle.
6, stability
With kit uncork according to the invention be placed on 2 ℃～8 ℃ preserved for 2 weeks after; It is the high value Quality Control thing of 30AU/mL and the low value Quality Control thing that the sign value is 10AU/mL that taking-up is measured the sign value by instance two said methods; Each replication 3 times, the relative deviation of calculating testing result and sign value.The result shows that the relative deviation of kit detected value according to the invention and sign value is all less than 10% after 2 weeks of uncork, and uncork stability better.
Place 2 ℃～8 ℃ preservations after 12 months kit according to the invention; It is the high value Quality Control thing of 30AU/mL and the low value Quality Control thing that the sign value is 10AU/mL that taking-up is measured the sign value by instance two said methods; Each replication 3 times, the relative deviation of calculating testing result and sign value.The result shows that the relative deviation of kit detected value according to the invention and sign value is all less than 10% after 12 months, and long-time stability are better.
Claims (10)
1. latex immunoreagent of measuring helicobacter pylori antibody is characterized in that: described reagent comprises that the pH value is 8.0 ± 0.3 damping fluid, helicobacter pylori specific antigen emulsion reagent, calibration object, low value Quality Control thing and high value Quality Control thing, wherein,
1) the pH value is that 8.0 ± 0.3 damping fluid comprises one or more in Tris/HCl damping fluid, phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid;
2) the contained antigen of helicobacter pylori specific antigen emulsion reagent comprises helicobacter pylori whole cell proteantigen, helicobacter Pylori urease antigen, helicobacter pylori cytotoxin associated protein A antigen or helicobacter pylori vacuolate cytotoxin A antigen;
3) calibration object, low value Quality Control thing and high value Quality Control thing are to be used for comparing with sample; Carry out the helicobacter pylori antibody solution of result's calculating and quality control, comprise PBS damping fluid, FBS, an amount of stabilizing agent, an amount of antiseptic and certain density helicobacter pylori specific antibody.
2. reagent according to claim 1 is characterized in that: described pH value is that 8.0 ± 0.3 damping fluid is the Tris/HCl damping fluid, and contains NaCl, PEG 6000 and Tween 80.
3. reagent according to claim 1 is characterized in that: the preparation of described helicobacter pylori specific antigen emulsion reagent comprises the steps:
1) step 1: the preparation of helicobacter pylori specific antigen;
2) step 2: at latex particle size is to add step 1 gained antigen in the latex solution of 50-150nm, stirring at room 8-15 hour, with 10, the centrifugal 10-20 of the speed of 000rpm minute, removes supernatant;
3) step 3: in step 2 gained deposition, add confining liquid, mix, stirring at room 0.5 hour with 10, the centrifugal 10-20 of the speed of 000rpm minute, is removed supernatant;
4) step 4: in step 3 gained deposition, add the damping fluid washing that contains stabilizing agent and antiseptic and disperse.
4. reagent according to claim 3 is characterized in that: the preparation of described helicobacter pylori specific antigen comprises the steps:
1) step 1: microbe growth comprises solid-state cultivation, the liquid cultivation in a small amount and liquid a large amount of the cultivation;
2) step 2: Bacteria Identification, fragmentation, centrifugal, extraction supernatant, purifying and determination of protein concentration.
5. reagent according to claim 3; It is characterized in that: the antigen that step 1 obtained is adjusted to neutrality with the 0.05M glycine buffer of pH=7.0; After being added in the said latex solution of step 2; Within 10 minutes,, form covalency antigen-latex compound with latex particle solution chemical crosslinking sensitization.
6. reagent according to claim 3 is characterized in that: the said latex solution of step 2 is with polyvinyl benzyl chloride latex particle mixing, centrifugal, cleaning back acquisition with PBS.
7. reagent according to claim 3 is characterized in that: with the covalency antigen-latex compound solution that forms, seal the not surface active groups methyl chloride of conjugated antigen of latex particle with the phosphate buffer of 0.05%BSA in the step 3.
8. reagent according to claim 3 is characterized in that: the said stabilizing agent of step 4 is selected from one or more in protein, amino acid, inorganic salts, surfactant, suspending agent or the antioxidant; Described antiseptic is selected from 0.1% Sodium azide, Proclin-300 or gentamicin; The concentration of calibration object can be high concentration single-point reference calibrations article; Be diluted to the reference calibrations article of a plurality of variable concentrations in use with physiological saline; Also can directly be prepared into the reference calibrations article of a plurality of variable concentrations; Can also be the single-point reference calibrations article of fixed concentration, draw calibration curve jointly with physiological saline.
9. reagent according to claim 8 is characterized in that: said stabilizing agent is the phosphate buffer that contains 0.05%BSA; The concentration of said calibration object, low value Quality Control thing and high value Quality Control thing is respectively 40AU/mL, 10AU/mL and 30AU/mL.
10. method of utilizing the described reagent of claim 1 to measure helicobacter pylori antibody, it is characterized in that: described method is following:
Selected sample is a human serum; Sample and pH value are that 8.0 ± 0.3 buffer solution preincubate is after 3-5 minute; Antibody combining site in the sample is fully exposed; Add helicobacter pylori specific antigen emulsion reagent; Continued to hatch 3-5 minute; Helicobacter pylori specific antibody in the human serum combines with helicobacter pylori specific antigen in the helicobacter pylori specific antigen emulsion reagent; Form insoluble antigen-antibody complex; Produce certain turbidity; Its turbidity height is directly proportional with specific anti bulk concentration in detecting sample; Under provision wavelengths, measure the absorbance of this insoluble antigen-antibody complex; Compare with the helicobacter pylori specific antibody calibration object of concentration known, then can calculate the concentration of helicobacter pylori antibody in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110219417A CN102305857A (en) | 2011-08-02 | 2011-08-02 | Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110219417A CN102305857A (en) | 2011-08-02 | 2011-08-02 | Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102305857A true CN102305857A (en) | 2012-01-04 |
Family
ID=45379738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110219417A Pending CN102305857A (en) | 2011-08-02 | 2011-08-02 | Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102305857A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102662059A (en) * | 2012-05-11 | 2012-09-12 | 北京美康生物技术研究中心 | Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof |
| CN102854314A (en) * | 2012-10-10 | 2013-01-02 | 深圳康美生物科技股份有限公司 | Kit for detecting helicobacter pylori antibody by using latex immunoturbidimetry assay |
| CN103558400A (en) * | 2013-11-25 | 2014-02-05 | 重庆中元生物技术有限公司 | Procalcitonin latex enhanced immunoturbidimetry detection kit |
| CN103961704A (en) * | 2014-04-25 | 2014-08-06 | 深圳雅臣生物科技有限公司 | Preparation method and preparation agent of nano lipidosome compound IgY resisting Helicobacter pylori, Pylori bacteria as well as related enzymes and adhesin of Helicobacter pylori and Pylori bacteria |
| CN104833804A (en) * | 2015-04-30 | 2015-08-12 | 必欧瀚生物技术（合肥）有限公司 | Helicobacter pylori IgG antibody ELISA semi-quantitative detection kit and application thereof |
| CN105606803A (en) * | 2016-01-18 | 2016-05-25 | 北京九强生物技术股份有限公司 | Latex enhanced immunoturbidimetry kit for content of helicobacter pylori antibodies |
| CN105891308A (en) * | 2016-03-30 | 2016-08-24 | 深圳三相生物传感科技有限公司 | Kit for diagnosing helicobacter pylori infection and method for detecting content of ammonia gas in human expiration |
| CN106771148A (en) * | 2016-12-28 | 2017-05-31 | 广州华弘生物科技有限公司 | A kind of immune globulin M detection reagent box and detection method |
| CN116693681A (en) * | 2023-07-18 | 2023-09-05 | 北京新兴四寰生物技术有限公司 | Monoclonal antibody for resisting helicobacter pylori cytotoxin related protein A and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5846751A (en) * | 1990-12-04 | 1998-12-08 | Quidel Corporation | Test kits and methods for detecting H. pylori |
| US5932430A (en) * | 1996-05-09 | 1999-08-03 | Meridian Diagnostics, Inc. | Immunoassay for H. pylori in fecal specimens |
| CN1786713A (en) * | 2004-12-09 | 2006-06-14 | 陈金华 | Application of enzyme labeling meter in latex enhancing immune transmittance turbidimetry |
| CN101435821A (en) * | 2008-12-19 | 2009-05-20 | 周炬华 | Immune detection kit of pylori spiral bacilli antibody and detection method thereof |
| CN101498732A (en) * | 2008-02-03 | 2009-08-05 | 北京九强生物技术有限公司 | Improved prealbumin detection kit |
| CN101573619A (en) * | 2006-10-16 | 2009-11-04 | 株式会社三菱化学药得论 | Immunological analysis reagent and immunological analysis method |
| CN101833010A (en) * | 2010-03-29 | 2010-09-15 | 上海太阳生物技术有限公司 | Kit for detecting fibrin (ogen) degradation products (FDP) (by using emulsion immunoturbidimetry) |
-
2011
- 2011-08-02 CN CN201110219417A patent/CN102305857A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5846751A (en) * | 1990-12-04 | 1998-12-08 | Quidel Corporation | Test kits and methods for detecting H. pylori |
| US5932430A (en) * | 1996-05-09 | 1999-08-03 | Meridian Diagnostics, Inc. | Immunoassay for H. pylori in fecal specimens |
| CN1786713A (en) * | 2004-12-09 | 2006-06-14 | 陈金华 | Application of enzyme labeling meter in latex enhancing immune transmittance turbidimetry |
| CN101573619A (en) * | 2006-10-16 | 2009-11-04 | 株式会社三菱化学药得论 | Immunological analysis reagent and immunological analysis method |
| CN101498732A (en) * | 2008-02-03 | 2009-08-05 | 北京九强生物技术有限公司 | Improved prealbumin detection kit |
| CN101435821A (en) * | 2008-12-19 | 2009-05-20 | 周炬华 | Immune detection kit of pylori spiral bacilli antibody and detection method thereof |
| CN101833010A (en) * | 2010-03-29 | 2010-09-15 | 上海太阳生物技术有限公司 | Kit for detecting fibrin (ogen) degradation products (FDP) (by using emulsion immunoturbidimetry) |
Non-Patent Citations (3)
| Title |
|---|
| T. ULF WESTBLOM: "Diagnosis of Helicobacter pylori infection in adult and pediatric patients by using Pyloriset，a rapid latex agglutination test", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| 王小众等: "幽门螺杆菌的血清学诊断", 《世界华人消化杂志》 * |
| 祖英秋: "基因干预所致机体生物学变化探索与胶乳增强免疫比浊方法检测梅毒抗体临床应用研究", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102662059A (en) * | 2012-05-11 | 2012-09-12 | 北京美康生物技术研究中心 | Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof |
| CN102662059B (en) * | 2012-05-11 | 2014-08-13 | 北京美康生物技术研究中心 | Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof |
| CN102854314A (en) * | 2012-10-10 | 2013-01-02 | 深圳康美生物科技股份有限公司 | Kit for detecting helicobacter pylori antibody by using latex immunoturbidimetry assay |
| CN102854314B (en) * | 2012-10-10 | 2014-12-24 | 深圳康美生物科技股份有限公司 | Kit for detecting helicobacter pylori antibody by using latex immunoturbidimetry assay |
| CN103558400A (en) * | 2013-11-25 | 2014-02-05 | 重庆中元生物技术有限公司 | Procalcitonin latex enhanced immunoturbidimetry detection kit |
| CN103961704A (en) * | 2014-04-25 | 2014-08-06 | 深圳雅臣生物科技有限公司 | Preparation method and preparation agent of nano lipidosome compound IgY resisting Helicobacter pylori, Pylori bacteria as well as related enzymes and adhesin of Helicobacter pylori and Pylori bacteria |
| CN104833804A (en) * | 2015-04-30 | 2015-08-12 | 必欧瀚生物技术（合肥）有限公司 | Helicobacter pylori IgG antibody ELISA semi-quantitative detection kit and application thereof |
| CN105606803A (en) * | 2016-01-18 | 2016-05-25 | 北京九强生物技术股份有限公司 | Latex enhanced immunoturbidimetry kit for content of helicobacter pylori antibodies |
| CN105891308A (en) * | 2016-03-30 | 2016-08-24 | 深圳三相生物传感科技有限公司 | Kit for diagnosing helicobacter pylori infection and method for detecting content of ammonia gas in human expiration |
| CN106771148A (en) * | 2016-12-28 | 2017-05-31 | 广州华弘生物科技有限公司 | A kind of immune globulin M detection reagent box and detection method |
| CN116693681A (en) * | 2023-07-18 | 2023-09-05 | 北京新兴四寰生物技术有限公司 | Monoclonal antibody for resisting helicobacter pylori cytotoxin related protein A and application thereof |
| CN116693681B (en) * | 2023-07-18 | 2023-12-01 | 北京新兴四寰生物技术有限公司 | Monoclonal antibody for resisting helicobacter pylori cytotoxin related protein A and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102305857A (en) | Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof | |
| CN102662059B (en) | Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof | |
| CN102854314B (en) | Kit for detecting helicobacter pylori antibody by using latex immunoturbidimetry assay | |
| CN103389381B (en) | Human epididymal secretory protein E4 chemiluminescence detection kit and preparation method thereof | |
| CN101592661B (en) | Brucellosis antibody competitive enzyme-linked immunosorbent assay reagent kit | |
| CN107192821B (en) | A kind of modified latex immunoturbidimetry assay kit improving helicobacter pylori pathogenicity bacterial strain Antibody positive rate | |
| CN101581726B (en) | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit | |
| CN105137073A (en) | Bovine Brucella colloidal gold antibody detection test paper strip | |
| CN101592660B (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit | |
| CN106093418A (en) | A kind of test kit measuring Troponin I and preparation method thereof | |
| CN106092942A (en) | Sialic test kit of a kind of mensuration and preparation method thereof | |
| CN106018829A (en) | Testing reagent for serum amyloid protein A and preparation method of testing reagent | |
| CN202916286U (en) | Latex enhanced turbidimetric immunoassay kit for quantitatively detecting procalcitonin (PCT) | |
| CN105181958A (en) | Method for quantitatively assaying 80nmAuNPs on the basis of indirect competitive enzyme-linked immunosorbent assay | |
| CN101699292A (en) | Rabbit monoclonal antibody based ciprofloxacin residue analysis enzyme-linked immune adsorption kit | |
| CN108570104A (en) | Recombinating adiponectin antigen, antibody and adiponectin nano rubber latex enhances immunoturbidimetry kit | |
| KR20170102344A (en) | How to Detect Active Tuberculosis Markers | |
| CN109738623A (en) | A kind of Serum A 1- acidoglycoprotein assay kit | |
| CN101799470A (en) | Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit | |
| US6187556B1 (en) | Composition, kit, and method for detecting Helicobacter pylori in biopsy | |
| CN107782902A (en) | A kind of myoglobins monoclonal antibody enzyme combination compound and the kit for detecting myoglobin content containing it | |
| CN111088312A (en) | Helicobacter pylori detection kit and preparation method thereof | |
| CA2676869A1 (en) | A diagnostic method for the determination of helicobacter pylori | |
| CN106405069A (en) | Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid | |
| CN106442987B (en) | The fluorescence detection reagent kit and its application method of staphylococcus aureus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120104 |