CN114019154A - Kit for complement detection based on flow cytometry, preparation method and application - Google Patents
Kit for complement detection based on flow cytometry, preparation method and application Download PDFInfo
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- CN114019154A CN114019154A CN202111317261.2A CN202111317261A CN114019154A CN 114019154 A CN114019154 A CN 114019154A CN 202111317261 A CN202111317261 A CN 202111317261A CN 114019154 A CN114019154 A CN 114019154A
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- buffer solution
- kit
- flow cytometry
- preparing
- complement
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- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 230000000295 complement effect Effects 0.000 title claims abstract description 21
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title description 12
- 239000004005 microsphere Substances 0.000 claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 22
- 102100027637 Plasma protease C1 inhibitor Human genes 0.000 claims abstract description 4
- 108010005642 Properdin Proteins 0.000 claims abstract description 4
- 102100038567 Properdin Human genes 0.000 claims abstract description 4
- 239000007853 buffer solution Substances 0.000 claims description 31
- 239000000843 powder Substances 0.000 claims description 11
- 210000004369 blood Anatomy 0.000 claims description 10
- 239000008280 blood Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000000654 additive Substances 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 230000000996 additive effect Effects 0.000 claims description 5
- 238000007664 blowing Methods 0.000 claims description 5
- 229960001484 edetic acid Drugs 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 238000003260 vortexing Methods 0.000 claims description 5
- 238000010009 beating Methods 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 108090000056 Complement factor B Proteins 0.000 claims description 2
- 102000003712 Complement factor B Human genes 0.000 claims description 2
- 230000010100 anticoagulation Effects 0.000 claims description 2
- 230000001112 coagulating effect Effects 0.000 claims description 2
- 238000007689 inspection Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 4
- 239000008176 lyophilized powder Substances 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000011095 buffer preparation Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000001000 lipidemic effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- 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
Abstract
The invention provides a kit for complement detection based on flow cytometry, which comprises: microspheres, a capture microsphere reagent and a detection antibody reagent, wherein the capture microsphere reagent at least contains anti-C3, C4, C1q, C1INH, C3d, C5b-9, Properdin and FactorB specific antibodies; the detection antibody reagent at least comprises SA-PE. The kit for complement detection based on flow cytometry is convenient to carry, and has the advantages of high detection speed, high precision, good accuracy and the like.
Description
Technical Field
The invention relates to a kit for complement detection based on flow cytometry, a preparation method and application thereof.
Background
The flow cytometry principle of operation is the multiparameter, rapid quantitative analysis of single cells or other biological particles by monoclonal antibodies at the cellular molecular level. The method can analyze tens of thousands of cells at high speed, can simultaneously measure a plurality of parameters from one cell, has the advantages of high speed, high precision and good accuracy, and is one of the most advanced cell quantitative analysis techniques in the present generation. The light source, the flow channel, the signal detection transmission and the data analysis system are the main components of the flow cytometer. At present, the detection of peripheral blood leucocyte, bone marrow cell, tumor cell and the like by using a flow cytometer is an important component of clinical detection in clinic.
Therefore, there is a need to develop a kit for complement detection based on flow cytometry.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a kit for complement detection based on flow cytometry, which is convenient to carry and has the advantages of high detection speed, high precision, good accuracy and the like.
In a first aspect, the present invention provides a kit for complement detection based on flow cytometry, comprising: microspheres, a capture microsphere reagent and a detection antibody reagent, wherein the capture microsphere reagent at least contains anti-C3, C4, C1q, C1INH, C3d, C5B-9, Properdin and Factor B specific antibodies; the detection antibody reagent at least comprises SA-PE.
The kit for complement detection based on flow cytometry is convenient to carry, and has the advantages of high detection speed, high precision, good accuracy and the like.
In a second aspect, the invention provides a preparation method of the kit for complement detection based on flow cytometry, which comprises the following steps:
s101: pretreating the microspheres;
s102: adjusting the 20 Xwashing buffer solution to room temperature, after the salt in the buffer solution is dissolved, adding 1ml of 20 Xwashing buffer solution into 19ml of deionized water to obtain a diluted buffer solution;
s103: adding 5mL of the diluted buffer solution into the matrix additive freeze-dried powder, standing for 13-18 min, and then, swirling to dissolve the buffer solution;
s104: preparing a calibrator;
s105: mixing the matrix additive, the calibrator and the capture microsphere reagent in a ratio of 1: 1: 1, adding a detection antibody with the same amount as the capture microsphere reagent, incubating for 1.8-2.2 h at room temperature, adding SA-PE with the same amount as the capture microsphere reagent, incubating for 0.4-0.6 h at room temperature, adding 1 multiplied by buffer solution 20 times the amount of the capture microsphere reagent, then vortexing for 3-10 s, taking 300-500 g, centrifuging for 3-7 min, adding 150-300 uL of the diluted buffer solution into each tube according to the sample requirement of a flow cytometer, vortexing for 8-12 s, and then resuspending the diluted buffer solution for detection.
As a specific embodiment of the present invention, the step S104 includes the following steps:
adding 250uL of the diluted buffer solution into a frozen powder glass bottle of a calibrator to completely dissolve the frozen powder attached to the wall of the bottle, marking the solution as C7 with the concentration of 10000pg/mL, and standing for 13-17 min;
preparing six EP tubes which are respectively marked as 6, 5, 4, 3, 2 and 1, adding 75uL of the diluted buffer solution into each tube, gradually diluting by 4 times, namely taking 25uL of the C7 solution into the EP tube 6, gently blowing and uniformly mixing for 30-40 times to obtain a C6 calibrator with the concentration of 2500pg/mL, repeating the steps, gradually diluting by 4 times, and respectively diluting to obtain the C5, C4, C3, C2 and C1 calibrators.
As a specific embodiment of the invention, the vial wall is rinsed several times with a pipette tip to aspirate the solution before using the calibrator.
As a specific embodiment of the invention, when the freeze-dried powder is unpacked, the bottle cap is slowly screwed to be half-opened, and the diluted buffer solution is respectively added into two pores on the side surface of the bottle cap.
As a specific embodiment of the present invention, in the step S101, the preprocessing includes the steps of: and (3) swirling the microspheres for 25-35 s, blowing and beating for 25-35 times by using a liquid transfer gun, and then swirling for 25-35 s.
In a specific embodiment of the present invention, in step S105, the rotation speed during incubation is 400 to 500 r/min.
In a third aspect, the invention provides application of the kit for detecting complement based on flow cytometry.
The application of the kit for detecting complement based on flow cytometry is characterized in that a detection sample is plasma or serum. Specifically, the detection sample is a non-hemolytic, lipemic or icteric blood sample.
As a specific embodiment of the present invention, the application further comprises the following steps:
collecting venous blood sample with standard test tube, naturally coagulating at room temperature for 30min, centrifuging at 1000g for 10 min, and collecting separated serum for inspection; and/or collecting venous blood samples by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation blood collection tube, centrifuging for 10 minutes at 1000g, and taking separated plasma for examination.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention in any way.
Example 1
Example 1 proposes a kit for complement detection based on flow cytometry, the preparation method of which comprises the following steps:
firstly, experimental operation steps:
1. preparation in the early stage of the experiment:
1.1 instrument consumable preparation: 50ul, 200ul, 1ml pipette, 1.5ml EP tube, flow tube, vortex mixer, incubation shaker, centrifuge, flow cytometer (APC and PE channels available), data analysis computer (win7 and above 64 systems)
1.2 reagent preparation: all reagents were allowed to return to room temperature naturally before use.
1.2.1 microsphere preparation: before the experiment, the microspheres are firstly vortexed for 30s, and are lightly blown by a pipette about 30 times, and are vortexed for 30s when in use.
1.2.2 Wash buffer preparation: after the 20 × wash buffer had returned to room temperature, 1ml of 10 × wash buffer was added to 19ml of deionized water until all the salts had dissolved.
1.2.3 preparation of matrix additives: slowly screwing the matrix additive freeze-dried powder glass bottle to half open, adding 5ml of experiment buffer solution into the glass bottle, standing for 15min, and vortexing to fully dissolve the experiment buffer solution. (when the lyophilized powder is opened, the bottle cap is slowly screwed to half-open in order to prevent the powder from flying out, 5ml of experiment buffer solution is added from a crack hole on the side surface of the bottle cap by a 1ml pipette gun in portions)
1.2.4 preparation of calibrators: slowly screwing the calibrator lyophilized powder glass bottle to half open, adding 250ul of experiment buffer solution into the glass bottle, slowly rotating the bottle to completely dissolve the lyophilized powder attached to the bottle wall, wherein the concentration of the solution is 10000pg/ml, the solution is marked as C7, standing for 15min, and sucking the solution by a pipette gun to wash the bottle wall for several times before use. (remark: when the lyophilized powder is unpacked, in order to prevent the powder from flying out, the bottle cap is slowly screwed to half-open, 125ul of experiment buffer solution is respectively added into two holes on the side surface of the bottle cap)
Preparing six EP tubes, respectively labeled as 6, 5, 4, 3, 2 and 1, adding 75ul of experiment buffer solution into each tube, diluting 4 times, namely adding 25ul of C7 solution into the EP tube 6, and gently blowing, beating and mixing for 30-40 times to obtain the C6 calibrator with the concentration of 2500 pg/ml.
Diluting by 4 times in the same manner as above to obtain calibrators of C5, C4, C3, C2 and C1.
1.3 preparation of samples to be tested
1.3.1 sample: serum or plasma
1.3.2 Collection:
serum: venous blood samples were collected in standard test tubes, allowed to spontaneously clot at room temperature for 30min, then centrifuged at 1000g for 10 min, and the separated serum was taken for examination.
Plasma: venous blood samples were collected with EDTA anticoagulant blood collection tubes, centrifuged at 1000g for 10 minutes, and the separated plasma was taken for testing.
2. The experimental procedures are shown in Table 1
TABLE 1 Experimental procedures
According to the detection result, if the value of the detection result exceeds the detection range, the sample needs to be diluted properly by using the experiment buffer solution, and then the detection is carried out, wherein the detection indexes are shown in table 2.
TABLE 2 test indexes
| Index (I) | Reference range |
| C3 | Males:880–2520mg/LFemales:880–2060mg/L |
| C4 | Males:120–720mg/LFemales:130–750mg/L |
| C1q | 560-2760mg/L |
| C1INH | 160-330mg/L |
| C3d | <12mg/L |
| C5b-9 | 106–263mg/L |
| Properdin | 15–41mg/L |
| FactorB | 74–286mg/L |
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (9)
1. A kit for complement detection based on flow cytometry, comprising: microspheres, a capture microsphere reagent and a detection antibody reagent, wherein the capture microsphere reagent at least contains anti-C3, C4, C1q, C1INH, C3d, C5B-9, Properdin and Factor B specific antibodies; the detection antibody reagent at least comprises SA-PE.
2. The method for preparing a kit for complement detection based on flow cytometry as described in claim 1, comprising the steps of:
s101: pretreating the microspheres;
s102: adjusting the 20 Xwashing buffer solution to room temperature, after the salt in the buffer solution is dissolved, adding 1ml of 20 Xwashing buffer solution into 19ml of deionized water to obtain a diluted buffer solution;
s103: adding 5mL of the diluted buffer solution into the matrix additive freeze-dried powder, standing for 13-18 min, and then, swirling to dissolve the buffer solution;
s104: preparing a calibrator;
s105: mixing the matrix additive, the calibrator and the capture microsphere reagent in a ratio of 1: 1: 1, adding a detection antibody with the same amount as the capture microsphere reagent, incubating for 1.8-2.2 h at room temperature, adding SA-PE with the same amount as the capture microsphere reagent, incubating for 0.4-0.6 h at room temperature, adding 1 multiplied by buffer solution 20 times the amount of the capture microsphere reagent, then vortexing for 3-10 s, taking 300-500 g, centrifuging for 3-7 min, adding 150-300 uL of the diluted buffer solution into each tube according to the sample requirement of a flow cytometer, vortexing for 8-12 s, and then resuspending the diluted buffer solution for detection.
3. The method for preparing a kit for complement detection based on flow cytometry according to claim 2, wherein the step S104 comprises the steps of:
adding 250uL of the diluted buffer solution into a frozen powder glass bottle of a calibrator to completely dissolve the frozen powder attached to the wall of the bottle, marking the solution as C7 with the concentration of 10000pg/mL, and standing for 13-17 min;
preparing six EP tubes which are respectively marked as 6, 5, 4, 3, 2 and 1, adding 75uL of the diluted buffer solution into each tube, gradually diluting by 4 times, namely taking 25uL of the C7 solution into the EP tube 6, gently blowing, beating and uniformly mixing for 30-40 times to obtain a C6 calibrator with the concentration of 2500pg/mL, repeating the steps, gradually diluting by 4 times, and respectively diluting to obtain C5, C4, C3, C2 and C1 calibrators.
4. The method of preparing a kit for complement detection based on flow cytometry according to claim 2 or 3, wherein the vial wall is washed several times with a pipette tip to aspirate the solution before using the calibrator.
5. The method for preparing a kit for complement detection based on flow cytometry according to claim 2 or 3, wherein when the freeze-dried powder is unpacked, a bottle cap is slowly screwed to be half-opened, and the diluted buffer solution is respectively added into two pores on the side surface of the bottle cap.
6. The method for preparing a kit for complement detection based on flow cytometry according to claim 2, wherein in the step S101, the pretreatment comprises the steps of: and (3) swirling the microspheres for 25-35 s, blowing and beating for 25-35 times by using a liquid transfer gun, and then swirling for 25-35 s.
7. The method for preparing a kit for complement detection according to any one of claims 2 to 5, wherein in step S105, the rotation speed during incubation is 400 to 500 r/min.
8. Use of a kit for complement detection based on flow cytometry according to any one of claims 2 to 7, wherein the sample to be detected is plasma or serum.
9. The use according to claim 8, further comprising the steps of:
collecting venous blood sample with standard test tube, naturally coagulating at room temperature for 30min, centrifuging at 1000g for 10 min, and collecting separated serum for inspection; and/or collecting venous blood samples by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation blood collection tube, centrifuging for 10 minutes at 1000g, and taking separated plasma for examination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111317261.2A CN114019154A (en) | 2021-11-09 | 2021-11-09 | Kit for complement detection based on flow cytometry, preparation method and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111317261.2A CN114019154A (en) | 2021-11-09 | 2021-11-09 | Kit for complement detection based on flow cytometry, preparation method and application |
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| Publication Number | Publication Date |
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| CN114019154A true CN114019154A (en) | 2022-02-08 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050277158A1 (en) * | 2004-06-10 | 2005-12-15 | Ge Chen | Method and kit for donor specific complement-fixing antibodies crossmatch |
| CN105308457A (en) * | 2013-03-14 | 2016-02-03 | 斯坦福大学托管董事会 | Methods of detecting donor-specific antibodies and systems for practicing the same |
| CN105548547A (en) * | 2016-02-18 | 2016-05-04 | 山东信力科生物科技有限公司 | Flow type array immunoassay kit for detecting lung cancer markers based on flow cytometry |
| CN107238711A (en) * | 2017-05-18 | 2017-10-10 | 无锡市精神卫生中心 | A kind of diagnostic kit and its detection method for detecting Alzheimer disease peripheral blood protein marker |
| US20180052158A1 (en) * | 2016-08-16 | 2018-02-22 | National University Corporation Asahikawa Medical University | Method and kit for examining complement system |
-
2021
- 2021-11-09 CN CN202111317261.2A patent/CN114019154A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050277158A1 (en) * | 2004-06-10 | 2005-12-15 | Ge Chen | Method and kit for donor specific complement-fixing antibodies crossmatch |
| CN105308457A (en) * | 2013-03-14 | 2016-02-03 | 斯坦福大学托管董事会 | Methods of detecting donor-specific antibodies and systems for practicing the same |
| CN105548547A (en) * | 2016-02-18 | 2016-05-04 | 山东信力科生物科技有限公司 | Flow type array immunoassay kit for detecting lung cancer markers based on flow cytometry |
| US20180052158A1 (en) * | 2016-08-16 | 2018-02-22 | National University Corporation Asahikawa Medical University | Method and kit for examining complement system |
| CN107238711A (en) * | 2017-05-18 | 2017-10-10 | 无锡市精神卫生中心 | A kind of diagnostic kit and its detection method for detecting Alzheimer disease peripheral blood protein marker |
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Application publication date: 20220208 |
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