CN1173182C - Albumen chip for detecting autoimmunity antibody of diabetes, as well as preparation and detection method - Google Patents
Albumen chip for detecting autoimmunity antibody of diabetes, as well as preparation and detection method Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to the technical fields of biomedical research and clinical application and particularly relates to a protein chip for detecting autoimmune antibodies of diabetes, a preparing method thereof and a detecting method thereof. The present invention aims to solve the problems of difficult confirmed diagnosis and high detection cost of the existing clinical diabetes analysis and has a scheme that the casing of the protein chip is a thin glass sheet with a plurality of reaction tanks on the upper surface, and a detecting microarray is arranged in each of the reaction tanks; each of detecting microarrays is composed of three protein miniature solid phase detecting sheets with different types and the same specification and size, and the protein miniature solid phase detecting pieces are respectively a GAD protein miniature solid phase detecting sheet, a PTP protein miniature solid phase detecting sheet and an insulin protein miniature solid phase detecting sheet. The preparing method comprises: the protein miniature solid phase detecting sheets are prepared and then are assembled. The detecting method comprises: an indirectly fluorescent method is adopted for detection, and then, a biological chip laser confocal scanner is utilized to judge experimental detection results.
Description
Affiliated technical field:
The present invention relates to biomedical research and clinical practice field, be specifically related to the diabetes autoimmune antibody and detect protein chip, its preparation and 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.The up-to-date prediction that the morbidity present situation and the The World Health Organization (WHO) of global diabetes makes the onset diabetes situation is pointed out that the whole world has diagnosed out the type ii diabetes patient to reach 1.3 hundred million people at present according to IDF (KDF), 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 change of growth in the living standard and 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 the phenomenon that insulinize was lost efficacy, must be relied in insecondary oral antidiabetic drug treatment.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 the report " definition of diabetes and complication thereof, diagnosis and 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: glutamic acid decarboxylase antibody (RHGAD 65KDA); Protein-tyrosine-phosphatase sample transmembrane protein antibody (human pancreatic island cell antigen 512 albumen autoantibodies); Insulin antibody (actrapid monotard's albumen autoantibody); Islet cells autoantibody (ICAs).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 in time predict the generation of following type i diabetes.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 be difficult for making a definite diagnosis and detecting the high problem of cost.
For solving existing technical matters, solution of the present invention is: a kind of diabetes autoimmune antibody detects protein 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 2 and detect microarray 3, each detects microarray 3 respectively by the miniature solid phase detection lug 4 of RHGAD 65KD albumen, the miniature solid phase detection lug of the albumen of the miniature solid phase detection lug 5 of recombined human pancreatic island cell antigen 512 albumen and 6 three same specifications of the miniature solid phase detection lug of rh-insulin's albumen is formed.
The area of the miniature solid phase detection lug of each albumen is at 0.1mm
2～9mm
2Between.
Described 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.
Described reaction tank 2 capacity are 10 μ l～100 μ l.
Include 2～10 corresponding proteins point of samples on the miniature solid phase detection lug of described albumen.
It is as follows that a kind of above-mentioned diabetes autoimmune antibody detects methods of making protein chips:
At first prepare the miniature solid phase detection lug of albumen, the present invention has three kinds of miniature solid phase detection lugs of albumen, be divided into the miniature detection lug of RHGAD 65KD albumen, the miniature detection lug of recombined human pancreatic island cell antigen 512 albumen and the miniature detection lug of rh-insulin's albumen according to the albumen kind difference on it, in the preparation of these three miniature solid phase detection lugs of albumen, detection lug adopts the transparent polystyrene cover glass or adopts glass cover slide and cellulose acetate film, and the preparation method 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.
The preparation scheme of the miniature solid phase detection lug of above-mentioned albumen is:
1. 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
2The miniature detection lug of rectangle albumen, each miniature detection lug contains 2～10 protein site sampling points.
2. 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
2The miniature detection lug of rectangle albumen, each miniature detection lug contains 2～10 protein site sampling points.
3. 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
2The miniature detection lug of rectangle albumen, each miniature detection lug contains 2～10 protein site sampling points.
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. the detection microarray is set in reaction tank, and this detection microarray sticks to be fixed in the reaction tank by the miniature solid phase detection lug arrangement of different types of albumen of three same specification sizes and with transparent binder to be made.
Above-mentioned preparation method also comprises packaging step, and chip surface is packed with masking foil, 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 protein chip is as follows: adopt the indirect fluorescent method to detect earlier, testing result is judged laboratory test results with the microarray scanner scanning of biochip laser confocal scanning instrument or other kinds again, also can only use common fluorescent microscope Direct observation, experimental result is manually judged; In the described testing process, any two that choose in the detection microarray are detected microarray and positive quality control detection microarray as negative Quality Control respectively.
In the above-mentioned indirect fluorescent method, carry out pre-treatment step earlier:
1. after chip being taken out, room temperature is placed 5min～15min, unpacks;
2. add 10 μ l～100 μ l phosphate buffers at each reaction tank, put into 37 ℃ of water-baths and hatch 15min～30min;
3. drip 3%H
2O
2Seal 10min under the 50 μ l room temperatures;
4. the phosphate buffer flushing is 2 times, each 5min;
5. drip under 1% bovine serum albumin(BSA), the 50 μ l room temperatures and seal 10min～20min.
Detect detecting microarray then:
1. a patient's test serum is diluted with 1: 5 usefulness phosphate buffer, directly drip, hatch 20min～1h for 37 ℃ in a detection microarray surface;
2. the phosphate buffer flushing is 3 times, each 5min;
3. add fluorescently-labeled mouse-anti human IgG monoclonal antibody, hatch 20min～1h for 37 ℃;
4. the phosphate buffer that contains 1% tween washs 3 times, each 10min;
5. drip envelope and mount the agent mounting.
The detecting operation step that Quality Control is detected microarray is:
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.
It is to be mixed with by 1: 1 by glycerine and 0.5% phosphate buffer to form that above-mentioned envelope is mounted agent.
Compared with prior art, the present invention has following characteristics:
1. utilize current up-to-date protein 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 three 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 albumen solid phase detection lug 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 detection 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.
Description of drawings:
Fig. 1 is the structural representation of chip;
Fig. 2 is the structural representation of single reaction tank.
The accompanying drawing drawing is described as follows:
The 1-shell, the 2-reaction tank, 3-detects microarray, the miniature solid phase detection lug of 4-RHGAD 65KD albumen, the miniature solid phase detection lug of 5-recombined human pancreatic island cell antigen 512 albumen, the miniature solid phase detection lug of 6-rh-insulin albumen.
Embodiment:
The present invention relates to protein detection chip, its preparation and detection method, be mainly used in three important indicators that detect on the diabetes auxiliary diagnosis: 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 embodiment but should not be construed as protection domain.
(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, and described reaction tank 2 is that the length of side is the square of 2.5mm.Be provided with one and detect microarray 3 in each reaction tank 2, this detection microarray 3 is 1mm by three areas
2, same specification size the miniature solid phase detection lug of albumen form, the miniature solid phase detection lug of said albumen is respectively the miniature solid phase detection lug 4 of RHGAD 65KD albumen, the miniature solid phase detection lug 5 of recombined human pancreatic island cell antigen 512 albumen and the miniature solid phase detection lug 6 of rh-insulin's albumen.Include 6 protein site sampling points on the miniature solid phase detection lug of each albumen.
(2) a kind of diabetes autoimmune antibody detection methods of making protein chips is as follows:
At first prepare the miniature solid phase detection lug of albumen, the miniature solid phase detection lug of said albumen is divided into 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. 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 commercialization reagent purification of recombinant human glutamate decarboxylase 65KD 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 sealed-20 ℃ storages with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid at 600 points/cm2 behind the point sample; Be cut into area when using and be 1mm
2, same specification size the miniature detection lug of rectangle, each miniature detection lug contains 6 protein site sampling points.
2. 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 the commercialization reagent reorganization purification of Recombinant human pancreatic island cell antigen 512 albumen β hypotypes 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 600 points/cm
2, seal with 0.5% bovine serum albumin(BSA) that contains L type polyglutamic acid behind the point sample ,-20 ℃ of storages.Be cut into area when using and be 1mm
2, same specification size the 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 rh-insulin's albumen, detection lug adopts the transparent polystyrene cover glass, thickness 0.1mm, reagent adopts the synthetic actrapid monotard's albumen of the commercialization reagent 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 area when using and be 1mm
2, same specification size the miniature detection lug of rectangle, each miniature detection lug contains 6 protein site sampling points.
Assemble then, select the glass flake that contains 78 reaction tanks for use, said reaction tank is that the length of side is the square of 2.5mm.The miniature solid phase detection lug of different types of albumen of three kinds of same specification sizes arranged and stick and be fixed in the reaction tank, make the detection microarray, contain the reaction tank that detects microarray by such 78 and form detection chip with transparent binder.Any two detection microarraies wherein are used for quality control, are called as Quality Control and detect microarray, and Quality Control detects microarray and is subdivided into negative Quality Control detection microarray and positive quality control detection microarray again, and other 76 detection microarraies are used to clinical detection.
Also comprise packaging step at last, the detection chip surface is packed with masking foil, 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 protein chip is as follows:
At first carry out the operation of indirect fluorescent method, be appointed as the Quality Control reaction tank for any two in said 78 reaction tanks, all the other 76 is the detection reaction pond.
The first step: pre-service 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-bath 15min;
3. drip 3%H
2O
2Seal 10min under the 50 μ l room temperatures;
4. the phosphate buffer flushing is 2 times, each 5min;
5. drip under 1% bovine serum albumin(BSA), the 50 μ l room temperatures and seal 20min.
Second step: be to the detecting operation step that detects microarray:
1. a patient's test serum is diluted with 1: 5 usefulness phosphate buffer, directly drip, hatch 1h for 37 ℃, wherein 76 detections that can be used for 76 parts of serum on this chip in a detection microarray 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 glycerine and 0.5% phosphate buffer and mount the agent mounting by the envelope that is mixed with at 1: 1.
The 3rd step: with the second step synchronous operation, the detecting operation step that Quality Control is detected microarray is: only used patient's test serum in the detecting operation step of second step detection microarray is changed to positive control and detects liquid or negative control detection 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, 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 make; 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 laser confocal scanning instrument, observations.
(4), the invention provides following criterion as a result at protein detection 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 a testing result that detects microarray, during actual detected result judges other to detect microarray decision methods identical: 1. when the average fluorescent strength ratio of protein site sampling point on the miniature solid phase detection lug of RHGAD 65KD65 albumen in the average fluorescent strength of protein site sampling point on the miniature solid phase detection lug of RHGAD 65KD65 albumen and the negative Quality Control detection microarray during above 50: 1, human glutamic acid decarboxylase autoantibody test positive then, otherwise negative; 2. when 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 negative Quality Control detect that the average fluorescent strength ratio of protein site sampling point surpasses 50: 1 on the miniature solid phase detection lug of recombined human pancreatic island cell antigen 512 albumen in the microarray, human pancreatic island cell antigen 512 albumen autoantibody test positive then, otherwise negative; 3. when the average fluorescent strength of protein site sampling point on the miniature solid phase detection lug of rh-insulin's albumen and negative Quality Control detect that the average fluorescent strength ratio of protein site sampling point surpasses 50: 1 on the miniature solid phase detection lug of rh-insulin's albumen in the microarray, actrapid monotard's albumen autoantibody test positive then, 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 positive quality control detects the average fluorescent strength of protein site sampling point on each detection lug of microarray and the ratio of background fluorescence intensity is higher than 50: 1, simultaneously negative Quality Control detects the average fluorescent strength of protein site sampling point on each detection lug of microarray and the ratio of background fluorescence intensity is lower than 50: 1, show that then this chip quality is good, this testing result is effective.
Claims (10)
1, a kind of diabetes autoimmune antibody detects protein 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 (2) and detect microarray (3), each detects microarray (3) respectively by the miniature solid phase detection lug of RHGAD 65KD albumen (4), the miniature solid phase detection lug of the albumen of (6) three same specifications of the miniature solid phase detection lug of the miniature solid phase detection lugs of recombined human pancreatic island cell antigen 512 albumen (5) and rh-insulin's albumen is formed.
2, diabetes autoimmune antibody as claimed in claim 1 detects protein chip, and it is characterized in that: the area of the miniature solid phase detection lug of described single albumen is at 0.1mm
2～9mm
2Between.
3, diabetes autoimmune antibody as claimed in claim 1 or 2 detects protein 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 protein 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 protein chip, it is characterized in that: include 2～10 corresponding proteins point of samples on the miniature solid phase detection lug of described albumen.
6, a kind of diabetes autoimmune antibody as claimed in claim 1 detects methods of making protein chips, it is characterized in that: at first prepare the miniature solid phase detection lug of albumen, the present invention has three kinds of miniature solid phase detection lugs of albumen, is divided into the miniature detection lug of RHGAD 65KD albumen, the miniature detection lug of recombined human pancreatic island cell antigen 512 albumen and the miniature detection lug of rh-insulin's albumen according to the albumen kind difference on it; In the preparation of these three miniature solid phase detection lugs of albumen, detection lug adopts the transparent polystyrene cover glass or adopts glass cover slide and cellulose acetate film, and the preparation method 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.
7, a kind of diabetes autoimmune antibody as claimed in claim 1 detects methods of making protein chips, it is characterized in that:
At first prepare the miniature solid phase detection lug of albumen, it comprises
1. 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
2The miniature detection lug of rectangle albumen, each miniature detection lug contains 2～10 protein site sampling points;
2. 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
2The miniature detection lug of rectangle albumen, each miniature detection lug contains 2～10 protein site sampling points;
3. 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 pH 7.5, and 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
2The miniature detection lug of rectangle albumen, each miniature detection lug contains 2～10 protein site sampling points;
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. the detection microarray is set in reaction tank, and this detection microarray sticks to be fixed in the reaction tank by the miniature solid phase detection lug arrangement of different types of albumen of three same specification sizes and with transparent binder to be made.
8, detect methods of making protein chips as claim 6 or 7 described diabetes autoimmune antibodies, it is characterized in that: also comprise packaging step, this step is that chip surface is packed with masking foil, is stored under-20 ℃ of conditions, places during transportation in the thermostatic container that is not higher than 4 ℃.
9, a kind of diabetes autoimmune antibody as claimed in claim 1 detects the detection method of protein chip, it is characterized in that: adopt the indirect fluorescent method to detect earlier, judge laboratory test results with the microarray scanner scanning of biochip laser confocal scanning instrument or other kinds again, also can only use common fluorescent microscope Direct observation, experimental result is manually judged; In the described testing process, any two that choose in the detection microarray are detected microarray and positive quality control detection microarray as negative Quality Control respectively.
10, diabetes autoimmune antibody as claimed in claim 9 detects the detection method of protein chip, it is characterized in that: in the described indirect fluorescent method, carry out pre-treatment step earlier:
1. after chip being taken out, room temperature is placed 5min～15min, unpacks;
2. add 10 μ l～100 μ l phosphate buffers at each reaction tank, put into 37 ℃ of water-baths and hatch 15min～30min;
3. drip 3%H
2O
2Seal 10min under the 50 μ l room temperatures;
4. the phosphate buffer flushing is 2 times, each 5min;
5. drip under 1% bovine serum albumin(BSA), the 50 μ l room temperatures and seal 10min～20min;
Detect detecting microarray then:
1. a patient's test serum is diluted with 1: 5 usefulness phosphate buffer, directly drip, hatch 20min～1h for 37 ℃ in a detection microarray surface;
2. the phosphate buffer flushing is 3 times, each 5min;
3. add fluorescently-labeled mouse-anti human IgG monoclonal antibody, hatch 20min～1h for 37 ℃;
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 are to be mixed with at 1: 1 to form that this envelope is mounted agent;
Again Quality Control being detected microarray detects:
Only used patient's test serum in the above-mentioned detection microarray operation steps is changed to positive control and detects liquid or negative control detection 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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| CNB02145535XA CN1173182C (en) | 2002-12-18 | 2002-12-18 | Albumen chip for detecting autoimmunity antibody of diabetes, as well as preparation and detection method |
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| CN1173182C true CN1173182C (en) | 2004-10-27 |
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