CN107436358A - Application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared - Google Patents
Application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared Download PDFInfo
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Abstract
The present invention relates to immunology and biological technical field, specifically application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared.The present invention also provides a kind of rheumatoid arthritis ELISA diagnostic reagents or kit.The present invention confirms diagnostic values of diagnosis, early diagnosis, Combining diagnosis, differential diagnosis value and synovia CTGFs of the change of serum C TGF to RA patient to RA patient first.The present invention is simple to operate, can detect the content of the CTGF albumen in rheumatoid arthritis human serum and synovia accurately, in high sensitivity, a kind of new means and method are provided for clinical examination and basic research.
Description
Technical field
The present invention relates to immunology and biological technical field, is a kind of human connective tissue growing factor specifically
(CTGF) new purposes, specifically, it is related to CTGF albumen in diagnosis rheumatoid arthritis and prepares rheumatoid joint
Application in scorching diagnostic reagent or kit.
Background technology
Rheumatoid arthritis (Rheumatoid Arthritis, abbreviation RA) be a kind of cause of disease it is extremely complex it is chronic enter
Row autoimmune disease.Early manifestation is migrans arthralgia and dysfunction, late period often may occur in which anchylosis and
Gross distortion, disability rate is high, seriously endangers the health of broad masses of the people." early to find, early diagnose, early treatment " is control
The main principle of RA progression of disease processed, thus early stage, Accurate Diagnosis RA can early intervention be received to patient and then delays disease
It is in progress significant.
RA clinical diagnosis Main Basiss joint involvement situation, symptom duration, acute phase reactant and blood at present
The Serum Indexes such as clear rheumatoid factor (RF), anti-citrulling protein antibodies (ACPA), but due to RA early clinic Symptoms
It is not true to type and diversity, and it has been often late period joint involvement occur, the non-specificity of acute phase reactant, therefore serum in addition
Index is learned just to be particularly important for RA early diagnosis, Accurate Diagnosis.
But the current guideline RF that is recommended and ACPA have some limitations.RF and ACPA can not only be in RA patient
In be detected, be also widely present in the Serum of Patients With Autoimmune Diseases such as osteoarthritis (OA), systemic loupus erythematosus (SLE),
Therefore its sensitiveness or specificity are relatively low, and respectively 69%, 85% and 67%, 95%, and both Combining diagnosis RA sensitiveness
And specificity then only has 78%, 82%, misdiagnosis rate is up to 19%, and the performance in early stage RA is also unsatisfactory in addition, positive rate
Only 57%, therefore there is an urgent need to find that early stage, Accurate Diagnosis and simple and convenient diagnosis can be carried out to RA patient for this area
Index.This respect research in RA patient for accomplishing early discovery, early diagnosis, early treatment, and so as to slow down disease process, control is faced
Bed symptom, it is significant to improve life quality.
Research object-human connective tissue growing factor (CTGF/CCN2) in the present invention belongs to the CCN of rich cysteine
Protein family is a kind of and the closely related secretion peptide of angiogenesis, molecular weight 4.5kb, including 4 domains, by aobvious outside 5
Son and 4 intrones form, and encode 349 amino acid, No. GENBANK is NP_001892.Research thinks that CTGF is thin at present
Certain facilitation is played in born of the same parents' proliferation apoptosis, angiogenesis and mitosis, the interaction of itself and TGF-β is even more to exist
Played an important role in the change of fibrosis.In recent years, more researchs report effects of the CTGF in RA pathogenic processes, such as
It can be metabolized by influenceing osteoclast, regulate and control the influence RA disease process such as TSP-1/TGF- β/CTGF signal paths.(with reference to text
Offer：1.Connective tissue growth factor promotes articular damage by increased
osteoclastogenesis in patients with rheumatoid arthritis.Arthritis Research&
Therapy 2009,11.2.Nishimura K,Sugiyama D,Kogata Y,Tsuji G,Nakazawa T,Kawano
S,et al.Meta-analysis:diagnostic accuracy of anti-cyclic citrullinated
peptide antibody and rheumatoid factor for rheumatoid arthritis.Ann Intern
Med.2007；146(11):797-808；3.Sun J,Zhang Y,Liu L,Liu G.Diagnostic accuracy of
combined tests of anti cyclic citrullinated peptide antibody and rheumatoid
factor for rheumatoid arthritis:a meta-analysis.Clin Exp Rheumatol.2014；32
(1):11-21；4.Infantino M,Manfredi M,Meacci F,Sarzi-Puttini P,Ricci C,Atzeni F,
et al.Anti-citrullinated peptide antibodies and rheumatoid factor isotypes in
the diagnosis of rheumatoid arthritis:an ssessment of combined tests.Clin
Chim Acta.2014；436:237-42.) we have found that CTGF is important stable in a RA disease process, lasting table
The albumen reached, and blood can be released into by synovia, it is diagnosed to RA, Combining diagnosis and antidiastole ability are more prominent.
However, so far, diagnosis, Combining diagnosis and the mirror there is no literature reported on CTGF albumen in RA patient both at home and abroad
Other diagnostic value, also there is no literature reported on the ELISA detection kit of CTGF albumen and such kit in RA serum or cunning
Application in liquid diagnosis.
The content of the invention
It is an object of the invention to provide the new purposes of CTGF albumen, particularly CTGF albumen in diagnosis rheumatoid joint
The application of scorching (RA)；It is a further object of the present invention to provide a kind of ELISA detection kit of CTGF albumen；The 3rd of the present invention
Purpose is to provide the detection method using mentioned reagent box；The fourth object of the present invention is to provide mentioned reagent box in RA serology
And the application in the diagnosis of articular cavity synovia.
It is contemplated that providing new serum and synovia index for clinical diagnosis RA, and it is clinical and test in laboratory RA
CTGF protein contents provide accurate, easy, high sensitivity and the detection hand that can be widely used in patients serum and synovial fluid samples
Section.
The present inventor by extensively and in-depth study, find first, using EUSA detect RA patient,
CTGF expressing quantities in other similar rheumatisants and normal human serum and synovia,
CTGF albumen relatively accurately can carry out diagnostic assessment to RA.Table based on CTGF albumen in serum, synovia
Up to amount and RA this correlation, detection is carried out to its expression quantity using the albumen as biomarker and can be used for instructing RA to suffer from
The diagnosis of person, at the same time, CTGF albumen can be used for preparing RA patient's diagnostic reagent or kit.
The first aspect of the present invention, there is provided human connective tissue growing factor (CTGF albumen) is preparing rheumatoid arthritis
(RA) application in diagnostic reagent or kit.
Described RA refers to the rheumatoid arthritis (RA) for meeting American society of rheumatism diagnostic criteria in 2010, including
Diagnose early stage RA, non-early stage RA；Diagnose RF negative RAs；Diagnose ACPA negative RAs；Differentiate and distinguish the positive non-RA diseases of RA and RF；
Differentiate and distinguish the positive non-RA diseases of RA and ACPA；Differentiate and distinguish the positive non-RA diseases of RA and RF, ACPA；
Described early stage RA refers to that symptom duration is less than 6 months；
Described RF or ACPA feminine genders refer to be less than or equal to normal upper limit；The described RF or ACPA positives refer to be more than normally
The upper limit；
Described non-RA diseases refer mainly to ankylosing spondylitis (AS), osteoarthritis (OA), systemic loupus erythematosus
(SLE), the rheumatism immunological diseases such as gout (GOUT) and dry syndrome (pSS).
Described diagnosis refers to the diagnosis to RA and normal population, and its diagnostic value is presented as the susceptibility of CTGF in serum
And specificity is respectively 82%, 91%, positive predictive value, negative predictive value are respectively then 0.85,0.90；CTGF is to RA in synovia
Susceptibility and specificity be respectively 95.71%, 90.70%, TG-AUC is up to 0.9668.
Further, described diagnostic reagent or kit are the ELISA diagnostic reagents or reagent of CTGF protein quantifications detection
Box.
Further, described diagnostic reagent or kit detect the expression quantity of CTGF albumen in serum or synovia.
Further, described serum is peripheral blood serum.Optimal critical value (the cut- of CTGF albumen in described serum
Off values) it is 87-90pg/ml.
Further, described synovia is articular cavity synovia；Optimal critical value (the cut- of CTGF albumen in described synovia
Off values) it is 103-105pg/ml.
Further, described diagnostic reagent or kit are to utilize people's connective group in ELISA method detection serum or synovia
Knit the expression quantity of growth factor.
Further, described diagnostic reagent includes human connective tissue growing factor standard items, quality-control product, Streptavidin bag
The microwell plate of quilt, the human connective tissue growing factor antibody of biotin labeling, the human connective tissue of horseradish peroxidase-labeled
Growth factor antibodies, dilution buffer, substrate solution, terminate liquid and cleaning buffer solution.
Further, described diagnostic reagent or kit are early stage RA diagnostic reagent or kit, and described early stage RA refers to
Symptom duration is less than 6 months.Described early stage RA diagnosis refer to the detection to early stage RA, and its diagnostic value is embodied in
CTGF does not have relevance with the RA courses of disease in serum, accordingly can be applied to RA early diagnosis.
Further, CTGF albumen and anti-citrulling protein antibodies (ACPA) Combining diagnosis in described serum.
Further, CTGF albumen, anti-citrulling protein antibodies (ACPA) and serum rheumatoid factor (SRF) in described serum
(RF) Combining diagnosis.
Described Combining diagnosis refers to joint CTGF and RF, CTGF and ACPA, the diagnosis of CTGF, ACPA and RF to RA, its
Diagnostic value is embodied as on the basis of accuracy and feasibility is considered, and CTGF and ACPA united mode are optimal.
Further, described diagnostic reagent or kit antidiastole rheumatoid arthritis (RA) and non-rheumatoid close
Other scorching rheumatism immunological diseases are saved, described non-RA refers to ankylosing spondylitis, osteoarthritis, systemic loupus erythematosus, gout
Or other rheumatism immunological diseases such as dry syndrome.
In serum, described antidiastole is primarily referred to as distinguishing RA and osteoarthritis (OA), dry syndrome (pSS), pain
Non- RA diseases, its diagnostic value such as wind (GOUT), ankylosing spondylitis (AS) and systemic loupus erythematosus (SLE) are embodied in
RA is above 0.85 to the TG-AUC of these diseases, therefore change of serum C TGF can accurately distinguish RA and non-RA patient.
In synovia, described antidiastole is primarily referred to as distinguishing RA and OA, and its diagnostic value is embodied in RA's and OA
Specificity, susceptibility are respectively 84.29%, 97.87%, and TG-AUC is up to 0.9535, therefore synovia CTGF can accurately distinguish RA
With OA patient.
The second aspect of the present invention, there is provided a kind of rheumatoid arthritis ELISA diagnostic reagents or kit, including：
A the ELISA ELISA Plates of CTGF antibody) are coated with, described coated antibody is the anti-human CTGF of mouse monoclonal antibody；
Coated antibody working concentration is 2 μ g/ml；
B the antibody of CTGF antigens) is detected, the polyclonal antibody for being rabbit-anti people CTGF；The working concentration for detecting antibody is 1 μ
g/ml；
C) ELIAS secondary antibody, it is the goat anti-rabbit igg of horseradish peroxidase-labeled；Diluted concentration is 1:2000；
D) standard protein, it is recombined human CTGF fusion proteins；
E) also include：Sample diluting liquid, coating buffer solution, confining liquid, ELISA ELISA Plates cleaning solution, the antibody of routine
Dilution, nitrite ion and terminate liquid.
Preferably, described kit, ELISA ELISA Plates are commercially available ELISA ELISA Plates, and preferably U.S. Corning is public
The ELISA ELISA Plates (the 96 removable ELISA Plate #2592 of hole wall scroll) of department；The anti-human CTGF of described mouse monoclonal antibody is purchased from
Abgent companies, #AM2095a；
Preferably, described rabbit-anti people CTGF polyclonal antibody is purchased from Abcam companies, #ab84244；
Preferably, the goat anti-rabbit igg of described HRP marks is purchased from Abcam companies, #ab6721；
Preferably, described recombined human CTGF albumen is purchased from R＆D companies, #Q5M8T4；
Preferably, described coating buffer solution, from 1 × PBS, pH:7.35-7.45；
Preferably, described confining liquid, from the PBS solution containing 2% bovine serum albumin(BSA)；
Preferably, described ELISA ELISA Plate cleaning solutions, for 1 × PBS solution containing 0.05%Tween-20；
Preferably, described sample diluting liquid：From 1 × PBS, pH:7.35-7.45；
Preferably, described antibody diluent, from 1 × PBS, pH:7.35-7.45；
Preferably, described nitrite ion, from the 3 of sigam companies #T322802,3 ', 5,5 '-tetramethyl benzidine
(TMB)；
Preferably, described terminate liquid, from 1.5-2mol/L sulfuric acid solution.
Third aspect present invention, there is provided a kind of to utilize above-mentioned rheumatoid arthritis ELISA diagnostic reagents or kit
Detection method, comprise the following steps：
A, detection ELISA ELISA Plates are prepared：Buffer solution dilution mouse anti-human CTGF monoclonal antibody is coated with to 2 μ g/ml, 4
DEG C overnight；
When b, detecting, sample dilutes certain multiple (serum 1 with sample diluting liquid：50；Synovia 1：50), it is loaded to ELISA
In ELISA Plate hole, 1-2 hours are incubated at room temperature；
C, people's recombinant C TGF albumen is diluted to various concentrations gradient with sample diluting liquid, is loaded into ELISA ELISA Plates hole,
It is incubated at room temperature 1-2 hours；The liquid in each hole is got rid of, adds ELISA ELISA Plate cleaning solutions, is washed 3-5 times, is dried；
D, detection antibody is diluted to 1 μ g/ml, added in ELISA ELISA Plates hole, be incubated at room temperature 1--2 hours；Get rid of each
Liquid in hole, ELISA ELISA Plate cleaning solutions are added, washed 3-5 times, dried；
E, the goat anti-rabbit igg of HRP marks is added, is incubated at room temperature 1--2 hours；The liquid in each hole is got rid of, is added
ELISA ELISA Plate cleaning solutions, wash 6-8 times, dry；
F, nitrite ion is added, room temperature lucifuge is incubated 10-20 minutes, adds terminate liquid terminating reaction；The 450nm on ELIASA
Determine OD values.
In the serum sample detection preferred embodiment of the present invention, described detection method is specially：
The previous day is detected, prepares detection ELISA ELISA Plates first：Buffer solution is coated with by antibody (the anti-human CTGF of mouse Dan Ke
Grand antibody) 1 μ g/ml are diluted to, 100 μ l are added in every hole of ELISA ELISA Plates, 4 DEG C of overnight incubations are placed in after shrouding.
During detection, serum sample sample diluting liquid is with 1:50 dilutions, are loaded with the volume in 100 μ l/ holes, incubation at room temperature 2
Hour；CTGF recombinant proteins are diluted to various concentrations gradient with sample diluting liquid, are loaded with the volume in 100 μ l/ holes, incubation at room temperature
2 hours；The liquid in each hole is got rid of, adds ELISA ELISA Plate cleaning solutions, per the μ l of hole 300, washs 3-5 times, dries；By CTGF
Detection antibody is diluted to 1 μ g/ml, per the μ l of hole 100, is incubated at room temperature 30 minutes 1 hour；The liquid in each hole is got rid of, adds ELISA
ELISA Plate cleaning solution, per the μ l of hole 300, wash 3-5 times, dry；The goat anti-rabbit igg of HRP marks is added, per the μ l of hole 100, room temperature
It is incubated 1 hour；The liquid in each hole is got rid of, adds ELISA ELISA Plate cleaning solutions, per the μ l of hole 300, washs 6-8 times, dries；Add
Enter TMB, per the μ l of hole 100, room temperature lucifuge is incubated 10-20 minutes, adds 2mol/L sulfuric acid solution terminating reactions, per the μ l of hole 50；
(450nm) determines OD values on ELIASA.
CTGF recombinant proteins are diluted to various concentrations gradient with sample diluting liquid, according to detection sets itself can be needed dense
Gradient is spent, in the serum of the present invention or the preferred embodiment of synovia sample, concentration gradient is：4000pg/ml, 2000pg/ml,
1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml.
Fourth aspect present invention, there is provided a kind of above-mentioned rheumatoid arthritis ELISA diagnostic reagents in RA serum and
Application in synovia diagnosis, described application specifically refer to be used to prepare RA serum and synovia diagnostic kit.
Beneficial effects of the present invention：
1) it is accurate：One side previous work finds CTGF albumen and RA height correlations, by retrospective cohort study and preceding
Zhan Xing cohort studies, it is found that CTGF albumen is higher to the value of RA diagnosis, Combining diagnosis and antidiastole, and be better than guide institute
RF, ACPA index of recommendation.On the other hand the ELISA detection reagents of humanized's CTGF albumen of commercial-free in the market
Box, this ELISA kit can accurately detect the content of the CTGF albumen in RA patients serums or synovia, as a result be quantified by ELIASA
Analysis, the subjectivity of the semi-quantitative methods such as SABC, immunofluorescence is eliminated, and confirmation is detected through clinical sample, it is in blood
Accuracy in clear and synovia is respectively up to 84.67%, 90%.
2) high sensitivity：The CTGF albumen that is detected with this method is minimum can be to 62.5pg/ml, and sensitiveness is apparently higher than general
The semi-quantitative method such as logical Western blot and SABC, and confirmation is detected through clinical sample, it is in serum and synovia
Sensitivity respectively up to 81.67%, 89.81%.
3) specificity is high：For kit after optimization, antigen-antibody reaction has no obvious non-specific responding, and through clinical sample
This detection confirms that its specificity in serum and synovia is respectively up to 90.67%, 90.32%.
4) it is simple and convenient：Agents useful for same and experiment consumptive material are commercially available commercially produced product in this method, are readily available；Detection
In only need pipettor and ELIASA is loaded and reading, common laboratory and hospital can carry out this detection.
5) cost is low：About 2000 yuan of totle drilling cost, every sample cost only needs 50 yuan, far below scientific research ELISA kit.
ELISA kit provided by the invention is simple to operate, can high specificity, detect patient RA in high sensitivity
The content of CTGF albumen in serum and synovia；It can also particularly differentiate that distinguishing the rheumatism such as RA and AS, OA, SLE, pSS, GOUT exempts from
Epidemic disease；A kind of new means and method are provided for clinical examination and basic research.
The kit of the present invention is mainly used in the quantitative inspection of CTGF albumen in the serum and articular cavity synovial fluid samples of patient RA
Survey, various biological samples (such as peripheral blood serum, articular cavity synovia, articular cavity synovial tissue, pass are can operate with basic research
Save chamber synovioblast) in CTGF albumen detection.
Complex instrument is not needed during present invention detection, is easy to the popularization and application in research institutions and medical institutions, can advise greatly
Mould detects clinical samples, quick to obtain people CTGF albumen related mass data and information, and combines a large amount of clinical information, it is determined that
The CTGF levels of serum and synovia have obvious diagnostic significance for diagnosis RA, have wide market prospects, larger warp
Ji and social benefit.
Brief description of the drawings
Fig. 1 be retrospective cohort study change of serum C TGF to RA diagnostic value, wherein A figures are RA patient and normal control blood
CTGF in clear is horizontal, and B figures are RA patient and normal control change of serum C TGF ROC curve figure.
Fig. 2 is retrospective cohort study synovia CTGF to RA diagnostic value, and wherein A figures are RA patient, OA patient and just
CTGF often in control synovia is horizontal, and B figures are RA patient and normal control synovia CTGF ROC curve figure.
Fig. 3 is differential diagnosis values of the retrospective cohort study synovia CTGF to RA.
Fig. 4 is early stage RA and the change of serum C TGF of non-early stage RA patient is horizontal.
Fig. 5 be different CTGF associated forms to RA diagnostic value, wherein A figures are joint CTGF and ACPA ROC curve
Figure, B figures are joint CTGF and RF ROC curve figure, and C figures are joint CTGF, ACPA and RF ROC curve figure.
Embodiment
Embodiment provided by the invention is elaborated with reference to embodiment.
Embodiment 1：Diagnostic values of the CTGF to RA in the clear and definite serum of retrospective cohort study
From 2010.1 to 2016.5.1, using retrospective cohort study, in The First Affiliated Hospital of Wenzhou Medical University, Shanghai
3 the brilliance hospitals of traditional Chinese and western medicine, central hospital of Kiamusze City centers include the serum of 98 RA patients and 103 normal healthy controls
(acquisition of sample has been examined by ethics), the present invention use GenWay Biotech companies of the U.S., #GWB-
Level of the SKR010ELISA ELISA kit detection CTGF albumen in RA patients serums, as a result as shown in Figure 1A, RA suffers from
For person's change of serum C TGF level obviously higher than normal healthy controls, difference has significant statistical significance (p<0.05).
Diagnostic value of the change of serum C TGF levels to RA patient is analyzed using ROC curve.As shown in Figure 1B, its cut-off value
For 88.66pg/ml, sensitiveness, specificity are respectively then 85.71%, 92.23%, and TG-AUC reaches 0.921, shows it
There is higher diagnostic value to RA.
Embodiment 2：Diagnostic values of the clear and definite synovia CTGF of retrospective cohort study to RA
From 2010.1 to 2016.5.1, using retrospective cohort study, in 3 central collections 70 described in embodiment 1
The synovia sample of RA patient and 43 normal controls (acquisition of sample has been examined by ethics), the present invention use embodiment 1
Expression of the ELISA kit detection CTGF albumen in synovia.As shown in Figure 2 A, in RA patient's synovia CTGF water
Put down apparently higher than normal healthy controls.Being found using ROC curve, CTGF is higher to the diagnostic value between RA and normal control, its
Cut-off values are 104.2pg/ml, and sensitiveness, specificity and TG-AUC are respectively then 95.71%, 90.70% He
0.9668, as shown in Fig. 2 B.
Embodiment 3：Differential diagnosis values of the clear and definite synovia CTGF of retrospective cohort study to RA
From 2010.1 to 2016.5.1, using retrospective cohort study, in 3 central collections 70 described in embodiment 1
The synovia sample of RA patient and 47 OA patients (acquisition of sample has been examined by ethics), the present invention use the institute of embodiment 1
State expression of the ELISA kit detection CTGF albumen in synovia.As shown in Figure 2 A, in RA patient's synovia CTGF level
Apparently higher than OA patient.Found using ROC curve, CTGF can extremely accurate distinguish RA and OA, and its cut-off value is
226.7pg/ml, sensitiveness, specificity and TG-AUC are respectively then 84.29%, 97.87% and 0.9535, see Fig. 3 institutes
Show.
Embodiment 4：Diagnostic values of the CTGF to RA in prospective cohort study's assessment serum
From 2016.5.16 to 2017.1.31, using prospective cohort study, continue at 3 centers described in embodiment 1
572 cases are collected, its change of serum C TGF concentration is detected using ELISA kit described in embodiment 1, are in CTGF optimal critical value
In the case of 88.66pg/ml, it is CTGF positive patients to share 209, is then tracked follow-up, finds 209 CTGF positives
178 are shared in patient and is diagnosed as RA, and shares 39 in 363 CTGF negative patients and is diagnosed as RA.Statistics finds that it is sensitive
Property, specificity, positive likelihood ratio, negative likelihood are respectively 82%, and 91%, 5.74,0.12, significantly larger than current index
RF, and in the case where specificity is not weaker than ACPA indexs, have more prominent sensitiveness, and CTGF is in ACPA feminine genders or RF
In the case of feminine gender, also there is high recall rate, further confirm that CTGF has high diagnostic value to RA.
The prospective cohort study of table 1 assesses CTGF diagnostic values
Embodiment 5：Diagnostic values of the change of serum C TGF to early stage RA
Using 2010 American society of rheumatism criteria for classifications, symptom duration≤June is defined as early stage RA, symptom is held
The continuous time>It is defined as non-early stage RA June, and the RA patient in embodiment 1 and embodiment 4 is grouped, analyzes this two groups
Change of serum C TGF is horizontal, as a result as shown in figure 4, early stage RA group CTGF is horizontal to be slightly below non-early stage RA groups, but no difference of science of statistics (P>
0.05), it was demonstrated that change of serum C TGF is equally applicable for early stage RA diagnosis.
Embodiment 6：Change of serum C TGF is worth to RA Combining diagnosis
Collect the RA patient in embodiment 1 and embodiment 4, using CTGF+ACPA, CTGF+RF and CTGF+ACPA+RF
Associated form, and calculate its TG-AUC using ROC curve and assess its diagnostic value, as a result as shown in figure 5, its AUC distinguishes
For 0.9642,0.9477,0.9654, the Combining diagnosis for the ACPA+RF that guide is recommended, and CTGF+ACPA connection are above
The TG-AUC of conjunction mode and CTGF+ACPA+RF associated form has no larger difference, therefore is considering cost and feasibility
On the basis of, recommend the Combining diagnosis pattern using CTGF+ACPA.
Embodiment 7：Differential diagnosis values of the change of serum C TGF to RA
Collect all cases in embodiment 1 and embodiment 4, analyzed based on ROC curve, (be specially by RA and non-RA
AS, GOUT, OA, pSS, SLE) in each single disease compare two-by-two, find in RA and AS groups, cut-off values, sensitivity
Property, specificity, AUC are respectively 73.35pg/ml, 86.18%, 93.48%, 0.9402；In RA and GOUT groups, cut-off
Value, sensitiveness, specificity, AUC are respectively 84.42pg/ml, 84.33%, 97.3%, 0.9539；In RA and OA groups, cut-
Off values, sensitiveness, specificity, AUC are respectively 46.75pg/ml, 88.48%, 90.38%, 0.9418；In RA and pSS groups,
Cut-off values, sensitiveness, specificity, AUC are respectively 151pg/ml, 75.12%, 90.77%, 0.8602；In RA and SLE groups
In, cut-off values, sensitiveness, specificity, AUC are respectively 79.64pg/ml, 85.71%, 95.83%, 0.9225, as a result such as
Shown in table 2.Thus, it can be known that CTGF has high differential diagnosis value to RA, RA and non-RA patient can be distinguished exactly, to facing
Bed diagnosis has certain reference value.
The CTGF differential diagnosis values of table 2
Embodiment 8：Kit optimizes
(1) optimization of coated antibody：The kit of the present invention, it is coated with respectively from different antibody：Rabbit-anti people CTGF's
Polyclonal antibody (Abcam companies, #ab84244), rabbit-anti people CTGF polyclonal antibody (Abcam companies, #ab109606), rabbit
Anti-human CTGF polyclonal antibody (GeneTex companies, #GTX52490), (Abcam is public for the anti-human CTGF of mouse monoclonal antibody
Department, #ab94939), the anti-human CTGF of mouse monoclonal antibody (Abgent companies, #AM2095a), detectable concentration is from 8 μ g/ml, 4
μ g/ml, 2 μ g/ml, 1 μ g/ml, 500ng/ml, 250ng/ml.As a result the polyclonal antibody from rabbit-anti people CTGF is found
(GeneTex companies, #GTX37727；Abcam companies, #ab84244) as coated antibody obvious non-specific responding be present,
And rabbit-anti people CTGF polyclonal antibody (Abcam companies, #ab109606), the anti-human CTGF of mouse monoclonal antibody (Abcam public affairs
Department, #ab94939) when being used as coated antibody, the concentration and absorption values of standard items do not have correlation；But use mouse anti-human
CTGF monoclonal antibody (Abgent companies, #AM2095a) then without obvious nonspecific reaction, the concentration of standard items and
Absorbance has preferable correlation.By optimizing repeatedly, the μ g/ml of antibody working concentration 2 are optimal, the concentration of standard items and suction
The correlation of luminosity numerical value is best.
(2) optimization of antibody is detected：The kit of the present invention, respectively from different antibody as detection antibody：Work as bag
By during monoclonal antibody (Abgent companies, #AM2095a) that antibody is the anti-human CTGF of mouse from more than three kinds grams of rabbit-anti people CTGF
Grand antibody (Abcam companies, #ab84244；Abcam companies, #ab109606；GeneTex companies, #GTX52490) respectively as
Detect antibody；Detectable concentration selects 4 μ g/ml, 2 μ g/ml, 1 μ g/ml, 500ng/ml, 250ng/ml, 125ng/ml.As a result find
Coated antibody rabbit-anti people CTGF polyclonal antibody (GeneTex companies, #GTX52490；Abcam companies, #ab109606) and mouse
Obvious non-specific responding is presented during anti-human CTGF monoclonal antibody (Abgent companies, #AM2095a) compatibility, standard items
Concentration and absorption values do not have correlation.Verified and found by multiple permutation and combination, only more grams in selection rabbit-anti people CTGF
When grand antibody (Abcam companies, #ab84244) is with the anti-human CTGF monoclonal antibodies of mouse (Abgent companies, #AM2095a) compatibility, standard items
Concentration and absorbance there is preferable correlation.Subsequently by optimizing repeatedly, when the working concentration of detection antibody is 1 μ g/ml
When, the concentration of standard items and the correlation of absorption values are best.
(3) optimization of confining liquid：Different confining liquids is selected respectively, containing 0.5% bovine serum albumin(BSA)+PBS solution, 1%
Bovine serum albumin(BSA)+PBS solution, containing 2% bovine serum albumin(BSA)+PBS solution, containing 3% bovine serum albumin(BSA)+PBS solution, contain
4% bovine serum albumin(BSA)+PBS solution, containing 5% bovine serum albumin(BSA)+PBS solution, off-period selection room temperature 1 hour, room temperature 2
Hour, 4 DEG C are overnight.As a result find that optimal confining liquid and off-period are the confining liquid containing 2% bovine serum albumin(BSA)+PBS solution
2 hours are best of breed with room temperature closing.
(4) optimization of ELIAS secondary antibody：The ELIAS secondary antibody of different genera is selected according to the difference of detection antibody, detection antibody is
Rabbit-anti people CTGF resists IgG antibody (Abcam companies, the # marked when (Abcam companies, #ab84244) from goat antirabbit HRP more
Ab6721) and goat antirabbit HRP mark IgG antibody (Abcam companies, #ab97051)；Antibody extension rate selection 1:1000,
1:2000,1:3000,1:5000,1:10000.IgG antibody (Abcam companies, the # that goat antirabbit HRP is marked in combined authentication
Ab97051 preferable correlation) is not shown, non-specific responding be present.Rabbit-anti people CTGF is more anti-(Abcam companies, #ab84244)
Result as detection antibody with ELIAS secondary antibody for IgG antibody (Abcam companies, #ab6721) compatibility of goat antirabbit HRP marks
Show preferable correlation, 1:2000 diluted concentration is optimal.
(5) optimization of sample diluting liquid：Select different sample diluting liquids respectively, sample diluting liquid is selected during pattern detection
1×PBS(pH:7.35-7.45) or containing 0.1%BSA, 0.5%Tween-20 1 × PBS solution.As a result optimal serum sample is found
This dilution is 1 × PBS (pH:7.35-7.45) it is better than containing 0.1%BSA, 0.5%Tween-20 1 × PBS solution.
Embodiment 9：The detection of serum sample
The previous day is detected, prepares detection ELISA ELISA Plates first：Buffer solution is coated with by antibody (the anti-human CTGF of mouse Dan Ke
Grand antibody) 2 μ g/ml are diluted to, 100 μ l are added in every hole of ELISA ELISA Plates, 4 DEG C of overnight incubations are placed in after shrouding.
During detection, serum sample sample diluting liquid is with 1：50 dilutions, are loaded with the volume in 100 μ l/ holes, incubation at room temperature 2
Hour；CTGF recombinant proteins are diluted to 4000pg/ml, 2000pg/ml, 1000pg/ml, 500pg/ml with sample diluting liquid,
250pg/ml, 125pg/ml, 62.5pg/ml difference gradient, it is loaded, is incubated at room temperature 2 hours with the volume in 100 μ l/ holes；Get rid of each
Liquid in hole, ELISA ELISA Plate cleaning solutions are added, per the μ l of hole 300, wash 3-5 times, dry；By CTGF detection antibody dilutions
Into 1 μ g/ml, per the μ l of hole 100, it is incubated at room temperature 90 minutes；The liquid in each hole is got rid of, adds ELISA ELISA Plate cleaning solutions, per hole
300 μ l, wash 3-5 times, dry；The goat anti-rabbit igg of HRP marks is added, per the μ l of hole 100, is incubated at room temperature 1 hour；Get rid of each
Liquid in hole, ELISA ELISA Plate cleaning solutions are added, per the μ l of hole 300, wash 6-8 times, dry；TMB is added, per the μ l of hole 100,
Room temperature lucifuge is incubated 10-20 minutes, adds 2mol/L sulfuric acid solution terminating reactions, per the μ l of hole 50；(450nm) is surveyed on ELIASA
Determine OD values.
Embodiment 10：The detection of synovia sample
The previous day is detected, prepares detection ELISA ELISA Plates first：Buffer solution is coated with by antibody (the anti-human CTGF of mouse Dan Ke
Grand antibody) 2 μ g/ml are diluted to, 100 μ l are added in every hole of ELISA ELISA Plates, 4 DEG C of overnight incubations are placed in after shrouding.
During detection, synovia sample sample diluting liquid is with 1：50 dilutions, are loaded with the volume in 100 μ l/ holes, incubation at room temperature 2
Hour；CTGF recombinant proteins are diluted to 4000pg/ml, 2000pg/ml, 1000pg/ml, 500pg/ml with sample diluting liquid,
250pg/ml, 125pg/ml, 62.5pg/ml difference gradient, it is loaded, is incubated at room temperature 2 hours with the volume in 100 μ l/ holes；Get rid of each
Liquid in hole, ELISA ELISA Plate cleaning solutions are added, per the μ l of hole 300, wash 3-5 times, dry；By CTGF detection antibody dilutions
Into 1 μ g/ml, per the μ l of hole 100, it is incubated at room temperature 90 minutes；The liquid in each hole is got rid of, adds ELISA ELISA Plate cleaning solutions, per hole
300 μ l, wash 3-5 times, dry；The goat anti-rabbit igg of HRP marks is added, per the μ l of hole 100, is incubated at room temperature 1 hour；Get rid of each
Liquid in hole, ELISA ELISA Plate cleaning solutions are added, per the μ l of hole 300, wash 6-8 times, dry；TMB is added, per the μ l of hole 100,
Room temperature lucifuge is incubated 10-20 minutes, adds 2mol/L sulfuric acid solution terminating reactions, per the μ l of hole 50；(450nm) is surveyed on ELIASA
Determine OD values.
Embodiment 11：The evaluation of CTGF protein ELISA kits
1st, the evaluation of clinical serum pattern detection
300 parts of serum samples for being diagnosed as patient RA and 150 parts of healthy blood donor's serum are collected from clinic, with embodiment 8
CTGF is horizontal in the ELISA method detection serum of foundation, using CTGF recombinant proteins as positive control, according to gained standard items OD
Value draws standard curve with corresponding concentration, calculates the serological levels of CTGF albumen in each crowd, and judges its positive and negative,
As a result as shown in table 3, it is 245 to find the positives sample size of RA patient, and the quantity of negative sample is 55, in healthy blood donor
Positive sample size is 14, and the quantity of negative sample is 136, and by being calculated, its specificity is 90.67%, is examined
Disconnected sensitiveness is 81.67%, accuracy 84.67%.
The clinical serum sample CTGF kits of table 3 detect
2nd, the evaluation of clinical synovia pattern detection
108 parts of synovia samples for being diagnosed as patient RA and 62 parts of normal synovia samples are collected from clinic, are established with embodiment 9
ELISA method detection serum in CTGF it is horizontal, regard CTGF recombinant proteins as positive control, according to gained standard items OD values and
Corresponding concentration draws standard curve, calculates horizontal in the synovia of CTGF albumen in each crowd, and judges its positive and negative, as a result
As shown in table 3, it is 97 to find the positives sample size of RA patient, and the quantity of negative sample is 11, and healthy blood donor is positives
Sample size be 6, the quantity of negative sample is 56, and by being calculated, its specificity is 90.32%, and diagnosis is sensitive
Property is 89.81%, accuracy 90%.
The clinical synovia sample CTGF kits detection of table 4
3rd, stabilization of kit is evaluated
All reagents are put into 37 DEG C to place 24 hours, testing result no significant difference (P=0.210), place 48
Hour, testing result no significant difference (P=0.043), the kit of 4 DEG C of preservations is placed 4 months, testing result difference
Not statistically significant (P=0.37), the kit of 4 DEG C of preservations are placed 6 months, and testing result has significant difference (P=0.049)
4th, Cost benefit assessment
CTGF ELISA kits are mostly scientific research kit at present, using GenWay Biotech companies CTGF kits as
Example, up to 7000 yuan of its unit price, if costly up to 175 yuan for clinic, each patient.And the present invention has significant price excellent
Gesture, single about 2000 yuan of kit totle drilling cost, and about 40 clinical samples are can detect per plate kit, every sample cost is only
50 yuan, easily by big well-established；In addition, it has high diagnostic value to RA.In summary, it is provided by the present invention
Kit has wide market prospects.
The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (11)
1. application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared.
2. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that described diagnostic reagent or kit is that human connective tissue growing factor quantitatively detects
ELISA diagnostic reagents or kit.
3. human connective tissue growing factor according to claim 2 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that described diagnostic reagent or kit detection serum or synovia in human connective tissue growth because
The expression quantity of son.
4. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that described diagnostic reagent or kit is to utilize ELISA method, is detected in serum or synovia
The expression quantity of human connective tissue growing factor.
5. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that the optimal critical value of human connective tissue growing factor is 87-90pg/ml in described serum；
The optimal critical value of human connective tissue growing factor is 103-105pg/ml in described synovia.
6. human connective tissue growing factor according to Claims 2 or 3 prepare diagnosis of rheumatoid arthritis reagent or
Application in kit, it is characterised in that described diagnostic reagent include human connective tissue growing factor standard items, quality-control product,
The coated microwell plate of Streptavidin, the human connective tissue growing factor antibody of biotin labeling, horseradish peroxidase-labeled
Human connective tissue growing factor antibody, dilution buffer, substrate solution, terminate liquid and cleaning buffer solution.
7. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that described diagnostic reagent or kit is early atage RA diagnostic reagent or examination
Agent box, described early atage RA referred to symptom duration less than 6 months.
8. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that human connective tissue growing factor and anti-citrulling protein antibodies, which are combined, in described serum examines
It is disconnected.
9. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or reagent
Application in box, it is characterised in that human connective tissue growing factor, anti-citrulling protein antibodies and serum class in described serum
Rheumatism agents diagnose.
10. human connective tissue growing factor according to claim 1 is preparing diagnosis of rheumatoid arthritis reagent or examination
Application in agent box, it is characterised in that described diagnostic reagent or kit antidiastole rheumatoid arthritis and non-class wind
Other rheumatism immunological diseases of wet arthritis, other rheumatism immunological diseases of described non-rheumatoid arthritis refer to tatanic
Rachitis, osteoarthritis, systemic loupus erythematosus, gout or dry syndrome.
11. a kind of rheumatoid arthritis ELISA diagnostic reagents or kit, it is characterised in that including：
A the ELISA ELISA Plates of CTGF antibody) are coated with, described coated antibody is the anti-human CTGF of mouse monoclonal antibody；Coating
Antibody working concentration is 2 μ g/ml；
B the antibody of CTGF antigens) is detected, the polyclonal antibody for being rabbit-anti people CTGF；The working concentration for detecting antibody is 1 μ g/ml；
C) ELIAS secondary antibody, it is the goat anti-rabbit igg of horseradish peroxidase-labeled；Diluted concentration is 1:2000；
D) standard protein, it is recombined human CTGF fusion proteins；
E) conventional sample diluting liquid, coating buffer solution, confining liquid, ELISA ELISA Plates cleaning solution, antibody diluent, nitrite ion
And terminate liquid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710508200.1A CN107436358A (en) | 2017-06-28 | 2017-06-28 | Application of the human connective tissue growing factor in diagnosis of rheumatoid arthritis reagent or kit is prepared |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402127A (en) * | 2018-09-29 | 2019-03-01 | 复旦大学附属眼耳鼻喉科医院 | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor |
| CN109959787A (en) * | 2019-04-18 | 2019-07-02 | 漳州卫生职业学院 | A kind of rheumatoid arthritis auxiliary diagnostic box and detection method |
| CN111007262A (en) * | 2019-12-25 | 2020-04-14 | 漳州卫生职业学院 | Kit for auxiliary diagnosis of RA and detection method |
| CN111060694A (en) * | 2019-12-19 | 2020-04-24 | 新疆维吾尔自治区人民医院 | Combined diagnosis kit for vasculitis hypertension and application thereof |
-
2017
- 2017-06-28 CN CN201710508200.1A patent/CN107436358A/en active Pending
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| Title |
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| KAZUHISA NOZAWA，ET AL.: "Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis", 《ARTHRITIS RESEARCH & THERAPY》 * |
| 丁爽等: "类风湿性关节炎患者关节滑膜液中结缔组织生长因子表达及其意义研究", 《中国实用内科杂志》 * |
| 丁爽等: "类风湿性关节炎患者血清中结缔组织生长因子水平分析", 《细胞与分子免疫学杂志》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402127A (en) * | 2018-09-29 | 2019-03-01 | 复旦大学附属眼耳鼻喉科医院 | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor |
| CN109959787A (en) * | 2019-04-18 | 2019-07-02 | 漳州卫生职业学院 | A kind of rheumatoid arthritis auxiliary diagnostic box and detection method |
| CN111060694A (en) * | 2019-12-19 | 2020-04-24 | 新疆维吾尔自治区人民医院 | Combined diagnosis kit for vasculitis hypertension and application thereof |
| CN111060694B (en) * | 2019-12-19 | 2024-02-06 | 新疆维吾尔自治区人民医院 | Combined diagnosis kit for vascular inflammatory hypertension and application thereof |
| CN111007262A (en) * | 2019-12-25 | 2020-04-14 | 漳州卫生职业学院 | Kit for auxiliary diagnosis of RA and detection method |
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