CN102937648A - 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 that diagnoses 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 the T cell.Under the normal physiological state, autoimmunity can in time be removed self cell of aging death, keeps 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 such 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 many kinds of skin diseases, chronic liver disease etc.
The complicated clinical manifestation of AID is changeable, often causes clinically and fails to pinpoint a disease in diagnosis and mistaken diagnosis; The normal chronic delay of disease, the aggravation of carrying out property causes irreversible organic or functional lesion at last, has a strong impact on Quality of Life, even threat to life.And the early diagnosis and therapy of AID will be controlled PD significantly, and patient's quality of life is significantly improved, and can live as the normal person.
Autoantibody detects all significant with diagnosis and the treatment monitoring of autoimmune disease, 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, and this patent has comprised totally 17 kinds of the target compositions of 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 simultaneously at present the diagnostic reagent of 17 kinds of antinuclear antibodies both at home and abroad.
Adopt indirect immunofluorescence analysis method (IFA), enzyme linked immunosorbent assay (ELISA) and Western blotting for the autoimmune disease diagnostic method more, but its deficiency is respectively arranged.The 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 fit to the examination test.But following shortcoming is arranged: can not be as making a definite diagnosis foundation; Result's judgement 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 make a definite diagnosis foundation, but also quantitative measurement detects 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, is existing great limitation aspect the diagnostic application popularization of autoimmune disease.Western blot(WB) is a kind of immunoassay technology that grows up on gel electrophoresis and immuno analytical method basis, high resolution and the high specific of solid-phase immunoassay and the advantage of susceptibility with SDS-PAGE relatively are fit to find unknown antibody.And for detecting known antibody, owing to used natural hybrid antigen, the problem of target stripe and band skew often appears in the experimentation can not find, and caused many difficulties for result's interpretation, very easily judge by accident and fail to judge.WB is long service time simultaneously, also is not suitable for promoting in clinical examination.Someone improves WB, directly purifying antigen is coated on the nitrocellulose filter, then carries out immune response and detects antibody, has formed the western blotting method of improvement.The existing film bar that detects simultaneously 15 autoantibodies by purifying antigen is parallel coated to nitrocellulose membrane, seals the nonspecific reaction district at present.In first time during incubation, the serum that has diluted with detect the reaction of film bar, 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 the incubation second time, then add zymolyte, hatch colour developing, add stop buffer, as a result interpretation.Advantage is to realize high-throughout detection owing to 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 that detects only has 15, and some antigen is the achievement of early stage research, at present existing new more significant target antigen.
2, employed purifying antigen 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, and is sometimes also dark than positive band, affected result's reliability.
4, different antigen strip width differs, and differs in the band interval, has affected result's accurate interpretation.
5, the disclosed kit of like product such as CN200810132304.8, do not have critical quality control band, result's interpretation need to be done a control film bar in addition, has increased the cost of experiment, owing to there is certain difference in reaction environment between groove and the groove, may cause false positive or false negative simultaneously.The Related product that has 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 provides 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 the technical scheme that adopts: a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum, comprise the film bar, enzyme mark liquid, substrate and thickening and washing Incubating Solution, the film bar is by slide glass and be fixed on successively antigen band on the slide glass, critical quality control band, the function nature controlling line consists of, and the 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 the nylon membrane among Mi-2 and the Ku form.
In the method, the function nature controlling line belongs to prior art, whether effectively its objective is for the primary first-order equation of judging this film bar, the feminine gender of step 1) and the positive of step 4) are the antigen in the relative antigen band, it is positive to have the antibody that these antigens can detect, and it is negative not have the antibody that these antigens can detect.In the antigen band of this programme all independent of each other be scoring on nitrocellulose filter or the nylon membrane of each antigen form an independently antigen detection line, these all antigen detection lines are referred to as the antigen band, " getting the film bar detects each sample; scanning obtains the gray-scale value of antibody that each antigen detects in the film bar " in the step 1, the gray-scale value here is for scanning the gray-scale value that obtains after each antigen detection line test sample.The inventive point of this programme is that critical quality control band is the contrast band of at least two antigens in the antigen band simultaneously; Can be simultaneously two and even more test strip (antigen band) be played the critical quality control band of interpretation by being provided with one, along with increasing of test strip, detected band of every increase, all to determine experiment to re-starting complete critical Quality Control value, test simultaneously difficulty and significantly strengthen.Usually, determine that a critical Quality Control value needs many times experiment, could finally determine.
The composition of described critical quality control band is the human IgG of 20ng ~ 20 μ g/mL.Have in the prior art and select the in contrast record of line of anti-sheep IgG, 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 not be used for the record of the antigen band interpretation more than 2 or 2 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 the antigen band more than 2 or 2 is carried out 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, has n antigen in the antigen band of film bar, getting the film bar detects each sample, scanning obtains the gray-scale value of antibody that each antigen detects in the film bar, the gray-scale value of antibody that antigen detects identical in m the sample is obtained n group numerical value as one group of 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) with 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) such as CV≤10%, then can obtain the critical Quality Control value CO of film bar, wherein CO=(M+2 * SD); Such 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 the film bar each sample is detected, scanning obtains the gray-scale value of antibody that each antigen detects in the film bar, with the critical Quality Control value CO comparison of itself and film bar, all be not less than CO such as all sample gray-scale values, then CO is effective; Less than CO, need again to measure checking if any one or above sample gray-scale value; As still have one or above sample gray-scale value less than CO, then adjust trace antigen amount repeating step 1), step 2), step 3) redefines the critical Quality Control value CO of film bar.In the method for determining 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 the film bar of having determined, regulate the concentration of the coated human IgG of critical quality control band.Concrete definite method is: certain density human IgG is dissolved in Tris or the Hepes damping fluid, then lines and be prepared into the finished film bar on the nitrocellulose filter, and only be coated with critical quality control band on the film bar; Choose at random 30 film bars and detect with this kit, and the scanning gray scale, the average M that measures for 30 times calculated
S, standard deviation SD
SWith coefficient of variation CV
S, CV wherein
S=M
S/ SD
SThe 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, then need readjust concentration and carry out described all experiments of this step, until obtain qualified critical quality control band, the coated concentration of the human IgG of this moment is 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 the film bar of determining, the technician can converse the concentration of suitable human IgG by ordinary skill in the art means, adopts existing coated technique, can prepare critical quality control band.
Sheep/mouse that described enzyme mark liquid is horseradish peroxidase-labeled/rabbit anti-human igg's antibody.
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 the interval is wide between each adjacent antigen band.
Described antigen strip width is 0.5mm-3mm, is spaced apart 2mm-20mm between the adjacent antigen band.
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 to 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), and the colour developing band is relatively got final product 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 the single reagent substrate, need not stop behind the adding substrate.
3, each antigen strip width homogeneous, the band interval is wide, film bar clean background, it is more simple and reliable that the result is judged.
Description of drawings
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
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited only to following embodiment.
Embodiment 1:
To shown in Figure 3, the CO nature controlling line among Fig. 2 is critical quality control band referring to Fig. 1; The kit of the detection autoimmunity disease related antinuclear antibodies spectrum of 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.
The film bar is by slide glass and be fixed on successively the antigen band on the slide glass, critical quality control band, function nature controlling line and consist of, the antigen band is formed by two independent of each other being scoring on nitrocellulose filter or the nylon membrane among 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 Ku at least, and the composition of the critical quality control band of present embodiment is the human IgG of 20ng ~ 20 μ g/mL.
This kit can carry out the critical quality control band that yin and yang attribute is judged to 17 tested antibody (antigen band) at most creationary the adding simultaneously.Concrete flow process is seen 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, has n antigen in the antigen band of film bar, getting the film bar detects each sample, scanning obtains the gray-scale value of antibody that each antigen detects in the film bar, the gray-scale value of antibody that antigen detects identical in m the sample is obtained n group numerical value as one group of 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) with 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) such as CV≤10%, then can obtain the critical Quality Control value CO of film bar, wherein CO=(M+2 * SD); Such 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 the film bar each sample is detected, scanning obtains the gray-scale value of antibody that each antigen detects in the film bar, with the critical Quality Control value CO comparison of itself and film bar, all be not less than CO such as all sample gray-scale values, then CO is effective; Less than CO, need again to measure checking if any one or above sample gray-scale value; As still have one or above sample gray-scale value less than CO, then 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, verify critical quality control band with positive sample, if the gray scale of twice certain sample of experiment needs again to do whole experiment so all less than critical Quality Control value.And along with increasing of test strip, detected band of every increase all will be determined experiment to re-starting complete critical Quality Control value, tests simultaneously difficulty and significantly strengthens.Usually, determine that a critical Quality Control value needs many times experiment, could finally determine.
Embodiment 2:
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 the 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 the result is judged, each antigen strip width homogeneous is arranged, the band interval is wide, and the antigen strip width is 0.5mm-3mm, is spaced apart 2mm-20mm between the adjacent antigen band, film bar clean background, and adopt the single reagent substrate of improvement, need not stop behind the adding substrate, substrate is the single agents that luminol 0.02%-2% or hydrogen peroxidase 10 .002%-0.2% consist of; All trace antigen all is highly purified, and has adopted international up-to-date coated technique, and concrete method for coating sees below to be stated.
Determining of critical Quality Control value:
1) determines 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 detection of present embodiment.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
pThe results are shown in following table, present embodiment m=125, n=p=17.
2) determine the critical Quality Control value of film bar
CO with above-mentioned each antibody
pProcess: calculate their mean value M and standard deviation SD and coefficient of variation CV(CV=M/SD), if CV＞10% should be adjusted the concentration of corresponding trace antigen, repeat steps 1), step 2); If CV≤10% then 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, compare with CO.Definite through testing, this gray-scale value of testing all positive sample is all above critical Quality Control value, and this critical Quality Control value is effective.
Embodiment 3:
Present embodiment be the film bar determined according to embodiment 2 critical Quality Control value (in Fig. 3 and all embodiment of the present invention, CO
pBe the critical Quality Control value of single antibody, CO is the critical Quality Control value of whole film bar), the concentration of regulating the coated human IgG of critical quality control band, finally coated concentration and the coated technique of definite critical quality control band.Concrete operations are: certain density human IgG is dissolved in Tris or the Hepes damping fluid, then line on the nitrocellulose filter with full-automatic point sample instrument, and be prepared into the finished film bar through techniques such as sealings, drying, cutting, and only be coated with critical quality control band on the film bar.Choose at random 30 film bars and detect with this kit, and the scanning gray scale, the average (M that measures for 30 times calculated
S), standard deviation (SD
S) and the coefficient of variation (CV
S), CV wherein
S=M
S/ SD
SThe standard that critical quality control band is qualified is: 1) the critical Quality Control value of 0.97 * CO(film bar)≤and M
S≤ 1.03 * CO; 2) CV
S＜5%.If do not meet wherein any one, then need readjust concentration or coated technique and carry out described all experiments of present embodiment, until obtain qualified critical quality control band, the coated concentration of the human IgG of this moment is 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 meet the requirements.The coated concentration of the critical quality control band of present embodiment is 20ng/mL.
Embodiment 4:
Adopt the technical scheme of embodiment 1, we are to by choosing at random 2 and be scoring to independently of one another and form the antigen band on nitrocellulose filter or the nylon membrane among 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 Ku, carry out test determination, its result is as follows again:
1) determines 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.The result: the gray-scale value of all positive sample is all above 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 meet the requirements.The coated concentration of the critical quality control band of this moment is 20 μ g/mL.
Embodiment 5:
Adopt the technical scheme of embodiment 1, we are to by choosing at random 3 and be scoring to independently of one another and form the antigen band on nitrocellulose filter or the nylon membrane among 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 Ku, carry out test determination, its result is as follows again:
1) determines 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.The result: the gray-scale value of all positive sample is all above 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 meet the requirements.The coated concentration of the critical quality control band of this moment is 2 μ g/mL.
Embodiment 6:
Adopt the technical scheme of embodiment 1, we are to by choosing at random 9 and be scoring to independently of one another and form the antigen band on nitrocellulose filter or the nylon membrane among 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 Ku, carry out test determination, its result is as follows again:
1) determines 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.The result: the gray-scale value of all positive sample is all above 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 meet the requirements.The coated concentration of the critical quality control band of this moment is 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, select at random 9 antigens among Mi-2 and the Ku, according to embodiment 2, the methods experiment of embodiment 3 10 times, the critical Quality Control value of the film bar of determining according to 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 the time, its coated critical quality control band can be used for the comparison of at least 2 antigen bands simultaneously, but the number of times that definite dense coated degree of critical quality control band need to be done experiment is more; In the scope of 200ng ~ 2 μ g/mL, experiment shows when determining critical quality control band in 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 use disclosed scheme of prior art, and enzyme mark liquid is the sheep/mouse of horseradish peroxidase-labeled/rabbit anti-human igg's 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 to make from natural tissues; Described SmD1 is artificial synthetic polypeptide.The innovative critical quality control band of the present invention, the colour developing band is relatively got final product 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.Totally 17 kinds of the antibody numbers that detects: 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 some project is the achievement of current research, such as SmD1, U1snRNP, ribosomes P0 albumen etc., than Sm, nRNPR/Sm and ribosomes P albumen better sensitivity and specificity has been arranged 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 that is applied in above-described embodiment comprises:
1) antigen is identified: answer by polyacrylamide gel electrophoresis (SDS-PAGE) evaluation antigen and purity thereof〉98%.
2) coated: 17 kinds of antigens of human IgG and present embodiment are dissolved in respectively tris damping fluid or the HEPES damping fluid of 0. 01-0. 1M, pH7.4, obtaining concentration is 1-100mg/mL solution.The mentioned solution of 1-l00ul is lined on the 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 the 0.05-0.5%Tween20 that contains of 10-100ml, 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 ℃ was sealed 3-12 hour, with hatching the lavation buffer solution washing.After drying, for subsequent use in 2-8 ℃.
4) film bar preparation: prepared nitrocellulose membrane is fixed on the slide glass, firmly after, be cut to the wide film bar into 1mm-3mm with fully automatic cutting machine, divide to install in the film barrel.
4) enzyme mark liquid: classical sodium periodate oxidation mark horseradish peroxidase (HRP) is to sheep/mouse/rabbit anti-human igg's antibody, component: sheep/mouse of mark HRP/rabbit anti-human igg's 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 need not to stop, and rear the drying for 3 times with the distilled water washing of film bar colour developing gets final product, and colour developing is steady in a long-term.
7) assembling of kit: with the 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 (10)
1. kit that detects autoimmunity disease related antinuclear antibodies spectrum, comprise the film bar, enzyme mark liquid, substrate and thickening and washing Incubating Solution, it is characterized in that, the film bar is by slide glass and be fixed on successively antigen band on the slide glass, critical quality control band, the function nature controlling line consists of, and the 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 the nylon membrane among Mi-2 and the Ku form.
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 20ng ~ 20 μ g/mL.
3. 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.
4. according to claim 1 to 3 each described a kind of kits that detect autoimmunity disease related antinuclear antibodies spectrum, it is characterized in that 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, has n antigen in the antigen band of film bar, getting the film bar detects each sample, scanning obtains the gray-scale value of antibody that each antigen detects in the film bar, the gray-scale value of antibody that antigen detects identical in m the sample is obtained n group numerical value as one group of 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) with 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) such as CV≤10%, then can obtain the critical Quality Control value CO of film bar, wherein CO=(M+2 * SD); Such 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 the film bar each sample is detected, scanning obtains the gray-scale value of antibody that each antigen detects in the film bar, with the critical Quality Control value CO comparison of itself and film bar, all be not less than CO such as all sample gray-scale values, then CO is effective; Less than CO, need again to measure checking if any one or above sample gray-scale value; As still have one or above sample gray-scale value less than CO, then adjust trace antigen amount repeating step 1), step 2), step 3) redefines the critical Quality Control value CO of film bar.
5. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 4 is characterized in that, described critical quality control band determines that method is: the concentration of regulating the coated human IgG of critical quality control band according to the critical Quality Control value of the film bar of determining.
6. according to claim 1 to 3 each described a kind of kits that detect autoimmunity disease related antinuclear antibodies spectrum, 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%.
7. according to claim 1 to 3 each described a kind of kits that detect autoimmunity disease related antinuclear antibodies spectrum, it is characterized in that described antigen band is parallel to each other and each antigen strip width homogeneous, the interval is wide between each adjacent antigen band.
8. a kind of kit that detects autoimmunity disease related antinuclear antibodies spectrum according to claim 7 is characterized in that, described antigen strip width is 0.5mm-3mm, is spaced apart 2mm-20mm between the adjacent antigen band.
9. according to claim 1 to 3 each described a kind of kits that detect autoimmunity disease related antinuclear antibodies spectrum, 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.
10. according to claim 1 to 3 each described a kind of kits that detect autoimmunity disease related antinuclear antibodies spectrum, it is characterized in that 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 to make from natural tissues; Described SmD1 is artificial synthetic polypeptide.
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