CN1176377C - Composite chip for detecting autoimmune antibody of diabetes mellitus, and preparation and detection method thereof - Google Patents
Composite chip for detecting autoimmune antibody of diabetes mellitus, and preparation and detection method thereof Download PDFInfo
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Abstract
The present invention relates to the field of biomedicine research and clinical application, more specifically to a composite chip for detecting autoimmune antibodies of diabetes mellitus, and a preparation method and a detection method thereof. The present invention aims to solve the problems of inaccurate accurate diagnosis and high detection cost of the existing clinical analysis of diabetes mellitus. The present invention has the solution that the upper surface of a shell of the composite chip for detecting autoimmune antibodies of diabetes mellitus is provided with a plurality of glass flakes of reaction tanks; detection micro arrays are arranged in the reaction tanks; each detection micro array is composed of four protein miniature detection sheets of a solid phase, wherein the detection sheets have different varieties, the same specification and the same size; the protein miniature detection sheets of the solid phase are respectively a miniature detection sheet of the solid phase of islet cells, a GAD protein miniature detection sheet of the solid phase, a PTP protein miniature detection sheet of the solid phase, and an insulin protein miniature detection sheet of the solid phase. The preparation method comprises: firstly, the miniature detection sheets of the solid phase are prepared; then, the detection sheets are assembled. The detection method comprises: firstly, the detection is carried out by an indirect fluorescent method; a result is judged by a laser confocal scanner of a biological chip.
Description
Affiliated technical field:
The present invention relates to biomedical research and clinical practice field, be specifically related to diabetes autoimmune antibody detection compound chip and preparation thereof, detection method.
Background technology:
Diabetes are one of diseases that present harm humans is healthy and quality of life is the most serious, and it increases with the blood sugar concentration continuation is the cardinal symptom performance, the health risk by multiple severe complication.Point out that according to the up-to-date prediction that IDF (KDF) makes the onset diabetes situation the morbidity present situation and the The World Health Organization (WHO) of global diabetes the whole world has diagnosed out the type ii diabetes patient to reach 1.3 hundred million people at present, China surpasses 4,000 ten thousand people.Expect 2025, global diabetic will break through 300,000,000, and China's diabetic's sum is near 100,000,000.In China, along with the raising of economic life level and the change of dietary structure, diabetic crowd just develops rapidly, and prevention and treatment diabetes have become the great social problem of China.
Diabetes can be divided into two classes according to the cause of disease, I type and II type, this diabetes cause difference of two types, all completely different to patient's harm and methods of treatment.As far back as the eighties, just the someone to observe a part of clinical diagnosis be that the type ii diabetes patient can detect specific islet cells autoantibody in blood, this class patient is prone to insecondary oral antidiabetic drug treatment and lost efficacy, and must rely on insulinize.Studies show that in a large number subsequently, the actual immune intermediary hypotype that belongs in the type i diabetes of this class patient is a kind of onset invisible type i diabetes of adult (LADA) in evening, onset is former because autoimmune disorder.But because this class patient's clinical manifestation and type ii diabetes are difficult to differentiate, most hospitals do not have the detection means of associated antibodies again, all are diagnosed as type ii diabetes usually, thereby use unsuitable type ii diabetes methods of treatment, delay treatment causes the loss of patient health and even life.
In 1999, the expert of The World Health Organization (WHO) appraised through discussion in report " definition of diabetes and complication thereof, diagnosis and the somatotype " first and has proposed the new somatotype of diabetes.Wherein, type i diabetes is divided into immune intermediary type and two hypotypes of special hair style, and points out, makes a definite diagnosis immune intermediary type i diabetes, mainly relies on the autoimmune antibody that detects among the diabetic at the various antigens of beta Cell of islet.People have found the multiple autoimmune antibody relevant with diabetes at present, and wherein most important autoimmune antibody comprises: islet cells autoantibody (ICAs); Glutamic acid decarboxylase antibody (GADA); Protein-tyrosine-phosphatase sample transmembrane protein antibody (ICA512/IA-2); Insulin antibody (IAA).Because of the appearance of islet cells autoantibody far early than clinical symptoms, in type i diabetes clinical early stage, multiple autoantibody just can detect at peripheral blood, its farthest the time can reach 8 years, can predict in time that following type i diabetes takes place.As taking the corresponding treatment measure, can delay disease time to this class patient greatly, even finally change the prognosis of disease.
Owing to situation, time and intensity and inconsistent occur, and the single recall rate of various autoimmune antibody all is no more than 80% to the diabetes autoimmune antibody in detection, so any single detection all causes failing to pinpoint a disease in diagnosis of actual patient probably.Therefore, the necessary joint-detection of diabetes autoimmune antibody just can be used for somatotype clinically and makes a definite diagnosis, and then the important practical of influence treatment and patient's prognosis is worth.At present conventional diabetes diagnosis is still with clinical symptoms and diagnoses in conjunction with relative lab index such as blood sugar concentration and the experiments of oral glucose tolerance; it has been not suitable for carrying out somatotype and has made a definite diagnosis; be subjected to simultaneously the interference of various factors because of himself reason regular meeting; produce the bigger testing result of fluctuation sometimes, that is to say the testing result instability.Existing in addition people attempts adopting specific sero-immunity method to assist classification diagnosis, but generally all be to detect separately at every kind of autoimmune antibody, therefore detect the cost height, general patient is difficult to adopt, also be difficult to simultaneously means, can not provide the present diabetic of adaptation to increase needed corresponding high flux standardization detection means as the health check-up examination.
Summary of the invention:
The present invention will solve existing diabetes clinical classification and make a definite diagnosis inaccurate and the high problem of detection cost.
For solving existing problem, solution of the present invention provides a kind of diabetes autoimmune antibody and detects compound chip, comprise shell 1 and detect microarray 3, its special character is, described shell 1 is the glass flake that upper surface is provided with a plurality of reaction tanks 2, be provided with one in the reaction tank and detect microarray 3, each detects microarray 3 is made up of the miniature solid phase detection lug of four kinds of same specification sizes respectively, and described miniature solid phase detection lug is respectively the miniature solid phase detection lug 4 of islet cells, the miniature solid phase detection lug 5 of RHGAD 65KD albumen, miniature solid phase detection lug 6 of recombined human pancreatic island cell antigen 512 albumen and the miniature solid phase detection lug 7 of rh-insulin's albumen.
Above-mentioned miniature solid phase detection lug size is at 0.1mm
2～9mm
2Between.
Above-mentioned glass flake specification is 22mm * 75mm, and thickness 1mm～3mm, its upper surface are provided with 3～200 reaction tanks 2, and described reaction tank 2 is that the length of side is the square of 0.2mm～18mm.
Above-mentioned reaction tank 2 capacity are 10 μ l～100 μ l.
Comprise on the miniature solid phase detection lug 4 of above-mentioned islet cells and have an appointment 1 * 10
3～1 * 10
6Individual islet cells; Include 2～10 corresponding proteins point of samples on the miniature solid phase detection lug of described each albumen.
The preparation method that a kind of above-mentioned diabetes autoimmune antibody detects compound chip is as follows:
At first prepare miniature solid phase detection lug, miniature solid phase detection lug among the present invention is divided into the miniature solid phase detection lug of islet cells, the miniature solid phase detection lug of RHGAD 65KD albumen, the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen and the miniature solid phase detection lug of rh-insulin's albumen according to the detection matrix species difference on it; In the preparation of these four miniature solid phase detection lugs, detection lug adopts glass cover slide or transparent polystyrene cover glass or the cellulose acetate film through silicidation, wherein the preparation of the miniature solid phase detection lug 4 of islet cells adopts the cells in vitro culture technique in conjunction with the cell climbing sheet technology, and the preparation method of other three kinds of miniature solid phase detection lugs of albumen comprises albumen is mixed the concentration that protein solution is regulated in the back with phosphate buffer successively; Protein solution is added 40% glycerine, mixing again; Adopt commercially available dna microarray instrument point sample instrument point sample on substrate plane; Hatched behind the point sample 3 hours; With the bovine serum albumin solution sealing, be cut into 0.1mm during use
2～9mm
2Miniature detection lug;
Assemble then: 1. select for use the surface to be provided with the glass flake of 3～200 reaction tanks, each reaction tank is the square that the length of side is 0.2mm～18mm, capacity 10 μ l～100 μ l; 2. in reaction tank, be provided with one and detect microarray, stick to be fixed in it by the different types of miniature solid phase detection lug arrangement of four same specification sizes and with transparent binder and make.
The preparation scheme of above-mentioned miniature solid phase detection lug is:
1. the preparation of the miniature solid phase detection lug of islet cells: at first choose cultured cell in vitro to islet cells autoantibody antibody sensitivity, described cultured cell in vitro to islet cells autoantibody antibody sensitivity comprises the beta Cell of islet oncocyte that islet-cell tumour excision tissue is cultivated, and islet cells in the mammalian pancreas tissue; Detect the cultivation of matrix then: cultural method be earlier with reference to the external cultivation of islet cells carry out former be commissioned to train foster, after going down to posterity, make adherent single-layer culturing cell grow in the surface of detection lug again, after treating that the whole detection lug of cell growth attaching surface surpasses 50%, take out detection lug, put into the phosphate buffer washing, drip fixedly 30min of immobile liquid in the detection lug surface again, fixing back is immersed phosphate buffer-glycerine mixed liquor and is placed-20 ℃ of storages, is cut into 0.1mm during use
2～9mm
2Miniature detection lug;
2. in the preparation of the miniature solid phase detection lug of RHGAD 65KD albumen, point sample reagent adopts the U.S. purification of recombinant human glutamate decarboxylase 65KD of sigma company albumen, the phosphate buffer dilution, pH7.5, the final concentration scope is at 10 μ g～200 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density is at 100～2000 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 0.1mm during use
2～9mm
2Miniature detection lug, each miniature detection lug contains 2～10 protein site sampling points;
3. in the preparation of the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen, reagent adopts U.S. sigma company reorganization purification of Recombinant human pancreatic island cell antigen 512 albumen β-hypotypes, the phosphate buffer dilution, pH7.5, the final concentration scope is at 10 μ g～200 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density is at 100～2000 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 0.1mm during use
2～9mm
2Miniature detection lug, each miniature detection lug contains 2～10 protein site sampling points;
In the preparation of 4. miniature rh-insulin's albumen solid phase detection lug, reagent adopts the synthetic actrapid monotard's albumen of U.S. sigma company, phosphate buffer dilution, pH7.5, the final concentration scope adds 40% glycerine at 10 μ g～200 μ g/ml, mixing adopts microarray point sample instrument point sample.Point sample density is at 100～2000 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 0.1mm during use
2～9mm
2Miniature detection lug, each miniature detection lug contains 2～10 protein site sampling points;
1., 2., 3. and detection lug 4. can adopt through the glass cover slide of silicidation (APES) or commercially available transparent polystyrene cover glass.
Above-mentioned preparation method also comprises packaging step, and chip surface is wrapped up with masking foil, packs again.
Chip is stored under-20 ℃ of conditions, places during transportation in the thermostatic container that is not higher than 4 ℃.
The detection method that a kind of above-mentioned diabetes autoimmune antibody detects compound chip is as follows: adopt the indirect fluorescent method to detect earlier, testing result is judged with the scanning of biochip laser confocal scanning instrument again, also can adopt the microarray scanner scanning laboratory test results of other kinds, carry out interpretation by software or professional person; Also can use common fluorescent microscope Direct observation, experimental result be judged by the professional person.In the above reaction tank any two as the Quality Control reaction tank, all the other are the detection reaction pond.
In the above-mentioned indirect fluorescent method,
At first carry out pre-service:
1. after chip being taken out, room temperature is placed 5min, unpacks;
2. add 50 μ l phosphate buffers at each reaction tank, put into 37 ℃ of water-baths and hatch 15min;
3. drip under 1% bovine serum albumin(BSA), the 50 μ l room temperatures and seal 20min;
To the detecting of detection reaction pond, operation steps is then:
1. a patient's test serum is diluted with 1: 5 usefulness phosphate buffer, directly drip, hatch 1h for 37 ℃ in a detection reaction pool surface;
2. the phosphate buffer flushing is 3 times, each 5min;
3. add fluorescently-labeled mouse-anti human IgG monoclonal antibody, hatch 1h for 37 ℃;
4. the phosphate buffer that contains 1% tween washs 3 times, each 10min;
5. drip envelope and mount the agent mounting;
To detecting of Quality Control microarray reaction tank, operation steps is:
Carry out synchronously with above-mentioned detection the detection reaction pond, only will detecting operation step to the detection reaction pond in used patient's test serum be changed to that positive control detects liquid or negative control detects liquid, all the other steps are identical; It is with health adult's serum that positive control detects liquid, leave heart 10min through 1500, human glutamic acid decarboxylase autoantibody, human pancreatic island cell antigen 512 albumen autoantibodies, actrapid monotard's albumen autoantibody each 5u of monoclonal antibody of purifying or the concentrated solution that is equivalent to this amount are dissolved in health adult's serum preparation form; It is with health adult's serum that negative control detects liquid, leaves heart 10min through 1500, gets supernatant and is prepared from.
Compared with prior art, the present invention has following advantage:
1. utilize current up-to-date protein chip and cell chip technology, by diabetes autoimmune antibody in the serum whether exist or its content what, differentiating for immune intermediary diabetic's somatotype provides important evidence, also can be widely used in type i diabetes preclinical phase and the early stage prediction of morbidity among the diabetes people at highest risk, therefore have important value for clinical application.
2. high flux.Use a detection chip can detect numerical digit simultaneously, the relevant information of three kinds of diabetes autoimmune antibodies in every part of serum once is provided, be highly suitable for and carry out sieving and diagnosis use in enormous quantities among the crowd to the tens place patients serum.
3. detect with low costly, economy is strong.A chip can detect numerical digit to the tens place patients serum, and four kinds of diabetes diagnosis marks of every part of serum are equivalent to conventional hundreds of result of experiment; But required detectable consumption only be equivalent to conventional sense reagent dosage 1/20～1/50, can reduce the detection cost greatly, have very strong economy, easily popularize.
4. high specificity, highly sensitive.With the purifying protein is to detect substrate, has avoided the interference in the conventional sense, has guaranteed the detection specificity of height, has effectively avoided the generation of false positive and false-negative testing result.Adopt up-to-date solid phase detection chip manufacture craft simultaneously, guaranteed the high sensitivity of its detection.
5. stabilized quality control.Highly purified antigen protein has guaranteed the stable of antigenic quality, batch interior difference between reducing to criticize, thus effectively realize standardized testing, reduce the fluctuation of testing result.Every chip all has independently Quality Control microarray simultaneously, has guaranteed the reliability of every chip quality.
6. testing result accurately and reliably.Chip adopts the indirect fluorescent method to detect, and testing result is judged by intelligentized analysis software with the special-purpose laser confocal scanning instrument scanning of biochip back, eliminated common laboratory and detected the factor that artificial result of determination may cause error.
7. by the cell chip technology, can the islet cells autoantibody islet cells autoantibody of difficult detection in the past be detected accurately.Multiple advantages such as the frozen section SABC method with respect to traditional islet cells autoantibody detects has high detection sensitivity, no background interference, little, the reliable in quality of differences between batches, and is easy and simple to handle can replace detection method in the past fully.
Description of drawings:
Fig. 1 is the surface structure synoptic diagram of chip;
Fig. 2 is the structural representation in single detection reaction pond.
Description of reference numerals is as follows:
The 1-shell, the 2-reaction tank, 3-detects microarray, the miniature solid phase detection lug of 4-islet cells, the miniature solid phase detection lug of 5-RHGAD 65KD albumen, the miniature solid phase detection lug of 6-recombined human pancreatic island cell antigen 512 albumen, the miniature solid phase detection lug of 7-rh-insulin albumen.
Embodiment:
The present invention relates to the diabetes autoimmune antibody and detect compound chip, its preparation and detection method, be mainly used in four important indicators that detect on the diabetes auxiliary diagnosis: islet cells autoantibody, human glutamic acid decarboxylase autoantibody, human pancreatic island cell antigen 512 albumen autoantibodies and actrapid monotard's albumen autoantibody.Below will the invention will be further described by embodiment, only limit to following but should not be construed as protection domain
Embodiment.
(1) a kind of diabetes autoimmune antibody detects protein chip, comprise shell 1 and detect microarray 3, described shell 1 is that specification is 22mm * 75mm, thickness is the glass flake of 1mm, described glass flake upper surface is provided with 78 reaction tanks 2, described reaction tank 2 is that the surperficial length of side is 2.5mm, base 2.0mm, the inversion platform shape of dark 0.5mm.Be provided with one and detect microarray 3 in each reaction tank 2, detecting microarray 3 is 1mm by four areas
2, square miniature solid phase detection lug forms: they are respectively the miniature solid phase detection lug 4 of islet cells, the miniature solid phase detection lug 5 of RHGAD 65KD albumen, the miniature solid phase detection lug 6 of recombined human pancreatic island cell antigen 512 albumen and the miniature solid phase detection lug 7 of rh-insulin's albumen.Include 1 * 10 in the miniature solid phase detection lug of each cell
5Individual monolayer adherence people embryo beta Cell of islet includes 6 protein site sampling points on said each miniature solid phase detection lug.
(2) a kind of preparation method of diabetes autoimmune antibody detection compound chip is as follows:
At first prepare miniature solid phase detection lug, said miniature solid phase detection lug is divided into the miniature solid phase detection lug of islet cells, the miniature solid phase detection lug of RHGAD 65KD albumen, the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen and the miniature solid phase detection lug of rh-insulin's albumen according to the albumen kind difference on it.
1. the preparation of the miniature solid phase detection lug of islet cells: detecting matrix is the beta Cell of islet oncocyte that islet-cell tumour excision tissue is cultivated, by the external cultivation of islet cells carry out former be commissioned to train foster, make adherent single-layer culturing cell grow in the surface of detection lug after going down to posterity, detection lug adopts the glass cover slide through silicidation (APES) in this example, after treating that cell attaching growth accounts for whole detection lug surface 50%, take out detection lug, put into the phosphate buffer washing, the immobile liquid that drips 4% paraformaldehyde again in detection lug surface is 30min fixedly, fixing back is immersed phosphate buffer-glycerine mixed liquor and is placed-20 ℃ of storages, the ratio of phosphate buffer-glycerine mixed liquor is 1: 1, PH7.4 is cut into 1mm during use
2The miniature detection lug of rectangle, include 1 * 10 in the miniature solid phase detection lug of each cell
5Individual monolayer adherence people embryo beta Cell of islet.Immobile liquid also can be 95% the ethanolic solution that contains 1% glacial acetic acid.
2. in the preparation of the miniature solid phase detection lug of RHGAD 65KD albumen, detection lug adopts the transparent polystyrene cover glass, thickness 0.1mm, point sample reagent adopts the U.S. purification of recombinant human glutamate decarboxylase 65KD of sigma company albumen, phosphate buffer dilution, PH7.5, final concentration 100 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density 600 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 1mm during use
2The miniature detection lug of rectangle, each miniature detection lug contains 6 protein site sampling points.
3. in the preparation of the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen, detection lug adopts the transparent polystyrene cover glass, thickness 0.1mm, reagent adopts U.S. sigma company reorganization purification of Recombinant human pancreatic island cell antigen 512 albumen β-hypotypes, phosphate buffer dilution, pH7.5, final concentration 100 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density 600 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 1mm during use
2The miniature detection lug of rectangle, each miniature detection lug contains 6 protein site sampling points.
4. in the preparation of the miniature solid phase detection lug of rh-insulin's albumen, detection lug adopts the transparent polystyrene cover glass, thickness 0.1mm, reagent adopts the synthetic actrapid monotard's albumen of U.S. sigma company, phosphate buffer dilution, pH7.5, the final concentration scope is at 100 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density is at 600 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 1mm during use
2The miniature detection lug of rectangle, each miniature detection lug contains 6 protein site sampling points.
Assemble then, 1. select the glass flake that contains 78 reaction tanks for use, said reaction tank is that the surperficial length of side is 2.5mm, base 2.0mm, the inversion platform shape of dark 0.5mm; 2. stick with the different types of miniature Protein Detection sheet arrangement of four kinds of same sizes and with transparent binder and be fixed in it, make chip.
Also comprise packaging step at last, chip surface is wrapped up with masking foil, pack again, be stored under-20 ℃ of conditions, place during transportation in the thermostatic container that is not higher than 4 ℃.
(3) a kind of detection method of diabetes autoimmune antibody detection compound chip is as follows:
At first carry out the indirect fluorescent method and detect, above in said 78 reaction tanks any two be appointed as the Quality Control reaction tank, all the other 76 is the detection reaction pond.
The first step: prepare before detecting:
1. after chip being taken out, room temperature is placed 5min, unpacks;
2. at each reaction tank, promptly all add 50 μ l phosphate buffers in 78 reaction tanks, put into 37 ℃ of water-baths and hatch 15min;
3. drip under 1% bovine serum albumin(BSA), the 50 μ l room temperatures and seal 20min.
Second step: the detecting operation step to the detection reaction pond is:
1. a patient's test serum is diluted with 1: 5 usefulness phosphate buffer, directly drip, hatch 1h for 37 ℃, 76 detections that can be used for 76 parts of serum on this chip in a reaction tank surface;
2. the phosphate buffer flushing is 3 times, each 5min;
3. add fluorescently-labeled mouse-anti human IgG monoclonal antibody, hatch 1h for 37 ℃, said fluorescence labeling can be selected commercially available FITC mark for use;
4. the phosphate buffer that contains 1% tween washs 3 times, each 10min;
5. drip envelope and mount the agent mounting, it is that glycerine and 0.5% phosphate buffer were mixed with PH7.4 with 1: 1 that said envelope is mounted agent.
The 3rd step: with the second step synchronous operation, to the detecting operation step of Quality Control reaction tank be: only used patient's test serum in the operation steps is changed to that positive control detects liquid or negative control detects liquid, all the other steps are identical.Said positive control detects liquid and prepares by following mode: health adult's serum, 1500 leave heart 10min, and the islet cells autoantibody positive serum (by international Juvenile Diabetes Foundation standard serum calibration) that human glutamic acid decarboxylase autoantibody, human pancreatic island cell antigen 512 albumen autoantibodies, each 5u of actrapid monotard's albumen autoantibody monoclonal antibody of purifying is dissolved in 160JDF unit is made; Said negative control detects liquid and prepares by following mode: health adult's serum, 1500 leave heart 10min, get supernatant and make.
Then with the scanning of biochip laser confocal scanning instrument, observations.
(4), the invention provides following criterion as a result at chip of the present invention.Select the burnt microarray scanner scanning of ScanArray copolymerization back observations in this example for use.
This standard is an example with the testing result in a detection reaction pond, during actual detected result judges other to detect microarray decision methods identical: 1. when the cell on the miniature detection lug of islet cells when average fluorescent strength ratio was above 20: 1 after experiment in average fluorescent strength and the negative Quality Control microarray after experiment of the cell on the miniature solid phase detection lug of islet cells, then ICA detects positive, otherwise it is negative, 2. when the average fluorescent strength ratio of protein site sampling point on the miniature solid phase detection lug of RHGAD 65KD albumen in the average fluorescent strength of protein site sampling point on the miniature solid phase detection lug of RHGAD 65KD albumen and the negative Quality Control microarray surpasses 50: 1, then human glutamic acid decarboxylase autoantibody detects the positive, otherwise negative; 3. when the average fluorescent strength ratio of protein site sampling point on the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen in the average fluorescent strength of protein site sampling point on the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen and the negative Quality Control microarray surpasses 50: 1, then human pancreatic island cell antigen 512 albumen autoantibodies detect the positive, otherwise negative; 4. when the average fluorescent strength ratio of protein site sampling point on the miniature solid phase detection lug of rh-insulin's albumen in the average fluorescent strength of protein site sampling point on the miniature solid phase detection lug of rh-insulin's albumen and the negative Quality Control microarray surpasses 50: 1, then actrapid monotard's albumen autoantibody detects the positive, otherwise negative.
Subsidiary two extra Quality Control reaction tanks can detect the quality of chip on the chip of the present invention.Criterion as a result is: if the ratio of cell average fluorescent strength and background fluorescence intensity is higher than 20: 1 in the positive quality control microarray minicell detection lug, the average fluorescent strength of protein site sampling point and the ratio of background fluorescence intensity are higher than 50: 1 on each miniature Protein Detection sheet; The ratio of cell average fluorescent strength and background fluorescence intensity is lower than 20: 1 in the negative Quality Control microarray minicell detection lug simultaneously, the ratio of the average fluorescent strength of protein site sampling point and background fluorescence intensity is lower than 50: 1 and shows and shows that then this chip quality is good on each detection lug, and this testing result is effective.
Claims (10)
1, a kind of diabetes autoimmune antibody detects compound chip, comprise shell (1) and detect microarray (3), it is characterized in that: described shell (1) is the glass flake that upper surface is provided with a plurality of reaction tanks (2), be provided with one in the reaction tank and detect microarray (3), each detects microarray (3) is made up of the miniature solid phase detection lug of four kinds of same specification sizes respectively, and described miniature solid phase detection lug is respectively the miniature solid phase detection lug of islet cells (4), the miniature solid phase detection lug of RHGAD 65KD albumen (5), the miniature solid phase detection lug of the miniature solid phase detection lugs of recombined human pancreatic island cell antigen 512 albumen (6) and rh-insulin's albumen (7).
2, diabetes autoimmune antibody as claimed in claim 1 detects compound chip, and it is characterized in that: described miniature solid phase detection lug size is at 0.1mm
2～9mm
2Between.
3, diabetes autoimmune antibody as claimed in claim 1 or 2 detects compound chip, it is characterized in that: described glass flake specification is 22mm * 75mm, thickness 1mm～3mm, its upper surface is provided with 3～200 reaction tanks (2), and described reaction tank (2) is that the length of side is the square of 0.2mm～18mm.
4, diabetes autoimmune antibody as claimed in claim 3 detects compound chip, and it is characterized in that: described reaction tank (2) capacity is 10 μ l～100 μ l.
5, diabetes autoimmune antibody as claimed in claim 4 detects compound chip, it is characterized in that: comprise on the miniature solid phase detection lug of described islet cells (4) and have an appointment 1 * 10
3～1 * 10
6Individual islet cells; Include 2～10 corresponding proteins point of samples on the miniature solid phase detection lug of described each albumen.
6, a kind of diabetes autoimmune antibody as claimed in claim 1 detects the preparation method of compound chip, it is characterized in that:
At first prepare miniature solid phase detection lug, miniature solid phase detection lug among the present invention is divided into the miniature solid phase detection lug of islet cells, the miniature solid phase detection lug of RHGAD 65KD albumen, the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen and the miniature solid phase detection lug of rh-insulin's albumen according to the detection matrix species difference on it; In the preparation of these four miniature solid phase detection lugs, detection lug adopts glass cover slide or transparent polystyrene cover glass or the cellulose acetate film through silicidation, wherein the preparation of the miniature solid phase detection lug of islet cells adopts the cells in vitro culture technique in conjunction with the cell climbing sheet technology, and the preparation method of other three kinds of miniature solid phase detection lugs of albumen comprises the concentration of albumen being mixed adjusting protein solution in back with phosphate buffer successively; Protein solution is added 40% glycerine, mixing again; Adopt commercially available dna microarray instrument point sample instrument point sample on substrate plane; Hatched behind the point sample 3 hours; Seal with bovine serum albumin solution; Be cut into 0.1mm in use
2～9mm
2Miniature detection lug;
Assemble then: 1. select for use the surface to be provided with the glass flake of 3～200 reaction tanks, each reaction tank is the square that the length of side is 0.2mm～18mm, capacity 10 μ l～100 μ l; 2. in reaction tank, be provided with one and detect microarray, stick to be fixed in it by the different types of miniature solid phase detection lug arrangement of four same specification sizes and with transparent binder and make.
7, diabetes autoimmune antibody as claimed in claim 6 detects the preparation method of compound chip, it is characterized in that: in the preparation of described miniature solid phase detection lug,
1. the preparation of the miniature solid phase detection lug of islet cells: at first choose the cultured cell in vitro to islet cells autoantibody antibody sensitivity, described cultured cell in vitro to islet cells autoantibody antibody sensitivity comprises beta Cell of islet oncocyte and the interior islet cells of mammalian pancreas tissue that islet-cell tumour excision tissue is cultivated; Detect the cultivation of matrix then: cultural method be earlier with reference to the external cultivation of islet cells carry out former be commissioned to train foster, after going down to posterity, make adherent single-layer culturing cell grow in the surface of detection lug again, after treating that the whole detection lug of cell growth attaching surface surpasses 50%, take out detection lug, put into the phosphate buffer washing, drip fixedly 30min of immobile liquid in the detection lug surface again, fixing back is immersed phosphate buffer-glycerine mixed liquor and is placed-20 ℃ of storages, is cut into 0.1mm during use
2～9mm
2Miniature detection lug;
2. in the preparation of the miniature solid phase detection lug of RHGAD 65KD albumen, point sample reagent adopts purification of recombinant human glutamate decarboxylase 65KD albumen, the phosphate buffer dilution, pH7.5, the final concentration scope is at 10 μ g～200 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density is at 100～2000 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 0.1mm during use
2～9mm
2Miniature detection lug, each miniature detection lug contains 2～10 protein site sampling points;
3. in the preparation of the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen, reagent adopts reorganization purification of Recombinant human pancreatic island cell antigen 512 albumen β-hypotypes, the phosphate buffer dilution, pH7.5, the final concentration scope is at 10 μ g～200 μ g/ml, add 40% glycerine, mixing adopts microarray point sample instrument point sample.Point sample density is at 100～2000 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 0.1mm during use
2～9mm
2Miniature detection lug, each miniature detection lug contains 2～10 protein site sampling points;
4. in the preparation of the miniature solid phase detection lug of rh-insulin's albumen, reagent adopts synthetic actrapid monotard's albumen, the phosphate buffer dilution, and pH7.5, the final concentration scope adds 40% glycerine at 10 μ g～200 μ g/ml, and mixing adopts microarray point sample instrument point sample.Point sample density is at 100～2000 points/cm
2, seal-20 ℃ of storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample.Be cut into 0.1mm during use
2～9mm
2Miniature detection lug, each miniature detection lug contains 2～10 protein site sampling points;
1., 2., 3. and the detection lug 4. adopt through the glass cover slide of silicidation (APES) or commercially available transparent polystyrene cover glass.
8, as the preparation method of claim 6 or 7 described diabetes autoimmune antibodies detection compound chips, it is characterized in that: described preparation method also comprises packaging step, and chip surface is wrapped up with masking foil, packs again.
9, a kind of detection method that adopts the diabetes autoimmune antibody detection compound chip of claim 1, it is characterized in that: adopt the indirect fluorescent method to detect earlier, testing result is judged with the scanning of biochip laser confocal scanning instrument again, or adopt the microarray scanner of other kinds to scan laboratory test results, carry out interpretation by software or professional person; Or only use common fluorescent microscope Direct observation, by the professional person experimental result is judged again; In the described reaction tank any two respectively as negative Quality Control reaction tank and positive quality control reaction tank, all the other are the detection reaction pond.
10, diabetes autoimmune antibody as claimed in claim 9 detects the detection method of compound chip, it is characterized in that:
In the described indirect fluorescent method, at first carry out pre-service,
1. after chip being taken out, room temperature is placed 5min, unpacks;
2. add 50 μ l phosphate buffers at each reaction tank, put into 37 ℃ of water-baths and hatch 15min;
3. drip under 1% bovine serum albumin(BSA), the 50 μ l room temperatures and seal 20min;
To the detecting of detection reaction pond, operation steps is then:
1. a patient's test serum is diluted with 1: 5 usefulness phosphate buffer, directly drip, hatch 1h for 37 ℃ in a detection reaction pool surface;
2. the phosphate buffer flushing is 3 times, each 5min;
3. add fluorescently-labeled mouse-anti human IgG monoclonal antibody, hatch 1h for 37 ℃;
4. the phosphate buffer that contains 1% tween washs 3 times, each 10min;
5. drip envelope and mount the agent mounting;
Detecting operation step to the Quality Control reaction tank is: with synchronous to the detection in detection reaction pond, only will detect in the microarray operation steps used patient's test serum and be changed to that positive control detects liquid or negative control detects liquid, all the other steps are identical; It is with health adult's serum that positive control detects liquid, leave heart 10min through 1500, human glutamic acid decarboxylase autoantibody, human pancreatic island cell antigen 512 albumen autoantibodies, actrapid monotard's albumen autoantibody each 5u of monoclonal antibody of purifying or the concentrated solution that is equivalent to this amount are dissolved in health adult's serum preparation form; It is with health adult's serum that negative control detects liquid, leaves heart 10min through 1500, gets supernatant and is prepared from.
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| CNB021455619A CN1176377C (en) | 2002-12-30 | 2002-12-30 | Composite chip for detecting autoimmune antibody of diabetes mellitus, and preparation and detection method thereof |
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|---|---|---|---|
| CNB021455619A CN1176377C (en) | 2002-12-30 | 2002-12-30 | Composite chip for detecting autoimmune antibody of diabetes mellitus, and preparation and detection method thereof |
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| CN1176377C true CN1176377C (en) | 2004-11-17 |
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| CN100342027C (en) * | 2005-09-22 | 2007-10-10 | 山东省医药生物技术研究中心 | Process for preparing protein, DNA and lipid microarray by using APES modified glass slide |
| CN100378458C (en) * | 2005-09-30 | 2008-04-02 | 中国科学院长春应用化学研究所 | Method for making sample cell of solid phase white amino reflective layer |
| CN101210928B (en) * | 2006-12-31 | 2012-07-25 | 陕西北美基因股份有限公司 | Method for analyzing carbohydrate-binding protein by functional sugar chip and flight mass spectrometer |
| CN103018434B (en) * | 2012-12-05 | 2016-01-20 | 博源诺信（北京）生物科技有限责任公司 | A kind of multiple determination device and a kind of kit, and application |
| CN103901218A (en) * | 2014-04-01 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Method for preparing positive serum of autoimmune antigen |
| CN103969234B (en) * | 2014-04-17 | 2017-02-15 | 山东东兴汇智生物科技有限公司 | Luciferase- poly-antigen fusion protein and protein A agarose-fusion protein-antibody complex |
| CN108398550B (en) * | 2018-03-07 | 2022-07-26 | 深圳市伯劳特生物制品有限公司 | Composition, chip, preparation method of chip and detection device comprising chip |
| CN108620144A (en) * | 2018-07-10 | 2018-10-09 | 南京宝沃生物科技有限公司 | A kind of micro-fluid chip being used in WB experiments |
| CN114034683A (en) * | 2021-12-01 | 2022-02-11 | 吉林大学 | Multiple immune chip for rapidly diagnosing type 1 diabetes and preparation method and application thereof |
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