CN101403749B - Reagent kit for auxiliary diagnosis of autoimmune disease through glucosan specificity antibody - Google Patents
Reagent kit for auxiliary diagnosis of autoimmune disease through glucosan specificity antibody Download PDFInfo
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Abstract
The invention discloses a kit used for helping to diagnose autoimmune diseases by a dextran specific antibody. Both an ELISA kit used for helping to diagnose systemic lupus erythematosus and an ELISA kit used for helping to diagnose rheumatoid arthritis which are discloses by the invention comprise antigens used for coating; the antigen used for coating can be Alpha-1 and 4-dextran, and astragalus polysaccharide is preferable; and the antigen used for coating can also be Alpha-1 and 6-dextran, and dextran is preferable. The kit can be used for helping to diagnose the systemic lupus erythematosus and the rheumatoid arthritis and has important application value.
Description
Technical field
The present invention relates to kit through the glucosan specificity antibody auxiliary diagnosis of autoimmune disease.
Background technology
The basic functions of immune system is identification oneself and nonego, and then reaches protection and self avoid the purpose that extraneous pathogenic microorganism is invaded and harassed.Autoimmunity is the ability of body immune system to self component generation immune response, and this phenomenon is present in all individualities.Autoimmune response does not under normal circumstances cause pathologic damage; But immune system still can produce excessive immune response to self cell or tissue composition under certain conditions; Cause autologous tissue or organ inflammatory injury, excessive immune response is taken place self component this body and the morbid state that causes is referred to as autoimmunity disease.Autoimmunity disease can be divided into organ specificity autoimmunity disease and general autoimmunity disease.Compare with other disease, the characteristics of autoimmunity disease comprise: 1. can detect autoantibody or autoreactive T cell in patient's body; 2. the process of the state of an illness and autoimmune response intensity are closely related; 3. mostly is chronic progressive disease, outbreak repeatedly, and chronic delay has a strong impact on patient's ability to work and quality of life; 4. obvious genetic tendency and sex trend are arranged, and female patients is artificially many than andropathy; 5. different inducements is arranged, and the sick form of expression is also complicated various.
Contain NAA in the healthy subjects serum, comprise IgM, IgG and IgA, its identifying object comprises DNA, actin and IgG etc.The generation of natural antibody does not also rely on the stimulation of exogenous antigen; These production of antibodies might stem from resident flora in the intestines and stomach and induce generation; Also maybe be relevant with the dead autoreactivity B1 cell of body itself, the affinity of these autoantibodies and autoantigen is relatively low and have a cross reactivity.Different with normal person's autoantibody, cause that autoimmunity disease causes the specificity of autoantibody of pathologic damage stronger, higher with autoantigen affinity, most antibody are IgG, they are produced by antigenic stimulus also.Research shows; Spontaneous antibody can make body cause pathology damage through number of mechanisms; These mechanism comprise: 1. antibody-mediated CDCC: autoantibody combines with the autoantigen of cell surface, makes target cell attach to phagocyte surface and then by phagic lysis through opsonic action; 2. the middle cooperation of antibody is used: bioactive antigen or receptors bind are arranged in spontaneous antibody and the body, make its deactivation or loss of function and cause the appearance of pathological reaction symptom.The anti-acetylcholine receptor antibodies that occurs in myasthenia gravis patient's body as one of autoimmunity disease is exactly typical case's representative of this mechanism of action.3. autoantibody combines with antigen to form immune complex and causes tissue damage: autoantibody combines to form immune complex with the antigen that is in free state, be deposited on part or whole body vascular wall basilar memebrane or tissue space under certain conditions; But immune complex activating complement and the reaction that causes inflammation.Systemic loupus erythematosus just embodies this characteristics of incidence.ADA is located deposition at subcutaneous, joint and GBM etc. and is caused inflammatory reaction in the Patients with SLE body; 4. active cell surface receptor and cause autoimmunity disease: it is the autoimmunity disease that is caused by thyrotropin receptor specific autoantibody in the serum that toxicity is filled the air capable goitre; Autoantibody in patient's body continuingly acts on the tsh receptor of thyroid cell, stimulates the thyroxine of thyroid cell hypersecretion and causes patient's hyperthyroidism.NAA possibly have certain physiological function, and autoantibody can cause autoimmunity disease under pathological state; Also have some autoantibodies itself generally not cause autoimmunity disease, but can offer help for the auxiliary diagnosis of some diseases, sclerosis has the auxiliary diagnosis meaning to the primary bile duct like self AMA.
The autoantibody level has become one of important indicator of diagnosis autoimmunity disease.Such as, (Systemic Lupus Erythematosus SLE) is a kind of autoimmune disease that relates to many systems and internal organs to systemic loupus erythematosus, because cell and humoral immune function obstacle produce multiple autoantibody.Can involve skin, serous coat, joint, kidney and central nervous system etc., and be characteristic with autoimmunity, have multiple autoantibody in patient's body, not only influence humoral immunity, also influence cellular immunity, complement system also changes; Pathogenesis mainly is because immune complex forms; The state of an illness is outbreak repeatedly and alleviates alternation procedure; This disease is seen with young women more; China's morbidity rate is higher than western countries, maybe be relevant with inherent cause.Autoantibody be the important diagnostic index of systemic loupus erythematosus unusually.
In the clinical department of internal medicine disease, the misdiagnosis rate and the rate of missed diagnosis of autoimmunity class disease are higher, and main cause mainly comprises the concealment of autoimmunity class disease incidence; Indeterminate being prone to obscured with other Medicine and Surgery disease mutually in clinical manifestation; The course of disease is long, and disease has arrived the serious stage when often making a definite diagnosis.The diagnostic criteria of autoimmunity class disease is main with grading type in addition, and certain subjective factor is arranged, shortage can be directly as the lab index of diagnostic criteria.Difficulty in these diagnosis directly causes the delay and the poor prognosis of treating.Therefore, sensitive and special lab index is extremely important for correct diagnosis, early detection and the prognosis evaluation of autoimmunity class disease.Such as; For systemic loupus erythematosus and rheumatoid arthritis; Laboratory diagnosis index commonly used at present is antinuclear antibodies and rheumatoid factor; But they still have very high loss and fallout ratio, so seek and identify that new lab index is used for diagnosing or auxiliary diagnosis autoimmunity class disease is very important.
Polysaccharide is the big molecule that is polymerized by 10 above monose, and molecular weight to millions of, is one of four big base substances that constitute vital movement by tens thousand of, and is closely related with different physiological roles.
Glucosan is a kind of polysaccharide of tool physiologically active, is made up of D-glucopyranose monomer, extensively is present in microorganism, the plant and animal.
For estimating the using value of an index in diagnosing the illness, need make check respectively to susceptibility, the specificity of this index, have only to possess very high susceptibility simultaneously and specificity could be as diagnosis index.Be the ROC curve with statistical method while detection sensitivity and specificity; The ROC curve is claimed experimenter's performance curve (receive operating characteristic curve) again, is the overall target that reflection detects index susceptibility and specificity continuous variable.Calculate ROC TG-AUC AUC and estimate diagnosis efficiency, area can think that greater than 0.5 this index has diagnostic significance, and area is big more, judges that value is high more.
Summary of the invention
The purpose of this invention is to provide kit through the glucosan specificity auxiliary diagnosis of autoimmune disease.
The ELISA kit of assistant diagnosis system property lupus erythematosus provided by the invention (SLE) comprises the antigen that is used to encapsulate; The said antigen that is used to encapsulate can be α-1,4-glucosan, preferred astragalus polyose; The said antigen that is used to encapsulate also can be α-1,6-glucosan, preferred dextran.
Said kit can comprise that also enzyme target two is anti-; It is anti-human IgG that said enzyme target two resists.
Said enzyme target two is anti-to can be any anti-human IgG, like goat anti human IgG, rabbit anti-human igg or mouse-anti human IgG.
Said kit also comprises the conventional reagent of ELISA, like following reagent: bovine serum albumin(BSA), PBS damping fluid, PBST solution, PBST-BSA solution, OPD-phosphoric acid citrate buffer solution, sulfuric acid and 96 hole ELISA Plates; The concentration of said PBS damping fluid is 0.15mol/L, and pH is 7.2; Said PBST solution is for adding the solution that Tween 20 obtains in said PBS damping fluid, the volume ratio of said Tween 20 and said PBS damping fluid is 0.05%; Said PBST-BSA solution is for adding the solution that bovine serum albumin(BSA) obtains in said PBST damping fluid, the final concentration of said bovine serum albumin(BSA) is 1 gram/L; Said OPD-phosphoric acid citrate buffer solution is for adding the solution that o-phenylenediamine obtains at the phosphoric acid citrate buffer solution; The mass percent concentration of said o-phenylenediamine is 0.06%; The pH of said phosphoric acid citrate buffer solution is 5.0; Concentration of phosphoric acid is 0.1mol/L, and the concentration of citric acid is 0.05mol/L.
The ELISA kit of auxiliary diagnosis rheumatoid arthritis provided by the invention (RA) comprises the antigen that is used to encapsulate; The said antigen that is used to encapsulate can be α-1,4-glucosan, preferred astragalus polyose; The said antigen that is used to encapsulate also can be α-1,6-glucosan, preferred dextran.
Said kit can comprise that also enzyme target two is anti-; It is anti-human IgG that said enzyme target two resists.
Said enzyme target two is anti-to can be any anti-human IgG, like goat anti human IgG, rabbit anti-human igg or mouse-anti human IgG.
When the said antigen that is used to encapsulate is α-1, during the 4-glucosan, said kit can comprise that also enzyme target two is anti-; It is anti-people IgA that said enzyme target two resists.
Said enzyme target two is anti-to can be any anti-people IgA, like goat anti people IgA, the anti-people IgA of rabbit or mouse-anti people IgA.
Said kit also comprises the conventional reagent of ELISA, like following reagent: bovine serum albumin(BSA), PBS damping fluid, PBST solution, PBST-BSA solution, OPD-phosphoric acid citrate buffer solution, sulfuric acid and 96 hole ELISA Plates; The concentration of said PBS damping fluid is 0.15mol/L, and pH is 7.2; Said PBST solution is for adding the solution that Tween 20 obtains in said PBS damping fluid, the volume ratio of said Tween 20 and said PBS damping fluid is 0.05%; Said PBST-BSA solution is for adding the solution that bovine serum albumin(BSA) obtains in said PBST damping fluid, the final concentration of said bovine serum albumin(BSA) is 1 gram/L; Said OPD-phosphoric acid citrate buffer solution is for adding the solution that o-phenylenediamine obtains at the phosphoric acid citrate buffer solution; The mass percent concentration of said o-phenylenediamine is 0.06%; The pH of said phosphoric acid citrate buffer solution is 5.0; Concentration of phosphoric acid is 0.1mol/L, and the concentration of citric acid is 0.05mol/L.
The present invention has studied serum antibody and cell surface receptor to alpha-glucans immunity recognition point; The result shows α-1; 4 glucosan specificity antibody IgG titre in patient SLE and patient RA obviously raises, and α-1,6 glucosan specificity antibody IgG titre in patient SLE and patient RA obviously raises; α-1,4 glucosan specificity antibody IgA titre in patient RA obviously raises.α-1 in the SLE patient body; The ROC TG-AUC AUC of 4 glucosan specificity antibody IgG reaches 0.9839, the α-1 in the RA patient body; The ROC TG-AUC AUC of 4 glucosan specificity antibody IgG reaches 0.7989, the α-1 in the SLE patient body; The ROC TG-AUC AUC of 6 glucosan specificity antibody IgG reaches 0.8633, the α-1 in the RA patient body; The ROC TG-AUC AUC of 6 glucosan specificity antibody IgG reaches 0.7800, the ROC TG-AUC AUC of α-1, the 4 glucosan specificity antibody IgA in the RA patient body reaches 0.67.Kit of the present invention can be used for assistant diagnosis system property lupus erythematosus and rheumatoid arthritis, has important use and is worth.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 is for detecting α-1, the 4 glucosan specificity antibody IgG in the SLE patient body.
Fig. 2 is the ROC curve of the kit of embodiment 1.
Fig. 3 is the sensitivity that the kit of embodiment 1 detects the SLE disease.
Fig. 4 is for detecting α-1, the 4 glucosan specificity antibody IgG in the RA patient body.
Fig. 5 is the ROC curve of the kit of embodiment 2.
Fig. 6 is the sensitivity that the kit of embodiment 2 detects the RA disease.
Fig. 7 is for detecting α-1, the 6 glucosan specificity antibody IgG in the SLE patient body.
Fig. 8 is the ROC curve of the kit of embodiment 3.
Fig. 9 is for detecting α-1, the 6 glucosan specificity antibody IgG in the RA patient body.
Figure 10 is the ROC curve of the kit of embodiment 4.
Figure 11 is for detecting α-1, the 4 glucosan specificity antibody IgA in the RA patient body.
Figure 12 is the ROC curve of the kit of embodiment 5.
Embodiment
Experimental technique among the following embodiment like no specified otherwise, is conventional method.
The preparation of embodiment 1, SLE detection kit and application
One, the preparation of kit
The composition of kit is following:
Astragalus polyose (APS): available from Pharmagenesi s company, be made up of D-glucose, main chain is through α-1, and the 4-glycoside bond connects, and side chain is through α-1, and the 6-glycoside bond connects.
Horseradish peroxidase (Horseradish peroxidase, HRP) the anti-human IgG of labelled goat: available from SBA company;
Bovine serum albumin(BSA) (BSA): available from Hyclone company;
The PBS damping fluid: the concentration of PBS damping fluid is 0.15mol/L, and pH is 7.2;
PBST solution: in above-mentioned PBS damping fluid, add Tween 20, the volume ratio of Tween 20 and PBS damping fluid is 0.05%;
PBST-BSA solution: in above-mentioned PBST solution, add bovine serum albumin(BSA) (BSA), the final concentration of bovine serum albumin(BSA) is 1 gram/L;
OPD-phosphoric acid citrate buffer solution (pH 9.6): add o-phenylenediamine at the phosphoric acid citrate buffer solution, the mass percent concentration of o-phenylenediamine is 0.06; The pH of said phosphoric acid citrate buffer solution is 5.0, and concentration of phosphoric acid is 0.1mol/L, and the concentration of citric acid is 0.05mol/L;
Sulfuric acid;
96 hole ELISA Plates: Costar company.
Two, the application of kit
The kit auxiliary diagnosis SLE disease of applying step one preparation.
1, serum collection
3 groups of crowds' serum below collecting respectively:
First group (SLE): 30 routine patients SLE that make a definite diagnosis meet American Rheumatism Association's diagnostic criteria, and are the active stage patient;
Second group (NS): picked at random 30 routine healthy subjects, as negative control;
The 3rd group (CA): the 30 routine tumour patients of making a definite diagnosis, wherein, lung cancer patient (7 people), leukaemia people (4 people), cervical carcinoma patient (6 people), oophoroma patient (7 people), carcinoma of endometrium patient (6 people).
2, detect
Encapsulate elisa plate with APS, detect 3 groups of APS specific antibody IgG in crowd's serum respectively.Concrete grammar is following:
1) with APS (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 2%BSA37 ℃ of sealing 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l respectively and uses the dilutability of PBST-BSA solution dilution to be the 30 routine tumour patient serum of 1:200, dilutability 30 routine SLE patients serums as 1:200 as the 30 routine healthy subjects serum (HDS) of 1:200, dilutability; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat anti-human igg of the HRP mark of 1:5000 dilution again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The OD reading of healthy subjects control group is controlled at 0.2-0.3 in all testing, and other absorbance reading of 2 groups all obtains association index (binding index) divided by the control group reading, and all display result are all represented with association index.Antibody horizontal is used relatively difference of Student ' s t test between each group crowd.In addition, set healthy subjects group average antibody level and add 3 times of readings after the standard deviation as judging positive cutoff value (cut-off value), and calculate with this and respectively to organize positive rate.Positive rate between each group crowd is used relatively difference of Fisher ' sexact check.
3 groups of crowds' APS specific antibody IgG level is seen Fig. 1.3 groups of crowds' positive rate is seen table 1.
Anti-α-1 in table 1 different crowd, 4-glucosan IgG antibody positive rate relatively
| The HS group | The SLE group | The CA group |
| 3.3％ | 86.7％ (<0.0001) | 6.7％ (0.388) |
The result shows: do not have IgG antibody in healthy subjects and the tumour patient serum, but the IgG antibody horizontal improves obviously among most SLE patients serum.SLE patients serum antibody horizontal has been compared significant difference (p < 0.0001) with healthy subjects serum antibody level, and tumour patient serum antibody level and the horizontal no difference of science of statistics of healthy subjects serum antibody.SLE patients serum antibody positive rate level has been compared significant difference with healthy subjects positive rate of antibody level, and tumour patient positive rate of antibody level and the horizontal no difference of science of statistics of healthy subjects positive rate of antibody.
3, the using value evaluation of kit
The ROC curve is claimed experimenter's performance curve (receive operating characteristiccurve) again, is the overall target that reflection detects index susceptibility and specificity continuous variable.Calculate ROC TG-AUC AUC and can estimate diagnosis efficiency, area can think that greater than 0.5 this index has diagnostic significance, and the big more judgement of area is worth high more.
Be the using value of kit in diagnosing SLE of evaluation procedure one preparation, draw the ROC curve, see Fig. 2 according to the antibody horizontal of patient group and control group.The result shows: TG-AUC AUC reaches 0.9839, proves that this kit has certain using value in diagnosing SLE.
Three, the sensitivity of kit is measured
From 30 routine SLE patients serums, choose three higher samples of identification intensity respectively, do the serum dilution test experience with 30 routine normal human serum samples.
1) with APS (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 2%BSA37 ℃ of sealing 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l respectively with 30 routine healthy subjects serum (HDS) of the different dilutabilitys (1/50,1/100,1/200,1/400,1/800,1/1600) of PBST-BSA solution dilution, three higher samples of identification intensity; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat anti-human igg of the HRP mark of 1:5000 dilution again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The result sees Fig. 3.
The result shows that SLE patients serum (SLE-2, SLE-21, SLE-30) still has tangible identification to APS when serum dilution is 1:800, forms marked difference with normal human serum (N40).
The preparation of embodiment 2, RA detection kit and application
One, the preparation of kit
Two, the application of kit
The kit auxiliary detection RA disease of applying step one preparation.
1, serum collection
3 groups of crowds' serum below collecting respectively:
First group (RA): 30 routine patients RA that make a definite diagnosis meet American Rheumatism Association's diagnostic criteria, and are the active stage patient;
Second group (HS): picked at random 30 routine healthy subjects, as negative control;
The 3rd group (CA): the 30 routine tumour patients of making a definite diagnosis, wherein, lung cancer patient (7 people), leukaemia people (4 people), cervical carcinoma patient (6 people), oophoroma patient (7 people), carcinoma of endometrium patient (6 people).
2, detect
Encapsulate elisa plate with APS, detect 3 groups of APS specific antibody IgG in crowd's serum respectively.Concrete grammar is following:
1) with APS (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 2%BSA37 ℃ of sealing 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l and uses the dilutability of PBST-BSA solution dilution to be the 30 routine tumour patient serum of 1:200, dilutability 30 routine RA patients serums as 1:200 as the 30 routine healthy subjects serum (HDS) of 1:200, dilutability; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat anti-human igg of the HRP mark of 1:5000 dilution again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The OD reading of healthy subjects control group is controlled at 0.2-0.3 in all testing, and other absorbance reading of 2 groups all obtains association index (binding index) divided by the control group reading, and all display result are all represented with association index.Antibody horizontal is used relatively difference of Student ' s t test between each group crowd.In addition, set healthy subjects group average antibody level and add 3 times of readings after the standard deviation as judging positive cutoff value (cut-off value), and calculate with this and respectively to organize positive rate.Positive rate between each group crowd is used relatively difference of Fisher ' sexact check.
3 groups of crowds' APS specific antibody IgG level is seen Fig. 4.3 groups of crowds' positive rate is seen table 2.
Anti-α-1 in table 2 different crowd, 4-glucosan IgG antibody positive rate relatively
| The HS group | The RA group | The CA group |
| 3.3％ | 50％ (<0.0005) | 6.7％ (0.388) |
The result shows: do not have IgG antibody in healthy subjects and the tumour patient serum, but the IgG antibody horizontal improves obviously among most RA patients serum.RA patients serum antibody horizontal is compared with the normal human serum antibody horizontal all has significant difference (p 0.0005), and tumour patient serum antibody level and normal human serum antibody horizontal no difference of science of statistics.RA patients serum antibody positive rate level has been compared significant difference with normal human serum antibody positive rate level, and tumour patient positive rate of antibody level and the horizontal no difference of science of statistics of normal human serum antibody positive rate.
3, the using value evaluation of kit
The ROC curve is claimed experimenter's performance curve (receive operating characteristiccurve) again, is the overall target that reflection detects index susceptibility and specificity continuous variable.Calculate ROC TG-AUC AUC and can estimate diagnosis efficiency, area can think that greater than 0.5 this index has diagnostic significance, and the big more judgement of area is worth high more.
For the using value of kit in diagnosis RA of evaluation procedure one preparation, draw the ROC curve according to the antibody horizontal of patient group and control group, see Fig. 5.The result shows: TG-AUC AUC reaches 0.7989, proves that this kit has certain using value in diagnosis among the RA.
Three, the sensitivity of kit is measured
From 30 routine RA patients serums, choose three higher samples of identification intensity respectively, do the serum dilution test experience with 30 routine normal human serum samples.
1) with APS (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 2%BSA37 ℃ of sealing 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l respectively with 30 routine healthy subjects serum (HDS) of the different dilutabilitys (1/50,1/100,1/200,1/400,1/800,1/1600) of PBST-BSA solution dilution, three higher samples of identification intensity; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat anti-human igg of the HRP mark of 1:5000 dilution again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The result sees Fig. 6.
The result shows that RA patients serum (RA-15, RA-22, RA-11) still has tangible identification to APS when serum dilution is 1:400, forms marked difference with normal human serum (N40).
The preparation of embodiment 3, SLE detection kit and application
One, the preparation of kit
Dextran (17-0280-01): available from Sigma company, dextran is by α-1, the straight chain type alpha-glucans that the 6-glycosidic bond connects to form.
Horseradish peroxidase (Horseradish peroxidase, HRP) the anti-human IgG of labelled goat: available from SBA company;
Bovine serum albumin(BSA) (BSA): available from Hyclone company;
The PBS damping fluid: the concentration of PBS damping fluid is 0.15mol/L, and pH is 7.2;
PBS-T solution: in above-mentioned PBS damping fluid, add Tween 20, the volume ratio of Tween 20 and PBS damping fluid is 0.05%;
PBST-BSA solution: in above-mentioned PBST solution, add bovine serum albumin(BSA) (BSA), the final concentration of bovine serum albumin(BSA) is 1 gram/L;
OPD-phosphoric acid citrate buffer solution (pH 9.6): add o-phenylenediamine at the phosphoric acid citrate buffer solution, the mass percent concentration of o-phenylenediamine is 0.06%; The pH of said phosphoric acid citrate buffer solution is 5.0, and concentration of phosphoric acid is 0.1mol/L, and the concentration of citric acid is 0.05mol/L;
Sulfuric acid;
96 hole ELISA Plates: Costar company.
Two, the application of kit
The kit auxiliary detection SLE disease of applying step one preparation.
1, serum collection
With 2 of the step 2 of embodiment 1.
2, detect
Encapsulate elisa plate with dextran, detect 3 groups of dextran specific antibody IgG in crowd's serum respectively.Concrete grammar is following:
1) with dextran (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 37 ℃ of sealings of 2%BSA 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l respectively to use the dilutability of PBST-BSA solution dilution is that 1: 200 30 routine healthy subjects serum, dilutability is that 1: 200 30 routine tumour patient serum, dilutability is 1: 200 30 routine SLE patients serums; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat anti-human igg of HRP mark of dilution in 1: 5000 again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The OD reading of healthy subjects control group is controlled at 0.2-0.3 in all testing, and other absorbance reading of 2 groups all obtains association index (binding index) divided by the control group reading, and all display result are all represented with association index.Antibody horizontal is used relatively difference of Student ' s t test between each group crowd.In addition, set healthy subjects group average antibody level and add 3 times of readings after the standard deviation as judging positive cutoff value (cut-off value), and calculate with this and respectively to organize positive rate.Positive rate between each group crowd is used relatively difference of Fisher ' sexact check.
3 groups of crowds' dextran specific antibody IgG level is seen Fig. 7.3 groups of crowds' positive rate is seen table 3.
Anti-α-1 in table 3 different crowd, 6-glucosan IgG antibody positive rate relatively
| The HS group | The SLE group | The CA group |
| 6.7％ | 73.3％ (<0.0001) | 20％ (0.288) |
The result shows: SLE patients serum antibody horizontal is compared with the normal human serum antibody horizontal all has significant difference, and tumour patient serum antibody level and normal human serum antibody horizontal no difference of science of statistics.SLE patients serum antibody positive rate level has been compared significant difference with normal human serum antibody positive rate level, and tumour patient positive rate of antibody level and the horizontal no difference of science of statistics of normal human serum antibody positive rate.
3, the using value evaluation of kit
The ROC curve is claimed experimenter's performance curve (receive operating characteristiccurve) again, is the overall target that reflection detects index susceptibility and specificity continuous variable.Calculate ROC TG-AUC AUC and can estimate diagnosis efficiency, area can think that greater than 0.5 this index has diagnostic significance, and the big more judgement of area is worth high more.
Be the using value of kit in diagnosing SLE of evaluation procedure one preparation, draw the ROC curve, see Fig. 8 according to the antibody horizontal of patient group and control group.The result shows: TG-AUC AUC reaches 0.8633, proves that this kit has certain using value in diagnosing SLE.
Embodiment 4, through α-1,6 glucosan specificity antibody IgG detection kit auxiliary detection RA disease
One, the preparation of kit
Two, the application of kit
The kit auxiliary detection RA disease of applying step one preparation.
1, serum collection
With 2 of the step 2 of embodiment 2.
2, detect
Encapsulate elisa plate with dextran, detect 3 groups of dextran specific antibody IgG in crowd's serum respectively.Concrete grammar is following:
1) with dextran (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 2%BSA37 ℃ of sealing 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l respectively and uses the dilutability of PBST-BSA solution dilution to be the 30 routine tumour patient serum of 1:200, dilutability 30 routine RA patients serums as 1:200 as the 30 routine healthy subjects serum of 1:200, dilutability; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat anti-human igg of the HRP mark of 1:5000 dilution again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The OD reading of healthy subjects control group is controlled at 0.2-0.3 in all testing, and other absorbance reading of 2 groups all obtains association index (binding index) divided by the control group reading, and all display result are all represented with association index.Antibody horizontal is used relatively difference of Student ' s ttest between each group crowd.In addition, set healthy subjects group average antibody level and add 3 times of readings after the standard deviation as judging positive cutoff value (cut-off value), and calculate with this and respectively to organize positive rate.Positive rate between each group crowd is used relatively difference of Fisher ' sexact check.
3 groups of crowds' dextran specific antibody IgG level is seen Fig. 9.3 groups of crowds' positive rate is seen table 4.
Anti-α-1 in table 4 different crowd, 6-glucosan IgG antibody positive rate relatively
| The HS group | The RA group | The CA group |
| 6.7％ | 60％ (<0.0005) | 20％ (0.288) |
The result shows: RA patients serum antibody horizontal is compared with the normal human serum antibody horizontal all has significant difference, and tumour patient serum antibody level and normal human serum antibody horizontal no difference of science of statistics.RA patients serum antibody positive rate level has been compared significant difference with normal human serum antibody positive rate level, and tumour patient positive rate of antibody level and the horizontal no difference of science of statistics of normal human serum antibody positive rate.
3, the using value evaluation of kit
The ROC curve is claimed experimenter's performance curve (receive operating characteristiccurve) again, is the overall target that reflection detects index susceptibility and specificity continuous variable.Calculate ROC TG-AUC AUC and can estimate diagnosis efficiency, area can think that greater than 0.5 this index has diagnostic significance, and the big more judgement of area is worth high more.
For the using value of kit in diagnosis RA of evaluation procedure one preparation, draw the ROC curve according to the antibody horizontal of patient group and control group, see Figure 10.The result shows: TG-AUC AUC reaches 0.7800, proves that this kit has certain using value in diagnosis among the RA.
Embodiment 5, through α-1,4 glucosan specificity antibody IgA detection kit auxiliary detection RA disease
One, the preparation of kit
The composition of kit is following:
Astragalus polyose (APS): available from Pharmagenesis company, be made up of D-glucose, main chain is through α-1, and the 4-glycoside bond connects, and side chain is through α-1, and the 6-glycoside bond connects.
Horseradish peroxidase (Horseradish peroxidase, HRP) the anti-people IgA of labelled goat: available from SBA company;
Bovine serum albumin(BSA) (BSA): available from Hyclone company;
The PBS damping fluid: the concentration of PBS damping fluid is 0.15mol/L, and pH is 7.2;
PBST solution: in above-mentioned PBS damping fluid, add Tween 20, the volume ratio of Tween 20 and PBS damping fluid is 0.05%;
PBST-BSA solution: in above-mentioned PBST solution, add bovine serum albumin(BSA) (BSA), the final concentration of bovine serum albumin(BSA) is 1 gram/L;
OPD-phosphoric acid citrate buffer solution (pH 9.6): add o-phenylenediamine at the phosphoric acid citrate buffer solution, the mass percent concentration of o-phenylenediamine is 0.06%; The pH of said phosphoric acid citrate buffer solution is 5.0, and concentration of phosphoric acid is 0.1mol/L, and the concentration of citric acid is 0.05mol/L;
Sulfuric acid;
96 hole ELISA Plates: Costar company.
Two, the application of kit
1, serum collection
With 2 of the step 2 of embodiment 2.
2, detect
The kit auxiliary detection RA disease of applying step one preparation.
Encapsulate elisa plate with dextran, detect 3 groups of dextran specific antibody IgA in crowd's serum respectively.Concrete grammar is following:
1) with dextran (50 μ g/ml) as antigen coated 96 hole ELISA Plates, every hole 100 μ l, 4 ℃ are spent the night, PBST solution is washed 5 times;
2) 37 ℃ of sealings of 2%BSA 2h, PBST solution is washed 5 times;
3) every hole adds 100 μ l respectively to use the dilutability of PBST-BSA solution dilution is that 1: 200 30 routine healthy subjects serum, dilutability is that 1: 200 30 routine tumour patient serum, dilutability is 1: 200 30 routine RA patients serums; Hatch 2h for 37 ℃, PBST solution is washed 5 times;
4) add the goat-anti people IgA of the HRP mark of 1:5000 dilution again, hatch 1h for 37 ℃, PBST solution is washed 5 times; The colour developing of OPD-phosphoric acid citrate buffer solution, 12.5% sulfuric acid solution cessation reaction reads the OD492nm absorbance.
Each sample is established 3 repetitions.The OD reading of healthy subjects control group is controlled at 0.2-0.3 in all testing, and other absorbance reading of 2 groups all obtains association index (binding index) divided by the control group reading, and all display result are all represented with association index.Antibody horizontal is used relatively difference of Student ' s t test between each group crowd.In addition, set healthy subjects group average antibody level and add 3 times of readings after the standard deviation as judging positive cutoff value (cut-off value), and calculate with this and respectively to organize positive rate.Positive rate between each group crowd is used relatively difference of Fisher ' sexact check.
3 groups of crowds' APS specific antibody IgG level is seen Figure 11.3 groups of crowds' positive rate is seen table 5.
Anti-α-1 in table 5 different crowd, 6-glucosan IgA antibody positive rate relatively
| The HS group | The RA group | The CA group |
| 6.7％ | 36.7％ (0.036) | 16.7％ (0.388) |
The result shows, patient's RA α-1,4 glucosan specificity antibody IgA raises (p < 0.05) than the normal person, and tumour patient group and normal person organize indifference.RA patients serum antibody positive rate level has been compared significant difference with normal human serum antibody positive rate level, and tumour patient positive rate of antibody level and the horizontal no difference of science of statistics of normal human serum antibody positive rate.
3, the using value evaluation of kit
The ROC curve is claimed experimenter's performance curve (receive operating characteristiccurve) again, is the overall target that reflection detects index susceptibility and specificity continuous variable.Calculate ROC TG-AUC AUC and can estimate diagnosis efficiency, area can think that greater than 0.5 this index has diagnostic significance, and the big more judgement of area is worth high more.
For the using value of kit in diagnosis RA of evaluation procedure one preparation, draw the ROC curve according to the antibody horizontal of patient group and control group, see Figure 12.The result shows: TG-AUC AUC reaches 0.67, proves that this kit has certain using value in diagnosis among the RA.
Claims (7)
1. the following antigen that is used for encapsulating is in the application of the ELISA kit of preparation assistant diagnosis system property lupus erythematosus or rheumatoid arthritis;
The said antigen that is used to encapsulate is α-1, the 4-glucosan; Or the said antigen that is used to encapsulate is α-1, the 6-glucosan.
2. application as claimed in claim 1 is characterized in that: the said antigen that is used to encapsulate is astragalus polyose; Or the said antigen that is used to encapsulate is dextran.
3. according to claim 1 or claim 2 application is characterized in that: said kit comprises that also enzyme target two is anti-; It is anti-human IgG that said enzyme target two resists.
4. application as claimed in claim 3 is characterized in that: it is goat anti human IgG, rabbit anti-human igg or mouse-anti human IgG that said enzyme target two resists.
5. application as claimed in claim 1 is characterized in that: said kit also comprises following reagent: bovine serum albumin(BSA), PBS damping fluid, PBST solution, PBST-BSA solution, OPD-phosphoric acid citrate buffer solution, sulfuric acid and 96 hole ELISA Plates; The concentration of said PBS damping fluid is 0.15mol/L, and pH is 7.2; Said PBST solution is for adding the solution that Tween 20 obtains in said PBS damping fluid, the volume ratio of said Tween 20 and said PBS damping fluid is 0.05%; Said PBST-BSA solution is for adding the solution that bovine serum albumin(BSA) obtains in said PBST damping fluid, the final concentration of said bovine serum albumin(BSA) is 1 gram/L; Said OPD-phosphoric acid citrate buffer solution is for adding the solution that o-phenylenediamine obtains at the phosphoric acid citrate buffer solution; The mass percent concentration of said o-phenylenediamine is 0.06%; The pH of said phosphoric acid citrate buffer solution is 5.0; Concentration of phosphoric acid is 0.1mol/L, and the concentration of citric acid is 0.05mol/L.
6. application as claimed in claim 1 is characterized in that: the said antigen that is used to encapsulate is α-1, and 4-glucosan, said kit comprise that also enzyme target two is anti-; It is anti-people IgA that said enzyme target two resists.
7. application as claimed in claim 6 is characterized in that: it is goat anti people IgA, the anti-people IgA of rabbit or mouse-anti people IgA that said enzyme target two resists.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1185161A (en) * | 1994-09-01 | 1998-06-17 | 生化学工业株式会社 | (1--3)-beta-d-glucan-binding protein, antidody that recognizes the prolein, and use of the protein and antibody |
| US20050142618A1 (en) * | 2003-12-03 | 2005-06-30 | Nir Dotan | Method for diagnosing diseases based on levels of anti-glycan antibodies |
| CN1735346A (en) * | 2002-09-27 | 2006-02-15 | 生物E股份有限公司 | Cell separation compositions methods |
| CN101063680A (en) * | 2006-04-29 | 2007-10-31 | 北京华大吉比爱生物技术有限公司 | Microarray-ELISA detecting reagent kit for detecting autoimmunity disease relevant antibody spectrum |
| CN101139395A (en) * | 2007-08-03 | 2008-03-12 | 广州甘蔗糖业研究所 | Preparation method of dextran monoclonal antibody |
-
2008
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1185161A (en) * | 1994-09-01 | 1998-06-17 | 生化学工业株式会社 | (1--3)-beta-d-glucan-binding protein, antidody that recognizes the prolein, and use of the protein and antibody |
| CN1735346A (en) * | 2002-09-27 | 2006-02-15 | 生物E股份有限公司 | Cell separation compositions methods |
| US20050142618A1 (en) * | 2003-12-03 | 2005-06-30 | Nir Dotan | Method for diagnosing diseases based on levels of anti-glycan antibodies |
| CN101063680A (en) * | 2006-04-29 | 2007-10-31 | 北京华大吉比爱生物技术有限公司 | Microarray-ELISA detecting reagent kit for detecting autoimmunity disease relevant antibody spectrum |
| CN101139395A (en) * | 2007-08-03 | 2008-03-12 | 广州甘蔗糖业研究所 | Preparation method of dextran monoclonal antibody |
Non-Patent Citations (3)
| Title |
|---|
| Douwes等.Measurement of b(133)-Glucans in Occupational and Home Environments with an Inhibition Enzyme Immunoassay.《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.1996,第62卷(第9期),3176–3182. * |
| 郭杰标等.以共价连接法使抗原固相化制备酶联免疫吸附测定试剂的研究.《中国职业医学》.2006,第33卷(第6期),460-461. * |
| 闻平等.(1-3)-beta-D-葡聚糖ELISA检测方法建立及初步临床应用.《现代检验医学杂志》.2003,第18卷(第5期),1-2. * |
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