CN102346190A - Double-antibody sandwich ELISA reagent kit and method for detecting hyperoxide reductase IV in biological sample and application of reagent kit - Google Patents
Double-antibody sandwich ELISA reagent kit and method for detecting hyperoxide reductase IV in biological sample and application of reagent kit Download PDFInfo
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Abstract
The invention relates to the field of medical detection, in particular to a double-antibody sandwich ELISA reagent kit for detecting hyperoxide reductase IV in a biological sample. The reagent kit comprises a trapping agent consisting of a specific monoclonal antibody and/or polyclonal antibody resisting against the hyperoxide reductase IV, an enzyme Labeling second antibody, sample diluents and a substrate. The reagent kit is especially used for detecting the content of the hyperoxide reductase IV and further judging whether the level of hyperoxide reductase IV in a human body exceeds the standard or not. The reagent kit has high specificity and sensitivity.
Description
Technical field
The present invention relates to field of medical examination, particularly be used for double antibodies sandwich ELISA kit and the method and the utilization of the superoxide reductase IV of detection of biological sample.
Background technology
Rheumatoid arthritis (RA) be a kind of be that basic pathology changes with the synovitis; With the chronic destructive arthropathy general autoimmunity disease that is characteristic; Mostly the state of an illness is carrying out property, finally causes joint stringiness or bony ankylosis and seriously disables, and has a strong impact on patients ' life quality.But, even cause joint deformity without the rheumatoid arthritis protracted course of disease of correct treatment.As autoimmune disease, the autoreactivity CD4 that pathogenic antigens drives
+The T cell activation is the core approach of rheumatoid arthritis morbidity.
For the diagnosis of rheumatoid arthritis, main flow is still continued to use the criteria for classification of the RA of U.S. rheumatology association in 1987 now, thereby it mainly leans on clinical manifestation and gets rid of other arthritis RA is judged.In addition, the X line changes and rheumatoid factor also is to judge the working standard of RA.But said method complex operation and specificity are not high, and are not suitable for early diagnosis.
As everyone knows, the pathological change of RA begins to develop to bone to cartilage gradually from synovial tissue.RA will be caused joint function disturbance even labour forfeiture, and cause the many internal organs of whole body to be got involved and then threat to life in early days if effectively treat.If can make diagnosis to the RA state of an illness as early as possible, and early treatment, thereby stop or delay its development, can effectively improve the result of treatment of RA and reduce the generation of its complication.And to RA high susceptibility and specific detection index are arranged in early days is to realize the prerequisite of RA early diagnosis and therapy.
In the early diagnosis index, IL-8, IL-6 level raise in the positive rheumatoid arthritis patients of cytomegalovirus dna and there is contact in the CMV genome between occurring, but specificity and susceptibility are low.At the RA autoantibody as detecting aspect the index: there is the low shortcoming of sensitivity in rheumatoid factor (RF); Patient's antiperinuclear factor (APF) that can 53.3% among the negative rheumatoid arthritis patient of RF is positive in early days, but the holding time of APF antigen sheet lack (only being 1～2 week), detect stable low; Sa antibody positive rate in rheumatoid arthritis is 40%, and specificity is 98.9%, but rheumatoid arthritis in early days only about 23%; The correlation research of superoxide reductase IV and rheumatoid arthritis someone is as yet reported.
Summary of the invention
One of the object of the invention is to provide a kind of kit, and the content of this kit ability fast detecting superoxide reductase IV is simple to operate, with low cost.
For realizing above-mentioned purpose, technical scheme of the present invention is:
1, the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample comprises specific monoclonal antibody and/or many anti-trapping agent, ELIAS secondary antibody, sample diluting liquid and substrates of forming with antiperoxide reductase IV.
2, further, said trapping agent is a mouse anti superoxide reductase IV IgG antibody.
3, further, mouse anti superoxide reductase IV IgG antibody is not more than 640 times of volume dilution, and its dilution is a trapping agent.
4, according to the 1 described double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample, said trapping agent invests on the solid support.
5, according to the 4 described double antibodies sandwich ELISA kits that are used for the superoxide reductase IV of detection of biological sample, said solid support is ELISA Plate or biochip.
6, according to the 1 described double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample, said biological sample is serum, urine, groups of cells fabric or blood plasma.
7, according to the 6 described double antibodies sandwich ELISA kits that are used for the superoxide reductase IV of detection of biological sample, said biological sample is a serum.
8, according to the 1 described double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample, said ELIAS secondary antibody is rat anti people's superoxide reductase IV IgG antibody of HRP mark.
9, according to the 8 described double antibodies sandwich ELISA kits that are used for the superoxide reductase IV of detection of biological sample; Rat anti people's superoxide reductase IV IgG antibody of said HRP mark is not more than 640 times of volume dilution liquid, and its dilution is an ELIAS secondary antibody.
10, according to the 1 described double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample, the amount ratio of said trapping agent and ELIAS secondary antibody is 1:1.
Two of the object of the invention is to provide the purposes of mentioned reagent box; This purposes can remedy the technological gap of superoxide reductase IV detection range; And, be specially adapted to the detection of biological sample for rheumatoid arthritis and other immunity disease provide diagnosis train of thought.
For realizing above-mentioned purpose, technical scheme of the present invention is:
11, each said double antibodies sandwich ELISA kit of 1-10 is in the application that detects superoxide reductase IV content.
12, use the application of said kit in the detection type rheumathritis according to 11 described double antibodies sandwich ELISA kits.
13, use according to 11 described double antibodies sandwich ELISA kits, said kit detects the content of the superoxide reductase IV of test serum and normal human serum, as the OD of test serum
450nmValue is that superoxide reductase IV is positive greater than 0.257, is less than or equal to 0.257 and is superoxide reductase IV feminine gender.
Three of the object of the invention is to provide the method for superoxide reductase IV content, and this method susceptibility is high, and is simple to operate.
For realizing above-mentioned purpose, technical scheme of the present invention is:
14, each said double antibodies sandwich ELISA kit of utilization 1-10 detects the method for superoxide reductase IV content, a, biological sample is contacted and is incubated with trapping agent in the double antibodies sandwich ELISA kit; B, unconjugated biological sample is separated, antigen-antibody complex; C, the ELIAS secondary antibody in the antigen-antibody complex adding double antibodies sandwich ELISA of step b gained kit get the double antibodies sandwich compound; D, detection are incorporated into the content of the superoxide reductase IV of trapping agent.
15, detect the method for superoxide reductase IV content according to the said double antibodies sandwich ELISA of 14 described utilizations kit, comprise that also content and the normal person with the superoxide reductase IV of steps d gained compares judgement.
16, according to 14 described methods, among the step a,, biological sample to be measured is contacted with trapping agent the trapping agent coated elisa plate, be incubated and seal with confining liquid; Said biological sample is the serum dilution that is not more than 200 times of volume dilution, and said trapping agent is the dilution that mouse anti superoxide reductase IV IgG antibody is not more than 640 times of volume dilution.
17, detect the method for superoxide reductase IV content according to the said double antibodies sandwich ELISA of 14 described utilizations kit; Among the step b; Wash unconjugated biological sample separation with insulation among the step a and with the ELISA Plate of confining liquid sealing, get antigen-antibody complex.
18, detect the method for superoxide reductase IV content according to the said double antibodies sandwich ELISA of 14 described utilizations kit; Among the step c; ELIAS secondary antibody in the antigen-antibody complex adding double antibodies sandwich ELISA of step b gained kit is carried out insulation reaction, gets the double antibodies sandwich compound; Said ELIAS secondary antibody is the dilution that rat anti people's superoxide reductase IV IgG antibody of HRP mark is not more than 640 times of volume dilution liquid.
19, detect the method for superoxide reductase IV content according to the said double antibodies sandwich ELISA of 14 described utilizations kit; In the steps d; After in the double antibodies sandwich compound of step c gained, adding the colour developing of chromogen substrate lucifuge; Add the stop buffer cessation reaction sample liquid that must develop the color, through surveying the OD of colour developing sample liquid
450nmValue detects the content of the superoxide reductase IV that is incorporated into trapping agent.
20, detect the method for superoxide reductase IV content according to the said double antibodies sandwich ELISA of 14 described utilizations kit, said method also comprises negative control group and blank group, and said negative control group is the biological sample that stems from the normal person.
21, detect the method for superoxide reductase IV content according to the said double antibodies sandwich ELISA of 20 described utilizations kit, the OD of said biological sample to be measured
450nmValue is greater than the OD of negative control group sample
450nmWhen value mean value and its 2 times of standard variance sums, be judged to be the superoxide reductase IV positive, be less than or equal to then negative.
Beneficial effect of the present invention is: the content of the specific detection superoxide reductase IV of this kit ability, and its cost is low, and susceptibility is good; Use this kit to detect the content of superoxide reductase IV; And compare with the level of normal person's superoxide reductase IV; But the diagnostics classes rheumathritis that the specificity religion is high so this kit can be prepared into the rheumatoid arthritis diagnostic kit, is particularly useful for early diagnosis.
Description of drawings
In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is described in further detail below, wherein:
Fig. 1 is superoxide reductase IV double antibody sandwich method ELISA testing result figure in the middle of the various disease group serum.
Fig. 2 is a superoxide reductase IV double antibody sandwich method ELISA typical curve, and horizontal ordinate is the dilute concentration of peroxidase IV standard items, and ordinate is the corresponding OD of each dilute concentration standard items
450Value.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer, the preferred embodiments of the present invention are carried out detailed description below in conjunction with accompanying drawing.Acquisition with said people's superoxide reductase IV in the present embodiment is through making up prokaryotic expression plasmid, transforming and express bacterium and fermented and cultured; Collect a large amount of bacterial sediments that contains superoxide reductase IV, obtain through ultrasonic disruption thalline, superoxide reductase IV inclusion body Xian Di, purifying and gradient dialysis again; With said people's superoxide reductase IV rat is carried out routine immunization program and purifying, obtain rat anti people superoxide reductase IV polyclonal antibody IgG,, resist as two of double antibody sandwich method ELISA at this antibody marked horseradish peroxidase (HRP); The optimum concentration range of mouse anti human superoxide reductase IV IgG antibody and rat anti people superoxide reductase IV IgG antibody and confirming of yin and yang attribute critical value have further been confirmed; Formulated the high rheumatoid arthritis antigen superoxide reductase IV detection method of species specificity and susceptibility, it can catch 7.8ng/mL superoxide reductase IV antigen.
Embodiment
The preparation of one IgG antibody
1 makes up people's superoxide reductase IV expression plasmid, prepares recombinant antigen TRX-superoxide reductase IV fusion with the pET prokaryotic expression system.
1.1 according to the people's superoxide reductase IV maturation protein sequential coding that retrieves among the GenBank, the DNA of design composite coding superoxide reductase IV albumen;
1.2 respectively at DNA5 ' end and 3 ' end design EcoR I and Hind III restriction enzyme site, through polymerase chain reaction (PCR), construction recombination plasmid pET superoxide reductase IV;
1.3 at BL21 (DE3) through the IPTG abduction delivering;
1.4 the Ni-NTA affinity chromatography prepares TRX-superoxide reductase IV;
1.5 the correctness of the superoxide reductase IV antigen of identifying TRX-superoxide reductase IV plasmid and expression through the relative molecular mass analysis and the Western trace of dna sequencing, fusion.
2 rat anti people superoxide reductase IV Polyclonal Antibody Preparation and purifying
2.1 Freund
The a incomplete Freund: 3 parts of saxols, 1 part in sheep oil mixes, and 115 ℃, gets incomplete Freund behind the 15min autoclaving.Heating and melting before using is cooled to about 50 ℃, adds antigen and carries out emulsification treatment; The b Freund's complete adjuvant: add Bacille Calmette-Guerin on the basis of the above and get final product to final concentration 1mg/mL,, fully emulsified during use with antigenic solution and its mixed in equal amounts.
2.2 the preparation and the purifying of rat anti people's superoxide reductase IV IgG antibody
2.2.1 the preparation of rat anti people's superoxide reductase IV antibody (repeat three these tests, each 1 week at interval is to prepare the antibody of three different batches)
The immunogenic preparation of a: with the superoxide reductase IV behind the expression and purification, head exempts to mix with the equivalent complete Freund's adjuvant also fully emulsified, and second and third exempts to mix with incomplete Freund's adjuvant also fully emulsified, and the 4th exempts from not add adjuvant.
The b immune programme for children: select 4 of healthy male Wistar rats, head exempts from the previous day and takes a blood sample respectively, and separation of serum is as negative control.Preparation rat anti people's superoxide reductase IV hyper-immune serum immune programme for children such as following table:
Table 1 preparation rat anti people superoxide reductase IV hyper-immune serum immune programme for children
Four exempt from back 14d arteria carotis bloodletting, collect blood.Place 1h, 4 ℃ of refrigerator overnight for 37 ℃.Separated out serum in second day, blood clot is collected serum once more with the centrifugal 15min of 4000r/min, and-20 ℃ of preservations are subsequent use.
The purifying of rat anti people's superoxide reductase IV IgG antibody
Except that using rat, also can adopt other animal to prepare anti-people's superoxide reductase IV IgG antibody.
A caprylic acid-ammonium sulfate method is slightly put forward rat anti people superoxide reductase IV IgG antibody: slightly put forward rat anti people superoxide reductase IV IgG antibody; Step is following: the 0.06mol/L pH4.8 acetate buffer of the 10mL serum adding equivalent of separation, mixing are got in (1); (2) dropwise add caprylic acid 75 μ g/mL serum under the stirring at room, stirring at room 30min, 4 ℃ leave standstill>3h, and make its abundant post precipitation, 13, the centrifugal 30min of 000r/min; (3) get the supernatant filter paper filtering, in filtrating, add the PBS of the 10mmol/L pH7.4 of 1/10 volume, regulate pH=7.4 with 2 mol/L NaOH; (4) in liquid, slowly add saturated ammonium sulfate 0.277g/mL under the ice bath and make its final concentration reach 45%, stir 30min, put 4 ℃ and leave standstill 3h or spend the night, 13,000r/min is centrifugal, and 30min abandons supernatant; (4) ratio in 1:4 adds PBS in deposition, and fully piping and druming is fully dissolved it; (5) lysate is packed in the bag filter, 4 ℃ down with 10mmol/L pH7.4 PBS dialysis, every 6h changes liquid once, until Nessler's reagent detect do not have the deposition generation in the dislysate till.
The anti-people's superoxide of b High Q post anion-exchange chromatography purification of rat reductase IV IgG antibody: antibody purification IgG; Step is following: (1) sample pre-treatments: will slightly carry IgG and remove impurity with 0.45 μ m membrane filtration; Under 4 ℃ of conditions, carrying out dialysis equilibrium with buffer A (pH8.0,20 mmol/L Tris-HCl damping fluids) spends the night; (2) chromatographic column pre-treatment: through pre-treatments such as 0.5mol/L NaOH, washing, 1.0mol/L NaCl, washings, and with buffer A balance HighQ chromatographic column; (3) go up appearance: that gets 5mL slightly carries the IgG sample; (4) clean: with 50 mL buffer A balances and wash unconjugated protein and impurity; (5) wash-out: (1.0 mol/L NaCl) carries out linear gradient ionic strength wash-out with buffer B, flow velocity 0.5 mL/min, and elution time is 200min, the fraction collection elution fraction.
C IgG antibody purity and concentration determination: after the eluting peak sample preparation of collecting; Carry out SDS-PAGE electrophoresis (Fig. 3-B and Fig. 5), measure the purity of IgG antibody, and measure the ultimate density of IgG antibody with the Bradford method; And with its dilution for 2mg/mL, subsequent use after the packing in-20 ℃ of preservations.
3 glutaraldehyde two step method HRP mark rat anti people superoxide reductase IV IgG antibody
A takes by weighing HRP25mg and is dissolved in 1.25% glutaraldehyde solution, in the room temperature standing over night.
The reacted enzyme solutions of b is used the physiological saline wash-out through Sephadex G-25 chromatographic column.Flow speed control was collected brown effluent at 1ml/1 minute.Greater than 5ml, then be concentrated into 5ml like volume with PEG.Place in the 25ml small beaker, slowly stir.
C is diluted to 5ml with antibody 12.5mg to be marked with physiological saline, stirs down dropwise to add in the enzyme solutions.
D continues to stir 3 hours with 1M PH9.5 carbonic acid buffer 0.25ml.
E adds 0.2M lysine 0.25ml, behind the mixing, puts room temperature 2 hours.
F under agitation dropwise adds the equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour.
G 3000rpm centrifugal half an hour, abandon supernatant.Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15M PH7.4.
H packs above-mentioned solution in the bag filter, to the PB buffer saline dialysis of 0.15M PH7.4, removes (detecting) behind the ammonium ion with Nai Shi reagent, and 10,000rpm removed and precipitates in centrifugal 30 minutes, and supernatant is enzyme conjugates, after the packing, stored frozen.
4 coated antibodies
Provide by Abcam company.
5 experimental techniques
The acquisition of test serum sample
All rheumatoid arthritis to be measured (RA), osteoarthritis (OA) serum are provided by attached southwestern hospital of Third Military Medical University department of traditional Chinese medicine; Systemic loupus erythematosus (SLE) serum is provided by the attached southwestern hospital dermatology department of Third Military Medical University; Normal human serum is provided by attached southwestern hospital of Third Military Medical University Physical Examination center.
The processing of test serum sample and preservation
4 ℃ are spent the night after 3, and centrifugal 30 minutes of 000rpm gets supernatant, 100ul packing ,-70 ℃ of preservations.
Best mouse anti human superoxide reductase IV IgG antibody encapsulates confirming of concentration and rat anti people superoxide reductase IV IgG antibody optium concentration
Adopt the square formation titrimetry; Concrete steps are following: (1) with mouse anti superoxide reductase IV IgG antibody with coating buffer doubling dilution (1:20,1:40,1:80,1:160,1:320,1:640); Coated elisa plate; The l00ul/ hole; Put 4 ℃ of wet box incubated overnight; Wash the plate machine washing and wash 3 times, 5min/ time, clap and do; (2) add 100 μ l/ hole confining liquids, 37 ℃ of wet box sealing 1h wash the plate machine washing and wash 3 times, 5min/ time, clap and do; (3) add the test serum sample, 100 μ l/ holes, 37 ℃ of wet box effect 1h wash the plate machine washing and wash 3 times, 5min/ time, clap and do; (4), add in the ELISA Plate 100 μ l/L with rat anti people's superoxide reductase IV IgG antibody doubling dilution (1:20,1:40,1:80,1:160,1:320,1:640) of HRP mark; 37 ℃ of wet boxes were hatched 30 minutes; Wash the plate machine washing and wash 3 times, 5min/ time, clap and do; (5) add tmb substrate 100 μ l/ holes, behind 37 ℃ of lucifuge colour developing 15min, add 100 μ l/ hole 2mol/L sulfuric acid cessation reactions; (6) detect: survey OD with microplate reader
450nmValue.Simultaneously making blank with 1% BSA/PBS solution, is best effort concentration with the mouse anti superoxide reductase IV IgG antibody of P/N value maximum and the dilutability of rat anti superoxide reductase IV IgG antibody.
Respectively mouse anti human superoxide reductase IV monoclonal antibody IgG and rat anti people superoxide reductase IV polyclonal antibody IgG are made serial dilution, carry out the square formation test, the result sees table 1.The square formation titration results shows that 1:40 doubly dilutes when mouse anti human superoxide reductase IV IgG antibody, when rat anti people's superoxide reductase IV IgG antibody is doubly diluted with 1:80, and OD
450nmValue is greater than 1.0, yin and yang attribute serum OD
450nmRatio (P/N) maximum.Therefore selecting the best of mouse anti human superoxide reductase IV IgG antibody to encapsulate concentration is 1:20, and promptly concentration is 7.5 μ g/mL; The optimum diluting multiple of rat anti people's superoxide reductase IV IgG antibody is l:40, and promptly to encapsulate concentration be 20 μ g/mL to the best, and above-mentioned the best encapsulates concentration and is threshold concentration, greater than threshold concentration the time, can realize identical effect equally, but financial cost increases.
Confirming of table 2 mouse-anti superoxide reductase IV IgG antibody and rabbit antiperoxide reductase IV IgG antibody best effort concentration
Test serum is confirming of the righttest extension rate
With concentration is 7.5 μ g/mL mouse anti human superoxide reductase IV antibody concentration coated elisa plates; Add 1:20,1:50,1:100,1:200 extension rate test serum; Adding concentration again is 20 μ g/mL rat anti people superoxide reductase IV antibody, and the serum dilute concentration when selecting the P/N value maximum is as best effort concentration.
According to mouse anti human superoxide reductase IV IgG antibody and the rat anti people superoxide reductase IV IgG antibody best effort concentration confirmed; Test serum is done the dilution of different multiples; Carrying out each dilutability of double-antibody sandwich elisa repeats 3 times; Obtain the mean P value and the N value of sample, confirm the best effort concentration of test serum.The result sees table 2, and when test serum was done the 1:50 dilution, the P/N value was 7.030 to the maximum, confirmed that therefore best serum diluting multiple is 1:50.Wherein 1:50 is the extension rate threshold value, when extension rate during less than 1:50, can realize identical effect equally.
The best dilution of table 3 test serum working concentration
Confirming of yin and yang attribute critical value
Get 27 portions of normal human serums, detect, make blank with 1% BSA/PBS solution simultaneously with the method based on the double-antibody sandwich elisa of antiperoxide reductase IV antibody of above-mentioned foundation.With 27 parts of normal human serum OD
450nmMean value (X) and 2 standard variances (SD) sum of value be as the upper limit of negative sample value, promptly during the OD of test serum sample value＞X+2SD, is judged as the positive; Otherwise it is negative.
Through 27 parts of normal human serum samples are detected (table 4), trying to achieve its X value is 0.136778, and the SD value is 0.060107, confirms that the yin and yang attribute critical point is X+2SD=0.136778+2 * 0.060107=0.257.Be the OD of testing sample
450nmValue is that superoxide reductase IV is positive greater than 0.257, is less than or equal to then negative.
27 parts of normal human serum double-antibody sandwich elisas of table 4 testing result
The specificity test
Get Osteoarthritis (OA), systemic loupus erythematosus (SLE), other autoimmune diseases (AD); Comprise pleomorphism sclerosis, chorionitis, dermatomyositis, rheumatoid arthritis (RA) and normal controls (NC) serum; Method according in the present embodiment detects, observations.
Sensitivity Detection result
To contain the recombinant expressed people's superoxide reductase IV of 2ug/ml according to following concentration gradient doubling dilution (2000ng/ml, 1000ng/ml, 500ng/ml, 250ng/ml, 125 ng/ml, 62.5ng/ml, 31.25 ng/ml, 15.625 ng/ml, 7.8125 ng/ml); 100 μ L/ holes are added in the ELISA Plate; Each dilutability repeats 3 times; Detect by the superoxide reductase IV double-antibodies sandwich ELISA of setting up, set blank (PBS) contrast simultaneously.
Superoxide reductase IV double antibody sandwich method ELISA method according to setting up will contain recombinant expressed people's superoxide reductase IV and make a series of doubling dilutions, and the result sees table 6 and Fig. 2.When people's superoxide reductase IV is diluted to 7.8125ng/ml, OD
450nmValue is 0.078, is a bit larger tham blank value 0.069, owing in each enzyme mark hole, add 100 μ L, therefore, detects in the serum that to contain superoxide reductase IV albumen minimum content be 0.78ng.
Table 6 superoxide reductase IV double antibody sandwich method ELISA sensitivity tests result
The replica test result
(1) criticize in repeated determination test: get the rheumatoid arthritis serum that 3 parts of different times are collected, press optimum dilution degree dilution, be added in the antigen coated hole, every duplicate samples is done and is repeated ELISA for 3 times and measure.OD by every duplicate samples
450nmValue calculates average OD
450nmValue (X) and standard variance (SD), and then calculate every duplicate samples variation within batch coefficient [CV=(SD/X) * 100%]; (2) criticize between repeated determination test: under the identical situation of other condition, with the mouse anti human superoxide reductase IV antibody sandwich ELISA Plate of the purifying of 3 parts of different batches preparation, the rheumatoid arthritis serum that 3 parts of different times are collected detects.OD by every duplicate samples
450nmValue calculates average OD
450nmBe worth (X) and SD, and then calculate the interassay coefficient of variation of every duplicate samples.
Through statistical analysis, between the rheumatoid arthritis serum that 3 parts of different times are collected batch in revision test OD
450nmThe coefficient of variation of value (CV) is 2.44%～2.96%, less than 5%; Revision test OD between 3 times batches of rheumatoid arthritis serums that 3 parts of different times are collected
450nmThe CV of value is 2.34%～3.01%, less than 5% (table 7).In the superoxide reductase IV double antibody sandwich method ELISA method that shows foundation has well batch and batch between repeatability.
Table 7 replica test result
5.9 superoxide reductase IV and RF and CCP discrimination are analyzed
The positive judgment value of superoxide reductase IV is passed through chi-square test of four-fold table with the rheumatoid disease factor (RF) and anti-cyclic citrullinated peptide (CCP) respectively, and superoxide reductase IV and RF compare, card side=5.6 (correction), P＜0.05; Superoxide reductase IV and CCP compare, card side=27.22, and P＜0.01 illustrates superoxide reductase IV as rheumatoid arthritis diagnostic detection index, its discrimination is higher than RF and CCP.
Table 8 superoxide reductase IV and RF chi-square test of four-fold table
To be example with serum as biological sample detect the content of superoxide reductase IV present embodiment, also can adopt like the content of other biological samples such as blood plasma, urine, synovial tissue to superoxide reductase IV and detect.
Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art is to be understood that; Can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (21)
1. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample is characterized in that comprising specific monoclonal antibody and/or many anti-trapping agent, ELIAS secondary antibody, sample diluting liquid and the substrates of forming with antiperoxide reductase IV.
2. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 1 is characterized in that: said trapping agent is a mouse anti human superoxide reductase IV IgG antibody.
3. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 2; It is characterized in that: mouse anti superoxide reductase IV IgG antibody is not more than 640 times of volume dilution, and its dilution is a trapping agent.
4. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 1, it is characterized in that: said trapping agent invests on the solid support.
5. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 4, it is characterized in that: said solid support is ELISA Plate or biochip.
6. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 1 is characterized in that: said biological sample is blood preparation, humoral specimen and groups of cells fabric.
7. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 6, it is characterized in that: said blood preparation is a serum.
8. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 1 is characterized in that: said ELIAS secondary antibody is rat anti people's superoxide reductase IV IgG antibody of HRP mark.
9. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 8; It is characterized in that: rat anti people's superoxide reductase IV IgG antibody of said HRP mark is not more than 640 times of volume dilution liquid, and its dilution is an ELIAS secondary antibody.
10. the double antibodies sandwich ELISA kit that is used for the superoxide reductase IV of detection of biological sample according to claim 1, it is characterized in that: the amount ratio of said trapping agent and ELIAS secondary antibody is 1:1.
11. each said double antibodies sandwich ELISA kit of claim 1-10 is in the application that detects superoxide reductase IV content.
12. double antibodies sandwich ELISA kit according to claim 11 is used, and it is characterized in that: the application of said kit in vegetation detection type rheumathritis kit.
13. double antibodies sandwich ELISA kit according to claim 11 is used, and it is characterized in that: said kit detects the content of the superoxide reductase IV of test serum and normal human serum, as the OD of test serum
450nmValue is that superoxide reductase IV is positive greater than 0.257, is less than or equal to 0.257 and is superoxide reductase IV feminine gender.
14. each said double antibodies sandwich ELISA kit of utilization claim 1-10 detects the method for superoxide reductase IV content, a, biological sample is contacted and is incubated with trapping agent in the double antibodies sandwich ELISA kit; B, unconjugated biological sample is separated, antigen-antibody complex; C, the ELIAS secondary antibody in the antigen-antibody complex adding double antibodies sandwich ELISA of step b gained kit get the double antibodies sandwich compound; D, detection are incorporated into the content of the superoxide reductase IV of trapping agent.
15. utilization double antibodies sandwich ELISA kit according to claim 14 detects the method for superoxide reductase IV content, it is characterized in that: said method comprises that also content and the normal person with the superoxide reductase IV of steps d gained compares judgement.
16. utilization double antibodies sandwich ELISA kit according to claim 14 detects the method for superoxide reductase IV content; It is characterized in that: among the step a; With the trapping agent coated elisa plate, biological sample to be measured is contacted with trapping agent, be incubated and seal with confining liquid; Said biological sample is the serum dilution that is not more than 200 times of volume dilution, and said trapping agent is the dilution that mouse anti superoxide reductase IV IgG antibody is not more than 640 times of volume dilution.
17. utilization double antibodies sandwich ELISA kit according to claim 14 detects the method for superoxide reductase IV content; It is characterized in that: among the step b; Wash unconjugated biological sample separation with insulation among the step a and with the ELISA Plate of confining liquid sealing, get antigen-antibody complex.
18. utilization double antibodies sandwich ELISA kit according to claim 14 detects the method for superoxide reductase IV content; It is characterized in that: among the step c; ELIAS secondary antibody in the antigen-antibody complex adding double antibodies sandwich ELISA of step b gained kit is carried out insulation reaction, gets the double antibodies sandwich compound; Said ELIAS secondary antibody is the dilution that rat anti people's superoxide reductase IV IgG antibody of HRP mark is not more than 640 times of volume dilution liquid.
19. utilization double antibodies sandwich ELISA kit according to claim 14 detects the method for superoxide reductase IV content; It is characterized in that: in the steps d; After in the double antibodies sandwich compound of step c gained, adding the colour developing of chromogen substrate lucifuge; Add the stop buffer cessation reaction sample liquid that must develop the color, through surveying the OD of colour developing sample liquid
450nmValue detects the content of the superoxide reductase IV that is incorporated into trapping agent.
20. utilization double antibodies sandwich ELISA kit according to claim 14 detects the method for superoxide reductase IV content; It is characterized in that: said method also comprises negative control group and blank group, and said negative control group is the biological sample that stems from the normal person.
21. utilization double antibodies sandwich ELISA kit according to claim 20 detects the method for superoxide reductase IV content, it is characterized in that: the OD of said biological sample to be measured
450nmValue is greater than the OD of negative control group sample
450nmWhen value mean value and its 2 times of standard variance sums, be judged to be the superoxide reductase IV positive, the OD of said biological sample to be measured
450nmValue is less than or equal to the OD of negative control group sample
450nmThen negative when value mean value and its 2 times of standard variance sums.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100256334A CN102346190A (en) | 2011-01-24 | 2011-01-24 | Double-antibody sandwich ELISA reagent kit and method for detecting hyperoxide reductase IV in biological sample and application of reagent kit |
| PCT/CN2011/083644 WO2012100600A1 (en) | 2011-01-24 | 2011-12-07 | Use of specific antibodies for peroxiredoxin iv in preparing in vitro diagnostic reagents for early-stage rheumatoid arthritis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100256334A CN102346190A (en) | 2011-01-24 | 2011-01-24 | Double-antibody sandwich ELISA reagent kit and method for detecting hyperoxide reductase IV in biological sample and application of reagent kit |
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| Publication Number | Publication Date |
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| CN102346190A true CN102346190A (en) | 2012-02-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2011100256334A Pending CN102346190A (en) | 2011-01-24 | 2011-01-24 | Double-antibody sandwich ELISA reagent kit and method for detecting hyperoxide reductase IV in biological sample and application of reagent kit |
Country Status (2)
| Country | Link |
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| CN (1) | CN102346190A (en) |
| WO (1) | WO2012100600A1 (en) |
Cited By (2)
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| CN103308674A (en) * | 2012-12-20 | 2013-09-18 | 重庆联佰博超医疗器械有限公司 | Circulating immune complex of peroxide reductase IV and application of complex |
| CN106370855A (en) * | 2016-09-28 | 2017-02-01 | 吉林大学 | Sheep peroxide redox enzyme 6 double antibody sandwich ELISA kit based on BSaBA signal amplifying system |
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| JP2011206049A (en) | 2010-03-08 | 2011-10-20 | Sumio Sugano | Necrosis marker and use thereof |
| RU2524616C1 (en) * | 2013-01-21 | 2014-07-27 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Владивостокский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ГОУ ВПО ВГМУ Минздрава России) | Diagnostic technique for early stage of rheumatoid arthritis |
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| WO2012100600A1 (en) | 2012-08-02 |
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Application publication date: 20120208 |