CN102937648B - Kit for detecting antinuclear antibody spectrum related to autoimmune diseases (AIDs) - Google Patents
Kit for detecting antinuclear antibody spectrum related to autoimmune diseases (AIDs) Download PDFInfo
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Abstract
The invention relates to a kit for detecting antinuclear antibody spectrum related to AIDs. The kit comprises a membrane strip, enzyme-labeled liquid, a substrate and concentrating and washing incubation liquid, wherein the membrane strip is composed of a carrier piece and an antigen band, a critical quality control band and a functional quality control line which are sequentially fixed on the carrier piece. The antigen band is composed of at least two of dsDNA, nucleosomes, SmD1, ribosome P0proteins, histones, U1snRNP, Ro/SS-A(52KD), Ro/SS-A(60KD), La/SS-B, Sc1-70, CENP-B, Jo-1, AMA-M2, PM-Sc1, PCNA, Mi-2 and Ku through independent marking to a nitrocellulose membrane or a nylon membrane. The kit is provided with the ingenious critical quality control band, one critical quality control band can play a role in interpreting two or even more detection bands (antigen bands), and result determination is simpler and more reliable.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of kit diagnosing the illness, particularly, relate to a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum.
Background technology
Autoimmunity is that body immune system reacts to autoantigen, produces autoantibody and self replys T cell.Under normal physiological state, autoimmunity can be removed self cell of aging death in time, maintains the stable of interior environment.Under the state of some pathology, may produce directly and self responsiveness T cell and autoantibody of collateral damage autologous tissue, and cause and pathology and the dysfunction of corresponding organ tissue be called autoimmune disease (Autoimmune disease, AID).Common autoimmune disease is as systemic loupus erythematosus, rheumatoid arthritis, systemic sclerosis, multiple dermatitis/myositis, mixed connective tissue disease, hyperthyroidism, juvenile diabetes, primary blood platelet purpura, autoimmune hemolytic anemia, ulcerative colitis and permitted multiple dermatosis, chronic liver disease etc.
The complicated clinical manifestation of AID is changeable, often causes and fails to pinpoint a disease in diagnosis and mistaken diagnosis clinically; The normal chronic delay of disease, the aggravation of carrying out property, finally causes irreversible organic or functional lesion, has a strong impact on patient's quality of life, even threat to life.And the early diagnosis and therapy of AID will be controlled PD significantly, patient's quality of life is significantly improved, can as normal person, live.
Autoantibody detects the diagnosis with autoimmune disease and treatment monitoring all significant, and a lot of autoantibodies have been written into the international standard of AID diagnosis, wherein the most important thing is antinuclear antibodies (antinuclear antibodies, ANA).ANA is the general name of the autoantibody of anti-cell nuclear antigen composition, totally 17 kinds of the target compositions that this patent has comprised common antinuclear antibodies: dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and Ku.These autoantibodies all common are clear and definite clinical meaning.Also do not detect at present the diagnostic reagent of 17 kinds of antinuclear antibodies both at home and abroad simultaneously.
For autoimmune disease diagnostic method, adopt indirect immunofluorescence analysis method (IFA), enzyme linked immunosorbent assay (ELISA) and Western blotting more, but respectively have its deficiency.Indirect immunofluorescence analysis method is to detect the common technology of autoantibody.Its experiment matrix is Hep-2 cell or different primate liver organization frozen section, contains complete spectrotype, is applicable to examination test.But there is following shortcoming: can not be as making a definite diagnosis foundation; The judgement of result need to have rich experience; The meaning of titre is greater than caryogram, but the judgement subjectivity of titre is strong; Sensitivity is lower, and specificity is not high yet.
Enzyme linked immunosorbent assay (ELISA) has highly sensitive, can be used as screening experiment, also can be used as and makes a definite diagnosis foundation, also can quantitative measurement, detect the state of an illness and result for the treatment of.But single test can only detect single index, and flux is low, and testing cost is higher, aspect the diagnostic application popularization of autoimmune disease, existing great limitation.Western blot(WB) be a kind of immunoassay technology growing up on gel electrophoresis and immuno analytical method basis, there is the high resolution of SDS-PAGE and the high specific of solid-phase immunoassay and susceptibility, be relatively applicable to finding unknown antibody.And for detecting known antibody, owing to having used natural hybrid antigen, in experimentation, often there is can not find the problem of target stripe and band skew, and caused many difficulties to the interpretation of result, very easily judge by accident and fail to judge.WB is long service time simultaneously, is also not suitable for promoting in clinical examination.Someone improves WB, directly purifying antigen is coated on nitrocellulose filter, then carries out immune response and detects antibody, has formed the western blotting method of improvement.The existing film bar that simultaneously detects 15 autoantibodies, by by the parallel nitrocellulose membrane that is coated with of purifying antigen, seals nonspecific reaction district at present.When incubation for the first time, the serum having diluted with detect film bar and react, different samples or Quality Control are carried out between different incubation slots.If sample is positive, specific IgG(also comprises IgA and IgM) be combined with corresponding antigens.For detecting the antibody of combination, add the anti-human IgG(enzyme conjugates of enzyme mark) carry out incubation for the second time, then add zymolyte, hatch colour developing, add stop buffer, result interpretation.Advantage is to realize high-throughout detection, owing to having used purifying antigen, the Western blot height that specificity and remolding sensitivity are traditional many.
For existing kit, there is following shortcoming:
1, the antibody detecting only has 15, and some antigen is the achievement of early stage research, at present existing new more significant target antigen.
2, the purifying antigen using mostly is from natural tissues and extracts, and the deficiency of purity (>=90%) has still affected the sensitivity and the specificity that detect.
3, film bar background is dark, sometimes also dark than positive band, has affected the reliability of result.
4, different antigen strip width differs, and differs in band interval, has affected the accurate interpretation of result.
5, like product kit as disclosed in CN200810132304.8, there is no critical quality control band, a control film bar need to be separately done in the interpretation of result, has increased the cost of experiment, because reaction environment between groove and groove exists certain difference, may cause false positive or false negative simultaneously.The Related product having at present also has critical quality control band, but a quality control band can only carry out interpretation to the result of a test strip or project, still not have at present with a critical quality control band simultaneously to two so that more band carry out the application of interpretation.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum, and the quality control band of this kit can be simultaneously plays the effect of interpretation to two and even more test strip.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum, comprise film bar, enzyme mark liquid, substrate and thickening and washing Incubating Solution, film bar is by slide glass and be fixed on successively the antigen band on slide glass, critical quality control band, function nature controlling line forms, antigen band is by dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, at least two independent of each other being scoring on nitrocellulose filter or nylon membrane in Mi-2 and Ku form.
In the method, function nature controlling line belongs to prior art, whether effectively its objective is for judging the primary first-order equation of this film bar, the feminine gender of step 1) and the positive of step 4) are antigen in relative antigen band, there is the antibody that these antigens can detect positive, do not there is the antibody that these antigens can detect negative.In the antigen band of this programme, all independent of each other being scoring to of each antigen forms an independently antigen detection line on nitrocellulose filter or nylon membrane, these all antigen detection lines are referred to as antigen band, " getting film bar detects each sample; scanning obtains the gray-scale value of antibody that each antigen detects in film bar " in step 1, the gray-scale value is here to scan the gray-scale value obtaining after each antigen detection line detects sample.The inventive point of this programme is that critical quality control band is the contrast band of at least two antigens in antigen band simultaneously; By being provided with one, can be simultaneously two and even more test strip (antigen band) be played to the critical quality control band of interpretation, along with increasing of test strip, detected band of every increase, all to determine experiment to re-starting complete critical Quality Control value, test difficulty simultaneously and significantly strengthen.Conventionally, determine that a critical Quality Control value need to many times test, could finally determine.
The composition of described critical quality control band is the human IgG of 20ng ~ 20 μ g/mL.In prior art, have and select the anti-sheep IgG record of line in contrast, in CN200810132304.8, but the control line that needs standardized anti-sheep IgG on each the antigen band in this patent, manufacture craft is more loaded down with trivial details like this, and cost is higher, in CN200810132304.8, can be for the record of 2 or 2 above antigen band interpretations about same control line.The present invention finds by creative experiments, selects the human IgG of finite concentration scope as critical quality control band, can be clear, accurately 2 or 2 above antigen bands are carried out to interpretation.
As preferably, the composition of described critical quality control band is the human IgG of 200ng ~ 2 μ g/mL.
The critical Quality Control value of described critical quality control band determines that method comprises the steps: 1) select m to be diagnosed as negative new blood sample as sample, in the antigen band of film bar, there is n antigen, getting film bar detects each sample, scanning obtains the gray-scale value of antibody that each antigen detects in film bar, using the gray-scale value of antibody that antigen detects identical in m sample as one group of numerical value, obtain n group numerical value, calculate respectively the mean value M of this n group numerical value
p, standard deviation SD
pcritical Quality Control value CO with antibody that each antigen detects
p, CO wherein
p=(M
p+ 2 * SD
p), m, n, p are natural number and m>=120, n>=2, n>=p>=1; 2) by the critical Quality Control value CO of antibody that each antigen detects
pprocess, calculate its mean value M, standard deviation SD, coefficient of variation CV; 3) as CV≤10%, can obtain the critical Quality Control value CO of film bar, wherein CO=(M+2 * SD); As CV > 10%, adjust trace antigen amount repeating step 1), step 2) redeterminate, until CV≤10%; 4) select to be diagnosed as positive new blood sample as sample, get film bar each sample is detected, scanning obtains the gray-scale value of antibody that each antigen detects in film bar, by the critical Quality Control value CO comparison of itself and film bar, as all sample gray-scale values are all not less than CO, CO is effective; If any one or above sample gray-scale value, be less than CO, need again measure checking; As still have one or above sample gray-scale value to be less than CO, adjust trace antigen amount repeating step 1), step 2), step 3) redefines the critical Quality Control value CO of film bar.In determining the method for critical Quality Control value, sample size and antigen number can be selected according to requirement of experiment.
According to the critical Quality Control value of definite film bar, regulate the concentration of the coated human IgG of critical quality control band.Concrete determine that method is: certain density human IgG is dissolved in Tris or Hepes damping fluid, then lines and on nitrocellulose filter, be prepared into finished film bar, and coated critical quality control band only on film bar; Choose at random 30 film bars and detect with this kit, and scan gray scale, calculate the average M measuring for 30 times
s, standard deviation SD
swith coefficient of variation CV
s, CV wherein
s=M
s/ SD
s.The standard that critical quality control band is qualified is: 1) 0.97 * CO≤M
s≤ 1.03 * CO; 2) CV
s< 5%; If do not meet wherein any one, need to readjust all experiments of concentration described in carry out this step, until obtain qualified critical quality control band, the coated concentration of human IgG is now the coated concentration of critical quality control band.Final coated concentration and the coated technique of determining critical quality control band.According to the critical Quality Control value of definite film bar, technician, by ordinary skill in the art means, can converse the concentration of suitable human IgG, adopts existing coated technique, can prepare critical quality control band.
Sheep/mouse/rabbit anti-human igg antibody that described enzyme mark liquid is horseradish peroxidase-labeled.
Described substrate is that mass percent concentration is the single agents of the luminol of 0.02%-2% or the hydrogen peroxide formation that mass percent concentration is 0.002%-0.2%.
Described antigen band is parallel to each other and each antigen strip width homogeneous, and between each adjacent antigen band, interval is wide.
Described antigen strip width is 0.5mm-3mm, between adjacent antigen band, is spaced apart 2mm-20mm.
Described autoimmune disease comprises systemic loupus erythematosus, mixed connective tissue disease, dry syndrome, systemic scleroderma, polymyositis/dermatomyositis, overlap syndrome.
Described ribosomes P0 albumen, U1 snRNP, Ro/SS-A (52KD), La/SS-B, Scl-70, CENP-B, Jo-1, PM-Scl, PCNA, Mi-2 and Ku are expressed by sf9 insect cell and purifying makes; DsDNA, nucleosome, histone, Ro/SS-A (60 KD) and AMA-M2 extract and make from natural tissues; SmD1 is artificial synthetic polypeptide.
Compared with prior art, the invention has the beneficial effects as follows:
1, the innovative critical quality control band of the present invention, critical quality control band can be simultaneously plays the effect of interpretation to two and even more test strip (antigen band), colour developing band is relatively got final product to judged result with the depth of the color of critical quality control band: the positive: color is more identical or dark than critical quality control band; Negative: color is more shallow than critical quality control band.
2, the present invention has also improved single reagent substrate, adds after substrate and does not need to stop.
3, each antigen strip width homogeneous, band interval is wide, and film bar clean background judges result more simple and reliable.
Accompanying drawing explanation
Fig. 1 is the structural representation of existing film bar;
Fig. 2 is the structural representation of film bar of the present invention;
Fig. 3 is that critical Quality Control value is determined process flow diagram.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited only to following embodiment.
Embodiment 1:
Referring to shown in Fig. 1 to Fig. 3, the CO nature controlling line in Fig. 2 is critical quality control band; The kit of the detection autoimmunity disease related antinuclear antibodies spectrum of the present embodiment comprises film bar, enzyme mark liquid, substrate and thickening and washing Incubating Solution, wherein the selection of enzyme mark liquid, substrate and thickening and washing Incubating Solution and scope all belong to prior art, for enzyme mark liquid, substrate and thickening and washing Incubating Solution, select the product of prior art also can realize technical scheme of the present invention.
Film bar is by slide glass and be fixed on successively the antigen band on slide glass, critical quality control band, function nature controlling line and form, at least two independent of each other are scoring to nitrocellulose filter or nylon membrane on of antigen band in dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and Ku form, and the composition of the critical quality control band of the present embodiment is the human IgG of 20ng ~ 20 μ g/mL.
This kit is creationary has added the critical quality control band that the most multipair 17 tested antibody (antigen band) carry out yin and yang attribute judgement simultaneously.Concrete flow process is shown in shown in the process flow diagram of Fig. 3.The critical Quality Control value of critical quality control band determines that method comprises the steps:
1) select m to be diagnosed as negative new blood sample as sample, in the antigen band of film bar, there is n antigen, getting film bar detects each sample, scanning obtains the gray-scale value of antibody that each antigen detects in film bar, using the gray-scale value of antibody that antigen detects identical in m sample as one group of numerical value, obtain n group numerical value, calculate respectively the mean value M of this n group numerical value
p, standard deviation SD
pcritical Quality Control value CO with antibody that each antigen detects
p, CO wherein
p=(M
p+ 2 * SD
p), m, n, p are natural number and m>=120, n>=2, n>=p>=1;
2) by the critical Quality Control value CO of antibody that each antigen detects
pprocess, calculate its mean value M, standard deviation SD, coefficient of variation CV;
3) as CV≤10%, can obtain the critical Quality Control value CO of film bar, wherein CO=(M+2 * SD); As CV > 10%, adjust trace antigen amount repeating step 1), step 2) redeterminate, until CV≤10%;
4) select to be diagnosed as positive new blood sample as sample, get film bar each sample is detected, scanning obtains the gray-scale value of antibody that each antigen detects in film bar, by the critical Quality Control value CO comparison of itself and film bar, as all sample gray-scale values are all not less than CO, CO is effective; If any one or above sample gray-scale value, be less than CO, need again measure checking; As still have one or above sample gray-scale value to be less than CO, adjust trace antigen amount repeating step 1), step 2), step 3) redefines the critical Quality Control value CO of film bar.
Generally speaking, critical Quality Control value define two key points, first is that the CV of the gray scale upper limit of 17 bands can not surpass 10%, otherwise tests after need to readjusting the trace concentration of corresponding antigens again.The secondth, by positive sample, verify critical quality control band, if the gray scale of twice certain sample of experiment is all less than critical Quality Control value, need so again to do whole experiment.And along with increasing of test strip, detected band of every increase, all will determine experiment to re-starting complete critical Quality Control value, tests difficulty simultaneously and significantly strengthens.Conventionally, determine that a critical Quality Control value need to many times test, could finally determine.
Embodiment 2:
The present embodiment is to carrying out test determination by dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and these 17 antigen bands that form on nitrocellulose filter or nylon membrane that are scoring to independent of each other of Ku.On the basis of embodiment 1, it is more simple and reliable for result is judged, each antigen strip width homogeneous is arranged, band interval is wide, and antigen strip width is 0.5mm-3mm, between adjacent antigen band, is spaced apart 2mm-20mm, film bar clean background, and adopt the single reagent substrate of improvement, do not need to stop after adding substrate, substrate is the single agents that luminol 0.02%-2% or hydrogen peroxidase 10 .002%-0.2% form; All trace antigen is all highly purified, and has adopted international up-to-date coated technique, and concrete method for coating sees below.
Determining of critical Quality Control value:
1) determine the critical Quality Control value of each antibody
Choose at random 125 parts of the negative fresh serum of clinical definite, plasma specimens, with the kit of the present embodiment, detect.Measure the gray-scale value of the corresponding antibodies that its antigen band detects, calculate the gray-scale value average M of each same antibody in 125 parts of samples
pwith standard deviation SD
p, can obtain the confidence upper limit (M of each antibody 95%
p+ 2 * SD
p), be the critical Quality Control value of each antibody, be denoted as CO
p.The results are shown in following table, the present embodiment m=125, n=p=17.
2) determine the critical Quality Control value of film bar
By the CO of above-mentioned each antibody
pprocess: the mean value M and standard deviation SD and the coefficient of variation CV(CV=M/SD that calculate them), if CV > 10% should adjust the concentration of corresponding trace antigen, repeat steps 1), step 2); If CV≤10%, can obtain the critical Quality Control value of film bar, be denoted as CO=(M+2 * SD).The results are shown in following table:
This tests CV=3.3%, and CO is effective, is 133.5.
3) validity of the critical Quality Control value of checking film bar.
Choose at random 520 parts of the positive fresh serum of clinical definite, plasma specimens, measure its corresponding antibodies gray-scale value, with CO comparison.Definite through testing, this gray-scale value of testing all positive sample is all over critical Quality Control value, and this critical Quality Control value is effective.
Embodiment 3:
The present embodiment be according to the critical Quality Control value of the definite film bar of embodiment 2 (in Fig. 3 and all embodiment of the present invention, CO
pfor the critical Quality Control value of single antibody, CO is the critical Quality Control value of whole film bar), regulate the concentration of the coated human IgG of critical quality control band, finally determine coated concentration and the coated technique of critical quality control band.Concrete operations are: certain density human IgG is dissolved in Tris or Hepes damping fluid, then with full-automatic point sample instrument, line on nitrocellulose filter, and be prepared into finished film bar through techniques such as sealing, dry, cuttings, and coated critical quality control band only on film bar.Choose at random 30 film bars and detect with this kit, and scan gray scale, calculate the average (M measuring for 30 times
s), standard deviation (SD
s) and the coefficient of variation (CV
s), CV wherein
s=M
s/ SD
s.The standard that critical quality control band is qualified is: 1) the critical Quality Control value of 0.97 * CO(film bar)≤M
s≤ 1.03 * CO; 2) CV
s< 5%.If do not meet wherein any one, need to readjust concentration or coated technique and carry out all experiments described in the present embodiment, until obtain qualified critical quality control band, the coated concentration of human IgG is now the coated concentration of critical quality control band.The results are shown in following table:
Wherein, M
s=135.1=1.01 * CO; CV
s=3.4%<5%, all meets the requirements.The coated concentration of the critical quality control band of the present embodiment is 20ng/mL.
Embodiment 4:
Adopt the technical scheme of embodiment 1, we are to choosing at random 2 and be scoring to independently of one another and form antigen band on nitrocellulose filter or nylon membrane in dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and Ku, carry out test determination, its result is as follows again:
1) determine the critical Quality Control value of each antibody
According to the method for embodiment 1 step 1), choose at random 120 parts of the positive fresh serums of clinical definite, plasma specimen and use this kit measurement, the critical Quality Control value CO of each antibody
pthe results are shown in following table.In the present embodiment, m=120, n=p=2.
2) determine the critical Quality Control value of film bar
According to embodiment 1 step 2) method, obtain M, CO and CV, the results are shown in following table.
This tests CV=2.9%, and CO is effective, is 151.5.
3) validity of the critical Quality Control value of checking film bar.
According to the method for embodiment 1 step 3), choose at random 63 parts of the positive fresh serums of clinical definite, plasma specimen and detect.Result: the gray-scale value of all positive sample is all over critical Quality Control value, and this critical Quality Control value is effective.
4) the coated concentration of critical quality control band determines.
According to the method for embodiment 2, calculate the average (M that 30 film bars are measured
s), standard deviation (SD
s) and the coefficient of variation (CV
s).The results are shown in following table:
Wherein, M
s=148.6=0.98 * CO; CV
s=2.5%<5%, all meets the requirements.The coated concentration of critical quality control band is now 20 μ g/mL.
Embodiment 5:
Adopt the technical scheme of embodiment 1, we are to choosing at random 3 and be scoring to independently of one another and form antigen band on nitrocellulose filter or nylon membrane in dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and Ku, carry out test determination, its result is as follows again:
1) determine the critical Quality Control value of each antibody
According to the method for embodiment 1 step 1), choose at random 122 parts of the positive fresh serums of clinical definite, plasma specimen and use this kit measurement, the critical Quality Control value CO of each antibody
pthe results are shown in following table.In the present embodiment, m=122, n=p=3.
2) determine the critical Quality Control value of film bar
According to embodiment 1 step 2) method, obtain CO and CV, the results are shown in following table.
This tests CV=6.2%, and CO is effective, is 138.0.
3) validity of the critical Quality Control value of checking film bar.
According to the method for embodiment 1 step 3), choose at random 95 parts of the positive fresh serums of clinical definite, plasma specimen and detect.Result: the gray-scale value of all positive sample is all over critical Quality Control value, and this critical Quality Control value is effective.
4) the coated concentration of critical quality control band determines.
According to the method for embodiment 2, calculate the average (M that 30 film bars are measured
s), standard deviation (SD
s) and the coefficient of variation (CV
s).The results are shown in following table:
Wherein, M
s=141.0=1.02 * CO; CV
s=4.0%<5%, all meets the requirements.The coated concentration of critical quality control band is now 2 μ g/mL.
Embodiment 6:
Adopt the technical scheme of embodiment 1, we are to choosing at random 9 and be scoring to independently of one another and form antigen band on nitrocellulose filter or nylon membrane in dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and Ku, carry out test determination, its result is as follows again:
1) determine the critical Quality Control value of each antibody
According to the method for embodiment 1 step 1), choose at random 125 parts of the positive fresh serums of clinical definite, plasma specimen and use this kit measurement, the critical Quality Control value CO of each antibody
pthe results are shown in following table.In the present embodiment, m=125, n=p=9.
2) determine the critical Quality Control value of film bar
According to embodiment 1 step 2) method, obtain CO and CV, the results are shown in following table.
This tests CV=3.0%, and CO is effective, is 135.1.
3) validity of the critical Quality Control value of checking film bar.
According to the method for embodiment 1 step 3), choose at random 282 parts of the positive fresh serums of clinical definite, plasma specimen and detect.Result: the gray-scale value of all positive sample is all over critical Quality Control value, and this critical Quality Control value is effective.
4) the coated concentration of critical quality control band determines.
According to the method for embodiment 2, calculate the average (M that 30 film bars are measured
s), standard deviation (SD
s) and the coefficient of variation (CV
s).The results are shown in following table:
Wherein, M
s=137.8=1.01 * CO; CV
s=4.1%<5%, all meets the requirements.The coated concentration of critical quality control band is now 200ng/mL.
By dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, 9 antigens of random selection in Mi-2 and Ku, according to embodiment 2, the methods experiment of embodiment 3 10 times, according to the critical Quality Control value of the definite film bar of embodiment 2, adopt the human IgG of variable concentrations to test, the coated concentration that obtains critical quality control band is respectively 130ng/ml, 210ng/mL, 400 ng/mL, 680 ng/mL, 820 ng/mL, 950 ng/mL, 1.3 μ g/ml, 2.1 μ g/ml, 5.5 μ g/ml, 13 μ g/ml.In above-mentioned whole embodiment, experimental result shows, human IgG concentration is in the scope of 20ng ~ 200ng/mL, 2 μ g ~ 20 μ g/mL time, its coated critical quality control band can, simultaneously for the comparison of at least 2 antigen bands, still determine that the dense coated degree of critical quality control band need to be done the number of times of testing more; In the scope of 200ng ~ 2 μ g/mL, when experiment shows the interior definite critical quality control band of this concentration range, needed experiment number significantly reduces, and can determine faster the coated concentration of critical quality control band, has reduced experimental cost and has improved the production efficiency of product.
In above-mentioned whole embodiment, enzyme mark liquid, substrate and thickening and washing Incubating Solution can be used disclosed scheme of prior art, and enzyme mark liquid is the sheep/mouse of horseradish peroxidase-labeled/rabbit anti-human igg antibody preferably.Above-mentioned autoimmune disease comprises systemic loupus erythematosus, mixed connective tissue disease, dry syndrome, systemic scleroderma, polymyositis/dermatomyositis, overlap syndrome etc.Described ribosomes P0 albumen, U1 snRNP, Ro/SS-A (52KD), La/SS-B, Scl-70, CENP-B, Jo-1, PM-Scl, PCNA, Mi-2 and Ku are expressed by sf9 insect cell and purifying makes; Described dsDNA, nucleosome, histone, Ro/SS-A (60 KD) and AMA-M2 extract and make from natural tissues; Described SmD1 is artificial synthetic polypeptide.The innovative critical quality control band of the present invention, relatively gets final product judged result by colour developing band with the depth of the color of critical quality control band: the positive: color is more identical or dark than critical quality control band; Negative: color is more shallow than critical quality control band.Totally 17 kinds of the antibody numbers detecting: dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, Ro/SS-A (52KD), Ro/SS-A (60 KD), La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and the corresponding autoantibody of Ku.And the achievement that some project is current research, as SmD1, U1snRNP, ribosomes P0 albumen etc., has had better sensitivity and specificity than Sm, nRNPR/Sm and ribosomes P albumen respectively.The antigen overwhelming majority used in the present invention is recombinant antigen, and all antigen purity are more than 98%, has improved significantly the sensitivity and the specificity that detect.
The technology being applied in above-described embodiment comprises:
1) antigen is identified: by polyacrylamide gel electrophoresis (SDS-PAGE) evaluation antigen and purity thereof, answer >98%.
2) coated: 17 kinds of antigens of human IgG and the present embodiment are dissolved in respectively to tris damping fluid or the HEPES damping fluid of 0. 01-0. 1M, pH7.4, obtaining concentration is 1-100mg/mL solution.The above-mentioned solution of 1-l00ul is lined on nitrocellulose filter with full-automatic point sample instrument, and strip width is 0.5mm-3mm, and band is spaced apart 2mm-20mm.4 ℃ of coated 24-48 hour.Putting in order of various antigens can be adjusted arbitrarily.
3) sealing: with 10-100ml containing 0.05-0.5%Tween20,0.5-5%BSA, 0.5-5% casein, the skimmed milk power of 0.5-5%, the tris damping fluid of 0. 01-0.1M of the sucrose of 5-10% or HEPES damping fluid, pH7.4,25-37 ℃ of sealing 3-12 hour, with hatching lavation buffer solution washing.After drying, standby in 2-8 ℃.
4) film bar preparation: prepared nitrocellulose membrane is fixed on slide glass, firmly after, with fully automatic cutting machine, be cut to the wide film bar into 1mm-3mm, minute install in film barrel.
4) enzyme mark liquid: classical sodium periodate oxidation mark horseradish peroxidase (HRP) is to sheep/mouse/rabbit anti-human igg antibody, component: sheep/mouse of mark HRP/rabbit anti-human igg antibody 0.01-0.1%, 0.2-2%BSA, 0.2-2% sucrose, Proclin 300 0.01-0.1%, Tween-20 0.01-0.1%, PBS0.01-0.1mol/L.
5) hatch lavation buffer solution: PBS0.01-0.1mol/L, 0.2-2%BSA, Proclin 300 0.01-0.1%, Tween-20 0.01-0.1%.Hatching lavation buffer solution has washing and hatches two large functions, can be used for diluted sample, hatches sample and washing film bar.
6) substrate preparation: luminol or derivatives thereof 0.02%-2%, hydrogen peroxidase 10 .002%-0.2%, Proclin 300 0.01-0.1%, Tween-20 0.01-0.1%.Substrate, without termination, dries for 3 times with distilled water washing after the colour developing of film bar, and colour developing is steady in a long-term.
7) assembling of kit: by film barrel, hatch lavation buffer solution, enzyme mark liquid, substrate and be packaged into kit.
As mentioned above, can implement preferably the present invention.
Claims (7)
1. a kit that detects autoimmunity disease related antinuclear antibodies spectrum, comprise film bar, enzyme mark liquid, substrate and thickening and washing Incubating Solution, it is characterized in that, film bar is by slide glass and be fixed on successively the antigen band on slide glass, critical quality control band, function nature controlling line forms, antigen band is by dsDNA, nucleosome, SmD1, ribosomes P0 albumen, histone, U1 snRNP, molecular weight is the Ro/SS-A of 52KD, molecular weight is the Ro/SS-A of 60KD, La/SS-B, Scl-70, CENP-B, Jo-1, AMA-M2, PM-Scl, PCNA, Mi-2 and Ku totally 17 independent of each other being scoring on nitrocellulose filter or nylon membrane form, the composition of described critical quality control band is the human IgG of 20ng ~ 20 μ g/mL, and described critical quality control band determines that method is: the concentration that regulates the coated human IgG of critical quality control band according to the critical Quality Control value of definite film bar, the critical Quality Control value of critical quality control band determines that method comprises the steps:
1) select m to be diagnosed as negative new blood sample as sample, in the antigen band of film bar, there is n antigen, getting film bar detects each sample, scanning obtains the gray-scale value of antibody that each antigen detects in film bar, using the gray-scale value of antibody that antigen detects identical in m sample as one group of numerical value, obtain n group numerical value, calculate respectively the mean value M of this n group numerical value
p, standard deviation SD
pcritical Quality Control value CO with antibody that each antigen detects
p, CO wherein
p=(M
p+ 2 * SD
p), m, n, p are natural number and m>=120, n>=2, n>=p>=1;
2) by the critical Quality Control value CO of antibody that each antigen detects
pprocess, calculate its mean value M, standard deviation SD, coefficient of variation CV;
3) as CV≤10%, can obtain the critical Quality Control value CO of film bar, wherein CO=(M+2 * SD); As CV > 10%, adjust trace antigen amount repeating step 1), step 2) redeterminate, until CV≤10%;
4) select to be diagnosed as positive new blood sample as sample, get film bar each sample is detected, scanning obtains the gray-scale value of antibody that each antigen detects in film bar, by the critical Quality Control value CO comparison of itself and film bar, as all sample gray-scale values are all not less than CO, CO is effective; If any one or above sample gray-scale value, be less than CO, need again measure checking; As still have one or above sample gray-scale value to be less than CO, adjust trace antigen amount repeating step 1), step 2), step 3) redefines the critical Quality Control value CO of film bar.
2. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 1, is characterized in that, the composition of described critical quality control band is the human IgG of 200ng ~ 2 μ g/mL.
3. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 1 and 2, it is characterized in that, described substrate is that mass percent concentration is the single agents of the luminol of 0.02%-2% or the hydrogen peroxide formation that mass percent concentration is 0.002%-0.2%.
4. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 1 and 2, is characterized in that, described antigen band is parallel to each other and each antigen strip width homogeneous, and between each adjacent antigen band, interval is wide.
5. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 4, is characterized in that, described antigen strip width is 0.5mm-3mm, between adjacent antigen band, is spaced apart 2mm-20mm.
6. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 1 and 2, it is characterized in that, described autoimmune disease comprises system property lupus erythematosus, mixed connective tissue disease, dry syndrome, systemic scleroderma, polymyositis/dermatomyositis, overlap syndrome.
7. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 1 and 2, it is characterized in that, Ro/SS-A, La/SS-B, Scl-70, CENP-B, Jo-1, PM-Scl, PCNA, Mi-2 and the Ku that described ribosomes P0 albumen, U1 snRNP, molecular weight are 52KD expressed by sf9 insect cell and purifying makes; The Ro/SS-A that described dsDNA, nucleosome, histone, molecular weight are 60KD and AMA-M2 extract and make from natural tissues; Described SmD1 is artificial synthetic polypeptide.
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