CN110297093A - A kind of method and kit detecting immunoglobulin G 4 - Google Patents
A kind of method and kit detecting immunoglobulin G 4 Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to a kind of methods and kit for detecting immunoglobulin G 4, belong to technical field of medical examination.A kind of method of immunoglobulin G 4 (IgG4) in detection sample, the monoclonal antibody of two high specific anti-igg 4Fc segments is used, in conjunction with solid phase carrier, second monoclonal antibody special to IgG4 is marked first monoclonal antibody special to IgG4 by a kind of peroxidase.By being formed with the bridge linking effect of IgG4 in sample to be tested on the compound and fixation and solid phase carrier of the 2nd IgG4 monoclonal antibody of the first IgG4 monoclonal antibody-IgG4- enzyme mark.The present invention is using nonspecific interference material composition in high-efficient cleaning washing lotion removal sample.The present invention realizes the high stability and high specific to IgG4 quantitative detection in biological sample, and concentration of the accurate quantitative analysis determinand IgG4 in people's biological sample to the detection method of biological sample IgG4 content.
Description
Technical field
The present invention relates to a kind of methods and kit for detecting immunoglobulin G 4, belong to technical field of medical examination.
Background technique
Immunoglobulin G 4 (abbreviation human IgG 4) is the 4th kind of four kinds of hypotypes of immunoglobulin G in human blood (IgG),
With its unique effect in autoimmune disease, the basis of mankind's various autoimmune disease is constituted, is ground in clinic
Study carefully and forms a new field in autoimmune disease.IgG4 correlation autoimmune disease is for special anti-in vivo
Original produces a kind of disease that IgG4 antibody is characterized.IgG4 correlation exempts from the pathogenesis and other immunoglobulins of disease certainly
There is significant difference.It is slow that IgG4 is immunoreacted compared with other immunoglobulins, has monovalent functional structure, anti-in contact
It is slowly formed after original and exempts from antibody certainly, and the antibody formed can be competed with other types IgG, block causing a disease for other types antibody
Mechanism.Since the blocking effect to target antigen proteinase activity can also form pathogenic, a part of autoimmune disease patient
IgG4 content in blood increases, therefore this kind of disease is named as IgG4 diseases related (IgG4-RD).
IgG4-RD is unknown chronic, the progressive autoimmunity disease of a kind of reason, the horizontal significant increasing of patients serum IgG4
Height, affected tissue and organ due to big amount lymphocyte and IgG4 positive plasmocyte infiltrating, while accompanied by tissue fibrosis and send out
Raw enlargement or nodositas/proliferative lesion.IgG4-RD afflicted organ is extensive, can lead to a variety of internal organs and is simultaneously or sequentially involved,
A kind of internal organs can only be involved.The organ of most commonly-occurring disease includes pancreas, liver and gall, salivary gland, lachrymal gland, retroperitoneal space and lymph
Knot.Due to the diverse clinical manifestations of IgG4-RD, it is related to clinical multiple subjects, with disease of digestive system, endocrine system disease
Disease especially is difficult to distinguish with afflicted organ's tumour.Therefore, clinically the rate of missed diagnosis of IgG4-RD and misdiagnosis rate are higher.At present
The diagnosis diseases related to IgG4 mainly includes clinical manifestation, the pathological characters of iconography and affected tissue.According to IgG4 phase
The characteristics of IgG4 increases in closing property disease blood serum, the serology mark diseases related as clinical diagnosis IgG4 of serum IgG 4
Will object has formd international consensus.Detection 4 concentration of serum IgG has been included in the diseases related diagnosis guide of international IgG4.
Detecting 4 concentration of serum IgG has certain diagnostic value, is alternatively arranged as judging that disease activity and prediction are early
Phase recurrence assesses curative effect in time and monitors the objective indicator of disease medication reaction.Version in 2015 " the diseases related management of IgG4 and
The international consensus guide for the treatment of " in specify serum IgG 4 raising be diagnosis diseases related three important indicators of IgG4 it
One.China's research diseases related to IgG4 is started late, less to disease and the research of serum IgG 4.With Domestic Medicine
Complete, diseases related to the IgG4 diagnosing and treating general of promotion and methods for clinical diagnosis to the attention degree of the disease
It has and significantly develops.
The immunoglobulin G 4 assay kit that a small number of Grade A hospitals domestic at present have used Siemens to produce.
The product has been registered in national Bureau of Drugs Supervision as import reagent.The products measure immunoglobulin G 4 is the side according to scattering turbidimetry
Method.The testing cost of this method is higher, needs special instruments and equipment and special agent, is accordingly difficult to universal in basic hospital.Together
When, this method uses polystyrene microsphere particle coating 4 antibody of anti-igg as solid phase, and the coated antibody of institute belongs to Anti-TNF-α
Body.Since human serum immunoglobulin is there are four hypotype, there is high similarity between hypotype molecule, polyclonal antibody is difficult to avoid
Cross jamming between hypotype；In addition sensitivity of polystyrene microsphere particle coating 4 antibody of anti-igg as Solid-phase Assay IgG4
It is lower, it shows as scattered light urbidmetry IgG4 definite value and is significantly higher than ELISA method using high specific IgG4 bispecific monoclonal antibody
Detection.A high possibility of 4 basic value of human serum IgG is that the specificity of detection is insufficient, may include other immune globulins
It is white.Therefore there are potential risks to the variation of IgG4 in specific recognition human serum.
Summary of the invention
The technical problems to be solved by the present invention are: firstly, providing a kind of easy, quick 4 Gao Te of immunoglobulin G
The opposite sex, high stability and highly sensitive quantitative detecting method；Second, it provides a kind of easy, quick, clinical practice high special
Property, the immue quantitative detection reagent box of high stability and high sensitivity.To achieve the above object, the present invention uses following technical scheme.
The method of immunoglobulin G 4 (IgG4), uses two 4 Fc pieces of high specific anti-igg in a kind of detection sample
The monoclonal antibody of section, for first monoclonal antibody special to IgG4 in conjunction with solid phase carrier, which is that the first IgG4 is mono-
Clonal antibody, second monoclonal antibody special to IgG4 are marked by a kind of peroxidase, which is the 2nd IgG4 Dan Ke
Grand antibody；First IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody with detection sample present in conjunction with IgG4, shape
At the 2nd IgG4 monoclonal antibody of the first IgG4 monoclonal antibody-IgG4- enzyme mark compound and be fixed on solid phase carrier；The
One IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody do not constitute competition to the combination of IgG4, and since two to IgG4 spy
It is different in conjunction with monoclonal antibody be incorporated into IgG4 molecule Fc section and have high stability；The first IgG4 on solid phase carrier is mono-
Dosage correlation is presented in clonal antibody in conjunction with the IgG4 in detected sample, and the IgG4 being combined still is kept and oxidizing ferment mark
The combination of 2nd IgG4 monoclonal antibody of note, and determine that the 2nd IgG4 of oxidation label is mono- according to the amount for the IgG4 being combined
The amount of clonal antibody is quantified in tested sample by the oxidizing ferment of the 2nd IgG4 labeling of monoclonal antibody and the color producing reaction of zymolyte
Existing IgG4.This item realizes the height to IgG4 quantitative detection in biological sample to the detection method of biological sample IgG4 content
Stability and high specific.
The first IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody body acupuncture are to Fc segment in IgG4 molecule.It is based on
High stable fusion protein is to improve the stability of drug in vivo, and according to protein drug in successful case and IgG4 Fc sections
Fusion improves protein stability.By detecting IgG4Fc sections come IgG4 in optimization monitoring people's blood in clinical IgG4 correlation disease
The concentration and variation of disease improve the stability of IgG4 in detection patient's blood.The first IgG4 monoclonal antibody of solid phase antibody of use
It is 4 heavy chain monoclonal antibody of anti-human igg (Abcam, ab1930) of antibody preparation company, Britain production；2nd IgG4 monoclonal is anti-
Body is the IgG4Fc section enzyme labeled monoclonal antibody (Abcam, ab99817) of Abcam company production.With double antibody combination pair
The detection of IgG4, evade due in biological sample IgG4 molecule Fab section be metabolized caused by measurement unstability.
The first and second Fc sections of monoclonal antibodies have to the highly selective of IgG4 protein molecular.The design is to be based on
IgG present in biological sample include there are four types of hypotype (IgG1, IgG2, IgG3, IgG4), the architectural differences of four kinds of hypotypes by
The position of IgG hinge section length and disulfide bond is constituted, and the position of IgG hinge area and disulfide bond is respectively positioned on Fc sections.
The progress of genetic engineering technology promotes the rapid development of pharmaceutical grade protein.But vivo protein enzyme denaturation or decomposition
Protein constitutes the unstability of albumen concentration in vivo.To improve the protein that the internal stability of pharmaceutical grade protein is established
The stability of protein can be improved by merging for pharmaceutical grade protein and other high stable protein moleculars in integration technology.At present at
The case of function is mainly merging between protein drug and immunoglobulin Fc section.
Immunoglobulin fc region is also responsible for effector in addition to antigen binding capacity (major function of immunoglobulin)
Function, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC)；Additionally, there are in the area Fc
FcRn sequence has the work for stablizing the IgG level in serum and being conjugated to internal FcRn receptor to increase Half-life in vivo
With.In the case that the sequence using immunoglobulin Fc prepares fused protein, IgG4 Fc is used widely.Accordingly, originally
Invention is devised by detecting IgG4Fc sections of concentration and change diseases related in clinical IgG4 come IgG4 in optimization monitoring people's blood
Change, keeps the stability for detecting IgG4 in patient's blood more preferable.The solid phase antibody of use is the anti-human of antibody preparation company, Britain production
IgG4 heavy chain monoclonal antibody (Abcam, ab1930).Since heavy chain immunoglobulin includes Fab and Fc sections, inventor couple
Ab1930 is screened.It has been determined that the antigenic determinant of ab1930 monoclonal antibody is located at Fc sections using Fc sections of antigens.In order to protect
Card maximizes the IgG1 for excluding have structural similarity with IgG4, and the non-specific binding of IgG2, IgG3 have selected Abcam public
The IgG4Fc section enzyme labeled monoclonal antibody (Abcam, ab99817) of production is taken charge of as the secondary antibody in reaction system.In this way
Combination to the detection of IgG4 constitute in current field of biotechnology it is highest specificity and stability.
First IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody described in this invention are directed to IgG4 molecule
Fc sections.For antibody, Fc segment has high stability, carrys out pharmacy public affairs in a variety of biological protein medicaments such as U.S. in the market
Take charge of the long-lasting hypoglycemic agents object Du Lalu peptide of (Eli Lily) productionIt is in glucagon-like peptide -1
The Fc segment of an IgG4 has been merged on (glucagon-like peptide 1, GLP-1) analog so that glucagon-like
- 1 biological half-life of peptide is from several minutes to a couple of days.Immunoglobulin G is mainly formed by Fab and Fc sections.Fc sections can combine it is a variety of
The Fc receptor of cell and it is more stable.Using IgG4 Fc monoclonal antibody specific is directed to, first is that the stabilization of detection can be increased
Property, even if the IgG4 in blood sample is metabolized, Fc sections in blood the retention time it is long, be conducive to track IgG4 gone out in disease
Existing variation；Second is that high specific of the invention is improved, although tetra- kinds of hypotypes (IgG1, IgG2, IgG3, IgG4) of IgG have
Two identical light chains and heavy chain form, and have 95% similitude between amino acid sequence, but each hypotype structure is main not
With the position of length and disulfide bond that place is hinge area, and these differences are placed exactly in Fc segment, therefore Fc fragments specific
Antibody more effectively avoids the cross reaction with other hypotypes to Fc sections of IgG4 of combination, ensure that the high specific to IgG4.
It is not formed and is vied each other between the first IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody.
Two monoclonal antibodies to IgG4 specific bond are produced by Abcam company, Britain, have claim 1
Described in property.The article No. of two antibody is respectively ab1930 and ab99817.
Two IgG4 monoclonal antibodies are produced by Abcam company.To determine that two monoclonal antibodies do not constitute Competition,
Screening test is carried out to two monoclonal antibody cocktails, although two specific antibodies are both for Fc segment, experiment knot
Fruit confirms that two antibody are different from the binding site of Fc segment of IgG4, and first antibody does not influence second after in conjunction with IgG4
The combination of the compound of a antibody and first antibody and IgG4.
The method for detecting immunoglobulin G 4 (IgG4) in sample, includes the following steps:
1) it handles sample to be tested: sample to be tested being diluted with volume ratio 1: 10000,10 μ L samples is taken to be added to 1mL sample
In dilution, concussion is mixed, and from wherein taking 10 μ L to be added in 1mL sample diluting liquid, concussion is mixed；
2) it is loaded: the IgG4 of various concentration being added into the solid phase carrier container for being coated with the first IgG4 monoclonal antibody
Standard solution and diluted sample to be tested, 100 holes μ L/ are stored at room temperature 30 minutes with 20 DEG C -28 DEG C of postposition of sealing plate film sealing plate,
Make it in conjunction with the first IgG4 monoclonal antibody Fc segment being fixed on solid phase carrier container；
3) wash: the sample component being not associated in cleaning removal solid phase carrier container, 300 μ L high-efficient cleanings are added in every hole every time
Washing lotion is cleaned 4-6 times, is patted dry on blotting paper；
4) add ELIAS secondary antibody: horseradish peroxidase (HRP) label of high specific being added into solid phase carrier container
2nd IgG4 monoclonal antibody solution, every hole are added 100 μ L, are stored at room temperature 30 points with 20 DEG C -28 DEG C of postposition of sealing plate film sealing plate
Clock, the 2nd IgG4 monoclonal antibody for marking enzyme is in conjunction with the IgG4 in sample to be tested, to make the 2nd IgG4 Dan Ke of enzyme mark
Grand antibody is fixed on microwell plate；
5) it washs: removing the 2nd IgG4 monoclonal antibody of enzyme mark being not associated in solid phase carrier, 300 μ L are added in every hole every time
High-efficient cleaning washing lotion is cleaned 4-6 times, is patted dry on blotting paper；
6) it develops the color: TMB (tetramethyl benzidine) colour developing being added into solid phase carrier container, colour developing A liquid and B liquid are pressed into 1:1
Ratio mixes, and 100 μ L are added in every hole, and room temperature is protected from light light shake 15 minutes；TMB forms the cation of blue under the catalysis of HRP enzyme
Product；
7) terminate: into solid phase carrier container, 50 μ L of terminate liquid is added in every hole, and light shake is mixed to terminate reaction, blue solution
It is changed into yellow；The OD value spectrophotometric determination in each hole is sequentially measured at Single wavelength 450nm or dual wavelength 450/630nm
The OD value of colored solutions；
8) value of IgG4 in sample to be tested is measured by calibration curve method:
A. it draws standard curve: the average value of each concentration standards duplicate hole OD value is calculated, with the concentration of standard items
It (ng/mL) is abscissa, corresponding OD value is ordinate, draws standard curve using Data Analysis Software (Origin8)；
B. result judges: according to normal population IgG4 concentration of specimens mean value+3SD as positive cutoff value (cut-off value,
0.212g/L), the sample of right >=0.212g/L is determined as positive sample；< 0.212g/L is determined as negative sample.Detected value exists
It is prompted within the scope of 0.173g/L-0.212g/L close to positive decision content, it is proposed that repetition measurement.
The method of immunoglobulin G 4 (IgG4) in the detection sample:
The solid phase carrier container is 96 holes or 48 hole polystyrene ELISA Plates；The sample to be tested is standard items, people's blood
Clear or blood plasma；
The first IgG4 monoclonal antibody refers to the monoclonal antibody of 4 Fc segment of mouse anti-human igg, and the 2nd IgG4 is mono-
Clonal antibody refers to the monoclonal antibody of 4 Fc segment of mouse anti-human igg coupling HRP.
The first IgG4 monoclonal antibody of the coating is in the method for coating of solid phase carrier are as follows: every 100ML coating buffer is by 0.05M
Carbonate buffer solution, the first IgG4 monoclonal antibody and purified water composition；Every hole of solid phase carrier container adds 100 μ L coating buffers,
Set 4 DEG C overnight.
The cleaning reagent that washing step of the present invention uses is for high-efficient cleaning washing lotion and applied to the step in method
3) it is cleaned with step 5) using a kind of high-efficiency washing test solution.
The ingredient and concentration of the high-efficient cleaning washing lotion are as follows: purified water 400mL, Na2HPO4·12H2O21.789g,
KCl5.78g, KH2PO42.916g, NaCl86.4g, Tween-20 7.2mL, Proclin132 μ L, each ingredient after completely dissolution,
Purified water is added to be settled to 540mL.
New cleaning formula of liquid has been used in the cleaning process of step 3) described in the method for the present invention and step 5), has included specific
The Tween-20 and Proclin132 of ratio.The formula is suitable for 4 Fc sections of monoclonal antibody of this method anti-human igg as solid phase
Ingredient (the method and step of non-IgG4 in serum is efficiently removed after in conjunction with IgG4 in 4 standard items of human IgG or human serum sample
(3))；And IgG4 conjugate is mono- with the IgG4 of the second label peroxidase again suitable for first antibody on solid phase carrier and sample
Clonal antibody combines the non-specific binding (method and step (5)) of rear scavenger enzyme mark secondary antibody.
The cleaning method of the step 3) and step 5) are as follows: board-washing machine washing plate, 5 times, each every 300 μ L high-efficiency washing of hole
Liquid pats dry on blotting paper.Cleaning method can rapidly and efficiently wash away unbonded ingredient, and background interference is effectively reduced, and improve
Sensitivity avoids false positive.
Method of the present invention is a kind of high specific obtained according to immunology principle and experimentation and highly sensitive
4 detection method of human serum IgG of degree.Related technological invention feature includes that (1) has selected the people with high stability feature
Technical indicator of the immunoglobulin Fc section as detection IgG4, verifying, which has selected two, has high specific to 4 molecule of human IgG,
But do not form the Fc sections of monoclonal antibodies of IgG4 vied each other；(2) it establishes and a kind of to reduce non-specific binding including using
Detection method including high-efficient cleaning washing lotion and cleaning technique.(3) experiment proves that, the method for the present invention can specific recognition human serum
Middle IgG4, to other immunoglobulin hypotypes (IgG1, IgG2, IgG3) in sample without identification, the height that detection IgG4 is presented is special
It is anisotropic.In the reaction system after additional quantitative human IgG 4, (Siemens are raw for IgG4 detection method in the market with other
Scattering turbidimetry detection method is immunized in the IgG4 of production) the parallel comparison recovery experiment discovery that carries out, this method can detect accurately
10 additional microgram IgG4 (the final concentration of 0.1g/L of reaction solution) in reaction system, and similar product (immune scattering turbidimetry side
Method) IgG4 of the concentration is not detected, show that this method produces the detection sensitivity of IgG4 better than Siemens
IgG4 be immunized scattering turbidimetry detection method.This method at home and abroad clinical use IgG4 assay kit (Germany west
Door subsidiary, scattered light urbidmetry) make the parallel comparison with batch clinical serum sample.The positive clinical pattern detection of two methods
Coincidence rate is more than 90%, prompts the detection of this method and the similar import reagent of Chinese Bureau of Drugs Supervision's verifying approval to human serum IgG 4
With equivalence.
The present invention provides a kind of kits of immunoglobulin G 4 (IgG4) in detection sample, including following reagent:
1) the first coated ELISA Plate of IgG4 monoclonal antibody: the first IgG4 monoclonal is anti-in the coating buffer of coated elisa plate
The concentration range of body is 0.1 μ g/mL-10 μ g/mL；
2) 4 standard items of human IgG: for 4 standard solution of human IgG (Abcam, ab90286) of 6 various concentrations, standard items A
For 50ng/mL, standard items B be 25ng/mL, standard items C is 12.5ng/mL, standard items D is 6.25ng/mL, standard items E is
3.125ng/mL, standard items F are 0ng/mL；Each standard items are 1mL/ bottles, and each 1 bottle；
3) reference substance: 1mL/ bottles of (Abcam, the ab90286) positive reference substance of human IgG 4, normal human serum (Jackson, goods
Number be 009-000-001) 1mL/ bottles of negative controls, 1 bottle；
4) the 2nd IgG4 monoclonal antibody solution of horseradish peroxidase-labeled: 150mL/ bottles, the 2nd IgG4 monoclonal
G/mL/ bottles, 1 bottle of 0.3 μ of antibody concentration；Abcam, article No. ab99817；
5) Sample dilution: 20 times of concentration PBST, 15mL/ bottles, 1 bottle；
6) developing solution: colour developing A liquid, by anhydrous sodium acetate 0.3142g, cycloheptaamylose 0.2025g, carbamide peroxide
0.0697g and purified water composition, 8mL/ bottles, 1 bottle；Develop the color B liquid, by 24.3 μ L of the 10MNaOH of 0.2355g containing citric acid, the third three
Alcohol 6.2mL and purified water composition, 8mL/ bottles, 1 bottle；
7) terminate liquid: concentration be 2mol/L sulfuric acid solution, 10mL/ bottles, 1 bottle；
8) cleaning solution: 20 times of concentration PBST, 50mL/ bottles, 1 bottle.
The kit also contains Fresco Bag, sealer plate and specification.
Technical principle of the invention is to form three compounds using bispecific monoclonal antibody and test substance IgG4, and selection is anti-
IgG4 antibody Fc section be solid phase carrier, by the high degree of specificity and stability to test substance IgG4, effectively prevent with
The non-specific binding of IgG4 structure similar IgG1, IgG2, IgG3.It is single by Fc sections of enzyme labels of second high specific IgG4
Clonal antibody strengthens the specificity of identification and the detection for IgG4.With nonspecific interference in high-efficient cleaning washing lotion removal sample
Material composition, by HRP zymolyte TMB colour developing is added in reaction solution, by serial known concentration IgG4 standard items in reactant
The concentration dependent OD value of IgG4 is presented in system.It is big that linear regression coeffficient R2 value is obtained within the scope of 3ng/mL-50ng/mL standard items
In 0.99 dose-response relationship (Fig. 1), concentration of the accurate quantitative analysis determinand IgG4 in people's biological sample.The method of the present invention
It is suitable for the detection of the clinically sample to " IgG4 diseases related (IgG4- RD) " with kit.The method of the present invention and reagent
Box applies also for the clinically research and pattern detection to other a variety of diseases related with IgG4.
The present invention provides one kind to have high specific, people's immune globulin in easy, quick, accurate detection human serum sample
The practical approach of white G4 content, and the kit suitable for basic scientific research and clinical examination.Kit has at low cost, time-consuming
It is short, it is accurately, easy to operate, specific apparatus and reagent are not needed, equal spies can be completed under the conditions of general Clinical Test Lab
Point.
Verifying using the method for the present invention has excellent corresponding relationship between IgG4 concentration and optical signal.Verification method is specific
It is described as a known IgG4 concentration of the curve peak that is near the mark is selected to do and is half-and-half diluted to 5 concentration and is detected, used
Standard curve determines the IgG4 concentration of each diluted sample and then does linear regression analysis.Linear regression coeffficient is greater than 0.99 (figure
2)。
Combination uncompetitive inhibiting effect of the selected two IgG4 monoclonal antibodies to human IgG 4；With other three kinds of people
1,2,3 no cross reaction (table 1) of immunoglobulin hypotype, presents to IgG4 high specific.Inside and outside add in reaction system 10 micro-
Gram IgG4 (concentration 0.1mg/mL), this method can accurately detect the amount of added IgG4.With domestic registered import IgG4
The discovery of assay kit (Siemens, scattered light urbidmetry) comparative experiments, import IgG4 assay kit are not detected
Inside and outside add 10 microgram IgG4 (concentration 0.1mg/mL) (table 2) in reaction system, shows that the method for the present invention is with higher sensitive
Degree.Same batch of clinical serum of the registered IgG4 assay kit (Siemens, scattered light urbidmetry) of this method and China
Positive clinical pattern detection coincidence rate is more than 90% (table 3) in sample parallel IgG4 detection comparison, illustrates this method and in
The imported product " immunoglobulin G 4 assay kit (scattered light urbidmetry) " of Bureau of Drugs Supervision, country, state verifying registration has to clinic
The equivalence of serum sample IgG4 detection.
Experimental data shows batch internal difference of this method less than 10%, and difference between batch is less than 15% (table 3).To human serum IgG 4
Detection be not necessarily to special experiment equipment, operating process is simple and efficient.All operationss can be completed in 2 hours.
Advantages of the present invention is as follows: providing one kind has high specific, easy, quick, accurate can detect human serum sample
The practical approach of 4 content of immunoglobulin G and the kit suitable for basic scientific research and clinical examination in this.It is at low cost, consumption
When it is short, it is accurately, easy to operate, do not need specific apparatus and reagent, can be completed under the conditions of general Clinical Test Lab, with
The commercial reagent box of clinical use compares with significant advantage.It can be clinical diagnosis extensively suitable for the universal use of clinic
It is general universal.
The present invention will be further described with attached drawing combined with specific embodiments below, does not limit the invention in any way
Protection scope, it is all according to the disclosure of invention carry out this field equivalent replacement, all belong to the scope of protection of the present invention.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 shows the canonical plotting using kit detection immunoglobulin G 4 of the invention
Fig. 2 shows the IgG4 concentration linear regression analyses of diluted sample
Specific embodiment
A kind of embodiment 1: kit detecting IgG4 antibody concentration
One, kit forms:
Table 1: kit forms
Two, preparation methods
1. the preparation of ELISA Plate
The preparation of 1.1 coating buffers
Table 2
* IgG4Fc antibody is the first IgG4 monoclonal antibody in table 2, is produced by Abcam company, article No. is respectively
ab1930。
The coating of 1.2 ELISA Plates
Coating room coating machine in 100,000 grades of areas adds 100 μ L coating buffers to every hole of 96 hole elisa Plates, sets 4 DEG C overnight.
The preparation of 1.3 confining liquids
Table 3
The closing of 1.4 ELISA Plates
Coating buffer is discarded, is washed 1 time (300uL1 × PBST is added in every hole, stands 2min, gets rid of solution in hole, pat dry).
Envelope 1.5h is stood to 200 μ L confining liquids, 25 DEG C of incubators are added in every hole of 96 hole elisa Plates with liquid-transfering gun.
The drying of 1.5 ELISA Plates
Confining liquid is discarded, is washed 1 time (1 × PBST of 300uL is added in every hole, stands 2min, gets rid of solution in hole, pat dry).
It is placed in 25 DEG C of oven drying 5h.
The preservation of 1.6 ELISA Plates
After being coated with plate drying, ELISA Plate is put into aluminium foil bag, then be put into one piece of desiccant thereto, vacuumizes modeling
Envelope.
4 DEG C of preservations.
2. the preparation of standard items and yin and yang attribute reference substance
The preparation of 2.1 standard items:
4 albumen of human IgG (Abcam, article No. ab90286) is taken out from -80 DEG C of ultra low temperature freezers, concentration 1.04mg/
ML, in the centrifuge tube that 45 μ L sample dilutions to 200 μ L are taken in the Biohazard Safety Equipment of ten thousand grades of negative pressure positive rooms, then to centrifuge tube
It is middle that 4 albumen of human IgG that 5 μ L concentration are 1.04mg/mL is added, with turbine mixer 7 grades of mixings 30s, final concentration of 104 μ g/
ml.The sample diluting liquid for taking 20mL to dilute is into the centrifuge tube of 50mL, then final concentration of 104 μ g/ml is added into centrifuge tube
4 albumen of human IgG, 9.6 μ L, with 7 grades of mixing 30s of turbine mixer, the concentration of 4 albumen of human IgG is 50ng/ in the solution at this time
ML, then 2 times of gradients successively dilute, standard curve since 50ng/mL twice of gradient dilution to 3.125ng/mL.
The preparation of 2.2 yin and yang attribute reference substances
2.2.1 the preparation of positive reference substance
4 albumen of positive reference substance human IgG (Abcam, ab90286) is taken out from -80 DEG C of ultra low temperature freezers, concentration is
1.04mg/mL, the sample diluting liquid for taking 35 μ L to dilute in the Biohazard Safety Equipment of ten thousand grades of negative pressure positive rooms to 200 μ L from
In heart pipe, then the positive reference substance human IgG 4 that 0.35 μ L concentration is 1.04mg/mL is added into centrifuge tube, with turbine mixer 7
Shelves mix 30s, final concentration of 10.4 μ g/ml.The sample diluting liquid for taking 10mL to dilute is into the centrifuge tube of 15mL, then to centrifugation
4 solution of final concentration of 10.4 μ g/ml positive reference substance human IgG, 34.6 μ L is added in pipe, with 7 grades of mixing 30s of turbine mixer, i.e.,
For positive reference substance.
2.2.2 the preparation of negative controls
Normal human serum (Jackson, article No. 009-000-001) is taken out from -80 DEG C of ultra low temperature freezers, it is negative at ten thousand grades
The sample diluting liquid for taking 10mL to dilute in the Biohazard Safety Equipment of positive room is pressed to add into the centrifuge tube of 15mL, then into centrifuge tube
Enter 1 μ L normal human serum, with turbine mixer 7 grades of mixings 30s, as negative controls.
2.3 packing
After quality inspection is qualified, dispensed respectively by 1mL/ bottles of loading amount requirement to white in the Biohazard Safety Equipment in ten thousand grades of areas
In small reagent bottle, standard items cover the small bottle cap of white, and positive reference substance covers red small bottle cap, and it is small that negative controls cover blue
Bottle cap.
3. the preparation of ELIAS secondary antibody liquid
The preparation of 3.1 ELIAS secondary antibody liquid
Table 4
* mouse anti-human igg 4Fc (HRP) is the 2nd IgG4 monoclonal antibody in table 4, is produced by Abcam company, article No. difference
For ab99817.
3.2 packing
After quality inspection is qualified, is dispensed by 15mL/ bottles of loading amount requirement to white reagent bottle, added 100,000 grades of differentiation dress rooms
Cover blue bottle cap.
4. the preparation of sample diluting liquid
The preparation of 4.1 sample diluting liquids
Table 5
4.2 packing
After quality inspection is qualified, is dispensed by 15mL/ bottles of loading amount requirement to white and tried in the liquid subpackage room in 100,000 grades of areas
Agent bottle covers yellow bottle cap.
5. the preparation of developing solution
The preparation of 5.1 developing solution A
Table 6
The preparation of 5.2 developing solution B
Table 7
5.3 packing
After quality inspection is qualified, dispensed by 8mL/ bottles of loading amount requirement to brown reagent bottle in the packing room in 100,000 grades of areas
In, cover brown bottle cap.
6. the preparation of terminate liquid
The preparation of 6.1 terminate liquids
The concentrated sulfuric acid for taking 10.87mL with graduated cylinder correct amount in 100,000 grades of areas liquor room, is slowly added to pure containing 50mL
Change and diluted in the beaker of water, be slowly stirred mixing with magnetic stirring apparatus, then measures 20mL purified water and rinse the amount for measuring the concentrated sulfuric acid
Cylinder is poured into 100mL measurer and adds a certain amount of purified water and be settled to 100mL, makes its final concentration of 2mol/L.Constant volume
Afterwards, solution is transferred to terminate liquid special agent bottle.
6.2 packing
After quality inspection is qualified, dispensed by 10mL/ bottles of loading amount requirement to white reagent bottle in the packing room in 100,000 grades of areas,
Cover red bottle cap.
7. the preparation of concentrated cleaning solution
The preparation of 7.1 concentrated cleaning solutions
Table 8
7.2 packing
After quality inspection is qualified, dispensed by 50mL/ bottles of loading amount requirement to the big reagent of white in the packing room in 100,000 grades of areas
Bottle covers the big bottle cap of white.
8. the assembling of kit
According to execution claimed below when kit package of the present invention:
8.1 assemble the box of kit and liner.
The standard items A, B dispensed, C, D, E, F, positive reference substance, negative controls sequence are successively put into reagent by 8.2
In 8 apertures of box liner.
ELIAS secondary antibody liquid, sample diluting liquid, colour developing A liquid, colour developing B liquid, terminate liquid sequence are put into kit liner by 8.3
In 5 mesoporous.
8.4 are put into cleaning solution in the macropore of kit liner.
8.5 lie directly in plastic packaging good ELISA Plate in kit.
8.6 lie against 3 sealing plate films in kit.
Specification is rolled well and is lain against in kit by 8.7.
Kit is covered and is put into 4 DEG C of refrigerator-freezers and saves by 8.8.
Three, the application method of kit
1. preparation
1.1 all reagents and sample to room temperature and are mixed using preceding recovery.
1.2 sample diluting liquids and concentrated cleaning solution are 20X, need to be diluted to 1X before use, will be dense according to experiment expense
Contracting cleaning solution or sample diluting liquid are diluted with distilled water by 1: 19 (v: v).
1.3 dilute sample to be tested with 1: 10000 (v: v).It is recommended that 10 μ L samples is taken to be added in 1mL sample diluting liquid,
Concussion mixes, and from wherein taking 10 μ L to be added in 1mL sample diluting liquid, concussion is mixed.Diluted sample at room temperature need to be in 8h
It uses.
1.4 should do standard curve when measuring serum every time simultaneously, preferably do multiple holes.If test substance too high levels in sample
(the OD value that sample OD value is greater than standard items A), measures again after please first diluting certain multiple (n times) with sample diluting liquid, makes its inspection
Measured value is in the standard curve range of linearity, multiplied by corresponding extension rate when calculating.
2. concrete operation step
2.1 prepare enough microplates, and dilute good patients serum to be measured in advance.
2.2 sample-adding
100 μ L various concentration IgG4 standard items and blood serum sample are added in microwell plate.The concentration of standard items is respectively
50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.125ng/mL、0ng/mL。
2.3 being incubated for
30 minutes are stood with (20 DEG C -28 DEG C) of room temperature of sealing plate film sealing plate postposition.
2.4 washing
Board-washing machine washing plate: 5 times, each every 300 μ L high-efficient cleaning washing lotion of hole finally pats dry on blotting paper.
2.5 add ELIAS secondary antibody
The 100 μ L of ELIAS secondary antibody liquid diluted is added in every hole.
2.6 being incubated for
Operation is the same as 2.3.
2.7 washing
Operation is the same as 2.4.
2.8 colour developing
Colour developing A liquid and B liquid are mixed in 1: 1 ratio, 100 μ L are added in every hole, and room temperature is protected from light light shake 15 minutes.
2.9 terminating
50 μ L of terminate liquid is added in every hole, and light shake is mixed to terminate reaction (color becomes yellow from blue in hole).
2.10 measurement
The OD value in each hole is sequentially measured at Single wavelength 450nm (or dual wavelength 450/630nm).
3. drawing standard curve
The average value of each concentration standards duplicate hole OD value is calculated, it is right with the concentration (ng/mL) of standard items for abscissa
The OD value answered is ordinate, draws standard curve using Data Analysis Software (Origin8).
4. positive cutoff value or reference interval
The serum of the healthy population largely without related disease is detected using the method for the present invention and product completion.Institute
Obtained sample concentration mean value+3SD is that positive cutoff value (cut-off value) is 0.212g/L, and >=0.212g/L can determine that as sun
Property；< 0.212g/L is determined as feminine gender.When detected value in 0.173g/L-0.212g/L region cue close to positive decision content, build
Discuss repetition measurement.
The method that embodiment 2. detects immunoglobulin G 4 in sample
One, material and method
1. material:
(1) kit of embodiment 1
(2) known dilution IgG4 concentration samples are clinical samples, which digests from No.1 Hospital, Shanxi Medical Univ
One male patient of internal medicine inpatient department, the concentration of IgG4 are measured as 0.401g/L by this kit.
(3) clinical samples to be measured: serum sample is provided by No.1 Hospital, Shanxi Medical Univ's gastroenterology, and Sample size is
49, Cuiping is artificially permitted in acquisition, and the acquisition date is in March, 2017 to July, and acquisition address disappears for No.1 Hospital, Shanxi Medical Univ
Change internal medicine inpatient department, phone 0351-4639020.
2. method:
(1) it handles sample to be tested: sample to be tested being diluted with volume ratio 1: 10000,10 μ L samples is taken to be added to 1 mL sample
In product dilution, concussion is mixed, and from wherein taking 10 μ L to be added in 1mL sample diluting liquid, concussion is mixed；
(2) it is loaded: being added into the solid phase carrier container (96 orifice plate) for being coated with the first IgG4 monoclonal antibody different dense
The IgG4 standard solution of degree and the sample to be detected diluted, 100 holes μ L/, with 20 DEG C of -28 DEG C of rooms of sealing plate film sealing plate postposition
Temperature stands 30 minutes, makes it in conjunction with the first IgG4 antibody Fc fragment being fixed on solid phase carrier container；
(3) wash: the sample component being not associated in cleaning removal solid phase carrier container, it is efficient that 300 μ L are added in every hole every time
Cleaning solution is cleaned 5 times, is patted dry on blotting paper；
(4) add ELIAS secondary antibody: horseradish peroxidase (HRP) label of high specific being added into solid phase carrier container
2nd IgG4 monoclonal antibody solution, every hole are added 100 μ L, are stored at room temperature 30 points with 20 DEG C -28 DEG C of postposition of sealing plate film sealing plate
Clock, the 2nd IgG4 monoclonal antibody for marking enzyme is in conjunction with the IgG4 in sample to be tested, to make the 2nd IgG4 Dan Ke of enzyme mark
Grand antibody is fixed on microwell plate；
(5) it washs: the 2nd IgG4 monoclonal antibody of enzyme mark being not associated in cleaning removal solid phase carrier container, each every hole
300 μ L high-efficient cleaning washing lotions are added, cleans 5 times, is patted dry on blotting paper；
(6) it develops the color: tetramethyl benzidine (TMB) colour developing being added into solid phase carrier container, colour developing A liquid and B liquid are pressed 1: 1
Ratio mixes, and 100 μ L are added in every hole, and room temperature is protected from light light shake 15 minutes；TMB forms the cation of blue under the catalysis of HRP enzyme
Product；
(7) terminate: into solid phase carrier container, 50 μ L of terminate liquid is added in every hole, and light shake is mixed to terminate reaction, and blue is molten
Liquid is changed into yellow；The OD value that each hole is sequentially measured at Single wavelength 450nm or dual wavelength 450/630nm is measured with spectrophotometric
Determine the OD value of colored solutions；
(8) value of IgG4 in sample to be tested is measured by calibration curve method:
A. it draws standard curve: the average value of each concentration standards duplicate hole OD value is calculated, with the concentration of standard items
It (ng/mL) is abscissa, corresponding OD value is ordinate, draws standard curve using Data Analysis Software (Origin8)；
B. result judges: obtained concentration of specimens mean value+3SD is 0.212g/L as positive cutoff value (cut-off value),
>=0.212g/L can determine that as the positive；< 0.212g/L is determined as feminine gender；When detected value is in 0.173g/L-0.212 g/L range
The close positive decision content of prompt, it is proposed that repetition measurement.
Two, results
1. the standard curve established
Fig. 1 display detects the canonical plotting of immunoglobulin G 4 using kit of the invention.The concentration of standard items
Respectively 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 0ng/mL.Calculate each concentration standard
The average value of product duplicate hole OD value, with the concentration (ng/mL) of standard items for abscissa, corresponding OD value is ordinate, uses number
Standard curve is drawn according to analysis software (Origin8).
2. the IgG4 concentration linear regression analysis of diluted sample
Doing double of dilution with known IgG4 concentration, (sample comes from No.1 Hospital, Shanxi Medical Univ's gastroenterology inpatient department
A male patient, the concentration of IgG4 is measured as 0.401g/L by this kit) 5 concentration (40ng/mL, 20ng/
ML, 10ng/mL, 5ng/mL and 3ng/mL) it is detected, the IgG4 concentration of each diluted sample is determined with standard curve, then
Do linear regression analysis.Linear regression coeffficient is also greater than 0.99.
Fig. 2 is the IgG4 concentration linear regression analysis result of diluted sample.
3. kit of embodiment and method specific experiment
One, material and method
1. material
(1) 1 kit of embodiment
(2) IgG1, IgG2, IgG3 sample, IgG1 are purchased from Abcam, article No. ab90283；IgG2 is purchased from Abcam, article No.
For ab90284；IgG3 is purchased from Sino.Biological Inc, article No. 13906-HNAH.
2. method
IgG1, IgG2, IgG3 are respectively diluted to the sample to be tested of following concentration: 1000ng/mL, 500ng/ respectively
ML, 250ng/mL, 100ng/mL and 50ng/mL；
Using kit of the present invention to the IgG1 of various concentration, IgG2, IgG3 (1000ng/mL, 500ng/mL, 250ng/
ML, 100ng/mL and 50ng/mL) measured value, detection method of the method with embodiment 2 are carried out respectively.
Two, results
Measurement result is shown in Table 9, and finds OD value close to blank well.According to as a result, can be confirmed that the present invention establishes
Method will not be combined with IgG hypotype 1,2,3, it is therefore contemplated that the present invention has high specific.
The measured value result of table 9. various concentration IgG1, IgG2, IgG3 on kit of the present invention
4. kit of embodiment and method sensitivity experiment
One, material and method
1. material
(1) 1 kit of embodiment
(2) sample to be tested is addressed to Shanxi Dean medical test center (third party's medical diagnosis mechanism), measures human IgG 4
Content and provide examining report list.Sample measured value is completed by third party's medical diagnosis mechanism, accurately and reliably.Dean uses
The immunoglobulin G 4 assay kit (scattered light urbidmetry) of Siemens.
(3) sample to be tested is as follows:
A. negative serum；
B. the additional 10 μ g IgG4 of negative serum；
C. the additional 10 μ g IgG4 of negative serum, and IgG1 is added, each 10 μ g of IgG2；
D. the additional 10 μ g IgG4 of negative serum, and IgG1 is added, each 10 μ g of IgG2, IgG3.
2. method
(1) detection method is the same as embodiment 3.
(2) Siemens's kit test method is referring to the kit specification.
Two, results
Following sample is detected with Siemens IgG4 kit using kit of the present invention, the results are shown in Table 10 institutes
Show.It is found after being compared by the kit of this method and Siemens:
(1) additional IgG1, IgG2, IgG3 in IgG4 sample, do not influence the measurement result of kit of the present invention, and this
Invention kit has the recycling for being significantly higher than negative serum to IgG4；
(2) measurement range of Siemens Company IgG4 kit specification mark is 0.055-3.3g/L, however, in feminine gender
When serum China and foreign countries plus concentration are the IgG4 of 0.1g/L, Siemens's kit measured result and negative serum value indifference are said
Bright kit of the present invention has higher sensitivity.
The kit of the present invention of table 10 is compared with Siemens's kit is to 4 detection sensitivity of human serum IgG
The clinical detection comparative experiments of embodiment 5. present invention and the prior art
One, material and method
1. material
(1) sample to be tested: 49 normal human serum samples or the doubtful IgG4-RD serum sample of clinical diagnosis.Serum sample
It is provided by No.1 Hospital, Shanxi Medical Univ's gastroenterology.It acquires people Xu Cuiping, acquire in March, 2017 to July on date, acquisition
People address gastroenterology inpatient department, the first affiliated hospital, Mountain Western Medicine S University, phone 0351-4639020. collect whole blood and pay attention to
Haemolysis is avoided, is centrifuged 10 minutes after being stored at room temperature 2 hours or 4 DEG C overnight in 3000~3500rpm/min, takes supernatant that can carry out
Measurement.
(2) 1 kit of the embodiment of the present invention is prepared by Shanxi Rui Hao Biotechnology Co., Ltd
(3) sample to be tested is addressed to Shanxi Dean medical test center (third party's medical diagnosis mechanism), measures human IgG 4
Content and provide examining report list.Sample measured value is completed by third party's medical diagnosis mechanism, accurately and reliably.Dean uses
The immunoglobulin G 4 assay kit (scattered light urbidmetry) of Siemens.
2. method
Using kit of the present invention and Siemens produce kit measure respectively 49 normal human serum samples or
The doubtful IgG4-RD serum sample of clinical diagnosis.
(1) kit measurement method of the present invention is double-monoclonal antibody sandwich with 2 method of embodiment, the method for the present invention
ELISA method.
(2) Siemens Company's kit detecting step is operated referring to the kit specification, and Siemens's kit is
Immune scatter turbidimetry.
Two, results
Testing result is shown in Table 11.
The kit of the present invention of table 11 is with Siemens's kit to the measurement result of 49 human serum samples
The method of the present invention and Siemens produce kit to the consistency of the measurement result of 49 human serum samples
It is 91.8%, the coincidence rate that two methods detect human serum IgG 4 is close to 98%.Illustrate the method for the present invention and in China national
The imported product " immunoglobulin G 4 assay kit (scattered light urbidmetry) " of Bureau of Drugs Supervision's verifying registration, which has, to be implemented to clinical blood
The equivalence of this IgG4 of final proof detection.
The series of detailed descriptions listed above only for feasible embodiment of the invention specifically
Protection scope bright, that they are not intended to limit the invention, those skilled in the art can be designed that a lot of other modification and
Embodiment, these modifications and implementations will be fallen within scope and spirit disclosed in the present application.More specifically, exist
The application discloses, in the range of drawings and claims, can building block to theme combination layout and/or layout carry out it is more
Kind variations and modifications.In addition to variations and improvements to the component parts and or layout, those skilled in the art are come
It says, other purposes also will be apparent.
Claims (10)
1. a kind of method of immunoglobulin G 4 (IgG4) in detection sample, it is characterised in that: anti-using two high specifics
The monoclonal antibody of IgG4Fc segment, for first monoclonal antibody special to IgG4 in conjunction with solid phase carrier, which is the
One IgG4 monoclonal antibody, second monoclonal antibody special to IgG4 are marked by a kind of peroxidase, which is second
IgG4 monoclonal antibody；First IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody with IgG4 present in detection sample
In conjunction with the compound of formation the 2nd IgG4 monoclonal antibody of the first IgG4 monoclonal antibody-IgG4- enzyme mark is simultaneously fixed on solid phase load
On body；First IgG4 monoclonal antibody and the 2nd IgG4 monoclonal antibody do not constitute competition to the combination of IgG4, and due to two
The Fc section of IgG4 molecule is incorporated into the monoclonal antibody of IgG4 specific bond and there is high stability；On solid phase carrier
One IgG4 monoclonal antibody is presented dosage correlation in conjunction with the IgG4 in detected sample, the IgG4 being combined still keep with
The combination of 2nd IgG4 monoclonal antibody of oxidase label, and the second of oxidation label is determined according to the amount for the IgG4 being combined
The amount of IgG4 monoclonal antibody is quantitatively tested by the oxidizing ferment of the 2nd IgG4 labeling of monoclonal antibody and the color producing reaction of zymolyte
IgG4 present in sample.
2. the method for immunoglobulin G 4 (IgG4) in detection sample according to claim 1, it is characterised in that: first
IgG4 monoclonal antibody is 4 heavy chain monoclonal antibody of anti-human igg (Abcam, ab1930) of antibody preparation company, Britain production；The
Two IgG4 monoclonal antibodies are the IgG4Fc section enzyme labeled monoclonal antibody (Abcam, ab99817) of Abcam company production.
3. the method for immunoglobulin G 4 (IgG4) in detection sample according to claim 1 or 2, it is characterised in that packet
Include following steps:
1) it handles sample to be tested: sample to be tested is diluted with volume ratio 1: 10000,10 μ L samples is taken to be added to the dilution of 1mL sample
In liquid, concussion is mixed, and from wherein taking 10 μ L to be added in 1mL sample diluting liquid, concussion is mixed；
2) it is loaded: the IgG4 standard items of various concentration being added into the solid phase carrier container for being coated with the first IgG4 monoclonal antibody
Solution and diluted sample to be tested, 100 holes μ L/ are stored at room temperature 30 minutes with 20 DEG C -28 DEG C of postposition of sealing plate film sealing plate, make its with
The first IgG4 monoclonal antibody Fc segment being fixed on solid phase carrier container combines；
3) wash: the sample component being not associated in cleaning removal solid phase carrier container, 300 μ L high-efficiency washings are added in every hole every time
Liquid is cleaned 4-6 times, is patted dry on blotting paper；
4) add ELIAS secondary antibody: the second of horseradish peroxidase (HRP) label of high specific being added into solid phase carrier container
IgG4 monoclonal antibody solution, every hole are added 100 μ L, are stored at room temperature 30 minutes with 20 DEG C -28 DEG C of postposition of sealing plate film sealing plate, make enzyme
2nd IgG4 monoclonal antibody of label is in conjunction with the IgG4 in sample to be tested, to keep the 2nd Ig64 monoclonal antibody of enzyme mark solid
It is scheduled on microwell plate；
5) it washs: removing the 2nd IgG4 monoclonal antibody of enzyme mark being not associated in solid phase carrier, it is efficient that 300 μ L are added in every hole every time
Cleaning solution is cleaned 4-6 times, is patted dry on blotting paper；
6) it develops the color: TMB (tetramethyl benzidine) colour developing being added into solid phase carrier container, by colour developing A liquid and B liquid in 1: 1 ratio
It mixes, 100 μ L are added in every hole, and room temperature is protected from light light shake 15 minutes；TMB forms the cationic product of blue under the catalysis of HRP enzyme；
7) terminate: into solid phase carrier container, 50 μ L of terminate liquid is added in every hole, and light shake is mixed to terminate reaction, blue solution transformation
For yellow；The OD value spectrophotometric determination that each hole is sequentially measured at Single wavelength 450nm or dual wavelength 450/630nm is coloured
The OD value of solution；
8) value of IgG4 in sample to be tested is measured by calibration curve method:
A. it draws standard curve: calculating the average value of each concentration standards duplicate hole OD value, with the concentration (ng/mL) of standard items
For abscissa, corresponding OD value is ordinate, draws standard curve using Data Analysis Software (Origin8)；
B. result judges: being positive cutoff value (cut-off value, 0.212g/ according to normal population IgG4 concentration of specimens mean value+3SD
L), the sample of right >=0.212g/L is determined as positive sample；< 0.212g/L is determined as negative sample.Detected value is in 0.173g/
It is prompted within the scope of L-0.212g/L close to positive decision content, it is proposed that repetition measurement.
4. the method for immunoglobulin G 4 (IgG4) in detection sample according to claim 3, it is characterised in that:
The solid phase carrier container is 96 holes or 48 hole polystyrene ELISA Plates；The sample to be tested be standard items, human serum or
Blood plasma；
The first IgG4 monoclonal antibody refers to the monoclonal antibody of mouse anti-human igg 4Fc segment, and the 2nd IgG4 monoclonal is anti-
Body refers to the monoclonal antibody of mouse anti-human igg 4Fc segment coupling HRP；
The first IgG4 monoclonal antibody of the coating is in the method for coating of solid phase carrier are as follows: every 100ML coating buffer is by 0.05M carbonic acid
Salt buffer, the first IgG4 monoclonal antibody and purified water composition；Every hole of solid phase carrier container adds 100 μ L coating buffers, sets 4 DEG C
Overnight.
5. the method for immunoglobulin G 4 (IgG4) in detection sample according to claim 3, it is characterised in that: described
Step 3) and step 5) are cleaned using a kind of high-efficient cleaning washing lotion.
6. the method for immunoglobulin G 4 (IgG4) in detection sample according to claim 5, it is characterised in that: described
The ingredient and concentration of high-efficient cleaning washing lotion are as follows: purified water 400mL, Na2HPO4·12H2O 21.789g, KCl5.78g,
KH2PO42.916g, NaCl86.4g, Tween-207.2mL, Proclin132 μ L, each ingredient after completely dissolution, add purified water fixed
Hold to 540mL.
7. the method for immunoglobulin G 4 (IgG4) in detection sample according to claim 3, which is characterized in that described
The cleaning method of step (3) and step (5) are as follows: board-washing machine washing plate, 5 times, each every 300 μ L cleaning solution of hole is clapped on blotting paper
It is dry.
8. the kit of immunoglobulin G 4 (IgG4) in a kind of detection sample, which is characterized in that including following reagent:
1) the first coated ELISA Plate of IgG4 monoclonal antibody: the first IgG4 monoclonal antibody in the coating buffer of coated elisa plate
Concentration range is 0.1 μ g/mL-10 μ g/mL；
2) 4 standard items of human IgG: for 4 standard solution of human IgG (Abcam, ab90286) of 6 various concentrations, standard items A is
50ng/mL, standard items B are 25ng/mL, standard items C is 12.5ng/mL, standard items D is 6.25ng/mL, standard items E is
3.125ng/mL, standard items F are 0ng/mL；Each standard items are 1mL/ bottles, and each 1 bottle；
3) reference substance: 1mL/ bottles of (Abcam, the ab90286) positive reference substance of human IgG 4, (Jackson, article No. are normal human serum
009-000-001) negative controls 1mL/ bottles, 1 bottle；
4) the 2nd IgG4 monoclonal antibody solution of horseradish peroxidase-labeled: 150mL/ bottles, the 2nd IgG4 monoclonal antibody
G/mL/ bottles, 1 bottle of 0.3 μ of concentration；
5) Sample dilution: 20 times of concentration PBST, 15mL/ bottles, 1 bottle；
6) developing solution: colour developing A liquid, by anhydrous sodium acetate 0.3142g, cycloheptaamylose 0.2025g, carbamide peroxide 0.0697g
It is formed with purified water, 8mL/ bottles, 1 bottle；Develop the color B liquid, by 24.3 μ L of 0.2355g10MNaOH containing citric acid, glycerine 6.2mL and
Purified water composition, 8mL/ bottles, 1 bottle；
7) terminate liquid: concentration be 2mol/L sulfuric acid solution, 10mL/ bottles, 1 bottle；
8) cleaning solution: 20 times of concentration PBST, 50mL/ bottles, 1 bottle.
9. the kit of immunoglobulin G 4 (IgG4) in detection sample according to claim 8, which is characterized in that institute
It states kit and also contains Fresco Bag, sealer plate and specification.
10. 8,9 institute of the method for immunoglobulin G 4 (IgG4) and claim in detection sample described in claim 1 to 7
In the detection sample stated the kit of immunoglobulin G 4 (IgG4) be used for IgG4 diseases related (IgG4-RD) " sample
Detection and research purposes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111707835A (en) * | 2020-07-01 | 2020-09-25 | 珠海丽珠试剂股份有限公司 | Composition for detecting IgG4, kit, application and detection method |
| CN112285361A (en) * | 2020-09-27 | 2021-01-29 | 中国人民解放军空军军医大学 | Reagent for eliminating interference of anti-CD 38 monoclonal antibody medicine to anti-human globulin detection |
| WO2023028186A1 (en) * | 2021-08-27 | 2023-03-02 | Abbott Laboratories | Methods for detecting immunoglobulin g, subclass 4 (igg4) in a biological sample |
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