CN103698534A - Rabbit serum immunoglobulin liquid-phase suspension chips and preparation and application thereof - Google Patents
Rabbit serum immunoglobulin liquid-phase suspension chips and preparation and application thereof Download PDFInfo
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- CN103698534A CN103698534A CN201310674237.3A CN201310674237A CN103698534A CN 103698534 A CN103698534 A CN 103698534A CN 201310674237 A CN201310674237 A CN 201310674237A CN 103698534 A CN103698534 A CN 103698534A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention relates to the technical field of biology, and particularly relates to rabbit serum immunoglobulin IgM, IgG and IgA liquid-phase suspension chips and a preparation and application thereof. The rabbit serum immunoglobulin liquid-phase suspension chips comprise 42#, 48# and 57# microspheres which are respectively coupled to IgG, IgM and IgA capture antibodies. In addition, the rabbit IgM, IgG and IgA liquid-phase chips are prepared by the method, the IgM, IgG and IgA contents in WHBE (White Hair and Black Eyes) rabbit and JW rabbit are synchronous detected, and a method for detecting the rabbit serum immunoglobulin IgM, IgG and IgA liquid-phase suspension chips is built. The method for detecting the rabbit serum immunoglobulin IgM, IgG and IgA liquid-phase suspension chips can be applied to research and development of immunology and a biological diagnostic kit, and experimental materials and new resources are provided for development of the bioengineering and pharmaceutical industry, especially research and development and production of biological diagnostic reagents.
Description
Technical field
The present invention relates to biological technical field, be specifically related to rabbit anteserum Immunoglobulin IgM, IgG, IgA liquid phase suspending chip and preparation and application.
Background technology
Experimental rabbit is one of the most frequently used animal used as test in current biomedical product research and development and medical scientific field, especially at aspects such as biological antibody preparation, ophthalmologic operation researchs, is bringing into play the large incomparable advantage of mouse.Serum immune globulin Ig level is the important indicator of reflection animal body immune state, and variation and the disease of Ig content are closely related, and wherein IgG, IgM and IgA are the main effects molecules of humoral immunity.
Liquid phase suspending chip technology is to carry out qualitative, quantitative novel chip technology to many indexs simultaneously, but the operative technique platform of emulating the advanced as biological information, and liquid-phase chip technology is still restricted in the application in life science fundamental research field.At present released the commercial liquid phase chip reagent box that can be applicable to Animal Experimental Study abroad, but be confined to the detection of large mouse, the detection of experimental rabbit Serum Indexes, cannot be applied.
Summary of the invention
For overcoming above problem, the object of the present invention is to provide a kind of rabbit anteserum Immunoglobulin IgM, IgG, IgA liquid phase suspending chip.
A rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip, comprises respectively No. 42, No. 48 and No. 57 microballoons with IgG, IgM and the coupling of IgA capture antibody.
In addition, the present invention also provides the preparation method of a kind of rabbit anteserum Immunoglobulin IgM, IgG, IgA liquid phase suspending chip.
The preparation method of rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip, comprises the steps:
1) get 100 μ No. l42, No. 48 and No. 57 carboxyl microballoons activate;
2) No. 42, No. 48 after activation and No. 57 microballoons respectively with IgG, IgM and the coupling of IgA capture antibody of the affinity purification of variable concentrations, room temperature lucifuge rotation coupling 2h;
3) the complete microballoon of coupling washs with PBST, obtains rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip.
Wherein, step 1), for getting 100 μ No. l42, No. 48 and No. 57 carboxyl microballoons, adds 80 μ l0.1MNaH2PO
4, 10 μ l50mg/ml S-NHS and 10 μ l50mg/ml EDC activation damping fluid, room temperature lucifuge is shaken slowly 20min and is activated; Described step 3) is that the complete microballoon of coupling is with after twice of 0.05%PBST washing, add 1ml1%PBSB room temperature lucifuge sealing 30min, with 1%PBSB, clean microballoon one time, remove supernatant, add 150 μ l1%PBSB to preserve microballoon, obtain rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip.
In addition, the present invention also provides the method for using rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip to detect the application of rabbit anteserum Immunoglobulin IgG, IgM, IgA and detecting rabbit anteserum Immunoglobulin IgG, IgM, IgA with liquid phase suspending chip.
The using method of rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip, comprises the steps:
(1) with the wetting pvdf membrane reaction plate of 100 μ l/ hole 1%PBSB, sealing 10min, each 1 * 104 of the microballoon that every hole adds respectively coupling rabbit igg, IgM and IgA, mixes suction filtration supernatant discarded;
(2) reacting hole adds the rabbit anteserum sample that 50 μ l4 doubly dilute, sealer, constant-temperature incubation shaking table 500r/m room temperature lucifuge reaction 60min;
(3) wash buffer washes 3 times, add IgG, IgM and the IgA of mixing to detect antibody, 50 μ l/ holes, 500r/m room temperature lucifuge reaction 60min, wash buffer washes 3 times, adds Streptavidin-PE antibody, 50 μ l/ holes, 500r/m reacts 30min, and suction filtration supernatant discarded, with the resuspended microballoon of 125 μ l1%PBSB;
(4) in Bio-Plex200 liquid phase suspension chip system, detect, after the start of liquid phase suspension chip system, must calibrate by the laser optical path of calibration kit and validation kit, can detect.
Described method adopts fluorescent microsphere and suspension chip system streaming technology.
Described detected object is WHBE rabbit anteserum and JW rabbit anteserum.
Described rabbit body weight is 2-2.5kg.
The present invention prepares rabbit igg, IgM and IgA liquid-phase chip, synchronously detects IgG, IgM and IgA content in WHBE rabbit and JW rabbit anteserum, sets up and detects rabbit anteserum Immunoglobulin IgG, IgM, IgA liquid phase suspension chip method.Set up the method that detects rabbit anteserum Immunoglobulin IgG, IgM, IgA liquid phase suspending chip, can be applicable to the research and development of immunology and biological diagnosis kit, development to Bio-pharmaceutical Industry, the especially research, development and production for biological diagnostic reagent provide experiment material and new resource.
Method therefor of the present invention if no special instructions, is conventional method.Needed material or reagent in following examples, be if no special instructions market and buy.The approach that obtains of the various biomaterials that are described in embodiment be only the approach that obtains of a kind of experiment to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.Described percent concentration is mass/volume (W/V) percent concentration or volume/volume (V/V) percent concentration unless otherwise noted.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, should be understood that following examples are only not used in and limit the scope of the invention for the present invention is described.
In the following example, method therefor if no special instructions, is conventional method.Needed material or reagent in following examples, be if no special instructions market and buy.The approach that obtains of the various biomaterials that are described in embodiment be only the approach that obtains of a kind of experiment to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.
Described percent concentration is mass/volume (W/V) percent concentration or volume/volume (V/V) percent concentration unless otherwise noted.
Embodiment
1: materials and methods
1.1 animals used as test: 20 of JW rabbits, 20 of WHBE rabbits, male and female half and half, the about 2-2.5k of body weight, is provided by Zhejiang University of Traditional Chinese Medicine's experimental rabbit production base [SCXK(Zhejiang) 2009-0042].
1.2 preparation of samples: from WHBE rabbit and JW rabbit ear medium sized artery, get appropriate blood respectively, be placed in 10ml glass tube, 4 ℃, 3000r/m, centrifugal 10min, separation of serum, is stored in-20 ℃.
1.3 experiment reagents: the IgG capture antibody of affinity purification, IgM capture antibody, IgA capture antibody are all purchased from U.S. Bethyl company, the IgG of biotin mark detects antibody, IgM detects antibody, IgA detection antibody all purchased from U.S. Bethyl company, Streptavidin-PE antibody is purchased from American I nvitrogen company, Sulfo-NHS, EDC are purchased from U.S. Thermo company, and Bio-Plex carboxyl microballoon, Bio-Plex calibration kit and validation kit are all purchased from U.S. Bio-Rad company.
1.4 experimental apparatuss: Bio-plex200 liquid phase suspension chip system, desk-top high-speed refrigerated centrifuge, constant-temperature incubation shaking table, vacuum filtration device, multi-functional microplate reader.
1.5 rabbit anteserum Immunoglobulin IgGs, IgM and the preparation of IgA liquid phase suspending chip: No. 42, No. 48 and No. 57 microballoons respectively with IgG, IgM and the coupling of IgA capture antibody, get 100 μ No. l42, No. 48 and No. 57 carboxyl microballoons (1.25 * 10
6individual/kind), add 80 μ l0.1M NaH
2pO
4(pH6.2), 10 μ l50mg/ml S-NHS and 10 μ l50mg/ml EDC activation damping fluid, room temperature lucifuge is shaken slowly 20min and is activated.After activation No. 42, No. 48 and No. 57 microballoons respectively with IgG, IgM and the coupling of IgA capture antibody of the affinity purification of 5,10,15,20 and 30 μ g, room temperature lucifuge rotation coupling 2h, the microballoon 0.05%PBST(pH7.4 that coupling is complete) wash after twice, add 1ml1%PBSB room temperature lucifuge sealing 30min.With 1%PBSB, clean microballoon one time, remove supernatant, add 150 μ l1%PBSB to preserve microballoon, available haemocytometer calculates the concentration of microballoon, the microballoon of 4 ℃ of couplings of keeping in Dark Place.Obtain rabbit anteserum Immunoglobulin IgG, IgM and IgA liquid phase suspending chip.
1.6 detect the foundation of the method for rabbit anteserum Immunoglobulin IgG, IgM, IgA liquid phase suspending chip: with rabbit igg, IgM and the IgA liquid-phase chip (rabbit anteserum Immunoglobulin IgG, IgM and IgA liquid phase suspending chip) of above-mentioned preparation, 20 WHBE rabbits and 20 JW rabbit anteserum samples are detected, duplicate detection twice, averages.Concrete operations are as follows: with the wetting pvdf membrane reaction plate in 100 μ l1%PBSB/ holes, sealing 10min, every hole adds respectively coupling rabbit igg, each 1 * 104 of the microballoon of IgM and IgA, mix, suction filtration supernatant discarded, the WHBE rabbit that reacting hole adds 50 μ l4 doubly to dilute, JW rabbit anteserum sample, sealer, constant-temperature incubation shaking table 500r/m room temperature lucifuge reaction 60min, wash buffer washes 3 times, the IgG that adds mixing, IgM and IgA detect antibody, 50 μ l/ holes, 500r/m room temperature lucifuge reaction 60min, wash buffer washes 3 times, add Streptavidin-PE antibody, 50 μ l/ holes, 500r/m reacts 30min, suction filtration supernatant discarded, with the resuspended microballoon of 125 μ l1%PBSB, in Bio-Plex200 liquid phase suspension chip system, detect.After the start of liquid phase suspension chip system, must calibrate by the laser optical path of calibration kit and validation kit, can detect.
2 results
To 20 WHBE rabbits and 20 JW rabbit anteserum samples, by liquid phase suspending chip technology, carry out serum immunoglobulin IgG, IgM, IgA detection, WHBE rabbit igg, IgM and IgA protein content are respectively 13836.83 ± 746.44FI, 18044.40 ± 606.27FI, 14816.00 ± 687.90FI, JW rabbit igg, IgM and IgA protein content are respectively 14469.85 ± 858.78FI, 18544.80 ± 600.53FI, 14186.68 ± 674.33FI, as shown in table 1, specifically in Table 1-3.
Table 1 rabbit anteserum Immunoglobulin IgG, IgM, IgA content
Table 2WHBE rabbit anteserum Immunoglobulin IgG, IgM, IgA content
Table 3JW rabbit anteserum Immunoglobulin IgG, IgM, IgA content
Conclusion
IgG, IgM and IgA that the suspension chip method of setting up can be applicable to experimental rabbit serum detect.
Claims (7)
1. a rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip, comprises respectively No. 42, No. 48 and No. 57 microballoons with rabbit anteserum Immunoglobulin IgG, IgM and the coupling of IgA capture antibody.
2. the preparation method of rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip, is characterized in that: comprise the steps:
1) get 100 μ No. l42, No. 48 and No. 57 carboxyl microballoons activate;
2) No. 42, No. 48 after activation and No. 57 microballoons respectively with IgG, IgM and the coupling of IgA capture antibody of the affinity purification of variable concentrations, room temperature lucifuge rotation coupling 2h;
3) the complete microballoon of coupling washs with PBST, obtains rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip.
3. the preparation method of rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip according to claim 2, is characterized in that: described step 1), for getting 100 μ No. l42, No. 48 and No. 57 carboxyl microballoons, adds 80 μ l0.1M NaH2PO
4, 10 μ l50mg/mlS-NHS and 10 μ l50mg/ml EDC activation damping fluid, room temperature lucifuge is shaken slowly 20min and is activated; Described step 3) is that the complete microballoon of coupling is with after twice of 0.05%PBST washing, add 1ml1%PBSB room temperature lucifuge sealing 30min, with 1%PBSB, clean microballoon one time, remove supernatant, add 150 μ l1%PBSB to preserve microballoon, obtain rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip.
4. use rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip to detect the application of rabbit anteserum Immunoglobulin IgG, IgM, IgA.
5. use rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip to detect the method for rabbit anteserum Immunoglobulin IgG, IgM, IgA, comprise the steps:
(1) with the wetting pvdf membrane reaction plate in 100 μ l1%PBSB/ holes, sealing 10min, each 1 * 104 of the microballoon that every hole adds respectively coupling rabbit igg, IgM and IgA, mixes suction filtration supernatant discarded;
(2) reacting hole adds the rabbit anteserum sample that 50 μ l4 doubly dilute, sealer, constant-temperature incubation shaking table 500r/m room temperature lucifuge reaction 60min;
(3) wash buffer washes 3 times, add IgG, IgM and the IgA of mixing to detect antibody, 50 μ l/ holes, 500r/m room temperature lucifuge reaction 60min, wash buffer washes 3 times, adds Streptavidin-PE antibody, 50 μ l/ holes, 500r/m reacts 30min, and suction filtration supernatant discarded, with the resuspended microballoon of 125 μ l1%PBSB;
(4) in Bio-Plex200 liquid phase suspension chip system, detect, after the start of liquid phase suspension chip system, must calibrate by the laser optical path of calibration kit and validation kit, can detect.
6. the method that use rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip according to claim 5 detects, is characterized in that: described method adopts fluorescent microsphere and suspension chip system streaming technology.
7. the method that use rabbit anteserum immunoglobulin (Ig) liquid phase suspending chip according to claim 5 detects, is characterized in that: described detected object is WHBE rabbit anteserum and JW rabbit anteserum.
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Cited By (1)
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| CN109060972A (en) * | 2018-05-16 | 2018-12-21 | 苏州药明泽康生物科技有限公司 | Application of the rabbit blood in preparation human disease's external diagnosis reagent case |
| CN109060972B (en) * | 2018-05-16 | 2021-09-24 | 苏州药明泽康生物科技有限公司 | Application of rabbit blood in preparing human disease in-vitro diagnosis kit |
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Application publication date: 20140402 |