CN105606803B - The latex enhancing immune turbidimetry kit of helicobacter pylori antibody content - Google Patents
The latex enhancing immune turbidimetry kit of helicobacter pylori antibody content Download PDFInfo
- Publication number
- CN105606803B CN105606803B CN201610029907.XA CN201610029907A CN105606803B CN 105606803 B CN105606803 B CN 105606803B CN 201610029907 A CN201610029907 A CN 201610029907A CN 105606803 B CN105606803 B CN 105606803B
- Authority
- CN
- China
- Prior art keywords
- helicobacter pylori
- kit
- antigen
- weight
- stereometer
- 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.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56922—Campylobacter
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application is related to the latex enhancing immune turbidimetry kit of helicobacter pylori antibody content.Specifically, the application is related to a kind of kit of helicobacter pylori antibody content in latex enhancing immune turbidimetry for Determination human serum, plasma sample, and it includes reaction buffer, is crosslinked with the latex particle and calibration object of Heliobacter pylori antigen.The kit of the application occurs immune response using helicobacter pylori antibody of the crosslinking in the Heliobacter pylori antigen and sample on latex particle surface and forms turbidity, and helicobacter pylori antibody content is detected by the rising degree of turbidity.The kit of the application can apply to clinical conventional biochemical analyzer, easy to operate, quick, high specificity.Stabilization of kit is improved, and sensitivity and detection range can meet the requirement of clinical practice.
Description
Technical field
This disclosure relates to medical immunology in-vitro diagnosis field；Provide it is a kind of using immunoturbidimetry method measure human serum,
The kit of helicobacter pylori antibody content in plasma sample.
Background technology
Helicobacter pylori (Helicobacter pylori, HP) by Barry Marshall (Barry J.Marshall) and
Guest sieve Warren (J.Robin Warren) reports discovery first in nineteen eighty-three.Therefore this two people obtains Nobel's life of 2005
Of science or Medicine.
Helicobacter pylori is a kind of monopole, more flagellums, end blunt circle, the gram-negative bacteria of helically bent, long 2.5-
It is 4.0 μm, wide 0.5-1.0 μm.It is in often typical helical form or arc on gastric epithelial cell surface.Helicobacter pylori is to nutrition
It is required that high, and need to grow under conditions of anaerobism, be difficult to cultivate in vitro, ambient oxygen requirement 5 to 8%, in air or definitely detest
It can not be grown under oxygen environment.
Helicobacter pylori can produce a variety of virulence factors, such as urease, adhesin, protease, cytotoxin and endotoxin.
Wherein, urease is considered as being not only important virulence factor, and is the maximally effective antigen of helicobacter pylori, can be in the infected
Very strong immune response is induced in vivo.
Helicobacter pylori is usually used in most of classical biochemical tests of identification enteric bacteria not in being tested to Clinical microorganism
React.This seven kinds of enzyme reactions of oxidizing ferment, catalase, urease, alkaline phosphatase, r- glutamyl transpeptidases, leucine peptase are
Foundation as helicobacter pylori biochemical identification.
After helicobacter pylori enters stomach, provide power by flagellum and pass through slime layer.Research shows that helicobacter pylori is viscous
There is extremely strong locomitivity, an important factor for strong power is helicobacter pylori pathogenicity under thick environment.Helicobacter pylori arrives
Up to after epithelial surface, firmly linked together, avoided with food by gastric emptying with epithelial cell by adhesin.And point
Superoxide dismutase (SOD) and catalase are secreted, to protect it from the lethal effect of neutrophil leucocyte.Helicobacter pylori
Rich in urease, ammonia is produced by urea enzyme hydrolysis urea, " ammonia cloud " protective layer is formed around thalline, to resist killing for hydrochloric acid in gastric juice
The effect of going out.After HP infection stomaches enter duodenum, constantly bred on normal mucosa, gradually encroach on mucous membrane, fold and fertilizer occur
Thickness, suppress the normal secretions of gastric juice and duodenal juice, destroy the normal defense function of mucous membrane.Urease can hydrolyze urine rapidly
A large amount of ammonia are produced after element, ammonia directly or indirectly can be damaged mucomembranous cell, cause stomach and duodenal lesion.
Helicobacter pylori infection generally existing, developing country 70-90% crowd carry, and developed country is 50%, lead to
Often in the Childhood, lesion can be occurred by carrying 1-10% in HP crowd.Helicobacter pylori infections are chronic actives
Property gastritis, peptic ulcer, the main pathogenic of gastric mucosa-associated lymphoid tissue (MALT) lymthoma and stomach cancer.Generation in 1994
Helicobacter pylori is set to I class procarcinogens by boundary's health organization/international cancer research institution (WHO/IARC).
The diagnosis detecting method of helicobacter pylori includes the invasive and major class of Noninvasive two at present.Invasive method need to lead to
Cross scope acquisition living tissue to be detected, main being separately cultured and direct smear, rapid urease test and medicine including bacterium
Quick experiment.And noninvasive method is then not required to carry out endoscopy, mainly include：
(1) C13 and C14 urea breath tests (UBT), urease caused by helicobacter pylori can be by endogenous or exogenous
Urea resolves into NH3 and CO2, and the latter absorbs in small intestine epimere, into blood after discharged with exhaling.Oral a certain amount of C13 and C14
After urea, the amount for determining C13 and C14 in exhaling respectively by high sensitivity mass spectrograph or liquid scintillation instrument can determine whether there is helicobacter pylorus
Bacterium infects.C13 is stable isotope, "dead", but expensive；C14 is radio isotope, and amount of radiation is smaller, is also had
Higher security, its price is more honest and cleaner, but pregnant woman and children should not still use, and large-scale use may be brought to environmental pollution
Hidden danger.Urea breath test has higher Sensitivity and Specificity, but still by other in such as medicine, UGB, stomach
Producing the factors such as urease bacterium influences, in fact it could happen that false negative or false positive.The method is also influenceed by critical value height, therefore
The determination of critical value is extremely important；In addition, also being influenceed by medication to expiration collection interval time length, matching somebody with somebody for examinee is obtained
Close also critically important.
(2) 15N urinary ammonias excretion test, orally after urea containing 15N, can be urinated using 15N urea caused by helicobacter pylori
Plain enzyme, which decomposes, produces NH3 and CO2, NH3 through absorbing the principle discharged in liver metabolism and through urine, is examined by application of gas chromatorgraphy/mass instrument
Survey 15N urinary ammonias in urine and judge whether helicobacter pylori infections.The method is noninvasive, "dead", and Sensitivity and Specificity is high, but examines
Survey result and many factors interference, and the equipment costliness such as absorbed, be metabolized, drain by body, clinical practice is by a definite limitation.
(3) Helicobacter pylori stool antigen detects, due to settling down the helicobacter pylori on gastric epithelial cell surface, with stomach
The quick renewal of mucous epithelium comes off, and helicobacter pylori also comes off therewith, and is discharged by intestines and stomach from excrement.Helicobacter pylori
Stool antigen test examination inspection, being capable of specificity using ELISA double antibody sandwich method and chemiluminescence immunoassay etc.
Diagnose helicobacter pylori infections in human body.This method behaviour is easy, time saving, is not required to expensive instrument, suitable for infant, children's pylorus
Epidemiology survey of the detection of pylori (Hp) infection, H. pylori eradication therapeutic evaluation, and helicobacter pylori infections etc..
(4) there are a variety of antigens in serum and secretion antibody test, helicobacter pylori phage surface, as urease, fat are more
Sugar, adhesin etc..These antigens can stimulation of host produce immune response, produce IgG, IgA, IgM antibody.Traditional serology
Mainly detection can IgG antibody of the long-term existence in serum for detection.Conventional method mainly has enzyme linked immunosorbent assay, is immunized
Enzyme test, immunoblot assay, latex agglutination test, chemiluminescence detection etc..The secretion such as urine, saliva antibody test side
Method, sampling is easy, no pain, and its sensitiveness, specificity are similar to serological test, but result may be by some factor shadows
Ring.
The method of traditional detection helicobacter pylori antibody, has that automaticity is low, hand-manipulated, time-consuming lacks
Point.Therefore it provides helicobacter pylori antibody in a kind of specific detection human serum, blood plasma, while it is applicable to automation instrument
Kit on device, which turns into, very to be needed.
The content of the invention
The first aspect of the disclosure is related to a kind of latex enhancing immune turbidimetry examination for determining helicobacter pylori antibody content
Agent box, it is included：
1) R1 reagents；
2) R2 reagents；And optionally
3) calibration object；
The R1 reagents include buffer solution, stabilizer, coagulant and preservative；
The R2 reagents include the coated latex particle of Heliobacter pylori antigen, buffer solution, stabilizer and preservative；
The calibration object includes helicobacter pylori antibody, buffer solution, stabilizer and preservative.
In some embodiments, latex particle is a kind of core shell structure；Newborn core is poly styrene polymer, and newborn shell is by benzene
Ethene, n-butyl acrylate and methacrylic acid copolymer composition.In some embodiments, latex particle is polystyrene colloidal
Newborn particle.
In some embodiments, the latex particle surface carries and is selected from following chemical group：Sulfate, sulfonic acid
Base, carboxyl, amino, hydroxyl, hydrazide group, chloromethyl and combinations thereof.
In some embodiments, the latex particle diameter range was 50 to 150nm (containing endpoint value)；Including but it is unlimited
Scope between 50,60,70,80,90,100,110,120,130,140,150nm and above-mentioned any two numerical value.
In some specific embodiments, the particle diameter of the latex particle is 150nm.Herein it is emphasized that in the upper of the application
Hereinafter " 150nm " does not imply that the particle diameter of each latex particle in reagent is just 150nm.In fact, 150nm is
Numerical value on one statistical significance；Because error, therefore the glue of 150nm specifications in the fabrication process be present in latex particle
Newborn particle refers to particle size range in the range of 150nm or so, such as 140 to 160nm, for another example in the range of 145 to 155nm.
In some embodiments, the concentration of the latex particle is 0.025% to 0.25% (by weight/stereometer).
For example, the scope between 0.025,0.05,0.1,0.15,0.2,0.25% and above-mentioned any two numerical value.It is specific at some
Embodiment in, the concentration of the coated polystyrene latex particles of Heliobacter pylori antigen is by weight/stereometer 0.15%.
In some embodiments, Heliobacter pylori antigen is selected from：Helicobacter pylori whole cell proteantigen, urease B
Subunit (UreB), Helicobacter pylori adhesin (HpaA), cavitating toxin (VacA), cytotoxin-associated protein (CagA), heat
Shock protein (HspB), flagellin A Asias egg position (FlaA), B subunits (FlaB) and combinations thereof.Can be by art-recognized
Bacteria Culture after extraction prepare above-mentioned antigen, can also buy the product of commercialization, for example, Calbioreagents,
The antigen of the offers such as Biospacific.
In some embodiments, the Heliobacter pylori antigen can be incorporated in glue by physical absorption or chemical coupling
Newborn particle surface.Heliobacter pylori antigen is incorporated on latex particle surface by optimization, chemical coupling cross-linking method.
In some embodiments, the buffer solution in described R1, R2 and calibration object is each independently selected from：3- [N, N- bis-
(ethoxy) amino] -2- hydroxy-propanesulfonic acids-sodium hydrate buffer solution, 4- (2- ethoxys) -1- piperazines propane sulfonic acid-sodium hydroxide
Buffer solution, trishydroxymethylaminomethane-HCl buffer solutions, phosphate buffer, glycine buffer, barbitol buffer solution and its
Combination.In some embodiments, the concentration of buffer solution is 10-200mM；Including but not limited to 10,20,30,40,50,60,
70th, 80,90,100,110,120,130,140,150,160,170,180,190,200mM and above-mentioned any two numerical value it
Between scope.In some embodiments, the pH of buffer solution is 7 to 8, such as 7.2,7.3,7.4,7.5.R1, R2 and calibration object
In buffer type can be with identical or different；Buffer concentration in R1, R2 and calibration object can be with identical or different.R1、R2
Can be with identical or different with the pH of buffer in calibration object.
In some embodiments, the stabilizer in reagent R1, R2 and calibration object is each independently selected from：By weight/body
Product meter 0.1%-5% bovine serum albumin(BSA) (preferably 0.5% to 1%), the sugarcane of by weight/stereometer 1%-10% concentration ranges
Sugar, the sodium chloride of by weight/stereometer 0.5%-1% concentration ranges, 2-50mM EDTA (preferably 10 to 20mM), by weight/
Tween-20 (the preferably 0.1%-0.5% of stereometer 0.1%-1% concentration ranges；More preferably 0.1%-0.2%), by weight/body
The glycerine of product meter 1%-10% concentration ranges, and combinations thereof.Stabilizer types in R1, R2 and calibration object can be with identical or not
Together；Stabilizer concentration in R1, R2 and calibration object can be with identical or different.
In some embodiments, the preservative in reagent R1, R2 and calibration object is each independently selected from：Sodium azide, sulphur
Willow mercury, phenol, ethyl mercury sodium thiosulfate and combinations thereof.In some embodiments, concentration of preservatives is 0.02%-0.1%
By weight/stereometer；Such as 0.05% to 0.15%.Preservative type in R1, R2 and calibration object can be with identical or different；
Concentration of preservatives in R1, R2 and calibration object can be with identical or different.
In some embodiments, the coagulant be selected from PEG-4000, PEG-6000, PEG-8000, PEG-10000,
Glucan T-40, glucan T-70, glucan T-500 and combinations thereof.Coagulant concentration range be 1%-10% by weight/volume
Meter；Such as the model between 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% and above-mentioned any two numerical value
Enclose.
In some embodiments, calibration is prepared by adding the helicobacter pylori antibody of concentration known in buffer solution
Product.In some embodiments, calibration object includes the helicobacter pylori antibody of at least two concentration；It is such as dense comprising at least five
The helicobacter pylori antibody of degree, such as, but not limited to 0,10,20,40,70,100U/ml.
In some specific embodiments, the latex enhancing immune of described measure helicobacter pylori antibody content is than turbid
Method kit includes：
R1 is included：
0.9% by weight/stereometer sodium chloride,
2% by weight/stereometer PEG-8000,
1% by weight/stereometer BSA,
10mM EDTA、
0.09% by weight/stereometer Sodium azide,
50mM HEPES buffer solutions pH7.2；
R2 is included：
The coated polystyrene latex particles 0.15% of Heliobacter pylori antigen by weight/stereometer,
150mM glycine buffer pH7.5,
0.9% by weight/stereometer sodium chloride,
0.5% by weight/stereometer BSA,
10mM EDTA、
0.1% by weight/stereometer polysorbas20,
By weight/sodium azide of stereometer 0.09%,
The wherein described a diameter of 150nm of polystyrene latex particles,
Described Heliobacter pylori antigen is helicobacter pylori whole cell proteantigen；
Calibration object includes：
Concentration is respectively 0,10,20,40,70,100U/ml helicobacter pylori antibody,
0.9% by weight/stereometer sodium chloride,
1% by weight/stereometer BSA,
10mM EDTA、
0.1% by weight/stereometer Sodium azide,
50mM HEPES buffer solutions pH7.2.
According to another aspect of the present disclosure, there is provided one kind prepares the coated polystyrene latex particles of Heliobacter pylori antigen
Method, including step：
1) polystyrene latex particles are provided；
2) Heliobacter pylori antigen is provided；
3) under the conditions of existing for PEG-20000, Heliobacter pylori antigen is cross-linked on polystyrene latex particles；
4) the coated polystyrene latex particles of Heliobacter pylori antigen are obtained；
Step 1) and step 2) are interchangeable.
In some embodiments, polystyrene latex particles are provided in buffer solution.In some embodiments, buffer
Liquid is pH 5 to 6.5 MES buffer solutions；Concentration is 0.02 to 0.5M, such as 0.05 to 0.01M；Such as 0.02,0.03,0.04,
0.05th, 0.06,0.07,0.08,0.09,0.1,0.15,0.2,0.25,0.3,0.35M buffer solution.In some embodiments
In, the polystyrene latex particles of by weight/stereometer 0.1% to 2.0% are provided in buffer solution；Such as 0.1%, 0.2%,
0.3%th, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%；It is preferred that
0.5% to 1.5%, more preferably 0.8% to 1.2%.In some embodiments, the diameter range of polystyrene latex particles is
50 to 150nm；Including but not limited to 50,60,70,80,90,100,110,120,130,140,150nm and above-mentioned any two
Scope between individual numerical value.
In some embodiments, Heliobacter pylori antigen is provided in buffer solution.In some embodiments, buffer solution
For pH 5 to 6.5 MES buffer solutions；Concentration is 0.02 to 0.5M, such as 0.05 to 0.01M；Such as 0.02,0.03,0.04,
0.05th, 0.06,0.07,0.08,0.09,0.1,0.15,0.2,0.25,0.3,0.35M buffer solution.In some embodiments
In, concentration of the Heliobacter pylori antigen in buffer solution is 0.05 to 0.5mg/ml, such as 0.05,0.06,0.07,0.08,
0.09th, 0.1,0.12,0.13,0.14,0.15,0.2,0.25,0.30,0.35mg/ml, preferably 0.05 to 0.2mg/ml.
In some embodiments, under the conditions of existing for PEG-20000, Heliobacter pylori antigen is cross-linked to polyphenyl second
On alkene latex particle.Described PEG-20000 is amido modified PEG-20000.Amido modified PEG-20000 concentration is
By weight/stereometer 0.05% to 0.15%, preferably 0.1%.
In some embodiments, Heliobacter pylori antigen and polystyrene latex particles are in 28 to 40 DEG C of cross-linking reactions 1
To 4 hours, preferably reacted 2 to 3.5 hours at 37 DEG C.
Optionally, cross-linking reaction is terminated after step 3)；Handed over it is preferred that being terminated by 0.1M pH7.0 glycine buffer
Connection reaction, preferably reaction are carried out 0.5 to 1.5 hour.
Optionally, after terminating reaction step, latex particle is rinsed.Preferably, delayed with 50mM pH8.0 glycine
Fliud flushing rinses latex particle, obtains the coated polystyrene latex particles of Heliobacter pylori antigen.
A kind of method for preparing the coated polystyrene latex particles of Heliobacter pylori antigen, including step are provided：
1) in 0.02M to 0.1M pH 5.0 to 6.5 MES buffer solutions, there is provided by weight/stereometer 0.5% to
1.5% polystyrene latex particles；
2) in the MES of the 0.02M to 0.1M pH5.0 to 6.5 containing 0.1% to 0.5% amido modified property PEG-20000
In buffer solution, there is provided 0.05mg/ml to 0.2mg/ml Heliobacter pylori antigen；
3) by the solution of step 1) and the solution of step 2) according to volume ratio 1:1 mixing, at 28 DEG C to 40 DEG C, helicobacter pylorus
Bacterium antigen and polystyrene latex particles cross-linking reaction 1 to 4 hour；
4) cross-linking reaction optionally, is terminated；
5) optionally, rinse cross-linking reaction after gained particle；
6) the coated polystyrene latex particles of Heliobacter pylori antigen are obtained.
In a particular embodiment, there is provided a kind of Heliobacter pylori antigen coated polystyrene latex particles of preparing
Method, including step：
1) diameter 150nm polystyrene colloidal milk solution is dissolved in 0.05M pH6.0 MES buffer solutions so that concentration is
By weight/stereometer 1%；
2) provide and resist containing the amido modified property PEG-20000 of by weight/stereometer 0.2% and 0.1mg/ml helicobacter pyloris
Former 0.05M pH6.0 MES buffer solutions,
3) by the solution obtained by step 1) and step 2) according to volume ratio 1:1 mixing, reacts 3 hours at 37 DEG C；
4) 0.1M pH7.0 glycine buffer terminating reaction 1h is added, supernatant is removed in centrifugation；
5) latex particle obtained by step 4) is washed 1 to 3 time with 50mM pH8.0 glycine buffer；
6) the coated polystyrene latex particles of Heliobacter pylori antigen are harvested；
Wherein step 1) and step 2) is interchangeable；
Wherein described polystyrene latex particles particle diameter is 150nm.
According to another aspect of the present disclosure, there is provided a kind of coated polystyrene latex particles of Heliobacter pylori antigen, its
Prepared by disclosed method.
According to another aspect of the present disclosure, there is provided a kind of detection reagent, its helicobacter pylori for including described in the disclosure resist
The polystyrene latex particles of primordial covering.
According to another aspect of the present disclosure, there is provided the coated polystyrene latex particles of Heliobacter pylori antigen of the disclosure
Purposes in detection reagent is prepared.
According to another aspect of the present disclosure, there is provided a kind of latex enhancing immune for determining helicobacter pylori antibody content is than turbid
Method kit, the kit are included using the coated polystyrene colloidal of Heliobacter pylori antigen prepared by disclosed method
Newborn particle.
Specific theory is not limited to, present disclose provides a kind of cross-linking method of optimization, by the amino for adding certain concentration
Change the PEG-20000 of modification, using PEG-20000 water absorption characters, in the hydrated sheath that latex surface has been formed, so that made
The R2 reagent reactings specificity and shelf stability of work are improved.
In specific reaction system, in the coated latex particle suspension of Heliobacter pylori antigen and human serum, blood plasma
Helicobacter pylori antibody occur antigen-antibody reaction form certain turbidity, the photometer that can be split detects, passes through survey
The rising degree of system turbidity carries out helicobacter pylori antibody quantitative analysis after fixed reaction.
Embodiment
The disclosure will be further illustrated by following non-limiting examples below, it is as well known to those skilled in the art, not
In the case of disclosure spirit, many modifications can be made to the disclosure, such modification also falls into the scope of the present disclosure.
Following experimental methods are conventional method unless otherwise instructed, used experiment material unless otherwise instructed,
Easily it can be obtained from commercial company.
Embodiment
Embodiment 1：The preparation of helicobacter pylori antibody kit 1
1. prepare R1 reagents
In 50mM pH7.2 HEPES buffer solution, sodium chloride, the by weight/body of by weight/stereometer 0.9% are added
PEG-8000, BS, 10mM EDTA, the by weight/Sodium azide of stereometer 0.1% of by weight/stereometer 1% of product meter 5%, are stirred
Mix is uniformly R1 reagents.
2. prepare R2 reagents
Diameter 150nm polystyrene colloidal milk solution (concentration 10% by weight/stereometer) (being purchased from Merck) 0.5mL, adds
Enter 4.5mL 0.05M MES buffer solutions (pH6.0), the crosslinking agent EDAC (being purchased from Merck) for adding 0.5mg/ml activates 30 points
Clock；
By 0.5mg Heliobacter pylori antigens (being purchased from Calbioreagents) 5mL 0.05M MES buffer solutions (pH6.0)
After dilution, it is added in above-mentioned latex solution, is reacted 3 hours at 37 DEG C immediately；
Glycine buffer (pH7.0) the stirring 1h for adding 1mL 0.1M carrys out terminating reaction, and supernatant is removed in centrifugation；
Washed 3 times with 33mL 50mM glycine buffer (pH8.0), harvest is coated with the latex particle of antigen；
With 33ml contain by weight/sodium chloride of stereometer 0.9%, 10mM EDTA, by weight/stereometer 0.5%BSA, by
The polysorbas20 of weight/volume meter 0.1%, by weight/Sodium azide of stereometer 0.09% 150mM pH7.5 glycine buffer,
The latex particle for being coated with antigen is dispersed into milk-white coloured suspension, is prepared into R2 reagents；
The coated polystyrene latex particles concentration of Heliobacter pylori antigen is by weight/stereometer in final R2 reagents
0.15%.
3. prepare calibration object
In 50mM pH7.2 HEPES buffer solution, add concentration be respectively 0,10,20,40,70,100U/ml pylorus
Pylori antibodies, add in addition by weight/sodium chloride of stereometer 0.9%, the BSA of by weight/stereometer 1%, 10mM EDTA,
By weight/sodium azide of stereometer 0.1%, is stirred for multiple spot calibration object.
Embodiment 2：The preparation of helicobacter pylori antibody kit 2
1. prepare R1
Reagent R1 preparation method is identical with embodiment 1.
2. prepare R2 reagents
Diameter 150nm polystyrene colloidal milk solution (concentration 10% by weight/stereometer) (being purchased from Merck) 0.5mL, adds
Enter 4.5mL 0.05M MES buffer solutions (pH6.0)；The crosslinking agent EDAC (being purchased from Merck) for adding 0.5mg/ml activates 30 points
Clock；
By the 0.5mg Heliobacter pylori antigens 5mL containing 0.2% amido modified PEG-20000 (being purchased from Pierce)
After 0.05M MES buffer solutions (pH6.0) dilution, it is added in above-mentioned latex solution, is reacted 3 hours at 37 DEG C immediately；
Glycine buffer (pH7.0) the stirring 1h for adding 1mL 0.1M carrys out terminating reaction, and supernatant is removed in centrifugation；
Washed 3 times with 33mL 50mM glycine buffer (pH8.0), harvest is coated with the latex particle of antigen；
With 33ml contain by weight/sodium chloride of stereometer 0.9%, 10mM EDTA, by weight/stereometer 0.5%BSA, by
The polysorbas20 of weight/volume meter 0.1%, by weight/Sodium azide of stereometer 0.09% 150mM pH7.5 glycine buffer,
The latex particle for being coated with antigen is dispersed into milk-white coloured suspension, is prepared into R2 reagents；
The coated polystyrene latex particles concentration of Heliobacter pylori antigen is 0.15% by weight/body in final R2 reagents
Product meter.
3. preparing for calibration object is same as Example 1.
Test case：
The usage of the detection kit of test case 1.
By taking the Biochemical Analyzer of Hitachi 7180 as an example：Wavelength 570nm is determined, is firstly added R1 reagents 180uL, 37 DEG C of reactions 30
Calibration object 3.5uL is added after second, then R2 reagent 60uL are added after reacting 60 seconds, measure reaction the 10th second, the absorbance of 70 seconds
(A1, A2), calculate absorbance difference △ A=A2-A1, often pipe replication 2 times, by each calibration 2 absorbances measured of QC
Difference △ A are ordinate, and corresponding concentration is abscissa, make " concentration-absorbance difference " calibration curve, take sample to be tested,
It is measured in the same method the absorbance difference of sample, substitutes into calibration curve, you can calculates containing for helicobacter pylori antibody in sample to be tested
Amount.
The disclosure kit of test case 2. is compared with the correlation of chemoluminescence method detection clinical sample
With the chemiluminescence detection kit of the kit of the disclosure and company A, the immunoturbidimetry detection kit of B companies
45 patient's samples are detected simultaneously respectively.
The kit fundamental analysis principle of company A is that it is horizontal to determine helicobacter pylori antibody in serum using prize law.With
Heliobacter pylori antigen coating microwell plate is made insolubilized antibody, and the helicobacter pylori antibody in serum sample can be captured to,
The antibody of anti-igg two of enzyme mark is added, solid-phase coating antigen-helicobacter pylori antibody-enzymic-labelled antibody is formed after incubation
Substrate is added after compound, fully washing, in determining its luminous intensity in 5-10 minutes, sample can be calculated according to standard curve
The content of middle helicobacter pylori antibody, the luminous intensity values of sample raise with the increase of helicobacter pylori antibody concentration.
The kit fundamental analysis principle of B companies is the coated latex particle of Heliobacter pylori antigen and imprisoning in sample
Helicobacter pylori antibody reacts, and forms turbidity, and helicobacter pylori antibody concentration is proportional in the elevated-levels and sample of turbidity.
Positive and negative coincidence rate is calculated compared with being detected with company A chemical illuminating reagent, testing result is shown in Tables 1 and 2.Compared to B
Company's kit, kit 2 prepared by method of disclosure is good with chemiluminescence method detection clinical sample measured value correlation, cloudy
Positive coincidence rate is 89%, the 80% of the kit 1 prepared higher than method of disclosure, higher than the 71% of B companies kit,
Connect and show that kit detection specificity prepared by this law is more preferable, the application of clinical diagnosis can be met.
The chemical luminescence reagent kit of the disclosure kit 2 of table 1. and company A detects sample correlations
Negative and positive coincidence rate：89%.
The chemical luminescence reagent kit of the disclosure kit 1 of table 2. and company A detects sample correlations
Negative and positive coincidence rate：80%.
The chemical luminescence reagent kit of table 3.B company's kits and company A detects sample correlations
Negative and positive coincidence rate：71%.
The disclosure kit of test case 3. is compared with the stability of B companies kits
With the kit 1, kit 2, B companies kit of the disclosure while the storage ability of monitoring reagent.Inspection
The blank absorbency and calibration absorbance of test agent, stable reagent is used as using blank and calibration absorbance change amplitude less than 10%
The qualified standard of property.
From following data as can be seen that blank absorbency becomes with calibration absorbance after disclosure kit 1 is deposited 12 months
Change blank absorbency and calibration absorbance change after being deposited 9 months more than 10%, B companies kit and be more than 10%, the disclosure is tried
Blank absorbency and calibration absorbance change are still less than 10% after agent box 2 is deposited 18 months.
The storage ability of 4. disclosure kit of table 1
The storage ability of 5. disclosure kit of table 2
Table 6.B companies kit storage ability
The technical scheme of the disclosure compared with prior art, has following features：
1) can apply on automated chemical analyser, detection speed is fast；
2) using native antigen coupling latex particle, with the existing method applied on Biochemical Analyzer and kit phase
Than detecting high specificity, there is very high correlation with chemiluminescence method；Helicobacter pylorus is crosslinked using the crosslinking method of optimization
For bacterium antigen on latex particle, reagent storage stability is more preferable.
Claims (1)
1. a kind of method for the coated polystyrene latex particles of Heliobacter pylori antigen for preparing natural origin, including step：
1) 0.5mL concentration is added 4.5mL's for the diameter 150nm polystyrene colloidals milk solution of by weight/stereometer 10%
In 0.05M MES pH of buffer 6.0；0.5mg/ml crosslinking agent EDAC is added, is activated 30 minutes；
2) the 0.5mg Heliobacter pylori antigens containing 0.2% amido modified PEG-20000 are buffered with 5mL 0.05M MES
Liquid pH6.0 dilutes；
3) step 2) resulting solution is added in step 1) resulting solution immediately, reacted 3 hours at 37 DEG C；
4) 1mL 0.1M glycine buffer pH7.0 stirring 1h terminating reactions are added, supernatant is removed in centrifugation；
5) washed 3 times with 33mL 50mM glycine buffer pH8.0；
6) harvest is coated with the latex particle of antigen；
The order of wherein described step 1) and step 2) is interchangeable.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610029907.XA CN105606803B (en) | 2016-01-18 | 2016-01-18 | The latex enhancing immune turbidimetry kit of helicobacter pylori antibody content |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610029907.XA CN105606803B (en) | 2016-01-18 | 2016-01-18 | The latex enhancing immune turbidimetry kit of helicobacter pylori antibody content |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105606803A CN105606803A (en) | 2016-05-25 |
| CN105606803B true CN105606803B (en) | 2018-02-09 |
Family
ID=55986895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610029907.XA Active CN105606803B (en) | 2016-01-18 | 2016-01-18 | The latex enhancing immune turbidimetry kit of helicobacter pylori antibody content |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105606803B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107224A (en) * | 2017-12-18 | 2018-06-01 | 郑州安图生物工程股份有限公司 | A kind of ProGRP assays liquid calibration object |
| CN110058028B (en) * | 2019-05-05 | 2021-03-02 | 苏州长光华医生物医学工程有限公司 | 24, 25-dihydroxyvitamin D immunoassay kit and application thereof |
| CN113281509A (en) * | 2021-05-29 | 2021-08-20 | 北京华宇亿康生物工程技术有限公司 | Preparation method of kit for detecting helicobacter pylori antigen with good stability |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1023600A4 (en) * | 1998-07-29 | 2002-05-08 | Syntron Biores Inc | Immunoassay device |
| DE602004020231D1 (en) * | 2003-07-28 | 2009-05-07 | Jsr Corp | SURFACE OF NON-SPECIFIC ADSORPTION INHIBITED BASE MATERIALS |
| JP2006177914A (en) * | 2004-12-20 | 2006-07-06 | Yukio Nagasaki | Surface having thick polymer layer, and preparation method therefor |
| JP5062410B2 (en) * | 2006-12-14 | 2012-10-31 | Ｊｓｒ株式会社 | Non-specific adsorption inhibitor, probe-binding particles and method for producing them |
| JPWO2010082681A1 (en) * | 2009-01-16 | 2012-07-12 | 国立大学法人 筑波大学 | Immune latex particles and production method thereof |
| CN102305857A (en) * | 2011-08-02 | 2012-01-04 | 深圳市亚辉龙生物科技有限公司 | Latex immunoreagent for determining helicobacter pylori antibody and detection method thereof |
| CN103063828B (en) * | 2011-10-24 | 2015-07-01 | 韩焕兴 | Method for utilizing covalence coupling to prepare photon point-antibody compound |
| CN102901810B (en) * | 2012-10-09 | 2014-08-27 | 北京九强生物技术股份有限公司 | Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit |
| 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 |
| CN105223348A (en) * | 2014-05-26 | 2016-01-06 | 北京九强生物技术股份有限公司 | The latex enhancing immune turbidimetry detection kit of people's Antithrombin III |
| CN104198724B (en) * | 2014-08-14 | 2016-05-11 | 上海睿康生物科技有限公司 | Fibrin or fibrinogen degradation product (FDP) detection kit |
| CN104360072B (en) * | 2014-12-05 | 2016-03-30 | 重庆中元生物技术有限公司 | A kind of ion stabilizer and suspension stabilizer with the use of D dimer latex strengthen immunoturbidimetry detection kit |
| CN104459139B (en) * | 2014-12-05 | 2016-03-30 | 重庆中元生物技术有限公司 | A kind of D dimer latex of surface functional group that utilizes strengthens immunoturbidimetry detection kit |
-
2016
- 2016-01-18 CN CN201610029907.XA patent/CN105606803B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105606803A (en) | 2016-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103940816B (en) | A kind of kit and preparation method measuring human body content of glycocholic acid | |
| CN107894508A (en) | A kind of solid phase competitive ELISA kit and its application for the detection of Senecan antiviral antibody | |
| CN102662059B (en) | Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof | |
| CN107192821B (en) | A kind of modified latex immunoturbidimetry assay kit improving helicobacter pylori pathogenicity bacterial strain Antibody positive rate | |
| CN105606803B (en) | The latex enhancing immune turbidimetry kit of helicobacter pylori antibody content | |
| CN105866407A (en) | Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof | |
| CN107045062B (en) | Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of human neutrophil genatinase associated lipocalin | |
| US6500629B1 (en) | Materials and methods for detection and quantitation of an analyte | |
| CN108614106A (en) | A kind of time-resolved fluoroimmunoassay chromatography card detecting vomitoxin and its acetyl derivatives | |
| CN111551715B (en) | Novel enzyme-linked immunosorbent assay detection kit for coronavirus IgM antibody | |
| CN105717308A (en) | Immunochromatography kit for fast and quantitatively detecting fecal lactoferrin | |
| CN101592660B (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit | |
| WO2001032908A1 (en) | Monoclonal antibody, hybridoma, immunoassay method and diagnosis kit | |
| CN101799470A (en) | Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit | |
| CN106442993A (en) | Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate | |
| US20080038760A1 (en) | Method for Detecting Anti-Transglutaminase Antibodies | |
| CN104833804A (en) | Helicobacter pylori IgG antibody ELISA semi-quantitative detection kit and application thereof | |
| CN107727861B (en) | A kind of pepsin assay kit and measuring method | |
| CN111458498A (en) | Hand-foot-mouth EV71 antigen detection kit | |
| CN108414742A (en) | Pepsinogen I and pepsinogen I I combined detection kits and preparation method thereof and detection method | |
| CN113109325A (en) | Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof | |
| CN106290899A (en) | Test kit for detection by quantitative CST4 | |
| CN105753982A (en) | Anti-human streptococcus pneumoniae fam1 family PspA protein antibodies and immunochromatography reagent kit for applying antibodies | |
| JP2017036917A (en) | Method of detecting helicobacter pylori | |
| JP3341629B2 (en) | Diagnostic reagent for Helicobacter pylori eradication therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Gao Aimin Inventor after: Lei Hong Inventor after: Liu Xi Inventor before: Gao Aimin Inventor before: Liu Xi |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |