CN108896538A - A kind of creatinine chemiluminescence immune detection reagent kit and each component preparation method - Google Patents
A kind of creatinine chemiluminescence immune detection reagent kit and each component preparation method Download PDFInfo
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- CN108896538A CN108896538A CN201810640306.1A CN201810640306A CN108896538A CN 108896538 A CN108896538 A CN 108896538A CN 201810640306 A CN201810640306 A CN 201810640306A CN 108896538 A CN108896538 A CN 108896538A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/54326—Magnetic particles
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Abstract
A kind of creatinine chemiluminescence immune detection reagent kit disclosed by the invention and each component preparation method, including following reagent：It is coated with working solution：It is placed in containing the coated anti-creatinine monoclonal antibody of the 0.025-0.25mg/ml magnetic particle in bovine serum albumin(BSA) and preservative buffer；Mark working liquid：It is placed in the creatinine conjugate antigen of the 0.001-0.01mg/ml acridinium ester label in the buffer containing bovine serum albumin(BSA) and preservative；Calibration solution 1：Calibration product containing low concentration creatinine antigen；Calibration solution 2：Calibration product containing high concentration creatinine antigen；Exciting liquid 1：The HNO3 of 0.1N containing 0.25%-1.25%H2O2；Exciting liquid 2：The NaOH of 0.25N containing 0.2%-2%Triton-X100.Background luminescence of the present invention is low, and signal-to-noise ratio is high, and the disturbing factor that shines is few, and luminous efficiency is high, and intensity is big, is easy to be coupled with albumen and does not influence luminous efficiency and luminous intensity, high sensitivity.
Description
Technical field:The invention belongs to technical field of immunoassay, specifically a kind of creatinine chemiluminescence immunoassay inspection
Test agent box and each component preparation method.
Background technique：Creatinine is product of the muscle in human body metabolism, is mainly excreted by glomerular filtration, in meat
When food intake dose is stablized, the generation of the not big variation of the muscle metabolism of body, creatinine will be more constant, and creatinine is almost complete
Portion enters crude urine through glomerular filtration, and not by reabsorption, when kidney damages, under glomerular filtration function
Drop, serum creatinine rise, therefore the degree of impairment of kidney can be diagnosed by detection blood, urine creatinine, provide accurately for treatment and prognosis
Foundation.
Picric acid method is relied primarily on currently on the market and enzymic creatinine assay method is detected, and wherein picric acid detection method is former
Reason is after proper manners originally remove removing protein, and creatinine occurs Jaffe with picric acid under alkaline environment and reacts, and generates orange-red picric acid
Creatinine compound measures absorbance between 510-520nm, and testing result is quick, low in cost, but the albumen in proper manners sheet
With alkaline picric acid nonspecific reaction can occur for matter, carbohydrate, acetone, Vc, pyruvic acid etc., and sensitivity is low, poor specificity, meeting
Cause the erroneous judgement of result.
Enzymic creatinine assay method is that creatinine generates sarcosine under the action of creatine amidino groups hydrolase, and sarcosine is in sarcosine
Hydrogen peroxide is generated under the action of oxidizing ferment, the latter reacts life with 4-AA, ESPAS under hydrogen peroxide enzyme effect
At aubergine quinone imines, absorbance value is detected at 546nm, such method high sensitivity, the range of linearity is wide, anti-bilirubin, blood
The ability of Lactoferrin and blood lipid is stronger, but the reaction time needs strict control, and reaction process is complex, higher cost.
Summary of the invention：
The object of the present invention is to provide a kind of creatinine chemiluminescence immune detection reagent kit and each component preparation methods, with solution
The deficiency of certainly existing detection.
A kind of creatinine chemiluminescence immune detection reagent kit of the invention, including following reagent：
1) it is coated with working solution：It is placed in micro- containing the 0.025-0.25mg/ml magnetic in bovine serum albumin(BSA) and preservative buffer
The coated anti-creatinine monoclonal antibody of grain；
2) mark working liquid：The 0.001-0.01mg/ml a word used for translation being placed in the buffer containing bovine serum albumin(BSA) and preservative
The creatinine conjugate antigen of pyridine ester label；
3) calibration solution 1：Calibration product containing low concentration creatinine antigen；
4) calibration solution 2：Calibration product containing high concentration creatinine antigen；
5) exciting liquid 1：The HNO3 of 0.1N containing 0.25%-1.25%H2O2；
6) exciting liquid 2：The NaOH of 0.25N containing 0.2%-2%Triton-X100.
Most preferably, described to pass through coating working solution, mark working liquid, calibration solution 1, calibration solution 2, exciting liquid 1 and excitation
The detection with the use of completion to creatinine of 2 six kinds of reagents of liquid.
The preparation method of coating working solution among the above, includes the following steps：
1) a certain amount of magnetic bead stoste is taken, magnetic bead is cleaned 2 times using magnetic bead cleaning solution, a certain amount of MES is then added
Buffer completes the cleaning dilution of magnetic bead stoste；
2) a certain amount of EDC and NHS is added in magnetic bead dilution, is placed on concussion in 37 DEG C of environment and mixes 30 points
Clock -60 minutes, complete the activation of magnetic bead；
3) activated magnetic bead is used into buffer solution for cleaning twice, a certain amount of MES is added, while working with antibody processed
Then liquid antibody working solution is added in magnetic bead solution, be placed on concussion in 37 DEG C of environment and mix -480 minutes 120 minutes, complete
It is coupled at antibody magnetic bead；
4) magnetic bead after coupling is rinsed twice using buffer, a certain amount of Block buffer is added, prevents at 37 DEG C
Environment in concussion mix 30 minutes to 60 minutes, complete the closing of magnetic bead；
5) magnetic bead after closing is used into buffer solution for cleaning twice, magnetic bead is added and saves liquid, is placed on 2-8 DEG C after mixing
It is saved in environment, completes the preservation of magnetic bead；
6) magnetic bead being coated with is diluted to 0.025-0.25mg/ml using buffer is saved, it is filling to be stored in 2-8 DEG C
In environment, the preparation of coating working solution is completed, i.e. magnetic bead is coated with creatinine monoclonal antibody.
Most preferably, the EDC is water-soluble carbodiimide, and NHS is n-hydroxysuccinimide.
The preparation method of mark working liquid among the above, includes the following steps：
1) acridinium ester solid powder is diluted to 5mg/ml using DMF, packing is saved into -20 DEG C of environment, takes 5mg/ml
Acridine ester solution be diluted to 1mg/ml using DMF, the working concentration that makes marks use；
2) creatinine monoclonal antibody is used into carbonic acid buffer dialysed overnight, completes antibody dialysis；
3) the antibody 1mg after taking dialysis good, is diluted to 1ml, is added into the 1mg/ml acridinium ester work also of 100ul, room
It is mixed -480 minutes 120 minutes in warm, completes acridinium ester and be coupled；
4) 10% lysine 100ul is added into the acridinium ester working solution after connection, room temperature is mixed 30 minutes, is quenched not complete
The acridinium ester of full response is completed to be quenched；
5) acridinium ester being quenched is purified using desalting column；
6) isometric phosphate buffer containing 5%BSA is added after purification, prevents from saving in -20 DEG C of environment；
7) acridinium ester marked is diluted to 0.001-0.01mg/ml using buffer is saved, it is filling to be stored in 2-8 DEG C
Environment in, complete acridinium ester label working solution configuration.
Most preferably, the DMF is n,N-Dimethylformamide.
The preparation method of calibration solution 1 and calibration solution 2 among the above, includes the following steps：
By creatinine antigen using calibration object diluted to 100-200umol/L and 1000-2000umolL concentration,
It dispenses and packs, be stored in 2-8 DEG C of environment.
The preparation method of exciting liquid 1 among the above, includes the following steps：
The concentrated nitric acid and hydrogenperoxide steam generator for taking corresponding amount are formulated into required concentration, and packing is stored in 2-8 DEG C of environment,
Complete the configuration of exciting liquid 1.
The preparation method of exciting liquid 2 among the above, includes the following steps：
Sodium hydroxide and the Triton X-100 mixing for taking corresponding amount, are formulated into required concentration, packing is stored in 2-8 DEG C of ring
In border, the configuration of exciting liquid 2 is completed.
The beneficial effects of the invention are as follows：The present invention is using acridinium ester shiner luminous efficiency not by substituent group and antigen-antibody
The advantages of influence of structure and the luminous dilute alkaline soln catalysis for only needing hydrogen peroxide, does chemiluminescence detection, has background
It shines low, signal-to-noise ratio is high, and the disturbing factor that shines is few, and luminous efficiency is high, and intensity is big, is easy to be coupled with albumen and do not influence the effect that shines
Rate and luminous intensity, high sensitivity combine with Myoglobin analysis particle, are conducive to full automatic working, save human time's cost
The advantages that.
Specific embodiment：
The present invention is further described below with reference to embodiment.
Embodiment
By comparing the creatinine detection reagent box of two kinds of testing principles, the results of performance analysis experiment of progress is of the invention
Creatinine detection reagent box is in sensitivity, precision, and the kit of comparison is superior in the range of linearity and anti-interference ability.
Experimental Comparison data are as follows：
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, realizes the purpose of creatinine chemiluminescence immunoassay detection, can also do
Several improvement and replacement out, these are improved and replacement also should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of creatinine chemiluminescence immune detection reagent kit, it is characterised in that：Including following reagent：
1) it is coated with working solution：It is placed in containing the 0.025-0.25mg/ml magnetic particle packet in bovine serum albumin(BSA) and preservative buffer
The anti-creatinine monoclonal antibody of quilt；
2) mark working liquid：The 0.001-0.01mg/ml acridinium ester being placed in the buffer containing bovine serum albumin(BSA) and preservative
The creatinine conjugate antigen of label；
3) calibration solution 1：Calibration product containing low concentration creatinine antigen；
4) calibration solution 2：Calibration product containing high concentration creatinine antigen；
5) exciting liquid 1：The HNO3 of 0.1N containing 0.25%-1.25%H2O2；
6) exciting liquid 2：The NaOH of 0.25N containing 0.2%-2%Triton-X100.
2. a kind of creatinine chemiluminescence immune detection reagent kit according to claim 1, wherein it is described by coating working solution,
The inspection with the use of completion to creatinine of mark working liquid, calibration solution 1,2 six kinds of calibration solution 2, exciting liquid 1 and exciting liquid reagents
It surveys.
3. being coated with the preparation method of working solution according to claim 1, include the following steps：
1) a certain amount of magnetic bead stoste is taken, is cleaned magnetic bead 2 times using magnetic bead cleaning solution, a certain amount of MES buffering is then added
Liquid completes the cleaning dilution of magnetic bead stoste；
2) a certain amount of EDC and NHS is added in magnetic bead dilution, is placed on concussion in 37 DEG C of environment and mixes 30 minutes -60
Minute, complete the activation of magnetic bead；
3) activated magnetic bead is used into buffer solution for cleaning twice, a certain amount of MES is added, while matching antibody processed working solution, so
Antibody working solution is added in magnetic bead solution afterwards, concussion in 37 DEG C of environment is placed on and mixes -480 minutes 120 minutes, complete antibody
Magnetic bead coupling；
4) magnetic bead after coupling is rinsed twice using buffer, a certain amount of Block buffer is added, prevents the ring at 37 DEG C
Concussion mixes 30 minutes to 60 minutes in border, completes the closing of magnetic bead；
5) magnetic bead after closing is used into buffer solution for cleaning twice, magnetic bead is added and saves liquid, 2-8 DEG C of environment is placed on after mixing
The preservation of magnetic bead is completed in middle preservation；
6) magnetic bead being coated with is diluted to 0.025-0.25mg/ml, the filling environment for being stored in 2-8 DEG C using buffer is saved
In, the preparation of coating working solution is completed, i.e. magnetic bead is coated with creatinine monoclonal antibody.
4. it is coated with the preparation method of working solution according to claim 3, wherein the EDC is water-soluble carbodiimide,
NHS is n-hydroxysuccinimide.
5. the preparation method of mark working liquid according to claim 1, includes the following steps：
1) acridinium ester solid powder is diluted to 5mg/ml using DMF, packing is saved into -20 DEG C of environment, takes a word used for translation of 5mg/ml
Pyridine ester solution is diluted to 1mg/ml using DMF, and the working concentration that makes marks is used；
2) creatinine monoclonal antibody is used into carbonic acid buffer dialysed overnight, completes antibody dialysis；
3) the antibody 1mg after taking dialysis good, is diluted to 1ml, is added into the 1mg/ml acridinium ester work also of 100ul, in room temperature
It mixes -480 minutes 120 minutes, completes acridinium ester and be coupled；
4) 10% lysine 100ul is added into the acridinium ester working solution after connection, room temperature mixes 30 minutes, is quenched completely not anti-
The acridinium ester answered is completed to be quenched；
5) acridinium ester being quenched is purified using desalting column；
6) isometric phosphate buffer containing 5%BSA is added after purification, is placed in -20 DEG C of environment and saves；
7) acridinium ester marked is diluted to 0.001-0.01mg/ml, the filling ring for being stored in 2-8 DEG C using buffer is saved
Within the border, the configuration of acridinium ester label working solution is completed.
6. the preparation method of mark working liquid according to claim 5, the DMF is n,N-Dimethylformamide.
7. the preparation method of calibration solution 1 and calibration solution 2 according to claim 1, includes the following steps：
It by creatinine antigen using calibration object diluted to the concentration of 150umol/L and 1500umolL, dispenses and packs, protect
There are in 2-8 DEG C of environment.
8. the preparation method of exciting liquid 1 according to claim 1, includes the following steps：
The concentrated nitric acid and hydrogenperoxide steam generator for taking corresponding amount are formulated into required concentration, and packing is stored in 2-8 DEG C of environment, complete
The configuration of exciting liquid 1.
9. the preparation method of exciting liquid 2 according to claim 1, includes the following steps：
Sodium hydroxide and the Triton X-100 mixing for taking corresponding amount, are formulated into required concentration, packing is stored in 2-8 DEG C of environment
In, complete the configuration of exciting liquid 2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113125740A (en) * | 2021-05-17 | 2021-07-16 | 郑州安图生物工程股份有限公司 | Method for identifying magnetic particle activation efficiency in chemiluminescence immunoassay technology |
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Application publication date: 20181127 |