CN107607713A - A kind of detection kit and its application - Google Patents
A kind of detection kit and its application Download PDFInfo
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- CN107607713A CN107607713A CN201710843965.0A CN201710843965A CN107607713A CN 107607713 A CN107607713 A CN 107607713A CN 201710843965 A CN201710843965 A CN 201710843965A CN 107607713 A CN107607713 A CN 107607713A
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Abstract
The present invention relates to a kind of detection kit and its application; more particularly to a kind of detection kit for being coated with compound antibody and its application in rheumatoid arthritis detection reagent; the kit includes solid phase carrier; the solid phase carrier is coated with compound antibody, and the compound antibody is the combination for resisting citrullinated protein antibodies and anti-carbamylation protein antibodies.The detection antibody of the application kit is the combination for resisting citrullinated protein antibodies and anti-carbamylation protein antibodies; being capable of a variety of RA autoantigens of one-time detection; it is sensitive insufficient with specificity to overcome single autoantigen detection; and a variety of autoantigen singles detect operationally cumbersome one by one, detection efficiency is substantially increased and accuracy that result judges.
Description
Technical field
The present invention relates to biological immune detection technique field, and in particular to a kind of detection kit and its application, especially relates to
And a kind of detection kit for being coated with compound antibody and its application in rheumatoid arthritis detection reagent.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of common and is difficult to the disease cured, and is drawn
The irreversible damage in joint is played, the morbidity later stage often leads to patient disabilities, influences quality of life, disablement.Its feature
It is lasting synovitis, systemic inflammation and autoantibody is present.The RA cause of disease and pathogenesis are still known little about it at present, and
When control for disease of diagnosis and early treatment have great importance.The method for being clinically used to diagnose RA at present is a lot, I
State is using the RA criteria for classifications of American society of rheumatism (ACR) revision in 1987, and its specificity is 89%, sensitivity 91%-
94%, but the standard easily fails to pinpoint a disease in diagnosis some early stages or atypical patient.
RF (rheumatoid factor) is a kind of in serological index, is less subject to the influence of the factors such as the course of disease, can be early in disease
Phase occurs, easy to detect, sensitiveness is high, but specificity is relatively low, and the RF positives can reach 5% in normal person.In addition anti-CCP resists
Body can occur in arthritis early stage, and related to the destruction of Bones and joints, but its sensitivity 60%, specificity 90%.Clinical trial
Detection the most frequently used Testing index of RA diseases is gone back except anti-cyclic citrulline antibody (anti-CCP antibody) and rheumatoid factor (RF) at present
There are AKA (AKA), Antifilaggrin antibody (AFA), the factor (APF), anti-waveform around anti-cell core
Albumen (AVA) antibody etc. is found to be clinical early diagnosis RA, guiding treatment, prediction prognosis offer important evidence.
Occur RA autoantigens inside RA patient at first, by running up to up to certain extent of reaction for autoantigen,
Autoantigen produces corresponding autoantibody through the T cell in stimulation oversaturation patient's body and memory cell.
The A of CN 101074951 disclose a kind of immune globulin G-glycosylation detection examination of rheumatic arthritis in early-stage diagnosis
Agent and detection method, this method are carried out with glycosylation of the agglutinin and the IgG antibody of mark specifically bound with sugar chain to IgG
Agglutinin-affine immune detection.The A of CN 1712964 disclose a kind of rheumatoid arthritis specific antigen mark ---
Citrullinated fiber connects albumen, and albumen is connected so as to detect rheumatoid arthritis by detecting citrullinated fiber.Above-mentioned side
Method is all detected using single antibody, and detection sensitivity is not high enough, and specificity is not strong, and testing result is not accurate enough.
But the current still early stage patient of some can not be diagnosed, and internationally recognized CCP2 methods detect RA, sensitivity
At most could be to 70%, and early stage patient can not be diagnosed, therefore, RA diagnosis need to develop the side of new more diagnostic significance
Method, to improve the accuracy of diagnostic result.
The content of the invention
It is an object of the invention to provide a kind of compound detection antibody and its application, the detection antibody is anti-citrullinated
The combination of protein antibodies and anti-carbamylation protein antibodies, can a variety of RA autoantibodies of one-time detection, overcome it is single itself
Antibody test is sensitive and specificity deficiency and a variety of autoantibody singles detect operationally cumbersome one by one, greatly improves
The accuracy that detection efficiency and result judge.
To reach the purpose of this invention, the present invention uses following technical scheme：
In a first aspect, the invention provides one kind detect antibody, it is described detection antibody be resist citrullinated protein antibodies and
The combinatorial antibody of anti-carbamylation protein antibodies.
In the present invention, inventor has found to carry out by using anti-citrullinated protein antibodies and anti-carbamylation protein antibodies
Combine to detect, compared to what is detected using single anti-citrullinated protein antibodies or single anti-carbamylation protein antibodies
Detection sensitivity significantly improves, two kinds of albumen synergies, common to detect various autoimmune antibody, compared to using wherein
A kind of albumen improves the efficiency of detection and the accuracy of result as detection antibody.
Preferably, the mass ratio of the anti-citrullinated protein antibodies and anti-carbamylation protein antibodies is (2-4):(1-
3), such as can be 2:1、2:2、2:3、3:1、3:2、3:3、4:1、4:2 or 4:3, preferably 3:2.
Inventor has found that anti-citrullinated protein antibodies and anti-carbamylation protein antibodies using aforementioned proportion can be with
The synergy for resisting citrullinated protein antibodies and anti-carbamylation protein antibodies is played to greatest extent, improves detection efficiency
With the accuracy of result.
Preferably, the working concentration of the combinatorial antibody is 0.1-20 μ g/mL, such as can be 0.1 μ g/mL, 0.2 μ g/
mL、0.3μg/mL、0.4μg/mL、0.5μg/mL、0.6μg/mL、0.8μg/mL、1μg/mL、2μg/mL、3μg/mL、4μg/mL、5
μg/mL、6μg/mL、7μg/mL、8μg/mL、9μg/mL、10μg/mL、11μg/mL、12μg/mL、13μg/mL、14μg/mL、15μ
G/mL, 16 μ g/mL, 17 μ g/mL, 18 μ g/mL, 19 μ g/mL or 20 μ g/mL, preferably 0.1-10 μ g/mL.
According to the present invention, it is described resist citrullinated protein antibodies be selected from, but not limited to, anti-citrullinated fibrin antibody,
Resist citrullinated vimentin antibodies, resist citrullinated II Collagen Type VIs antibody, resist citrullinated core week factor antibody, anti-citrulling
Change silk polyprotein antibody, resist citrullinated bovine albumin antibody, resist citrullinated histamine receptor antibody, anti-citrullinated α-anti-pancreas
Protease antibody, resist the citrullinated antibody of kinesin heavy chain 3, resist citrullinated fibrin original β chain antibodies, be anti-citrullinated
Keratin antibody, resist citrullinated tubulin β chain antibodies, resist citrullinated fibrin antibody, resist citrullinated fiber to glue
Even protein antibodies, anti-citrullinated soluble human HLA I antibody, anti-citrullinated α-enolase antibody, anti-melon ammonia
ASPN antibody is acidified, resists citrullinated cathepsin D's antibody, resist citrullinated beta-actin antibody, anti-citrulling
Change CapZa-1 antibody, resist citrullinated protein disulfide isomerase ER60 precursor antibodies, resist citrullinated mitochondria acetaldehyde to take off
Hydrogen enzyme antibody or resist in citrullinated Sp α receptor antibodies any one or at least two combination, it is preferably anti-citrullinated
Fibrin antibody, resist citrullinated vimentin antibodies, anti-citrullinated α-enolase antibody or resist citrullinated silk to gather
The combination of any one or two kinds in protein antibodies.
In the present invention, the anti-carbamylation protein antibodies are selected from, but not limited to, anti-carbamylation bovine albumin antibody, resisted
Carbamylation fibrin antibody, anti-carbamylation vimentin antibodies, anti-carbamylation collagen antibody or anti-carbamyl
Change enol enzyme antibody in any one or at least two combination, preferably anti-carbamylation bovine albumin antibody, anti-ammonia first
In acylated cellobiose protein antibodies or anti-carbamylation enol enzyme antibody any one or at least two combination.
In the present invention, if there are at least two kinds of anti-citrullinated protein antibodies in combinatorial antibody, specifically resist citrullinated
The mass ratio of protein antibodies is identical, such as the anti-citrullinated protein antibodies are to resist citrullinated fibrin antibody and anti-melon
Propylhomoserin vimentin antibodies, the then mass ratio for resisting citrullinated fibrin antibody and anti-citrullinated vimentin antibodies are
1:1；If similarly there is the anti-carbamylation protein antibodies at least 2 in combinatorial antibody, specific anti-carbamylation protein antibodies
Mass ratio be 1:1.
It is described to include direct coated according to the present invention.
According to the present invention, the solid phase carrier is for any one in ELISA Plate, magnetic bead, affinity membrane or liquid-phase chip or extremely
Few two kinds combination.
According to the present invention, the detection kit also includes first antibody, and the first antibody is selected from, but not limited to, fiber
Albumen original antibody, vimentin antibodies, II Collagen Type VIs antibody, core week factor antibody, silk polyprotein antibody, histamine receptor antibody,
α-antitrypsin antibody, the antibody of kinesin heavy chain 3, fibrinogen β chain antibodies, AKA, tubulin β chains
Antibody, fibrin antibody, fibronectin splicing variants antibody, α-enolase antibody, cathepsin D's antibody, beta-actin
Antibody, CapZa-1 antibody, human IgG antibody, protein disulfide isomerase ER60 precursor antibodies, mitochondria acetaldehyde dehydrogenase resist
In body or Sp α receptor antibodies any one or at least two combination, preferably antibody against fibrinogen, vimentin resist
In body, II Collagen Type VIs antibody, α-enolase antibody or anti-human IgG antibodies any one or at least two combination；
In the present invention, if there is at least two kinds of antibody in first antibody, the quality of specific antibody compares people in the art
Member can be adjusted as needed, and the application is using antibody mass than identical, such as the antibody is combined as fiber
Albumen original antibody, vimentin antibodies, II Collagen Type VIs antibody, α-enolase antibody and the combination of anti-human IgG antibodies, then with fibre
Fibrillarin original antibody, vimentin antibodies, II Collagen Type VIs antibody, α-enolase antibody and the mass ratio 1 of anti-human IgG antibodies:
1:1:1:1 is mixed, and forms compound first antibody.
According to the present invention, the first antibody is marked using biotin, and the biotin (biontin, B) is divided extensively
It is distributed in animal and plant tissue, is often extracted from content higher yolk and hepatic tissue, molecular weight 244.31kD, biotin molecule
There are two cyclic structures, wherein I ring is imidazolone ring, it is the main youth position combined with Avidin；II rings are thiphene ring, on C2
There is a pentanoic acid side chain, its terminal carboxyl group is the only structure of binding antibody and other biological macromolecular, after chemical modification, biology
Element can turn into derivative --- the activated biotin with various active group：1. the activated biotin of labelled protein amino is 2.
The activated biotin of the activated biotin of labelled protein aldehyde radical 3. labelled protein sulfydryl.The biotin of activation is typically used and rubbed
You are linked on antibody than 5 to 10 ratios than 1.
In the present invention, using the compound first antibody of biotin labeling the enzymes such as Multiple Antibodies HRP can be avoided to mark tired
Difficulty, (antibody of separate sources needs with a variety of secondary antibodies, a variety of enzyme marks two difficulty of secondary antibody for also avoiding using multiple HRP marks
The anti-non-specificity that can increase detection and inter-species intersect non-specific identification, can also increase the input and error of experiment).
According to the present invention, the detection kit also includes the Streptavidin of enzyme mark.
According to the present invention, the enzyme is horseradish peroxidase and/or phosphate (AP enzymes).
According to the present invention, the extension rate of the Streptavidin of the enzyme mark is 1000-40000, the enzyme mark
The work quality concentration of Streptavidin is 0.1-1 μ g/mL, and the concentration of the enzyme labelled antibody is pure according to the batch of antibody, concentration
Degree, affinity are just different.
The extension rate of the Streptavidin of enzyme mark for example can be 1000,1200,1500,1800,2000,
2500、3000、3500、4000、5000、6000、7000、8000、9000、10000、11000、13000、15000、18000、
20000th, 23000,25000,28000,30000,33000,35000,38000 or 40000, preferably 10000.
The mass concentration of the Streptavidin of enzyme mark for example can be 0.1 μ g/mL, 0.2 μ g/mL, 0.3 μ g/mL,
0.4 μ g/mL, 0.5 μ g/mL, 0.6 μ g/mL, 0.7 μ g/mL, 0.8 μ g/mL, 0.9 μ g/mL or 1 μ g/mL.
As kit, antibody and enzyme mark detection antibody are its nucleuses, as long as there is both compositions to can be achieved with base
This antibody-antibody association reaction, as auxiliary element, such as it is sample diluting liquid, cleaning solution, substrate solution, terminate liquid, critical
Reference substance, positive control and negative control, can with above two composition is supporting is assembled in a kit, can also be independent
There is provided, therefore kit can include these auxiliary elements in the present invention, can not also include, it is auxiliary present invention preferably comprises these
Co-ingredients, with convenient use.
According to the present invention, the kit also includes negative controls, positive reference substance, critical reference substance, sample dilution
Liquid, confining liquid, cleaning solution, nitrite ion, substrate solution and terminate liquid.
In one particular embodiment of the present invention, the concrete component in the kit is as follows：
(1) it is coated with buffer solution：0.01M-0.5M PBSs, 0.01%-0.5%ProClin300, pH 6.9-9.4；
(2) confining liquid：0.5-10%BSA, 0.01M-0.5M PBS, 0.01%-0.5%ProClin300, pH
6.9-9.4；
(3) Sample dilution：0.5-10%BSA, 0.01M-0.5M Tri-HCL buffer solutions, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(4) yin and yang attribute reference substance dilution：0.5-10%BSA, 0.01M-0.5M PBS, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(5) cleaning solution：0.01%-0.5%Tween-20,0.01M-0.5M PBS, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(6) enzyme labelled antibody dilution：0.5-10%BSA, 0.01M-0.5M PBS, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(7) colorbuffer：0.01M-1M phosphate-citrate buffers, pH 3.0-5.0；
(8) nitrite ion：TMB；
(9) terminate liquid：Sulfuric acid.
Second aspect, the present invention provide the application method of detection kit as described in relation to the first aspect, comprised the following steps：
(1) testing sample is diluted in equilibrium at room temperature 0.5-1h by kit with sample diluting liquid；
(2) testing sample diluted, negative controls and positive reference substance are added in the corresponding hole of ELISA Plate,
Shrouding, it is incubated, is washed；
(3) first antibody is added in ELISA Plate, shrouding, be incubated, washing；
(4) enzyme labelled antibody is added in ELISA Plate, shrouding, is incubated, washs, colour developing, the light for detecting the testing sample is inhaled
The absorbance value of receipts value and the negative controls, the absorbance value of testing sample subtract the absorbance value of negative control, for institute
State the result of testing sample.
Heretofore described incubation and washing are all the ordinary skill in the art, and those skilled in the art can be as needed
The temperature for selecting incubation, the time being incubated, the number of washing, are not particularly limited herein.
According to the present invention, the wavelength of step (3) described detection is 450nm and 630nm double UV checks.
According to the present invention, step (3) absorbance value-negative control for obtaining value for antibody=testing sample
The absorbance value of product.
According to the present invention, the result judgement after the detection is：
During the absorbance value of the absorbance value of the testing sample/critical reference substance >=1, the positive is judged to；
During the absorbance value ＜ 1 of the absorbance value of the testing sample/critical reference substance, feminine gender is judged to.
In one particular embodiment of the present invention, the application method of the detection kit, comprises the following steps：
(1 ') pre-process：Kit each group is placed in equilibrium at room temperature 0.5-1h, with Sample dilution carry out 3-100 times it is dilute
Release；
(2 ') it is loaded：The testing sample diluted is added in the corresponding hole of ELISA Plate, yin and yang attribute compares each 100 μ
L/ holes, with shrouding film shrouding, the ELISA Plate being loaded is put into 200rpm oscillators incubation at room temperature 30-60min, the enzyme that will be incubated
Target, abandon liquid and pat dry, washed 4-6 times with cleaning solution；
(3 ') the compound primary antibody of biotin labeling is added：Enzyme labelled antibody is added in ELISA Plate according to 100 μ l/ holes, with shrouding film
Shrouding, the ELISA Plate being loaded is put into 200rpm oscillators incubation at room temperature 30-60min, the ELISA Plate that will be incubated, abandons liquid bat
It is dry, washed 4-6 times with cleaning solution；
(4 ') enzyme-added mark streptavidin (HRP and/or AP enzymes)：By the enzyme mark streptavidin diluted by 100 μ l/ holes
Add in ELISA Plate, with shrouding film shrouding, the ELISA Plate being loaded is put into 200rpm oscillators incubation at room temperature 30-60min, will be incubated
The ELISA Plate cultivated, abandon liquid and pat dry, washed 4-6 times with cleaning solution；
(5 ') develop the color：Substrate A is with substrate B according to 1:10 be diluted it is well mixed, according to 100 μ l/ holes add ELISA Plate
In, put 400rpm oscillators incubation at room temperature 10-15min；
(6 ') measured value：Terminate liquid is added in ELISA Plate according to 100 μ l/ holes, mixing is patted, sets ELIASA wavelength
450nm and 630nm carries out the OD values that dual wavelength determines each hole.
The detection and analysis principle of kit of the present invention：
This product is using enzyme-linked immunologic diagnosis technology, two kinds of antibody complexes being coated on ELISA Plate：Anti- melon ammonia
Acidified protein antibody and anti-carbamylation protein antibodies, can specifically bind the autoantigen of RA in serum, then pass through biology
The first antibody of element mark is specific to catch the autoantigen that can be combined with first antibody in serum, be not bound with other into
Point can be washed off after washing, with reference to autoantigen washed again after the Streptavidin that HRP and/or AP marks is added,
Through HRP and/or AP catalyzed coloration agent A, developer B, the light wave that redox reaction produces special wavelength occurs, through containing 450nm/
It can be qualitatively judged after the ELIASA reading of 630nm wavelength.
Compared with prior art, the present invention has the advantages that：
(1) the detection antibody of the application kit is the group for resisting citrullinated protein antibodies and anti-carbamylation protein antibodies
Close, can a variety of RA autoantigens of one-time detection, overcome that the detection of single autoantigen is sensitive and specificity deficiency, and a variety of
Autoantigen single detects operationally cumbersome one by one, substantially increases detection efficiency and accuracy that result judges；
(2) two kinds of antibody in the application kit：Resist citrullinated protein antibodies and anti-carbamylation protein antibodies energy
Enough synergies, work in coordination so that the sensitivity of the application kit is 90.0%, and specificity is 97.3%, sensitiveness and
Specificity is above the kit of other existing antibody；
(3) the effect of kit of the present invention can be additionally used in after the clinical examination and treatment of rheumatoid arthritis examines, and has
There are wide market prospects and huge economic benefit.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with being preferable to carry out for the present invention
Example further illustrates technical scheme, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
Embodiment 1：The assembling of kit
1st, main agents：
(1) it is coated with buffer solution：0.01M-0.5M PBSs, 0.01%-0.5%ProClin300, pH 6.9-9.4；
(2) confining liquid：0.5-10%BSA, 0.01M-0.5M PBS, 0.01%-0.5%ProClin300, pH
6.9-9.4；
(3) Sample dilution：0.5-10%BSA, 0.01M-0.5M Tri-HCL buffer solutions, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(4) yin and yang attribute reference substance dilution：0.5-10%BSA, 0.01M-0.5M PBS, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(5) cleaning solution：0.01%-0.5%Tween-20,0.01M-0.5M PBS, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(6) enzyme labelled antibody dilution：0.5-10%BSA, 0.01M-0.5M PBS, 0.01%-0.5%
ProClin300, pH 6.9-9.4；
(7) colorbuffer：0.01M-1M phosphate-citrate buffers, pH 3.0-5.0；
(8) nitrite ion：TMB；
(9) terminate liquid：Sulfuric acid.
2nd, reaction plate working solution is prepared
It is coated with using the protein antibodies of two class posttranslational modifications：Citrullinated fibrin antibody will be resisted citrullinated with resisting
Vimentin antibodies in mass ratio 1:1 mixing, by anti-carbamylation bovine albumin antibody and anti-carbamylation fibrin antibody
In mass ratio 1:1 mixing, by anti-citrullinated protein antibodies and anti-carbamylation protein antibodies according to optimal proportions 3：2 are mixed
Close uniformly, be then diluted combinatorial antibody according to the μ g/mL of best effort concentration 5 with coating buffer, be configured to reaction plate work
Liquid.The reaction plate working solution prepared is added in 96 hole elisa Plates by 100 μ l/ holes, room temperature concussion 15min, puts 4 DEG C overnight
Waste liquid, which is abandoned, after (16-20h) pats dry reaction plate.Cleaning solution is added in 96 hole reaction plates by 250 μ l/ holes, 2min is stored at room temperature, abandons
Waste liquid pats dry reaction plate, board-washing 3 times, the confining liquid prepared is added in 96 hole reaction plates by 200 μ l/ holes, room temperature concussion is incubated
Educate to outwell after 2-4h and pat dry reaction plate.Reaction plate after patting dry, it is put into after 37 DEG C of incubators dry 3h and reaction plate is put into dry
Drying prescription uses aluminium foil bag hermetic package.
3rd, the preparation of the compound first antibody of biotin labeling：
Antibody against fibrinogen, vimentin antibodies, II Collagen Type VIs antibody, the α-enolase antibody purified is chosen,
Each 1mg of anti-human IgG antibodies, carries out biotinylation mark respectively, and long-chain activated biotin is dissolved in dimethyl by concentration 1mg/ml
In sulfoxide；It will wait to be coupled and purified antibody is dissolved in 0.1mol/L pH9.0 cold sodium bicarbonate solution by concentration 1mg/ml
In；By activated biotin liquid and antibody-solutions to be coupled by 1：8 mixing, incubate 4h at room temperature；To 0.05mol/ at 4 DEG C
L pH7.2 PBS 24h, wherein changing liquid 4 times, to remove uncombined free biotin, the antibody marked is by 1：1：1：
1：1 ratio mixes, and forms compound primary antibody.
4th, the preparation of enzyme mark streptavidin
The streptavidin that horseradish peroxidase (HRP) marks is diluted 10000 using enzyme combination diluent
Enzyme mark streptavidin is configured to again, and its work quality concentration is 0.6 μ g/mL.
5th, yin and yang attribute control is prepared
The serum and normal human serum for being diagnosed as patient with rheumatoid arthritis using being collected from hospital, are used after treatment
Yin and yang attribute reference substance dilution is diluted according to proper proportion.
The kit of establishment includes the following aspects：96 hole elisa Plates, negative control, positive control, critical control, sample
This dilution, cleaning solution, enzyme labelled antibody, nitrite ion, terminate liquid, shrouding film and specification.
Comparative example 1：CCP diagnostic kits
With artificial synthesized citrullinated polypeptide coated elisa plate, Sample serum is pressed after dilution and added instead per the μ l of hole 100
Answer plate sample aerial, choose remaining hole and add standard items or reference material, react at room temperature 1h, the anti-of HRP marks is added after scrubbed
Human IgG antibody, 30min is reacted at room temperature, tmb substrate is added after scrubbed, reacts at room temperature 15min, add terminate liquid terminating reaction,
Absorbance value is detected under 450nm and 630nm with spectrophotometer, the result of calculation compared with standard items or reference material.
Comparative example 2：
With anti-citrulling antibody coated elisa plate, Sample serum is pressed after dilution adds reaction plate sample sky per the μ l of hole 100
In, choose remaining hole and add standard items or reference material, react at room temperature 1h, the compound primary antibody of biotin labeling is added after scrubbed,
1h is reacted at room temperature, it is scrubbed to add the enzyme mark streptavidin diluted afterwards, 30min is reacted at room temperature, TMB bottoms are added after scrubbed
Thing, 15min is reacted at room temperature, adds terminate liquid terminating reaction, absorbance value is detected under 450nm and 630nm with spectrophotometer,
The result of calculation compared with standard items or reference material.
Comparative example 3：
With anti-carbamyl amine antibody coated elisa plate, Sample serum is pressed after dilution and reaction plate sample is added per the μ l of hole 100
In the air, choose remaining hole and add standard items or reference material, react at room temperature 1h, compound the one of biotin labeling is added after scrubbed
It is anti-, 1h is reacted at room temperature, it is scrubbed to add the enzyme mark streptavidin diluted afterwards, 30min is reacted at room temperature, is added after scrubbed
Tmb substrate, 15min is reacted at room temperature, add terminate liquid terminating reaction, detection light is inhaled under 450nm and 630nm with spectrophotometer
Receipts value, the result of calculation compared with standard items or reference material.
The application method of kit
(1) pre-process：Kit each group is placed in equilibrium at room temperature 0.5-1h, carried out sample with Sample dilution dilute
Release；
(2) it is loaded：The testing sample diluted is added in the corresponding hole of ELISA Plate, yin and yang attribute compares each 100 μ l/
Hole, with shrouding film shrouding；
(3) it is incubated：The ELISA Plate being loaded is put into 200rpm oscillators incubation at room temperature 30-60min；
(4) wash：The ELISA Plate that will be incubated, abandon liquid and pat dry, washed 4-6 times with cleaning solution；
(5) the compound primary antibody of biotin labeling is added：Enzyme labelled antibody is added in ELISA Plate according to 100 μ l/ holes, sealed with shrouding film
Plate；
(6) it is incubated：The ELISA Plate being loaded is put into 200rpm oscillators incubation at room temperature 30-60min；
(7) wash：The ELISA Plate that will be incubated, abandon liquid and pat dry, washed 4-6 times with cleaning solution；
(8) HRP enzyme mark streptavidins are added：The enzyme mark streptavidin diluted is added into ELISA Plate by 100 μ l/ holes
In, with shrouding film shrouding；
(9) it is incubated：The ELISA Plate being loaded is put into 200rpm oscillators incubation at room temperature 30-60min；
(10) wash：The ELISA Plate that will be incubated, abandon liquid and pat dry, washed 4-6 times with cleaning solution；
(11) develop the color：Substrate A is with substrate B according to 1:10 be diluted it is well mixed, according to 100 μ l/ holes add ELISA Plate
In, put 400rpm oscillators incubation at room temperature 10-15min；
(12) measured value：Terminate liquid is added in ELISA Plate according to 100 μ l/ holes, mixing is patted, sets ELIASA wavelength
450nm and 630nm carries out the OD values that dual wavelength determines each hole.
By using the kit progress detection sensitivity in kit of the present invention (embodiment 1) and comparative example 1-3 and specifically
Property：
Sample size and type：210 clinical serums altogether, wherein normal person 110, RA patient 60, systemic red
Spot lupus patient serum 40.Experimental result such as following table：
The kit clinical diagnosis result statistical form of 1 embodiment of table 1
Positive coincidence rate=54/ (54+6) × 100%=90.0%；
Negative match-rate=146/ (150) × 100%=97.3%；
Crude agreement=(54+146)/210 × 100%=95.2%；
Youden index=54/ (54+6)+146/ (146+4) -1=0.97；
The CCP kits (comparative example 1) of table 2 and clinical diagnosis result statistical form
Positive coincidence rate=43/ (43+17) × 100%=71.7%；
Negative match-rate=136/ (136+14) × 100%=90.7%；
Crude agreement=(43+136)/210 × 100%=85.2%；
Youden index=43/ (43+17)+136/ (136+14) -1=0.624；
Kit clinical diagnosis result statistical form in the comparative example 2 of table 3
Positive coincidence rate=42/ (42+18) × 100%=70%；
Negative match-rate=130/ (130+20) × 100%=86.7%；
Crude agreement=(42+130)/210 × 100%=81.9%；
Youden index=42/ (42+18)+130/ (130+20) -1=0.567；
Kit clinical diagnosis result statistical form in the comparative example 3 of table 4
Positive coincidence rate=41/ (41+19) × 100%=68.3%；
Negative match-rate=136/ (136+14) × 100%=90.7%；
Crude agreement=(41+136)/210 × 100%=84.2%；
Kit of the application using the preparation of a variety of RA Idiotypes antigen detection methods is can be seen that from the result of table 1- tables 4
Either sensitivity or specificity are superior to other and use detection kit.The sensitivity of the application kit is 90.0%, special
The opposite sex is 97.3%.Originally test result indicates that, there is spy using the kit prepared using new RA Idiotypes antigen detection method
It is different in nature strong, the features such as high sensitivity.
Embodiment 2
Compared with Example 1, except the quality for resisting citrullinated fibrin antibody and anti-carbamylation bovine albumin antibody
Than for 3:It is other same as Example 1 outside 2.
Embodiment 3
Compared with Example 1, except resisting citrullinated α-antitrypsin antibody and anti-carbamylation collagen antibody
Mass ratio is 3:It is other same as Example 1 outside 2.
Embodiment 4
Compared with Example 1, except the quality for resisting citrullinated vimentin antibodies and anti-carbamylation fibrin antibody
Than for 1:1, the work quality concentration of the combinatorial antibody is 10 μ g/mL, and first antibody is antibody against fibrinogen, vimentin
Antibody and II Collagen Type VI antibody, mass ratio 1:1:It is other same as Example 1 outside 1.
Embodiment 5
Compared with Example 1, except the quality of anti-citrullinated α-enolase antibody and anti-carbamylation enol enzyme antibody
Than for 2:1, the work quality concentration of the combinatorial antibody is 3 μ g/mL, and first antibody is histamine receptor antibody, α-anti-tryptose
Enzyme antibody and the antibody of kinesin heavy chain 3, mass ratio 1:1:It is other same as Example 1 outside 1.
Embodiment 6
Compared with Example 1, except the mass ratio for resisting citrullinated silk polyprotein antibody and anti-carbamylation enol enzyme antibody
For 4:1, the work quality concentration of the combinatorial antibody is 0.1 μ g/mL, and first antibody is silk polyprotein antibody, its work quality
Concentration be 1 μ g/mL outside, it is other same as Example 1.
Embodiment 7
Compared with Example 1, except the mass ratio of citrullinated fiber Fibronectin and carbamylation enolase is 2:3, institute
The work quality concentration for stating combinatorial antibody is 20 μ g/mL, and first antibody is mitochondria acetaldehyde-dehydrogenase enzyme antibody, and its work quality is dense
Spend for outside 0.1 μ g/mL, it is other same as Example 1.
Comparative example 4
Compared with Example 1, it is 5 except mass ratio of the citrullinated protein antibodies with anti-carbamylation protein antibodies is resisted:1 it
Outside, it is other same as Example 1.
Comparative example 5
Compared with Example 1, it is 1 except mass ratio of the citrullinated protein antibodies with anti-carbamylation protein antibodies is resisted:2 it
Outside, it is other same as Example 1.
Comparative example 6
Compared with Example 1, in addition to the work quality concentration of the combinatorial antibody is 23 μ g/mL, other and embodiment 1
It is identical.
Comparative example 7
Compared with Example 1, it is other same as Example 1 in addition to the first antibody of biotin labeling is not used.
The sensitivity and specificity detection of kit
Embodiment 2-7 and comparative example 4-7 is carried out to the detection of sensitivity and specificity, it is as a result as shown in table 5 below：
Table 5
| Positive coincidence rate | Negative match-rate | Crude agreement | |
| Embodiment 1 | 90.0% | 97.3% | 97.1% |
| Embodiment 2 | 89.5% | 94.1% | 92.4% |
| Embodiment 3 | 88.2% | 94.4% | 91.6% |
| Embodiment 4 | 89.4% | 89.4% | 90.4% |
| Embodiment 5 | 87.8% | 90.7% | 88.2% |
| Embodiment 6 | 85.9% | 86.7% | 89.6% |
| Embodiment 7 | 84.3% | 88.5% | 90.2% |
| Comparative example 1 | 71.7% | 90.7% | 85.2% |
| Comparative example 2 | 75% | 86.7% | 83.3% |
| Comparative example 3 | 68.3% | 90.7% | 84.2% |
| Comparative example 4 | 80.1% | 86.7% | 85.4% |
| Comparative example 5 | 79% | 89.2% | 85.6% |
| Comparative example 6 | 81.5% | 90.3% | 88.7% |
| Comparative example 7 | 70.4% | 83.1% | 86.8% |
As can be seen from Table 5, from embodiment as can be seen that when anti-citrullinated protein antibodies and anti-carbamylation albumen resist
The mass ratio 3 of body:2, and Detection results are most when resisting citrullinated protein antibodies and anti-carbamylation protein antibodies to be mixed antibody
Good, with the change for the mass ratio for resisting citrullinated protein antibodies and anti-carbamylation protein antibodies, its Detection results is under
Drop.
By embodiment 1 and comparative example 1-3 contrast as can be seen that but coated antibody be single antibody when, its detect imitate
Fruit is not as the present invention；Contrasted by embodiment 1 and comparative example 4-5, resist citrullinated protein antibodies and anti-carbamylation albumen to resist
The change of the mass ratio of body make it that detecting precision and specificity declines, especially citrullinated Vimentin and carbamyl chemical fibre
The former white mass ratio of fibrillarin is not at (2-4):In the range of (1-3), precision and specificity are detected and at (2-4):(1-3) scope
Inside it is very different, it is seen that resist the combinatorial antibody of citrullinated protein antibodies and anti-carbamylation protein antibodies to pass through specific quality
Than the effect for achieving precision and specificity raising；Contrasted by embodiment with comparative example 6-7 as can be seen that being combined when changing
The work quality concentration of antibody or the first antibody of biotin labeling is not used all to influence testing result.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
Claims (10)
1. a kind of detection kit, it is characterised in that the kit includes solid phase carrier, and the solid phase carrier is coated with compound
Antibody, the compound antibody are the combination for resisting citrullinated protein antibodies and anti-carbamylation protein antibodies.
2. detection kit according to claim 1, it is characterised in that described to resist citrullinated protein antibodies and anti-ammonia first
The mass ratio of acylated protein antibody is (2-4):(1-3), preferably 3:2；
Preferably, the work quality concentration of the combinatorial antibody is 0.1-20 μ g/mL, preferably 0.1-10 μ g/mL.
3. detection kit according to claim 1 or 2, it is characterised in that described to resist citrullinated protein antibodies to be anti-
Citrullinated fibrin antibody, resist citrullinated vimentin antibodies, resist citrullinated II Collagen Type VIs antibody, be anti-citrullinated
Core week factor antibody, resist citrullinated silk polyprotein antibody, resist citrullinated bovine albumin antibody, resist citrullinated histamine receptor
Antibody, resist citrullinated α-antitrypsin antibody, resist the citrullinated antibody of kinesin heavy chain 3, resist citrullinated fiber egg
White former β chain antibodies, resist citrullinated keratin antibody, resist citrullinated tubulin β chain antibodies, resist citrullinated fibrin
Antibody, resist citrullinated fiber Fibronectin antibody, resist citrullinated soluble human HLA I antibody, be anti-citrullinated
α-enolase antibody, anti-citrullinated ASPN antibody, anti-citrullinated cathepsin D's antibody, anti-citrullinated β-
Actin antibodies, resist citrullinated CapZa-1 antibody, resist citrullinated protein disulfide isomerase ER60 precursor antibodies,
Resist citrullinated mitochondria acetaldehyde-dehydrogenase enzyme antibody or resist any one in citrullinated Sp α receptor antibodies or at least two
Combination, preferably resist citrullinated fibrin antibody, resist citrullinated vimentin antibodies, resist citrullinated α-enolase
Antibody or the combination for resisting any one or two kinds in citrullinated silk polyprotein antibody；
Preferably, the anti-carbamylation protein antibodies are anti-carbamylation bovine albumin antibody, anti-carbamylation fibrin
In antibody, anti-carbamylation vimentin antibodies, anti-carbamylation collagen antibody or anti-carbamylation enol enzyme antibody
Any one or at least two combination, preferably anti-carbamylation bovine albumin antibody, anti-carbamylation fibrin antibody
In anti-carbamylation enol enzyme antibody any one or at least two combination.
4. according to the detection kit any one of claim 1-3, it is characterised in that the coating includes directly wrapping
Quilt；
Preferably, the solid phase carrier is any one in ELISA Plate, magnetic bead, affinity membrane or liquid-phase chip or at least two
Combination.
5. according to the detection kit any one of claim 1-4, it is characterised in that the detection kit also includes
First antibody；
Preferably, the first antibody includes antibody against fibrinogen, vimentin antibodies, II Collagen Type VIs antibody, core Zhou Yinzi
Antibody, silk polyprotein antibody, histamine receptor antibody, α-antitrypsin antibody, the antibody of kinesin heavy chain 3, fibrinogen β
Chain antibody, AKA, tubulin β chain antibodies, fibrin antibody, fibronectin splicing variants antibody, α-enolase
Antibody, cathepsin D's antibody, beta-actin antibody, CapZa-1 antibody, human IgG antibody, protein disulfide isomerase
In ER60 precursor antibodies, mitochondria acetaldehyde-dehydrogenase enzyme antibody or Sp α receptor antibodies any one or at least two combination, it is excellent
Elect as in antibody against fibrinogen, vimentin antibodies, II Collagen Type VIs antibody, α-enolase antibody or anti-human IgG antibodies
Any one or at least two combination；
Preferably, the first antibody is marked using biotin.
6. according to the detection kit any one of claim 1-5, it is characterised in that the detection kit also includes
The Streptavidin of Avidin, preferably enzyme mark；
Preferably, the enzyme is horseradish peroxidase and/or phosphate；
Preferably, the extension rate of the Streptavidin of the enzyme mark is 1000-40000, preferably 10000；
Preferably, the work quality concentration of the Streptavidin of the enzyme mark is 0.1-1 μ g/mL；
Preferably, the detection kit also includes negative controls, positive reference substance, critical reference substance, sample diluting liquid, envelope
Close liquid, cleaning solution, nitrite ion, substrate solution and terminate liquid.
7. the application method of the detection kit according to any one of claim 1-6, it is characterised in that including following step
Suddenly：
(1) testing sample is diluted in equilibrium at room temperature 0.5-1h by kit with sample diluting liquid；
(2) testing sample diluted, negative controls and positive reference substance are added in the corresponding hole of ELISA Plate, is sealed
Plate, it is incubated, is washed；
(3) first antibody is added in ELISA Plate, shrouding, be incubated, washing；
(4) enzyme labelled antibody is added in ELISA Plate, shrouding, is incubated, washs, colour developing, detect the absorbance value of the testing sample
With the absorbance value of the negative controls, the absorbance value of testing sample subtracts the absorbance value of negative control, is treated to be described
The result of test sample product.
8. application method according to claim 7, it is characterised in that the multiple of the dilution described in step (1) is 3-100,
Preferably 20；
Preferably, the wavelength of step (4) described detection is 450nm and 630nm double UV checks；
Preferably, the absorbance value of step (4) described testing sample subtracts the absorbance value of the negative controls.
9. the application method according to claim 7 or 8, it is characterised in that the result judgement of the testing sample is：
During the absorbance value of the absorbance value of the testing sample/critical reference substance >=1, the positive is judged to；
During the absorbance value ＜ 1 of the absorbance value of the testing sample/critical reference substance, feminine gender is judged to.
10. detection kit according to any one of claim 1-6 prepare the detection reagent of rheumatoid arthritis and/
Or the application in detection medicine.
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