CN1362623A - Multiple immunological microsphere and its prepn techn and detection method - Google Patents
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Abstract
The antigenic substance of specific antibody can be adsorbed on the emulsoid microspheres with different sizes to obtain multicomponent immunological microsphere, said multicomponent immunological microsphere can be mixed with sample to be tested, and the antigen and antibody reaction can be used to make specific antigen or antibody being in sample to be tested be adsorbed on the multicomponent immunologial microsphere, then the flow cytometer can be used to analyze related antigen and antibody contents being in tested sample.
Description
Affiliated technical field:
The present invention relates to biomedical research and clinical practice field, be specifically related to a kind of detect specific antigen or the polynary immune microsphere of antibody content, the technology of preparing of this polynary immune microsphere and the method that this polynary immune microsphere is detected in the body fluid.
Background technology:
Discover, some marks relevant with tumour can appear in the different phase patients serum of tumor growth, all can the content of tumor markers be detected by enzyme-linked immuno assay (ELISA) or radiommunoassay methods such as (RIA), be used for diagnosis, antidiastole or the early detection of tumour then, 1981, by Dunn and Tyrer, studies confirm that the latex beads of FITC-mark uses flow cytometer to can be used for detecting phagocytosis.After 1 year, study and in Cytometry 2,238, delivered the report of relevant use fluorescent microsphere as the standard detecting method of blood count through Stenart and Steinkamp.But every kind of technology all has corresponding deficiency, all only can detect an index as ELISA, RIA and fluorescent microsphere technology at every turn; So, detect the various marks relevant and will carry out a large amount of repetitive operation with tumour, also to spend a large amount of time, human and material resources and financial resources, and the experiment condition disunity, error is bigger between batch, so limited the correlative study of early diagnosis of tumor greatly, cause the tumor cases of present early detection clinically still less, thereby make clinical treatment face big pressure.
One of purpose of the present invention provides at double a kind of even becomes hundred times raising can hold the polynary immune microsphere of information content, two of purpose provides a kind of technology of preparing of simple polynary immune microsphere, three of purpose provide a kind of easy and simple to handle, can accurately promptly detect specific antigen in serum or other body fluid or the method that polynary immune microsphere is detected, specific antigen comprises various diseases mark, cell factor and soluble cell surface indicia etc., and specific antibody comprises self anti-tumour antibody, self antinuclear antibodies etc.
To achieve these goals, the technical scheme that the present invention takes is: a kind of polynary immune microsphere, comprise latex beads, its special character is: the diameter of described latex beads is between 1 micron～30 microns, make more than or equal to 0.3 micron at interval according to diameter, and its expoeridium has different specific antigens or specific antibody.
Above-mentioned specific antigen or specific antibody can be the marks relevant with various diseases, as tumor markers, autoantibody, various pathogenic microorganism antigen or antibody, various cell factor, the solubility surface indicia of various cells, various soluble recepter molecules etc.
A kind of technology of preparing of polynary immune microsphere, its special character is: choose diameter and at interval make the different latex beads of diameter more than or equal to 0.3 micron, the latex beads of diameter between 1 micron～30 microns, make a kind of immune microsphere at pan coating one species-specific antigen or the specific antibody of the latex beads of same diameter; With the immune microsphere for preparing with the PBS washing after, have the immune microsphere of different specific antigens or antibody to mix pan coating, promptly make polynary immune microsphere.
A kind of method that polynary immune microsphere is detected, its special character is: 1. polynary immune microsphere and testing sample are mixed the specificity of carrying out antigen and antibody and combine, more polynary immune microsphere is washed with PBS; 2. polynary immune microsphere is carried out the secondary association reaction, again with suspending again after the PBS washing; 3. analyze, detect with flow cytometer, the size by detecting immune microsphere and the power of luminous intensity or fluorescence intensity thereof draw the content of corresponding determined antigen or antibody.
In the such scheme, at polynary immune microsphere with antigen coated mistake, the 2. in the step secondary association reaction be meant the corresponding antiantibody that adding is crossed through chemiluminescent substance or fluorochrome mark, react as anti-people Ig antibody.
In the such scheme, 1. surface indicia there is the polynary immune microsphere of different specific antigens to put into centrifuge tube, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, the centrifugal back abandoning supernatant that finishes adds capacity test serum or other samples to be measured and carries out resuspended, hatched 30 minutes～2 hours for 37 ℃, in course of reaction, can use shaking table or regularly shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, with PBS washing 2 times, abandoning supernatant is carried out resuspended with PBS; 2. add the anti-Ig antibody that is marked with the specific fluorescent element, hatched 10 minutes～60 minutes for 37 ℃, in course of reaction, should use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins, centrifugal 3 minutes～5 minutes, clean 2 times with PBS, after the abandoning supernatant, carry out resuspended with PBS once more; 3. adding flow cytometer detects and analyzes.This scheme is applicable to and detects each strain specific antibodies of body fluid kind.
In the such scheme, at the polynary immune microsphere of antibody sandwich, the 2. in the step secondary association reaction be meant add earlier with microballoon on the antibody that adsorbs have mutually homospecific antibody, react as mouse monoclonal antibody, resuspended with PBS washing back; And then add the anti-Ig antibody be marked with the specific fluorescent element and react.
In the such scheme, 1. surface indicia there is the polynary immune microsphere of different specific antibodies to put into centrifuge tube, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, abandoning supernatant adds capacity test serum or other samples to be measured and carries out resuspended, hatched 30 minutes～2 hours for 37 ℃, in course of reaction, should use shaking table or regularly shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, with PBS washing 2 times, abandoning supernatant is carried out resuspended with PBS; 2. the identical mouse monoclonal antibody of specificity of institute's labelled antibody on first adding and the immune microsphere, hatched 10 minutes～60 minutes for 37 ℃, in course of reaction, to use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, with PBS washing 2 times, abandoning supernatant, carry out resuspended with PBS, add the anti-mouse Ig antibody that is marked with the specific fluorescent element then, hatched 10 minutes～60 minutes for 37 ℃, in course of reaction, use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, clean 2 times with PBS, after the abandoning supernatant, carry out resuspended with PBS once more; 3. adding flow cytometer detects and analyzes.This scheme is applicable to and detects each species-specific antigen of body fluid kind.
Use the polynary immune microsphere of above method preparation to can be used for various diseases is relevant in people and the various animal body fluid the specific antigen or the detection of antibody content, as tumor markers, autoantibody, various pathogenic microorganism antigen or antibody, various cell factor, the solubility surface indicia of various cells, various soluble recepter molecules etc.Detect people's sample, with regard to antibody with anti-people; Detect the sample of animal, with regard to antibody with anti-animal.
Compared with prior art, advantage of the present invention is:
1, makes simply, contain much information, can hold tens of kinds, hundreds of antigen or the information of specific antibody on one group of polynary immune microsphere, the information content of its testing result at double even become hundred times raising only can obtain the content of multiple antigen in the testing sample or antibody with one-time detection;
2, detection method is easy fast: owing to utilize on the polynary immune microsphere tens of kinds, hundreds of antigen or specific antibody that sample is detected, its operation steps only needed for two steps can finish, and can provide testing result in 1 hour;
3, susceptibility height: can detect the antigen extremely micro-in the testing sample or the content of antibody;
4, high specificity:, thereby have very high specificity because be specific antigen and antibody response;
5, good reliability: every experiment process is easy to standardization, once finishes detection, and test error is little, and good reproducibility can guarantee effectively that testing result accurately and reliably;
6, low, the relieve patient ' s burden of cost: reagent consumption is few, and the repeated use of equipment reduces, and effectively reduces and detects cost; The serum consumption is little, helps patient's body and restores, and therefore can be widely used in clinical detection and studies.
Embodiment:
The inventor has provided following embodiment, but the invention is not restricted to these embodiment.
In the preparation of the polynary immune microsphere of the present invention,, can prepare voluntarily or outsourcing as the latex beads of raw material and not homospecific sheep or rabbit polyclonal antibody.Diameter differs by more than or equals 0.3 micron and gets final product between the latex beads adjacent-specification, and the upper limit should guarantee that the different size quantity of the latex beads of diameter between 1 micron～30 microns should be able to satisfy the detection needs; In the detection method of the present invention, analyzing, detecting through final step adding flow cytometer, after calculating the content of various marks relevant in the testing sample according to the power of the size of microballoon and fluorescence signal, can and change according to the different of its content, having or not of auxiliary judgment in-vivo tumour, the development of types of organization, the state of an illness, change, lapse to, prognosis, the selection of therapeutic modality and judgement of result of treatment etc.
Embodiment 1, the polynary immune microsphere of use carry out the detection of cancer of the stomach mark:
Prepare the latex beads of the 25 kind specifications of diameter between 5 μ m～25 μ m, prepare 25 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the different latex beadses of these polyclonal antibodies difference marks, these polyclonal antibodies comprise: anti-CA50 antibody, anti-CA199 antibody again, anti-CA724 antibody, anti-SA antibody, anti-Fer antibody, anti-CA494 antibody, anti-CAMD1 antibody, anti-IAPP antibody, anti-CA19-5 antibody, anti-CEA antibody, anti-CD44 antibody, anti-gamma glutamyltransferase antibody, antizymohexase antibody, anti-glutathione transferase antibody, anti-creatine kinase antibody, anti-α-L fucoside enzyme antibody, anti-RNA (ribonucleic acid) enzyme antibody, anti-hexokinase antibody, anti-TGF antibody, anti-β
2-microglobulin antibody, anti-pyruvate kinase antibody, anti-5 '-nucleotidase antibody, anti-C polypeptide antibody, anti-CER antibody, anti-SIMA antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects cancer of the stomach correlating markings thing content.
Detection method is: 1. go on foot that these polynary immune microspheres are put into 3000 rev/mins of microcentrifugal tubes is centrifugal 5 minutes, abandoning supernatant, adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS, the mouse monoclonal antibody that adds anti-above-mentioned cancer of the stomach correlating markings thing was hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS, the 2. the step adds the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ lPBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 2, the polynary immune microsphere of use carry out the detection of lung cancer marker:
Prepare the latex beads of the 31 kind specifications of diameter between 1 μ m～30 μ m, prepare 31 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the latex beads of these polyclonal antibodies difference mark different sizes, these polyclonal antibodies comprise: anti-P53 albumen, nm23 albumen, CD44v6, neuronspecific enolase, cytokeratin 19 soluble fragments, tissue polypeptide antigen, cytokeratin 18 soluble fragments M again
3Antigenic determinant, carcinomebryonic antigen, epidermal growth factor, EGF-R ELISA, isoenzymes of creatine kinase, total sialoprotein, calcitonin, Fer, CEA, aldolase, glutathione transferase, creatine kinase, ribonuclease, hexokinase, TGF, pyruvate kinase, 5 '-nucleotidase, CER, tsgf, cd34, st-4-39, grp, CK20, TPS, CYFRA21-1 antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed the polynary immune microsphere that promptly forms detection of lung cancer correlating markings thing content.
Detection method is: the 1. the step above-mentioned polynary immune microsphere was put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, abandoning supernatant, adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step mouse monoclonal antibody that adds anti-above-mentioned lung cancer correlating markings thing earlier hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The sheep anti mouse polyclonal antibody that adds fluorochrome mark was then hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 3, the polynary immune microsphere of use carry out the detection of liver cancer marker:
Prepare the latex beads of the 26 kind specifications of diameter between 5 μ m～25 μ m, prepare 26 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the latex beads of these polyclonal antibodies difference mark different sizes, these polyclonal antibodies comprise: anti-P53 albumen again, nm23 albumen, CD44v6, carcinomebryonic antigen, epidermal growth factor, EGF-R ELISA, isoenzymes of creatine kinase, total sialoprotein, Fer, CEA, aldolase, glutathione transferase, creatine kinase, ribonuclease, hexokinase, TGF, pyruvate kinase, 5 '-nucleotidase, CER, tsgf, cd34, st-4-39, grp, AFP, gamma glutamyltransferase and isoenzymes 2 thereof, α-L fucoside enzyme antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects liver cancer correlating markings thing content.
Detection method is: 1. go on foot these polynary immune microspheres put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, abandoning supernatant, and adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step mouse monoclonal antibody that adds anti-above-mentioned liver cancer correlating markings thing earlier hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; And then add the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 4, the polynary immune microsphere of use carry out the detection of markers for breast cancer:
Prepare the latex beads of the 25 kind specifications of diameter between 5 μ m～25 μ m, prepare 25 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the latex beads of these polyclonal antibodies difference mark different sizes, these polyclonal antibodies comprise: anti-p16 albumen again, C-erb-2 albumen, CA-153, CEA, CD44, p53 albumen, CA549, HCG, nm23 albumen, CD44v6, carcinomebryonic antigen, epidermal growth factor, EGF-R ELISA, isoenzymes of creatine kinase, total sialoprotein, Fer, aldolase, glutathione transferase, creatine kinase, ribonuclease, hexokinase, TGF, pyruvate kinase, 5 '-nucleotidase, CER antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects breast cancer correlating markings thing content.
Detection method is: 1. go on foot these polynary immune microspheres put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, abandoning supernatant, and adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step mouse monoclonal antibody that adds anti-above-mentioned breast cancer correlating markings thing earlier hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; And then add the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 5, the polynary immune microsphere of use carry out the detection of cancer of pancreas mark:
Prepare the latex beads of the 23 kind specifications of diameter between 5 μ m～25 μ m, prepare 23 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, distinguish the latex beads of mark different sizes again with these polyclonal antibodies, these polyclonal antibodies comprise: anti-CA19-9, CA494, CA242, CAMD1, IAPP, CA50, p16 albumen, CD44, p53 albumen, nm23 albumen, CD44v6, carcinomebryonic antigen, isoenzymes of creatine kinase, total sialoprotein, Fer, aldolase, glutathione transferase, creatine kinase, ribonuclease, hexokinase, TGF, pyruvate kinase, 5 '-nucleotidase antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects cancer of pancreas correlating markings thing content.
Detection method is: 1. go on foot these polynary immune microspheres put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, abandoning supernatant, and adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step mouse monoclonal antibody that adds anti-above-mentioned cancer of pancreas correlating markings thing earlier hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; And then add the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 6, the polynary immune microsphere of use carry out the detection of oophoroma mark:
Prepare the 20 kind latex beadses of diameter between 5 μ m～25 μ m, prepare 20 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the latex beads of these polyclonal antibodies difference mark different sizes, these polyclonal antibodies comprise: anti-CA 125, IAPP, p16 albumen, β again
2-microglobulin, CD44, p53 albumen, nm23 albumen, CD44v6, carcinomebryonic antigen, isoenzymes of creatine kinase, total sialoprotein, Fer, aldolase, glutathione transferase, creatine kinase, ribonuclease, hexokinase, TGF, pyruvate kinase, 5 '-nucleotidase antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects oophoroma correlating markings thing content.
Detection method is: 1. go on foot these polynary immune microspheres put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, abandoning supernatant, and adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step mouse monoclonal antibody that adds anti-above-mentioned oophoroma correlating markings thing earlier hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; And then add the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 7, the polynary immune microsphere of use carry out the detection of kinds of tumors mark:
Prepare the latex beads of the 51 kind specifications of diameter between 1 μ m～30 μ m, prepare 51 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the latex beads of these polyclonal antibodies difference mark different sizes, these polyclonal antibodies comprise: anti-CA50 again, CA19-9, CA724, SA, Fer, POA, PSA, CA494, CA27.29, DU-PAN-2, CA242, CAMD1, IAPP, C polypeptide monoclonal antibody, CER, CK19, tsgf, cd34, st-4-39, grp, p53 albumen, CK20, TPA, TPS, NSE, CYFRA21-1, p16 albumen, p21 albumen, p15 albumen, C-erb-2 albumen, CA-153, CA19-5, CEA, CD44, CA549, AFP, SIMA, gamma glutamyltransferase, aldolase, glutathione transferase, creatine kinase, α-L fucosidase, ribonuclease, hexokinase, TGF, β
2-microglobulin, pyruvate kinase, 5 '-nucleotidase, CA125, HCG, SIXE, CA19 antibody.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects various tumor markers content in the body fluid.
Detection method is: 1. go on foot these polynary immune microspheres put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, abandoning supernatant, and adding 100 μ l test serums carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step add the mouse monoclonal antibody of various tumor markerses in the anti-above-mentioned body fluid earlier, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; And then add the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Embodiment 8, the polynary immune microsphere of use carry out the detection of cytokine content:
Prepare diameter between 5 μ m～25 μ m, diameter differs the latex beads of 30 kinds of specifications of 0.3 micron each other, prepare 30 kinds of not homospecific sheep or rabbit polyclonal antibody simultaneously, with the latex beads of these polyclonal antibodies difference mark different sizes, these polyclonal antibodies comprise: anti-IL-1, IL-2, IL-3 are up to IL-20, IFN, TNF, various growth factor antibodies again.The latex beads of different size is promptly formed not homospecific immune microsphere afterwards by above-mentioned different polyclonal antibody bag, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects various cytokine content in the body fluid.
Detection method is: these polynary immune microspheres were put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, and abandoning supernatant adds 100 μ l test serums and carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; The 2. the step add the mouse monoclonal antibody of various cell factors in the anti-above-mentioned body fluid earlier, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS; And then add the sheep anti mouse polyclonal antibody of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step add flow cytometer and detect and analyze.
Embodiment 9, the polynary immune microsphere of use carry out the detection of autoimmune antibody:
Prepare the latex beads of the 30 kind different sizes of diameter between 1 μ m～30 μ m, prepare 30 species-specific antigens simultaneously, distinguish the latex beads of mark different sizes again with these antigens, these antigens comprise: various microbial antigens, self nuclear antigen etc.The latex beads of different size different is promptly formed different immune microspheres after antigen coated by above-mentioned, these immune microspheres is mixed promptly forming the polynary immune microsphere that detects various autoantibody content in body fluid.
Detection method is: these polynary immune microspheres were put into 3000 rev/mins of microcentrifugal tubes centrifugal 5 minutes, and abandoning supernatant adds 100 μ l test serums and carries out resuspended, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times abandoning supernatant with PBS, the 2. the step adds the anti-people's antibody of rabbit of fluorochrome mark, hatched 30 minutes for 37 ℃, 3000 rev/mins centrifugal 5 minutes, clean 2 times with PBS, abandoning supernatant is carried out resuspended with 100 μ l PBS; The 3. the step adds flow cytometer and detects and analyze.
Claims (8)
1, a kind of polynary immune microsphere comprises latex beads, it is characterized in that: the diameter of described latex beads is between 1 micron～30 microns, make greater than 0.3 micron at interval according to diameter, and its expoeridium different specific antigens or specific antibody.
2, as claims 1 described a kind of polynary immune microsphere, it is characterized in that: the antigen of described bag quilt or specific antibody are the marks relevant with various diseases, as the solubility surface indicia or the various soluble recepter molecule of tumor markers, autoantibody, various pathogenic microorganism antigen or antibody, various cell factor, various cells.
3, as the technology of preparing of claims 1 described a kind of polynary immune microsphere, it is characterized in that: choose diameter and at interval make the different latex beads of diameter, make a kind of immune microsphere at pan coating one species-specific antigen or the antibody of the latex beads of same diameter greater than 0.3 micron, the latex beads of diameter between 1 micron～30 microns; With the immune microsphere for preparing with the PBS washing after, have the immune microsphere of different specific antigens or antibody to mix pan coating, promptly make polynary immune microsphere.
4, as the detection method of claims 1 described a kind of polynary immune microsphere, it is characterized in that: 1. polynary immune microsphere and testing sample are mixed the specificity of carrying out antigen and antibody and combine, more polynary immune microsphere is washed with PBS; 2. polynary immune microsphere is carried out the secondary association reaction, again with suspending again after the PBS washing; 3. detect, analyze with flow cytometer, the size by detecting immune microsphere and the power of luminous intensity or fluorescence intensity thereof draw the content of corresponding determined antigen or antibody.
5, as the detection method of claims 4 described a kind of polynary immune microspheres, it is characterized in that: at polynary immune microsphere with antigen coated mistake, the 2. in the step secondary association reaction be meant that the corresponding antiantibody that adding is crossed through chemiluminescent substance or fluorochrome mark reacts.
6, detection method as claims 5 described a kind of polynary immune microspheres, it is characterized in that: described the 1. the step is to have the polynary immune microsphere of different specific antigens to put into centrifuge tube pan coating, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, the centrifugal back abandoning supernatant that finishes, adding capacity test serum or other samples to be measured carries out resuspended, hatched 30 minutes～2 hours for 37 ℃, in course of reaction, can use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, with PBS washing 2 times, abandoning supernatant is carried out resuspended with PBS; Described the 2. the step be to add the anti-Ig antibody that is marked with the specific fluorescent element, hatched 10 minutes～60 minutes for 37 ℃; Should use shaking table or timing to shake in the course of reaction, 3000 rev/mins～5000 rev/mins, centrifugal 3 minutes～5 minutes, clean 2 times with PBS, after the abandoning supernatant, carry out resuspended with PBS again.
7, as the detection method of claims 4 described a kind of polynary immune microspheres, it is characterized in that: described the 2. in the step at polynary immune microsphere with antibody sandwich, the secondary association reaction be meant add earlier with microballoon on the antibody that adsorbs have mutually homospecific antibody and react, resuspended with PBS washing back; Adding the anti-Ig antibody that is marked with the specific fluorescent element then reacts.
8, detection method as claims 7 described a kind of polynary immune microspheres, it is characterized in that: described the 1. the step is to have the polynary immune microsphere of different specific antibodies to put into centrifuge tube surface indicia, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, abandoning supernatant, adding capacity test serum or other samples to be measured carries out resuspended, hatched 30 minutes～2 hours for 37 ℃, in course of reaction, should use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, with PBS washing 2 times, abandoning supernatant is carried out resuspended with PBS; Described the 2. the step add earlier with immune microsphere on the identical mouse monoclonal antibody of specificity of institute's labelled antibody, hatched 10 minutes～60 minutes for 37 ℃, in course of reaction, to use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, with PBS washing 2 times, abandoning supernatant, carry out resuspended with PBS, add the anti-mouse Ig antibody that is marked with the specific fluorescent element then, hatched 10 minutes～60 minutes for 37 ℃, in course of reaction, use shaking table or timing to shake, 3000 rev/mins～5000 rev/mins centrifugal 3 minutes～5 minutes, clean 2 times with PBS, after the abandoning supernatant, carry out resuspended with PBS once more; Described the 3. the step is to add flow cytometer to detect and analyze.
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