CN103439517A - Systemic lupus erythematosus (SLE) autoantibody detector - Google Patents
Systemic lupus erythematosus (SLE) autoantibody detector Download PDFInfo
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- CN103439517A CN103439517A CN2013104114632A CN201310411463A CN103439517A CN 103439517 A CN103439517 A CN 103439517A CN 2013104114632 A CN2013104114632 A CN 2013104114632A CN 201310411463 A CN201310411463 A CN 201310411463A CN 103439517 A CN103439517 A CN 103439517A
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Abstract
The invention provides a systemic lupus erythematosus (SLE) autoantibody detector which comprises chips and a kit, wherein a porous plate is arranged in the kit; the chips are positioned in the plate holes of the porous plate; reference substances and antigen protein are fixed on the chips according to a dot-matrix array. The detector provided by the invention can shorten the SLE detection time, reduce the detection cost, reduce the serum use for a patient, and improve the clinical diagnosis efficiency of the SLE.
Description
Technical field
The present invention relates to biomedical sector, specifically, relate to a kind of systemic loupus erythematosus autoimmune antibody pick-up unit.
Background technology
Systemic loupus erythematosus (Systemic Lupus Erythematosus is called for short SLE) is a kind of common chronic autoimmune disease.This sick feature is that body can produce autoantibody, thereby causes skin injury and organ dysfunction, and the organ that can involve comprises heart, liver, kidney, lung, skin, blood vessel, joint and nervous system etc.SLE often needs the time of several years from falling ill to making a definite diagnosis, although can't cure fully at present, early diagnosis is very important to slowing down its development.Because SLE is caused by many factors, and often be attended by other autoimmune disease, therefore diagnose this disease need to carry out multinomial detection, wherein, the detection of SLE autoantibody has very important significance.
The autoantibody relevant to SLE mainly contains anti-dsDNA antibody and anti-Sm antibody, in addition, also comprises anti-ribosomes P albumen, the antibody such as anti-PCNA, U1-nRNP, SS-A, SS-B.Wherein, the target antigen of anti-dsDNA antibody is duplex DNA, and this antibody can be combined into immune complex with DNA and deposit in glomerular basement membrane, or directly acts on glomerulus antigen and cause injury of kidney.At present, anti-dsDNA antibody has higher specificity, and parallels with the activity of SLE, has been listed in one of diagnostic criteria of SLE.Anti-Sm antibody belongs to antinuclear antibodies, is also the antibody with high specificity of a kind of SLE.Anti-ribosomes P protein antibodies is the specific antibody of SLE, rarely found in other diseases and normal person.Anti-PCNA antibody (anti-proliferating cell nuclear antigen antibody) is a kind of nucleoprotein of coordinating DNA replication dna, and the diagnosis of SLE is had to very high value.Anti-U1-nRNP antibody (anti-micronuclear ribonucleoprotein compound antibody) can detect in the SLE patient of 30-40%, and mostly in the patients serum simultaneously with the appearance of anti-Sm antibody.Anti SS-A antibody is relevant to all kinds of autoimmune diseases, in 100% neonate SLE patient, can occur.Anti SS-B antibody sees the women SLE patient of 10-20%.
At present, the diagnosis of SLE adopts immunofluorescence or ELISA method usually, chooses respectively some above-mentioned SLE autoantibodies as detecting index, more rule of thumb result is analyzed.There is following shortcoming in the method: 1, detect index choose with interpretation of result in, have more human factor, do not there is ubiquity, accuracy also has much room for improvement; 2, once can only analyze a kind of detection index, efficiency is low, and Diagnostic Time is long, and expense is higher.
Summary of the invention
The purpose of this invention is to provide a kind of precise and high efficiency, the Autoantibodies of Systemic Lupus Erythematosus pick-up unit with low cost.
For realizing the object of the invention, the invention provides a kind of the Autoantibodies of Systemic Lupus Erythematosus pick-up unit, comprise: chip, kit, be provided with porous plate in described kit, described chip is arranged in the plate hole of porous plate, on described chip, according to dot matrix, arranges and is fixed with reference substance, antigen protein.
Reference substance of the present invention comprises the goat anti-human igg that antigenic dilution, content are 40-60 μ g/mL, the human IgG that content is 28-35 μ g/mL, the human IgG that content is 10-14 μ g/mL; Described antigen protein comprises dsDNA, sm, P
0, P
1, P
2, PCNA, U1-snRNP68/70, U-snRNP BB ', U1-snRNP C, U1-snRNP A, Ro/SS-A 52KD, Ro/SS-A 60KD, La/SS-B, nucleolin.
Antigen protein of the present invention, its content is as follows: dsDNA 0.3-0.4mg/mL, sm 0.1-0.2 mg/mL, P
00.3-0.4mg/ mL, P
10.12-0.18mg/mL, P
20.3-0.5 mg/ mL, PCNA 0.25-0.3mg/ mL, U1-snRNP68/70 0.16-0.28 mg/mL, U-snRNP BB ' 0.13-0.28mg/mL, U1-snRNP C 0.15-0.2mg/ mL, U1-snRNP A 0.25-0.35 mg/mL, Ro/SS-A 52KD 0.15-0.2 mg/mL, Ro/SS-A 60KD 0.1-0.15 mg/ mL, La/SS-B 0.15-0.2 mg/ mL, nucleolin 0.1-0.15 mg/ mL
Also dispose sample dilution, concentrated washing lotion, ELIAS secondary antibody, developer in kit of the present invention.
The phosphate buffer that sample dilution of the present invention is pH7.4, wherein the contain mass concentration BSA that is 1%, the Proclin300 that mass concentration is 0.05%; Described concentrated washing lotion, its composition is counted with mass volume ratio concentration: NaCl8%, KCl0.2%, Na
2hPO
42.135%, KH
2pO
40.24%; The mountain goat anti-human igg that described ELIAS secondary antibody is the HRP mark, described developer is sedimentation type TMB.
Chip of the present invention, its material is preferably cellulose acetate membrane.
Antigen protein of the present invention, when detecting the SLE autoantibody, has good specificity and sensitivity.The present invention is with the negative control point of antigenic dilution, the positive reaction control point of goat anti-human igg, the positive demonstration control point of high concentration human IgG, the negative demonstration control point of low concentration human IgG; During experiment, negative control point does not react with serum sample, does not finally develop the color; And, for the positive reaction control point, no matter in serum sample, whether contain the SLE associated antibodies, as long as add serum, IgG wherein just can react with the goat anti-human igg final colour developing.Positive and negative demonstration control point is the interpretation standard point of chip, and the colour developing degree shows that lower than feminine gender control point is negative, higher than the positive, shows that control point is positive, and the colour developing degree falls between and is the weak positive.When only a kind of antibody positive being arranged, corresponding diagnosis is done in other clinical manifestations of suggestion consideration.The setting of reference substance of the present invention makes the easier interpretation of testing result, thereby can draw easily the expression of SLE autoantibody in sample serum.During detection, to after the dilution of sample serum, it be reacted with chip, antigen protein on chip can be caught corresponding SLE autoantibody in serum, by adding ELIAS secondary antibody, with the SLE autoantibody be trapped on chip, be combined, add again the developer that can react with ELIAS secondary antibody, just the situation of the SLE autoantibody in serum can be presented on the film chip.Accompanying drawing 3-5 is shown in by the reaction principle schematic diagram.
Chip of the present invention has high integration, take enzyme linked immunoassay as basis, and 14 kinds of SLE associated protein o'clock on a chip, can be obtained to Measuring Several Indexes simultaneously, for the clinical diagnosis of SLE provides more reference.Compare with detecting respectively indices, reduced experimental implementation, shortened detection time, be convenient to carry out the examination of large quantities of samples, saved cost simultaneously, also reduced patients serum's use amount, thereby greatly improved the efficiency of SLE clinical diagnosis.
The accompanying drawing explanation
The distribution schematic diagram that Fig. 1 is chip internal reference product and antigen protein.
The implication of in Fig. 1, numeral 1 ~ 14 and Chinese character sun, sun ', cloudy, the moon ' is: sun: positive reaction contrast-goat anti-human igg; Cloudy: negative reaction contrast-antigenic dilution; 1:dsDNA; 2:sm; 3:P
0; 4:P
1; 5:P
2; 6:PCNA; 7:U1-snRNP 68/70; 8:U-snRNP BB '; 9:U1-snRNP C; 10:U1-snRNP A; 11:Ro/SS-A(60KD); 12:Ro/SS-A(52KD); 13:La/SS-B; 14:nucleolin; Sun ': the positive contrast-high concentration human IgG that shows; Cloudy ': the negative contrast-low concentration human IgG that shows.
The schematic diagram of arranging that Fig. 2 is chip array on every cellulose acetate membrane.
Fig. 3 is that the SLE autoantibody detects principle schematic.
The positive reaction contrast of Fig. 4 principle schematic.
Fig. 5 is for showing the contrast principle schematic.
In Fig. 3 ~ 5: 1, ELIAS secondary antibody, 2, membrane carrier, 3, measured antibody, 4, antigen, 5, human IgG, 6, the goat anti-human igg.
Fig. 6 is porous plate chips distribution schematic diagram.
In Fig. 6: 7, porous reaction plate, 8, the film chip.
Fig. 7 is SLE patients serum testing result schematic diagram.
Embodiment
Below with specific embodiment, further illustrate content of the present invention, but and mean and limit the invention never in any form.In the following example, method therefor is conventional method if no special instructions.
(1) making of chip:
Step 1: the cellulose acetate membrane in 0.2 μ m aperture is cut to 25cm * 75cm, is placed in the draw-in groove of point sample instrument.
Step 2: under the condition of room temperature, relative humidity 60%, use the German Gesim NanoPlotter2.1 of company type point sample instrument, by 14 kinds of antigen proteins (dsDNA, sm, P
0, P
1, P
2, PCNA, U1-snRNP68/70, U-snRNP BB ', U1-snRNP C, U1-snRNP A, Ro/SS-A 52KD, Ro/SS-A 60KD, La/SS-B, nucleolin) and 4 kinds of reference substances (negative reaction contrast-antigenic dilution, positive reaction contrast-goat anti-human igg, positive contrast-high concentration human IgG, the negative demonstration contrast-low concentration human IgG of showing) according to array arrangement point on cellulose acetate membrane.Accompanying drawing 1 is seen in the point sample position of each antigen protein and reference substance, dot spacing 0.7mm, and some system is measured about 10nL, and every kind of sample repeats specking 3 times, two kinds of albumen of every horizontally-arranged point system, array is 6 * 9 arranges, and forms a chip, sees accompanying drawing 1.16 chips of some system on every cellulose acetate membrane, chip position distributes and sees accompanying drawing 2.After point has been made, in the room temperature placement, spend the night albumen fully is fixed on cellulose acetate membrane.
Step 3: the cellulose acetate membrane after point sample is placed in and contains 1%BSA, and in the confining liquid of 2% sucrose, the 1.5h that at room temperature vibrates, take out drying at room temperature.
Step 4: under chip is cut out according to a position processed, obtain the chip of 1.0cm * 1.0cm, then the gained chip is placed on respectively in the plate hole of 24 hole porous plates, see accompanying drawing 6, preserve under 4 ℃.
On said chip, the content of described antigen protein is as follows: dsDNA 0.3-0.4mg/mL, sm 0.1-0.2 mg/mL, P
00.3-0.4mg/ mL, P
10.12-0.18mg/mL, P
20.3-0.5 mg/ mL, PCNA 0.25-0.3mg/ mL, U1-snRNP68/70 0.16-0.28 mg/mL, U-snRNP BB ' 0.13-0.28mg/mL, U1-snRNP C 0.15-0.2mg/ mL, U1-snRNP A 0.25-0.35 mg/mL, Ro/SS-A 52KD 0.15-0.2 mg/mL, Ro/SS-A 60KD 0.1-0.15 mg/ mL, La/SS-B 0.15-0.2 mg/ mL, nucleolin 0.1-0.15 mg/ mL.The content of described reference substance is as follows: positive reaction contrast-goat anti-human igg 40-60 μ g/mL, positive contrast-high concentration human IgG 28-35 μ g/mL, the negative contrast-low concentration human IgG 10-14 μ g/mL that shows of showing.
(2) preparation of reagent:
Sample dilution: containing the phosphate buffer (pH7.4) of 1%BSA+0.05 antiseptic (Proclin 300).Each kit is equipped with 25mL.
Concentrated washing lotion: NaCl 8g, KCl 0.2g, Na
2hPO
42.135g, KH
2pO
40.24g, being dissolved in 100mL water, each kit is equipped with 20mL.During use, dilution is 10 times.
ELIAS secondary antibody: the mountain goat anti-human igg of HRP mark.
Chromogenic reagent: sedimentation type TMB.
Above-mentioned porous plate, sample dilution, washing lotion, ELIAS secondary antibody, chromogenic reagent are positioned in kit, obtain the Autoantibodies of Systemic Lupus Erythematosus pick-up unit of the present invention.
The detection of SLE autoantibody:
Sample is prepared: the sample dilution in the Autoantibodies of Systemic Lupus Erythematosus pick-up unit that adopts embodiment 1 to prepare, the serum sample of Patients with SLE is pressed to 1:500 dilution, the sample after being diluted;
Sample reaction: get the sample 1mL after dilution, add in the plate hole of porous plate, with the chip incubation reaction, add a cover, in room temperature vibration 1.5h;
Rinsing: will concentrate 10 times of distilled water dilutings for washing lotion, and obtain rinsing liquid, the liquid in plate hole is siphoned away, and add rinsing liquid 1mL, and after vibration 5min, siphon away liquid, and repeat twice of rinsing;
The ELIAS secondary antibody combination: adopt above-mentioned rinsing liquid, after HRP-mountain goat anti-human igg is diluted with 1:1000, in each plate hole, add 1mL, room temperature vibration 1.5h, siphon away liquid, then add rinsing liquid 1mL, room temperature vibration rinsing 3 times;
Colour developing: add sedimentation type TMB developer, room temperature reaction 10min, observing response result.Fig. 7 is shown in by the schematic diagram of testing result.Wherein, the positive reaction control point shows darker blueness, and the negative reaction control point does not develop the color, the positive darker blueness (identical with the positive reaction control point) of control point demonstration that shows, and feminine gender shows that control point shows weak blueness.If other check point Show Colors >=positive shows that contrasting interpretation is strong positive, if Show Color interpretation between feminine gender demonstration contrast and positive demonstration contrast is the weak positive, it is negative that the monitoring point Show Color≤feminine gender demonstration contrasts interpretation.On chip, front 6 kinds of antigen protein specificitys are higher, great to the diagnostic significance of systemic loupus erythematosus, and antibody corresponding to rear 6 kinds of antigens also occurs in other autoimmune diseases, can be used as the reference index of auxiliary diagnosis.
Collect the SLE patient's that rheumatism immunity section of certain front three hospital makes a definite diagnosis with SLICC systemic loupus erythematosus standard 23 parts of serum, 30 parts of health check-up normal person's serum, apply the Autoantibodies of Systemic Lupus Erythematosus pick-up unit of the present invention, detected with reference to the described method of embodiment 2.The testing result demonstration, the recall rate of positive sample is 19/23=82.6%, the detection coincidence rate 30-2/30=93.3%(two routine sample standard deviations of negative sample detect the dsDNA positive).In the systemic lupus erythematosus diagnosis standard of formulating in international rheumatism association, only having 2 in 11 reference indexs, to relate to antibody abnormal, the diagnosis of disease also needs with reference to multinomial patient clinical symptom, but therefore by pick-up unit examination of the present invention, go out the Disease more than 80%, show that this pick-up unit can provide effectively auxiliary for clinical diagnosis.
The setting of antigen protein point sample concentration:
By 14 kinds of antigen proteins of the present invention and human IgG and goat anti-human igg respectively gradient dilution become 7 concentration (0.1-1.2mg/mL does not wait), put respectively on the film chip, with 20 parts of SLE positive serums, react afterwards, obtain gray-scale value after colour developing.For every kind of antigen protein, the mean value of each antigen protein concentration value 20 gray-scale values corresponding with it can be made regression straight line.On 14 parts of antigen protein concentration-grey scale curve, choose the total a certain optimum gradation value of the range of linearity separately and show the gray scale Cutoff value of control point as the positive, the antigen protein concentration that this gray-scale value is corresponding is the best point sample concentration of 14 kinds of antigen proteins, this gray-scale value correspondence, on the concentration-grey scale curve of human IgG, can obtain the positive point sample concentration that shows contrast.In like manner obtain the point sample concentration of positive reaction contrast.
Show the definite with reference to following methods of contrast concentration for feminine gender: with the above-mentioned antigen protein concentration point coremaking sheet determined, and the human IgG of some gradient concentration processed, use the serum of 20 parts of people taking physical examinations to carry out the chip reaction, obtain the colour developing gray-scale value of negative reaction, computation of mean values, consider in the situation of standard deviation and the coefficient of variation and draw negative Cutoff value, by the concentration-grey scale curve of corresponding human IgG, obtain the negative point sample concentration that shows the control point human IgG.
Claims (6)
1. a Autoantibodies of Systemic Lupus Erythematosus pick-up unit, it is characterized in that comprising: chip, kit, be provided with porous plate in described kit, described chip is arranged in the plate hole of porous plate, on described chip, according to dot matrix, arranges and is fixed with reference substance, antigen protein.
2. the Autoantibodies of Systemic Lupus Erythematosus pick-up unit according to claim 1, it is characterized in that: described reference substance comprises the goat anti-human igg that antigenic dilution, content are 40-60 μ g/mL, the human IgG that content is 28-35 μ g/mL, the human IgG that content is 10-14 μ g/mL; Described antigen protein comprises dsDNA, sm, P
0, P
1, P
2, PCNA, U1-snRNP68/70, U-snRNP BB ', U1-snRNP C, U1-snRNP A, Ro/SS-A 52KD, Ro/SS-A 60KD, La/SS-B, nucleolin.
3. the Autoantibodies of Systemic Lupus Erythematosus pick-up unit according to claim 2, it is characterized in that: described antigen protein, its content is as follows: dsDNA 0.3-0.4mg/mL, sm 0.1-0.2 mg/mL, P
00.3-0.4mg/ mL, P
10.12-0.18mg/mL, P
20.3-0.5 mg/ mL, PCNA 0.25-0.3mg/ mL, U1-snRNP68/70 0.16-0.28 mg/mL, U-snRNP BB ' 0.13-0.28mg/mL, U1-snRNP C 0.15-0.2mg/ mL, U1-snRNP A 0.25-0.35 mg/mL, Ro/SS-A 52KD 0.15-0.2 mg/mL, Ro/SS-A 60KD 0.1-0.15 mg/ mL, La/SS-B 0.15-0.2 mg/ mL, nucleolin 0.1-0.15 mg/ mL.
4. according to the described the Autoantibodies of Systemic Lupus Erythematosus pick-up unit of any one in claim 1 ~ 3, it is characterized in that: also dispose sample dilution, concentrated washing lotion, ELIAS secondary antibody, developer in described kit.
5. the Autoantibodies of Systemic Lupus Erythematosus pick-up unit according to claim 4 is characterized in that: the phosphate buffer that described sample dilution is pH7.4, wherein the contain mass concentration BSA that is 1%, the Proclin300 that mass concentration is 0.05%; Described concentrated washing lotion, its composition is counted with mass volume ratio concentration: NaCl 8%, KCl 0.2%, Na
2hPO
42.135%, KH
2pO
40.24%; The mountain goat anti-human igg that described ELIAS secondary antibody is the HRP mark, described developer is sedimentation type TMB.
6. the Autoantibodies of Systemic Lupus Erythematosus pick-up unit according to claim 5, it is characterized in that: described chip, its material is cellulose acetate membrane.
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|---|---|---|---|
| CN2013104114632A CN103439517A (en) | 2013-09-11 | 2013-09-11 | Systemic lupus erythematosus (SLE) autoantibody detector |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106596919A (en) * | 2016-06-30 | 2017-04-26 | 深圳市亚辉龙生物科技股份有限公司 | Kit (chemiluminiscence method) for determining anti-ribonucleoprotein 70 antibody IgG and manufacturing method thereof |
| CN107110861A (en) * | 2014-08-08 | 2017-08-29 | 阿勒格尼-辛格研究所 | It is used as the autoantibody of the antilymphocyte of diagnostic biomarker |
| CN113567660A (en) * | 2021-07-22 | 2021-10-29 | 中南大学湘雅二医院 | Method for detecting autoantibodies of systemic lupus erythematosus patient |
| CN114137224A (en) * | 2021-11-29 | 2022-03-04 | 中南大学湘雅二医院 | Systemic lupus erythematosus autoantibody detection device based on big data |
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Cited By (5)
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Application publication date: 20131211 |