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 PDF

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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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温旭
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Hebei Boyin Biotechnology Co ltd
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Hebei Boyin Biotechnology Co ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; 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 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

Kit for complement detection based on flow cytometry, preparation method and application
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.
Figure BDA0003344146460000031
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
Figure BDA0003344146460000041
Figure BDA0003344146460000051
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.
CN202111317261.2A 2021-11-09 2021-11-09 Kit for complement detection based on flow cytometry, preparation method and application Pending CN114019154A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
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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