CN1314967C - Inspection and related kit for nexin of citrulline chemical fibre - Google Patents
Inspection and related kit for nexin of citrulline chemical fibre Download PDFInfo
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- CN1314967C CN1314967C CNB2005100440801A CN200510044080A CN1314967C CN 1314967 C CN1314967 C CN 1314967C CN B2005100440801 A CNB2005100440801 A CN B2005100440801A CN 200510044080 A CN200510044080 A CN 200510044080A CN 1314967 C CN1314967 C CN 1314967C
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Abstract
The present invention discloses a detection method and a related reagent kit for nexin of citrulline chemical fibers. The detection reagent kit comprises a carrier pre-coated by an antibody of resisting fiber nexin, labeled antibodies of resisting citrulline, related sample dilute solution, washing solution, color developing agents and stop solution. The detection method for nexin of citrulline chemical fibers comprises: a plate (strip) which is coated with the antibody of resisting fiber nexin in advance is taken; blood plasma is added in each hole for a reaction; the reactant is washed after taken out; the labeled antibodies of resisting citrulline are added in each hole; the labeled antibodies react and are washed; substrates-color developing agents are added in each hole for a color developing reaction; an enzyme labeling instrument or a calculating instrument or a fluorescent detector or a luminous instrument is selected according to the labeling types of the labeled antibodies of resisting citrulline for detection; detection data of sample holes and detection data of contrast holes are compared for calculating a result. The method of the present invention has the advantages of simplicity, convenience, rapidness, good repeatability, good generalization value and good application value; particularly, the sensitivity and the specificity of the present invention are largely higher than those of a conventional method.
Description
Technical field
The present invention relates to a kind of detection method of albumen, relate in particular to a kind of method for quick and related kit of fibronectin of citrullineization.
Technical background
Rheumatoid arthritis is a kind of self-immunological diseases.This disease shows as chronic polyarthirtis, the pathology painful swelling of joints, and late period, joint tissue was subjected to havoc, the function of joint forfeiture.Rheumatoid arthritis people's body contains various self-immune antiboidies, and wherein most important is rheumatoid factor.Rheumatoid factor is a kind of immunoglobulin (Ig) of anti-G type immunoglobulin (Ig).Nearly seventy percent patient blood has rheumatoid factor.Therefore, whether the existence of rheumatoid factor is one of common counter of diagnostics classes rheumatic arthritis clinically.But, rheumatoid factor also is present in the patients'blood such as systemic lupus erythematosus, xerosis, scleroderma and dermatomyositis, and often come across in other many chronic inflammation patient bloods, comprise chronic hepatitis, tumour, tuberculosis, subacute bacterial endocarditis etc.Therefore rheumatoid factor is not the special self-immune antiboidy of rheumatoid arthritis.In addition, clinically also often by anti-keratin microfilament protein antibody in the detection blood, antikeratin antibody, the level of antiperinuclear factor and anti-cyclic citrulline antibody is come diagnostics classes rheumatic arthritis.But above mensuration all is antibody in patient's blood but not antigen mark; And the generation of self-immune antiboidy is non-specific often.Therefore, the research and development of new rheumatoid arthritis special mark, especially antigenic mark and it is carried out fast detecting will be the primary developing direction of this disease of timely and effective diagnosis and its reagent for clinical diagnosis of preparation and medicine.
(Peptidylarginine deiminase is to be present in a kind of enzyme in the tissue PAD) to the Peptidyl arginine deiminase type IV enzyme, at present, has found five kinds of PAD enzymes (being PAD1,2,3,4 and 6) altogether.It can carry out the back translation to some other histone and modify (post-translational modification) under the situation that calcium ion exists.This enzyme can be catalyzed into carbonyl with the amino of the arginine in the polypeptied chain (arginine), thereby makes arginine change into citrulline (citrulline).This is referred to as citrullineization (citrullination) by the process that arginine in the polypeptide of PAD catalysis changes into citrulline.In recent years, by the research of immunology, cellular biochemistry and molecular genetics, PAD4 enzyme (also it is called PADI4 or PAD5 in some article) is proved to be in human rheumatoid arthritis pathology generating process and plays important effect.2004, Chang Xiaotian finds when the work of Japanese RIKEN: the PAD4 enzyme has in rheumatoid arthritis synovial tissue of joint widely expressed, and the fibronectin in the synovial tissue is by citrullineization simultaneously; And to the rheumatoid arthritis patient, the systemic lupus erythematosus patient, the further detection back that normal person's blood plasma carries out is found, fibronectin (fibronectin) in the rheumatoid arthritis patient blood plasma also presents obvious citrullineization, the a large amount of PAD4 enzyme that exists in this and the rheumatoid arthritis synovial membrane chamber is onrelevant not, and this citrulline fibronectin has the specifically expressing and the existence of height in rheumatoid arthritis patient's arthritis synovial tissue and blood, the comparing result highly significant, its specificity is considerably beyond traditional rheumatoid arthritis index of correlation---rheumatoid factor, with histogenic immunity chemistry and Western blot checking, also confirmed this result, therefore, the citrulline fibronectin will be expected to become a kind of new specific antigenic mark for rheumatoid arthritis.
The citrulline fibronectin will be to quick diagnosis rheumatoid arthritis and its reagent for clinical diagnosis of preparation and medicine are significant clinically as specific antigenic mark for rheumatoid arthritis.But the detection method and the matched reagent that also do not have by retrieval, disclosed citrulline fibronectin at present.
Summary of the invention
In prior art, have nonspecific defective with rheumatoid factor diagnostics classes rheumatic arthritis, the problem to be solved in the present invention is to utilize the citrulline fibronectin as specific antigenic mark for rheumatoid arthritis, and a kind of detection method and related kit of fibronectin of citrullineization is provided.Quick to realize, accurate, as to make things convenient for diagnostics classes rheumatic arthritis purpose.
The detection kit of the citrulline fibronectin that the present invention relates to comprises that anti-fibronectin antibody wraps good anti-citrulline antibody of the carrier of quilt, mark and relevant sample diluting liquid, washing lotion, developer, stop buffer in advance.
Wherein, the carrier that above-mentioned anti-fibronectin antibody wraps quilt in advance is meant anti-fibronectin antibody is diluted to 5～150ug/ml with damping fluid, the ELISA Plate of conventional bag quilt, enzyme mark bar, microballoon or film.
Wherein, the anti-citrulline antibody that above-mentioned mark is good is meant with the anti-citrulline antibody of conventional method through enzyme labeling, fluorescein-labelled, isotope labeling, golden mark or chemiluminescent substance mark.
The present invention also provides a kind of detection kit of citrulline fibronectin, comprise by anti-fibronectin antibody or anti-citrulline antibody point and being marked on the nitrocellulose filter, the diafiltration plate made from conventional method and with the anti-citrulline antibody and the washing lotion of colloid gold label.
The present invention provides a kind of detection kit of citrulline fibronectin again, comprise with anti-fibronectin antibody or anti-citrulline antibody lining in proportion on the nitrocellulose filter fast detecting citrulline fibronectin gold test strip bar of making by conventional method.
Use the method that kit of the present invention detects the citrulline fibronectin, step is as follows:
When detecting blood preparation, get the ELISA Plate that is coated with anti-fibronectin antibody in advance or the enzyme mark bar of institute's expense, every hole adds through the sample diluting liquid blood plasma 100ul of dilution proportion routinely, putting 30～37 ℃ reacted 1～4 hour down, take out the back with containing the PBS washing lotion that percent by volume is 0.01%～0.05%Tween-20, wash 3～5 times, every hole adds the good anti-citrulline antibody 100ul of mark of dilution in proportion, putting 30～37 ℃ reacted 1～2 hour down, wash 3～5 times, every hole adds substrate---developer 100ul, 30～37 ℃ of chromogenic reactions 10～60 minutes, according to anti-citrulline antibody labeling type selecting microplate reader, calculating instrument, fluorescence detector or light-emitting appearance detect, and are compared by the detection data of sample well and the detection data of control wells, calculate the result.
Wherein, described developer is according to the conventional developer of anti-citrulline antibody labeling type corresponding selection.
Use the method that diafiltration plate kit of the present invention detects the citrulline fibronectin, step is as follows:
Get the diafiltration plate for preparing, blood plasma to be checked be added drop-wise in the diafiltration plate hole 2～3, treat that diafiltration is intact after, drip 2～3 of the anti-citrulline antibody of colloid gold label, treat that diafiltration is intact after, drip 2～5 of washing lotions, the intact back of diafiltration is the visual inspection result directly.
Use citrulline chemical fibre dimension of the present invention and connect the method for Protein Detection with gold test strip bar detection citrulline fibronectin, step is as follows:
Get " citrulline chemical fibre dimension connects Protein Detection gold test strip bar " that prepared, its particular end is directly inserted in the blood plasma to be checked taken out after 1～2 minute, 5～15 minutes, according to the red precipitate line judged result that occurs on the test strips.
In the detection method of the citrulline fibronectin that the present invention relates to, the detection method of most preferred citrulline fibronectin is made up of following steps:
A. dilute 2000～5000 times of antifibrin monoclonal antibodies with 0.05M, PH9.6 carbonic acid/heavy carbonic damping fluid, drip the 100ul antibody-solutions in the sample well of 96 hole ELISA reaction plate, flat board is placed under 4 ℃, 10～12 hours, make antibody with dull and stereotyped crosslinked;
B. clean the ELISA reaction plate 3～5 times with PBS-Tween, the antibody that flush away is uncrosslinked;
C. drip 100ul with the percentage by weight of PBS damping fluid preparation be 5% skimmed milk power in the sample well of ELISA reaction plate, reacted under the room temperature 1～2 hour, adhere on the crosslinked antibody to stop nonspecific proteins;
D. wash the ELISA reaction plate 3 times with PBS-Tween, the above-mentioned unnecessary skimmed milk power of flush away;
E. use 3000～8000 times of PBS damping fluid dilute sample blood plasma, the above-mentioned sample that drips 100ul places flat board under 30～37 ℃ in ELISA reaction plate sample well, reacts 1～4 hour, makes antibody linked on fibronectin in the blood plasma and the flat board;
F. wash the ELISA reaction plate 3～5 times with PBS-Tween, the albumen that flush away is uncrosslinked;
G. with 3000～5000 times of the anti-citrulline antibody of above-mentioned 5% skimmed milk power dilution rabbit, the antibody that drips 100ul is in ELISA reaction plate sample well, described antibody is labeled alkaline phosphatase in advance, flat board is placed 30～37 ℃ of down reactions 1～2 hour, the citrulline chemical fibre that has adsorbed on anti-citrulline antibody and the flat board is tieed up be connected protein-crosslinking;
H. wash the ELISA reaction plate 3～5 times with PBS-Tween, the antibody that flush away is uncrosslinked;
I. drip 100ul and contain alkaline phosphatase substrate reagent A lkaline phosphatase yellow, make dull and stereotyped anti-citrulline antibody colour developing of going up absorption, under the room temperature, chromogenic reaction 10-60 minute, till reaction color is significantly;
J. detect absorbance value with spectrophotometer under 405 nanometers, absorbance value is the concentration of citrulline fibronectin in the representative sample as a result;
Wherein:
Above-mentioned PBS buffer formulation is: 8g/L NaCl, 0.2g/L KCl, 1.15g/L NaHPO
4And 0.2g/LKH2PO
4, pH7.4-7.6;
Above-mentioned PBS-Tween eluent: be meant to contain the PBS liquid that percent by volume is 0.01%～0.05%Tween-20.
Above-mentioned blood plasma is to be the patient blood of 3.8% sodium citrate (sodium citrate) by final concentration, under 500X to 1000Xg centrifugal 30-60 minute, and to collect supernatant and obtain, described blood plasma can be preserved down at-80 ℃.
The detection kit of the citrulline fibronectin that the present invention proposes and this kit of application detect the citrulline fibronectin, and the methods for clinical diagnosis and the designing and developing all of novel diagnostic reagent and medicine of setting up novel rheumatoid arthritis disease had very important significance.The detection method of the citrulline fibronectin that the present invention relates to is to come diagnostics classes rheumathritis disease with the notion that detects antigen for the first time on the clinicopathologia, also has important directive significance.
The detection kit of the citrulline fibronectin that the present invention relates to is formed simply, and convenient and practical, cost is low, and is easy and simple to handle, flexible, for clinical quick diagnosis provides simple and direct, testing tool easily, has application value widely.
The detection method of the citrulline fibronectin that the present invention relates to is a kind of very sensitive and detection method fast.The citrulline fibronectin that can be widely used in clinically in the detection type rheumathritis patient blood changes, to diagnose the illness and to understand the patient's condition.The detection method of the citrulline fibronectin that the present invention relates to has advantage, especially its sensitivity and specificity easy and simple to handle, quick, good reproducibility and is much higher than conventional method, has good practical value, and is easy to promote the use of.
Description of drawings
Fig. 1 Sandwich ELISA method detects the fibronectin of citrullineization in the blood plasma.
The fibronectin concentration of citrullineization in absorbance value (O.D.) representative sample.The result shows the content of the content of citrulline in arthritic (RA) blood plasma apparently higher than the citrulline of health group (healthy controls) and erythematosus lupus group (SLE).
Fig. 2. the expression of citrulline fibronectin in rheumatoid arthritis synovial tissue.
Method with the histogenic immunity chemistry, anti-fibronectin antibody and anti-citrulline protein antibodies are respectively at the serial section (A1 of rheumatoid arthritis synovial tissue, A2 and B1 detect the coexpression of two kinds of albumen on B2), illustrate that the fibronectin of pathological tissues is citrullineization.But (C1 C2), does not observe the existence that tangible extracellular fiber connects albumen in Osteoarthritis synovial tissue.
Embodiment
Embodiment 1: the fibronectin of measuring citrullineization in patient's blood
(1) blood plasma preparation.
With containing the blood collection tube collection rheumatoid arthritis patient that final concentration is 3.8% sodium citrate (sodium eitrate), systemic lupus erythematosus patient, normal person's new blood.Under 1000X g centrifugal 30 minutes.Collect supernatant.Blood plasma can be preserved down at-80 ℃.
(2) solution is prepared
The PBS buffer formulation is: 8g/L NaCl, 0.2g/L KCl, 1.15g/L NaHPO
4With 0.2g/L KH2PO
4PH7.5;
PBS-Tween eluent: Tween 20 is added the preparation of PBS damping fluid, stirring and evenly mixing in 1: 1000 ratio.
(3) step:
A. use 0.05M, pH9.6 carbonic acid/heavy carbonic (carbonate-bicarbonate) damping fluid dilution antifibrin monoclonal antibody (Abcam company product) 4000 times, making its its concentration is 50ug/ml, drip the 100ul antibody-solutions in the sample well of 96 hole ELISA reaction plate, flat board is placed under 4 ℃, 12 hours, make antibody with dull and stereotyped crosslinked;
B. clean the ELISA reaction plate 3 times with PBS-Tween, the antibody that flush away is uncrosslinked;
C. drip 100ul with the percentage by weight of PBS damping fluid preparation be 5% skimmed milk power in the sample well of ELISA reaction plate, reaction is 2 hours under the room temperature, adheres on the crosslinked antibody to stop nonspecific proteins;
D. wash the ELISA reaction plate 3 times with PBS-Tween, the above-mentioned unnecessary skimmed milk power of flush away;
E. use 4000 times of PBS damping fluid dilute sample blood plasma, the above-mentioned sample that drips 100ul places flat board under 37 ℃ in ELISA reaction plate sample well, reacts 2 hours, makes antibody linked on fibronectin in the blood plasma and the flat board;
F. wash the ELISA reaction plate 3 times with PBS-Tween, the albumen that flush away is uncrosslinked;
G. with the above-mentioned 5% skimmed milk power dilution anti-citrulline antibody of rabbit (Biogenesis company product) 5000 times, the antibody that drips 100ul is in ELISA reaction plate sample well, described antibody is labeled alkaline phosphatase (APlabeling kit in advance, Roche produces), flat board is placed 37 ℃ of down reactions 2 hours, the citrulline chemical fibre that has adsorbed on anti-citrulline antibody and the flat board is tieed up be connected protein-crosslinking;
H. wash the ELISA reaction plate 3 times with PBS-Tween, the antibody that flush away is uncrosslinked;
I. drip 100ul and contain alkaline phosphatase substrate reagent A lkaline phosphatase yellow (Sigma product), make dull and stereotyped anti-citrulline antibody colour developing of going up absorption, under the room temperature, chromogenic reaction 30 minutes is till reaction color significantly;
J. detect absorbance value with spectrophotometer under 405 nanometers, absorbance value is the concentration of citrulline fibronectin in the representative sample as a result; The result shows that the fibronectin of citrullineization only is present in (see figure 1) in rheumatoid arthritis patient's the blood.
Embodiment 2: the fibronectin of measuring citrullineization in patient's blood
(1) blood plasma preparation.
With containing the blood collection tube collection rheumatoid arthritis patient that final concentration is 3.8% sodium citrate (sodium citrate), systemic lupus erythematosus patient, normal person's new blood.Under 8000Xg centrifugal 30 minutes.Collect supernatant.Blood plasma can be preserved down at-80 ℃.
(2) solution is prepared
The PBS buffer formulation is: 8g/L NaCl, 0.2g/L KCl, 1.15g/L NaHPO
4With 0.2g/L KH2PO
4, pH7.6;
PBS-Tween eluent: Tween 20 is added the preparation of PBS damping fluid, stirring and evenly mixing in 1: 1000 ratio.
(3) step:
A. use 0.05M, PH9.6 carbonic acid/heavy carbonic (carbonate-bicarbonate) damping fluid dilution antifibrin monoclonal antibody (Abcam company product) 3000 times, drip the 100ul antibody-solutions in the sample well of 96 hole ELISA reaction plate, flat board is placed under 4 ℃, 10～12 hours, make antibody with dull and stereotyped crosslinked;
B. clean the ELISA reaction plate 5 times with PBS-Tween, the antibody that flush away is uncrosslinked;
C. drip 100ul with the percentage by weight of PBS damping fluid preparation be 5% skimmed milk power in the sample well of ELISA reaction plate, reaction is 1 hour under the room temperature, adheres on the crosslinked antibody to stop nonspecific proteins;
D. wash the ELISA reaction plate 3 times with PBS-Tween, the above-mentioned unnecessary skimmed milk power of flush away;
E. use 5000 times of PBS damping fluid dilute sample blood plasma, the above-mentioned sample that drips 100ul places flat board under 34 ℃ in ELISA reaction plate sample well, reacts 3 hours, makes antibody linked on fibronectin in the blood plasma and the flat board;
F. wash the ELISA reaction plate 4 times with PBS-Tween, the albumen that flush away is uncrosslinked;
G. with the above-mentioned 5% skimmed milk power dilution anti-citrulline antibody of rabbit (Biogenesis company product) 4000 times, the antibody that drips 100ul is in ELISA reaction plate sample well, described antibody is labeled alkaline phosphatase (APlabeling kit in advance, Roche produces), flat board is placed 35 ℃ of down reactions 1.5 hours, the citrulline chemical fibre that has adsorbed on anti-citrulline antibody and the flat board is tieed up be connected protein-crosslinking;
H. wash the ELISA reaction plate 4 times with PBS-Tween, the antibody that flush away is uncrosslinked;
I. drip 100ul and contain alkaline phosphatase substrate reagent A lkaline phosphatase yellow (Sigma product), make dull and stereotyped anti-citrulline antibody colour developing of going up absorption, under the room temperature, chromogenic reaction 40 minutes is till reaction color significantly;
J. detect absorbance value with spectrophotometer under 405 nanometers, absorbance value is the concentration of citrulline fibronectin in the representative sample as a result; The result shows that the fibronectin of citrullineization only is present in (see figure 1) in rheumatoid arthritis patient's the blood.
Embodiment 3: kit is formed
1. anti-fibronectin pre-coated elisa plate (bar)
Get anti-fibronectin antibody in 1: 3000 ratio, after the dilution of PH9.6 carbonate buffer solution, every hole adds 100ul, put room temperature after 4 hours, after containing 0.05%Tween-20PBS washing 3 times, every hole add 200ul2%BSA put 37 ℃ 2 hours, after button is done, dry up, in tin platinum bag on the vacuum seal, 4 ℃ of preservations.
2. anti-citrulline antibody is with the conventional mark of horseradish peroxidase.
3. product dilution: contain 2%BSA, PBS.
4. wash liquid: contain 0.05%Tween-20PBS.
5. developer: 0.4%TMB.
6. stop buffer: 2%H
2SO
4
Embodiment 4: detect and use:
1. the pre-bag of getting institute's expense is by plate (bar), with blood plasma to be checked with sample diluting liquid 4000 dilutions after, every hole adds 100ul, put 37 ℃ 1 hour;
2. wash 5 times with cleansing solution;
3. the anti-citrulline antibody of the enzyme-added mark in every hole 100ul, 37 ℃ 1 hour;
4. wash 5 times with cleansing solution;
5. every hole adds developer 100ul, 37 ℃ 15 minutes;
6. every hole adds stop buffer 50ul, surveys the absorbance in each hole in microplate reader 405nm place;
7. according to each hole absorbance size, compare result of calculation with the control wells absorbance.
Embodiment 5: with detection type rheumathritis synovial tissue of immunohistochemical method citrulline fibronectin
1. 0.3 cubic centimetre of left and right sides arthritis synovial membrane is fixed 12 hours in 10% formalin buffer (buffered formalin).Piece of tissue after fixing is dewatered according to standard program, and embedding is also made the paraffin section of 10 micron thickness.
2. the microslide that will be loaded with paraffin section dewaxes in dimethylbenzene and alcohol according to standard program, rehydration, and suspend in distilled water.
3. histotomy is placed the citrate buffer solution item for disposal 15 minutes of 95 ℃ of degree, the citrate buffer solution that will be loaded with section is then transferred to and was cooled off under the normal temperature 15 minutes again. and citrate buffer solution can strengthen the tissue antigen of albumen greatly.
4. histotomy is placed again distilled water water logging bubble twice, each 3 minutes.
5. histotomy is placed the PBS damping fluid to soak twice, each 5 minutes.
6. 3% skimmed milk power with the preparation of PBS damping fluid dilutes anti-fibronectin antibody (Abcam company product) and anti-citrulline protein antibodies (Upstate company product).The antibody of debita spissitudo is dripped on histotomy.Under 4 ℃ of degree, carry out antigen-antibody reaction 12 hours.
7. histotomy is placed the PBS damping fluid to soak each 5 minutes 3 times.
8. dilute the second anti-mouse or the rabbit igg antibody (for example Zemed company product) of phosphorylase mark with above skimmed milk power.The antibody of debita spissitudo dropped in add on the histotomy.Reaction was at room temperature carried out 1 hour.
9. histotomy is placed the PBS damping fluid to soak each 5 minutes three times.
10. handle histotomy, the position of display target albumen with suitable colour former.
11. histotomy is compared dyeing with the brazilwood extract dyeing agent.
12. dehydration, mounting.
13. microscopy.
The result shows: the fibronectin of citrullineization only is present in (see figure 2) in rheumatoid arthritis patient's the synovial tissue.
Claims (6)
1. the detection kit of a citrulline fibronectin comprises that anti-fibronectin antibody wraps good anti-citrulline antibody of the carrier of quilt, mark and relevant sample diluting liquid, washing lotion, developer, stop buffer in advance; Wherein:
After the carrier that described anti-fibronectin antibody wraps quilt in advance is meant anti-fibronectin antibody is diluted to 5～150 mcg/ml with damping fluid, the ELISA Plate of conventional bag quilt, enzyme mark bar, microballoon or film;
The anti-citrulline antibody that described mark is good is meant with the anti-citrulline antibody of conventional method through enzyme labeling, isotope labeling, golden mark or chemiluminescent substance mark.
2. the detection kit of citrulline fibronectin as claimed in claim 1 is characterized in that: described chemiluminescent substance mark is fluorescein-labelled.
3. the detection kit of a citrulline fibronectin comprises the nitrocellulose filter that a little indicates anti-fibronectin antibody, the diafiltration plate made from conventional method and with the anti-citrulline antibody and the washing lotion of colloid gold label.
4. application rights requires 1 described kit to detect the method for citrulline fibronectin, and step is as follows:
When detecting blood preparation, get the ELISA Plate that is coated with anti-fibronectin antibody in advance or the enzyme mark bar of institute's expense, every hole adds through the sample diluting liquid blood plasma 100ul of dilution proportion routinely, putting 30～37 ℃ reacted 1～4 hour down, take out the back with containing the PBS washing lotion that percent by volume is 0.01%～0.05%Tween-20, wash 3～5 times, every hole adds the good anti-citrulline antibody 100ul of mark of dilution in proportion, putting 30～37 ℃ reacted 1～2 hour down, wash 3～5 times, every hole adds substrate---developer 100ul, 30～37 ℃ of chromogenic reactions 10～60 minutes, according to anti-citrulline antibody labeling type selecting microplate reader, calculating instrument, fluorescence detector or light-emitting appearance detect, and are compared by the detection data of sample well and the detection data of control wells, calculate the result.
5. application kit as claimed in claim 4 detects the method for citrulline fibronectin, it is characterized in that, described developer is according to the conventional developer of anti-citrulline antibody labeling type corresponding selection.
6. application rights requires 3 described kits to detect the method for citrulline fibronectin, and step is as follows:
Get the diafiltration plate for preparing, blood plasma to be checked be added drop-wise in the diafiltration plate hole 2～3, treat that diafiltration is intact after, drip 2～3 of the anti-citrulline antibody of colloid gold label, treat that diafiltration is intact after, drip 2～5 of washing lotions, the intact back of diafiltration is the visual inspection result directly.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100440801A CN1314967C (en) | 2005-07-18 | 2005-07-18 | Inspection and related kit for nexin of citrulline chemical fibre |
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|---|---|---|---|
| CNB2005100440801A CN1314967C (en) | 2005-07-18 | 2005-07-18 | Inspection and related kit for nexin of citrulline chemical fibre |
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| CN1314967C true CN1314967C (en) | 2007-05-09 |
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Families Citing this family (2)
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| KR101968779B1 (en) * | 2011-05-27 | 2019-04-12 | 노르딕 바이오사이언스 에이/에스 | Detection of diagnostic peptides |
| CN107490698A (en) * | 2017-09-15 | 2017-12-19 | 广州市雷德生物科技有限公司 | A kind of detection kit and its application |
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Non-Patent Citations (4)
| Title |
|---|
| Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity. Karin Lundberg,et al,Arthritis Research & Therapy,Vol.7 No.3 2005 * |
| Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity. Karin Lundberg,et al,Arthritis Research & Therapy,Vol.7 No.3 2005;Citrullination of synovial proteins in murine models of rheumatoid arthritis. Vossenaar ER,et al,Arthritis Rheum,Vol.48 No.9 2003;Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis X,Chang,et al,Rheumatology,Vol.44 No.1 2005 * |
| Citrullination of synovial proteins in murine models of rheumatoid arthritis. Vossenaar ER,et al,Arthritis Rheum,Vol.48 No.9 2003 * |
| Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis X,Chang,et al,Rheumatology,Vol.44 No.1 2005 * |
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