CN107942072A - 1 detection kit of the injury of kidney factor - Google Patents

1 detection kit of the injury of kidney factor Download PDF

Info

Publication number
CN107942072A
CN107942072A CN201711145688.2A CN201711145688A CN107942072A CN 107942072 A CN107942072 A CN 107942072A CN 201711145688 A CN201711145688 A CN 201711145688A CN 107942072 A CN107942072 A CN 107942072A
Authority
CN
China
Prior art keywords
solution
kim
magnetic bead
injury
liquid storage
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201711145688.2A
Other languages
Chinese (zh)
Inventor
王保君
汤双双
欧卫军
唐启伟
徐艳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NANTONG EGENS BIOTECHNOLOGY CO Ltd
Original Assignee
NANTONG EGENS BIOTECHNOLOGY CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by NANTONG EGENS BIOTECHNOLOGY CO Ltd filed Critical NANTONG EGENS BIOTECHNOLOGY CO Ltd
Priority to CN201711145688.2A priority Critical patent/CN107942072A/en
Publication of CN107942072A publication Critical patent/CN107942072A/en
Pending legal-status Critical Current

Links

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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of 1 detection kit of the injury of kidney factor, including seven storage assemblies, is respectively used to 1 calibration objects of storage KIM, 1 quality-control products of KIM, enzyme conjugates working solution, magnetic bead working solution, cleaning solution, substrate solution and pre-treatment reagent；Magnetic bead working solution includes the carboxyl magnetic bead for being marked with 1 antibody of KIM, and enzyme conjugates working solution includes 1 antibody of anti-KIM of alkali phosphatase enzyme mark.The invention also discloses the detection method of the detection injury of kidney factor 1.Detection kit disclosed by the invention, lowest detection are limited to 0.1ng/ml；The range of linearity is 0.1 1000ng/ml, and detection sensitivity is high, and the range of linearity is wide, and detection duration foreshortens to 15 minutes, and simplifies detecting step.

Description

1 detection kit of the injury of kidney factor
Technical field
The present invention relates to field of biological detection, and in particular to a kind of 1 detection kit of the injury of kidney factor.
Background technology
Acute injury of kidney (Acutekidneyinjury, AKI) is as caused by many reasons, can be occurred in various clinics In the case of a kind of complicated renal hypofunction syndrome.It can show as the horizontal slight rise of serum creatinine, can also table It is now acute renal failure.Research finds that the generation of AKI and the increase of case fatality rate are closely related.This requires doctor in clinical work It can early diagnose in work, preferably be begun to decline in glomerular filtration rate(GFR, or only tissue damage and glomerular filtration rate(GFR be still just The normal stage finds and intervenes in time, to improve prognosis, reduce case fatality rate.Serum creatinine and urine volume are the diagnosis indexes of current AKI, But influenced be subject to several factors, Sensitivity and Specificity is not high.Lagged because AKI is diagnosed, often affect the best period for the treatment of adversely.Seek Ask and have become one of current research hot spot with hypersensitivity, specificity, the new biomarker that easily detects.
The injury of kidney factor 1 (Kidneyinjurymolec μ le-1, KIM-1) is a kind of adhesion molecule, it may be possible to Yi Zhongxin Immunoglobulin superfamily a member.Find that KIM-1 genes are ischemia/reperfusions using biochip technology research Most significant gene is expressed in model kidney.In recent years zoopery shows that KIM-1 may be more sensitive compared with other markers. Under physiological conditions, KIM-1 only has faint expression in liver,kidney,spleen, and its expression significantly increases in AKI.The mankind and rodent After the proximal tubular damage of animal, urine is shedded into positioned at extracellular KIM-1 albumen.More and more study each Confirmed in the animal model of AKI caused by kind of ischemic, renal toxicity and kidney transplant etc. KIM-1 can be used as in early days, it is noninvasive and special AKI diagnosis markers.KIM-1 has its advantage as new candidate's AKI early diagnosis markers：1. sensitiveness is high, it is normal Tissue is not expressed, and when AKI occurs, it is just significantly raised in the expression of proximal tubular epithelial cells, and is continued until that cell is complete Full rehabilitation；2. specificity is very high, particularly to ischemic or renal toxicity AKI specificity highers, in other organs (in document in recent years In only find KIM-1 elevated reports when having clear cell carcinoma of ovary) seldom expression；After 3. its extracellular portion sheds into urine Nature comparison is stablized, easily detection；4. seldom influenced be subject to chronic renal disease and urinary tract infections.
Chinese patent CN104730250A discloses a kind of enzyme linked immunological kit of people's kidney injury molecule, including identification people The monoclonal antibody of kidney injury molecule different epitopes is respectively as capture antibody and detection antibody, with people's injury of kidney of recombination expression Molecule is as antigen standard.Detection method is：Urine specimen elder generation centrifugal treating, takes supernatant, capture antibody and supernatant knot Close, form capture antibody-KIM1 compounds, antibody is detected adding, forming detection antibody-capture with above-mentioned mixed binding resists Body-KIM1 compounds, add nitrite ion and develop the color, and luminous value is detected under 450nm wavelength by microplate reader, is calculated in sample The content of KIM-1.Kit detection duration at least three hours, detection duration is too long, can not meet current clinical demand.
The content of the invention
Therefore, it is too long of the technical problem to be solved in the present invention is the detection duration of the injury of kidney factor 1 is detected in the prior art Defect.And then provide a kind of detection kit for detecting quick and high sensitivity the detection injury of kidney factor 1.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of 1 detection kit of the injury of kidney factor, including
First liquid storage component, for storing KIM-1 calibration objects；
Second liquid storage component, for storing enzyme conjugates working solution；
3rd liquid storage component, for storing magnetic bead working solution；
4th liquid storage component, for storing cleaning solution；
5th liquid storage component, for storing substrate solution；
6th liquid storage component, for storing pre-treatment reagent；
7th liquid storage component, for storing KIM-1 quality-control products.
Optionally, the magnetic bead working solution includes the carboxyl magnetic bead for being marked with KIM-1 antibody；The particle diameter of the magnetic bead is 1- 5μm。
Optionally, it is 0.1~1mg/ml that the mark, which has the concentration of the carboxyl magnetic bead of antibody,.
Optionally, the enzyme conjugates working solution includes the anti-KIM-1 antibody of alkali phosphatase enzyme mark.
Optionally, the concentration of the anti-KIM-1 antibody of the alkali phosphatase enzyme mark is 0.5~2 μ g/ml.
Optionally, the KIM-1 calibration objects and the KIM-1 quality-control products are the HEPES solution containing KIM-1 antigens.
Optionally, the substrate solution is enzyme-catalyzed chemical luminescence substrate solution.
Optionally, the pre-treatment reagent is the buffer solution for including NaCl, sucrose, glycerine, polysorbas20 and preservative.
The present invention also provides a kind of method for detecting the injury of kidney factor 1,1 detection kit of the injury of kidney factor is used Detection, includes the following steps：
(1) sample to be tested, KIM-1 calibration objects and KIM-1 quality-control products are added separately in pre-treatment reagent, after mixing Magnetic bead working solution, enzyme conjugates working solution are added, 37 DEG C of -42 DEG C of reaction 5min-10min, obtain the first reaction solution after mixing；
(2) first reaction solution is subjected to Magneto separate, collects magnetic bead；The magnetic bead is cleaned by cleaning solution；
(3) substrate solution is added in the magnetic bead after cleaning, obtains mixed liquor；Detect the luminous strong of the mixed liquor Degree.
Optionally, the sample to be tested, the KIM-1 calibration objects or the KIM-1 quality-control products and the pre-treatment reagent Ratio be 1：1.
Optionally, the ratio of the magnetic bead working solution, the enzyme conjugates working solution and the substrate solution is 1:1:2.
The above technical solution of the present invention has the following advantages over the prior art：
1. detection kit provided in an embodiment of the present invention is combined detection injury of kidney with magnetic bead using chemiluminescence law technology The factor 1, passes through pre-treatment reagent, the cooperation of enzyme conjugates working solution, magnetic bead working solution and substrate solution so that detection kidney damage The lowest detection for hindering the factor 1 is limited to 0.1ng/ml；The range of linearity is 0.1-1000ng/ml, linearly dependent coefficient r>0.99；； High sensitivity, the range of linearity are wide.Compared with prior art, detection time can foreshorten to 15min, substantially reduce detection duration.
2. detection kit provided in an embodiment of the present invention includes pre-treatment reagent, enzyme conjugates working solution and magnetic bead work Liquid, without the processing such as being filtered, being centrifuged to urine specimen, adds pre-treatment reagent, be directly added into enzyme conjugates working solution and Magnetic bead working solution, simplifies detecting step, improves detection efficiency.
3. detection kit provided in an embodiment of the present invention provides KIM-1 calibration objects and KIM-1 quality-control products, inspection ensure that Survey the accuracy of result.
4. the method for the detection injury of kidney factor 1 provided in an embodiment of the present invention, easy to operate, detection time is short, sensitivity Height, the range of linearity are wide.
Embodiment
In order to make the object, technical solutions and advantages of the present invention clearer, illustrate this below by way of specific embodiment The embodiment of invention, unless otherwise stated, disclosed in this invention experimental method use the art routine techniques.
Embodiment 1
The present embodiment provides a kind of method for preparing enzyme conjugates, comprise the following steps：
1. weighing appropriate Traut ' s reagents, it is made into antibody activation buffer solution (100mM Triethanolamine buffers, pH8.5) Into 1.376mg/mL solution.
2. weighing appropriate Sulfo-SMCC reagents, it is 17.5mg/mL solution to be made into concentration with dimethylformamide DMF.
3. KIM-1 labelled antibodies are stored in antibody activation buffer solution (100mM Triethanolamine buffers, pH8.5), concentration For 2mg/mL；Traut ' s the solution that step 1 is prepared, KIM-1 labelled antibodies and Traut ' s reagents mole are added into the solution Than for 1:15, mix at once, 25 DEG C stand reaction 15 minutes.
It is molten that 4. the Sulfo-SMCC that step 2 is prepared is added into alkaline phosphatase enzyme solutions (AP solution, concentration 20mg/ml) Liquid, alkaline phosphatase and Sulfo-SMCC molar ratios 1：10, mix at once, 25 DEG C stand reaction 15 minutes.
5. in the antibody-solutions terminated to step 3 priming reaction add 1M glycine solution, glycine with The molar ratio of Traut ' s reagents is 1:10, mix at once, 25 DEG C stand reaction 10 minutes.
6. the glycine solution of 1M, glycine and Sulfo- are added in the AP solution terminated to step 4 priming reaction The molar ratio of SMCC reagents is 1:10, mix at once, 25 DEG C stand reaction 10 minutes.
7. step 5 to be reacted to the antibody-solutions desalination at once of end, (100mM triethanolamines are replaced in cross-linking buffer Buffer solution, pH7.3), ultraviolet spectrophotometry measures antibody concentration；
8. step 6 to be reacted to the AP solution desalination at once of end, replace in cross-linking buffer that (100mM triethanolamines delay Fliud flushing, pH7.3), ultraviolet spectrophotometry measures AP concentration；
9. the antibody-solutions that step 7 has activated are mixed with the AP solution that step 8 has activated, the KIM-1 marks of activation are anti- The molar ratio of body and the alkaline phosphatase of activation is 1：1, fully mix, when 2~8 DEG C of standing reactions 18 are small.
10. weighing appropriate NEM reagents, it is made into antibody activation buffer solution (100mM Triethanolamine buffers, pH7.3) 12.5mg/mL solution.
11. adding the NEM solution that 1 percent debulking steps 10 are prepared in the solution terminated to step 9 cross-linking reaction, fill Divide and mix, 25 DEG C stand reaction 30 minutes to close unnecessary sulfydryl.
12. the cross-linking agent solution that step 11 is terminated to reaction end is purified by modes such as dialysis, desalination or ultrafiltration concentrations.
13. will enzyme conjugates concentrate measured concentration after purification, add isometric glycerine, fully mix, -20 DEG C of preservations are standby With.
Embodiment 2
The present embodiment provides a kind of method for preparing enzyme conjugates, comprise the following steps：
1. weighing appropriate Traut ' s reagents, it is made into antibody activation buffer solution (100mM Triethanolamine buffers, pH8.5) Into 1.376mg/mL solution.
2. weighing appropriate Sulfo-SMCC reagents, it is 17.5mg/mL solution to be made into concentration with dimethylformamide DMF.
3. KIM-1 labelled antibodies are stored in antibody activation buffer solution (100mM Triethanolamine buffers, pH8.5), concentration For 5mg/mL；Traut ' s the solution that step 1 is prepared, KIM-1 labelled antibodies and Traut ' s reagents mole are added into the solution Than for 1:30, mix at once, 25 DEG C stand reaction 15 minutes.
It is molten that 4. the Sulfo-SMCC that step 2 is prepared is added into alkaline phosphatase enzyme solutions (AP solution, concentration 20mg/ml) Liquid, alkaline phosphatase and Sulfo-SMCC molar ratios 1：15, mix at once, 25 DEG C stand reaction 15 minutes.
5. in the antibody-solutions terminated to step 3 priming reaction add 1M glycine solution, glycine with The molar ratio of Traut ' s reagents is 1:20, mix at once, 25 DEG C stand reaction 10 minutes.
6. the glycine solution of 1M, glycine and Sulfo- are added in the AP solution terminated to step 4 priming reaction The molar ratio of SMCC reagents is 1:20, mix at once, 25 DEG C stand reaction 10 minutes.
7. step 5 to be reacted to the antibody-solutions desalination at once of end, (100mM triethanolamines are replaced in cross-linking buffer Buffer solution, pH7.3), ultraviolet spectrophotometry measures antibody concentration；
8. step 6 to be reacted to the AP solution desalination at once of end, replace in cross-linking buffer that (100mM triethanolamines delay Fliud flushing, pH7.3), ultraviolet spectrophotometry measures AP concentration；
9. the antibody-solutions that step 7 has activated are mixed with the AP solution that step 8 has activated, the KIM-1 marks of activation are anti- The molar ratio of body and the alkaline phosphatase of activation is 1：2, fully mix, when 2~8 DEG C of standing reactions 24 are small.
10. weighing appropriate NEM reagents, it is made into antibody activation buffer solution (100mM Triethanolamine buffers, pH7.3) 12.5mg/mL solution.
11. adding the NEM solution that 1 percent debulking steps 10 are prepared in the solution terminated to step 9 cross-linking reaction, fill Divide and mix, 25 DEG C stand reaction 30 minutes to close unnecessary sulfydryl.
12. the cross-linking agent solution that step 11 is terminated to reaction end is purified by modes such as dialysis, desalination or ultrafiltration concentrations.
13. will enzyme conjugates concentrate measured concentration after purification, add isometric glycerine, fully mix, -20 DEG C of preservations are standby With.
Embodiment 3
The present embodiment provides a kind of coated method of magnetic bead, comprise the following steps：
1. KIM-1 coated antibodies are stored in the 100mM MES buffer solutions of pH5.0, concentration 1mg/mL；
2. taking the carboxyl magnetic bead solution of 100 times of antibody weight, carboxyl magnetic bead particle diameter is 3 μm, and Magneto separate abandons supernatant；
3. washing：The magnetic bead that above-mentioned steps obtain is dissolved again with the 100mM MES buffer solutions of pH5.0, and Magneto separate is abandoned Clearly.
4. repeat step 3 is once
5. add the magnetic bead that appropriate 100mM MES buffer solution steps 4 obtain.
6. appropriate NHS reagents are weighed, the solution for being 10mg/mL into concentration with the 100mM MES buffer solutions of pH5.0；
7. appropriate EDC reagents are weighed, the solution for being 10mg/mL into concentration with the 100mM MES buffer solutions of pH5.0；
8. the magnetic bead solution obtained to step 5 adds the NHS solution of the step 6 of 0.23 times of magnetic bead weight and enters 0.1 times of magnetic The EDC solution of the step 7 of pearl weight, reacts at room temperature 30 minutes on blending instrument.
9. magnetic bead solution Magneto separate after reaction, abandons supernatant.
10. add the magnetic bead that appropriate 100mM MES buffer solution steps 9 obtain.
11. the KIM-1 coated antibodies of step 1 are added in the magnetic bead that step 10 obtains, KIM-1 coated antibodies and carboxyl Magnetic bead solution quality ratio is 1:100, when room temperature cross-linking reaction 16 is small on blending instrument).
12. after crosslinking, Magneto separate, supernatant, which is collected, surveys crosslinking rate.Repeat step 3 is twice.
13. the magnetic bead obtained to step 12 adds magnetic bead confining liquid, when room temperature reaction 16 is small on blending instrument.
14. above-mentioned magnetic bead solution Magneto separate after reaction, abandons supernatant.
15. wash magnetic bead 3 times with magnetic bead cleaning solution.
16. the magnetic bead after washing is resuspended in magnetic bead and preserves in liquid, 2~8 DEG C save backup.
Embodiment 4
The present embodiment provides a kind of coated method of magnetic bead, comprise the following steps：
1. KIM-1 coated antibodies are stored in the 100mM MES buffer solutions of pH5.0, concentration 5mg/mL；
2. taking the carboxyl magnetic bead solution of 200 times of antibody weight, carboxyl magnetic bead particle diameter is 1.5 μm, and Magneto separate abandons supernatant；
3. washing：The magnetic bead that above-mentioned steps obtain is dissolved again with the 100mM MES buffer solutions of pH5.0, and Magneto separate is abandoned Clearly.
4. repeat step 3 is once
5. add the magnetic bead that appropriate 100mM MES buffer solution steps 4 obtain.
6. appropriate NHS reagents are weighed, the solution for being 10mg/mL into concentration with the 100mM MES buffer solutions of pH5.0；
7. appropriate EDC reagents are weighed, the solution for being 10mg/mL into concentration with the 100mM MES buffer solutions of pH5.0；
8. the magnetic bead solution obtained to step 5 adds the NHS solution of the step 6 of 0.23 times of magnetic bead weight and enters 0.1 times of magnetic The EDC solution of the step 7 of pearl weight, reacts at room temperature 30 minutes on blending instrument.
9. magnetic bead solution Magneto separate after reaction, abandons supernatant.
10. add the magnetic bead that appropriate 100mM MES buffer solution steps 9 obtain.
11. the KIM-1 coated antibodies of step 1 are added in the magnetic bead that step 10 obtains, KIM-1 coated antibodies and carboxyl Magnetic bead solution quality ratio is 1:200, when room temperature cross-linking reaction 24 is small on blending instrument.
12. after crosslinking, Magneto separate, supernatant, which is collected, surveys crosslinking rate.Repeat step 3 is twice.
13. the magnetic bead obtained to step 12 adds magnetic bead confining liquid, when room temperature reaction 24 is small on blending instrument.
14. above-mentioned magnetic bead solution Magneto separate after reaction, abandons supernatant.
15. wash magnetic bead 3 times with magnetic bead cleaning solution.
16. the magnetic bead after washing is resuspended in magnetic bead and preserves in liquid, 2~8 DEG C save backup.
Embodiment 5
The present embodiment provides a kind of detection kit, including the first liquid storage component, the second liquid storage component, the 3rd liquid storage group Part, the 4th liquid storage component, the 5th liquid storage component, the 6th liquid storage component, the 7th liquid storage component.Wherein, the first liquid storage component is used for KIM-1 calibration objects are stored, the second liquid storage component is used to store enzyme conjugates working solution, and the 3rd liquid storage component is used to store magnetic bead work Make liquid, the 4th liquid storage component is used to store cleaning solution, and the 5th liquid storage component is used to store substrate solution, and the 6th liquid storage component is used for Pre-treatment reagent is stored, the 7th liquid storage component is used to store KIM-1 quality-control products.
As one embodiment of the present of invention, magnetic bead working solution includes the carboxyl magnetic bead for being marked with KIM-1 antibody；Magnetic bead Particle diameter is 3 μm, and the concentration for being marked with the carboxyl magnetic bead of KIM-1 antibody is 0.5mg/ml；Enzyme conjugates working solution includes alkaline phosphorus The anti-KIM-1 antibody of sour enzyme mark, the concentration of the anti-KIM-1 antibody of alkali phosphatase enzyme mark is 2 μ g/ml；KIM-1 calibration objects and KIM-1 quality-control products are the HEPES solution containing KIM-1 antigens；Substrate solution is enzyme-catalyzed chemical luminescence substrate solution；Pre-treatment tries Agent is to include the buffer solution of NaCl, sucrose, glycerine, polysorbas20 and preservative.
It is specific as follows the present embodiment provides a kind of 1 detection kit component collocation method of the injury of kidney factor：
1. calibration object and quality-control product configuration
By KIM-1 antigens by definite concentration of tracing to the source, KIM-1 calibration objects and KIM-1 are prepared with calibration object diluted Quality-control product.Such as the concentration of calibration object is respectively：1,10,100,200,400,800ng/ml, quality-control product concentration is 100ng/ml.
The specific component of calibration object dilution is：50mM HEPES, 250mmol/L NaCl, 1%BSA and 1% preservative, PH is 7.5；Preservative is preferably Proclin-300.
2. pre-treatment reagent
Pre-treatment reagent is buffer solution, contains 100mM Tris；150mM NaCl；5% sucrose；5% glycerine；0.1%BSA With 0.15% preservative；Preservative is preferably Proclin-300.
3. enzyme conjugates working solution
Enzyme conjugates concentrate in embodiment 1 or embodiment 2 is diluted with enzyme combination diluent, is configured to suitable dense The kit of degree enzyme conjugates working solution, enzyme conjugates working solution concentration are 2 μ g/ml.The specific component of enzyme combination diluent For：50mM MES, 250mmol/L NaCl, 1%BSA, 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.15mmol/L ZnCl2、0.15mmol/L MgCl2With 0.15% preservative, pH pH6.5；Preservative is preferably Proclin-300.
4. magnetic bead working solution (M)
Magnetic bead in embodiment 3 or embodiment 4 is preserved into liquid magnetic bead diluted, is configured to the reagent of suitable concentration Box magnetic bead working solution, magnetic bead working solution concentration are 0.5mg/ml.The specific component of magnetic bead dilution is：The HEPES of 50mM, 250mmol/L NaCl, 1%BSA, 1% sucrose, 1% gelatin, 0.1% polysorbas20,1%PVP360 and 3% preservative, pH are 8.0；Preservative is preferably Proclin-300.
5. substrate solution
Accurately weigh 3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6. cleaning solution
Trishydroxymethylaminomethane 1211.4mg, NaCl 9g, polysorbas20 1g, water 900mL are added into container successively, is mixed It is even to be dissolved to each component, pH value of solution is adjusted to 8.0 ± 0.5 with sodium hydroxide solution, is added water and is settled to 1000mL, mixes 30 points Clock, 0.22 μm of filtering obtain cleaning solution.
Embodiment 6
The present embodiment provides a kind of detection kit, including the first liquid storage component, the second liquid storage component, the 3rd liquid storage group Part, the 4th liquid storage component, the 5th liquid storage component, the 6th liquid storage component, the 7th liquid storage component.Wherein, the first liquid storage component is used for KIM-1 calibration objects are stored, the second liquid storage component is used to store enzyme conjugates working solution, and the 3rd liquid storage component is used to store magnetic bead work Make liquid, the 4th liquid storage component is used to store cleaning solution, and the 5th liquid storage component is used to store substrate solution, and the 6th liquid storage component is used for Pre-treatment reagent is stored, the 7th liquid storage component is used to store KIM-1 quality-control products.
As one embodiment of the present of invention, magnetic bead working solution includes the carboxyl magnetic bead for being marked with KIM-1 antibody；Magnetic bead Particle diameter is 3 μm, and the concentration for being marked with the carboxyl magnetic bead of KIM-1 antibody is 0.1mg/ml；Enzyme conjugates working solution includes alkaline phosphorus The anti-KIM-1 antibody of sour enzyme mark, the concentration of the anti-KIM-1 antibody of alkali phosphatase enzyme mark is 0.5 μ g/ml；KIM-1 calibration objects It is the HEPES solution containing KIM-1 antigens with KIM-1 quality-control products；Substrate solution is enzyme-catalyzed chemical luminescence substrate solution；Pre-treatment Reagent is the buffer solution for including NaCl, sucrose, glycerine, polysorbas20 and preservative.
It is specific as follows the present embodiment provides a kind of 1 detection kit component collocation method of the injury of kidney factor：
1. calibration object and quality-control product configuration
By KIM-1 antigens by definite concentration of tracing to the source, KIM-1 calibration objects and KIM-1 are prepared with calibration object diluted Quality-control product.Such as the concentration of calibration object is respectively 1,10,100,200,400,800ng/ml, quality-control product concentration is 100ng/ml.
The specific component of calibration object dilution is：50mM HEPES, 250mmol/L NaCl, 1%BSA and 1% preservative, PH is 7.5；Preservative is preferably Proclin-300.
2. pre-treatment reagent
Pre-treatment reagent is buffer solution, contains 100mM Tris；150mM NaCl；5% sucrose；5% glycerine；0.1%BSA With 0.15% preservative；Preservative is preferably Proclin-300.
3. enzyme conjugates working solution
Enzyme conjugates concentrate in embodiment 1 or embodiment 2 is diluted with enzyme combination diluent, is configured to suitable dense The kit of degree enzyme conjugates working solution, enzyme conjugates working solution concentration are 0.5 μ g/ml.Enzyme combination diluent specifically into It is divided into：20mM MES, 250mmol/L NaCl, 1%BSA, 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.15mmol/L ZnCl2、0.15mmol/L MgCl2With 0.15% preservative, pH pH6.5；Preservative is preferably Proclin-300.
4. magnetic bead working solution (M)
Magnetic bead in embodiment 2 is preserved into liquid magnetic bead diluted, is configured to the kit magnetic bead of suitable concentration Working solution, magnetic bead working solution concentration are 0.1mg/ml.The specific component of magnetic bead dilution is：HEPES, 250mmol/L of 20mM NaCl, 1%BSA, 1% sucrose, 1% gelatin, 0.1% polysorbas20,1%PVP360 and 3% preservative, pH 8.0；Preservative is excellent Elect Proclin-300 as.
5. substrate solution
Accurately weigh 3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6. cleaning solution
Trishydroxymethylaminomethane 1211.4mg, NaCl9g, polysorbas20 1g, water 900mL are added into container successively, is mixed Dissolved to each component, adjust pH value of solution to 8.0 ± 0.5 with sodium hydroxide solution, add water and be settled to 1000mL, mix 30 points Clock, 0.22 μm of filtering obtain cleaning solution.
Embodiment 7
The present embodiment provides a kind of detection kit, including the first liquid storage component, the second liquid storage component, the 3rd liquid storage group Part, the 4th liquid storage component, the 5th liquid storage component, the 6th liquid storage component, the 7th liquid storage component.Wherein, the first liquid storage component is used for KIM-1 calibration objects are stored, the second liquid storage component is used to store enzyme conjugates working solution, and the 3rd liquid storage component is used to store magnetic bead work Make liquid, the 4th liquid storage component is used to store cleaning solution, and the 5th liquid storage component is used to store substrate solution, and the 6th liquid storage component is used for Pre-treatment reagent is stored, the 7th liquid storage component is used to store KIM-1 quality-control products.
As one embodiment of the present of invention, magnetic bead working solution includes the carboxyl magnetic bead for being marked with KIM-1 antibody；Magnetic bead Particle diameter is 3 μm, and the concentration for being marked with the carboxyl magnetic bead of KIM-1 antibody is 1mg/ml；Enzyme conjugates working solution includes alkaline phosphatase The anti-KIM-1 antibody of enzyme mark, the concentration of the anti-KIM-1 antibody of alkali phosphatase enzyme mark is 1 μ g/ml；KIM-1 calibration objects and KIM-1 quality-control products are the HEPES solution containing KIM-1 antigens；Substrate solution is enzyme-catalyzed chemical luminescence substrate solution；Pre-treatment tries Agent is to include the buffer solution of NaCl, sucrose, glycerine, polysorbas20 and preservative.
It is specific as follows the present embodiment provides a kind of 1 detection kit component collocation method of the injury of kidney factor：
1. calibration object and quality-control product configuration
By KIM-1 antigens by definite concentration of tracing to the source, KIM-1 calibration objects and KIM-1 are prepared with calibration object diluted Quality-control product.Such as the concentration of calibration object is respectively：1,10,100,200,400,800ng/ml, quality-control product concentration is 100ng/ml.
The specific component of calibration object dilution is：50mM HEPES, 250mmol/L NaCl, 1%BSA and 1% preservative, PH is 7.5；Preservative is preferably Proclin-300.
2. pre-treatment reagent
Pre-treatment reagent is buffer solution, contains 100mM Tris；150mM NaCl；5% sucrose；5% glycerine；0.1%BSA With 0.15% preservative；Preservative is preferably Proclin-300.
3. enzyme conjugates working solution
Enzyme conjugates concentrate in embodiment 1 or embodiment 2 is diluted with enzyme combination diluent, is configured to suitable dense The kit of degree enzyme conjugates working solution, enzyme conjugates working solution concentration are 1 μ g/ml.The specific component of enzyme combination diluent For：30mM MES, 250mmol/L NaCl, 1%BSA, 5% sucrose, 5% glycerine, 0.1% polysorbas20,0.15mmol/L ZnCl2、0.15mmol/L MgCl2With 0.15% preservative, pH pH6.5；Preservative is preferably Proclin-300.
4. magnetic bead working solution (M)
Magnetic bead in embodiment 2 is preserved into liquid magnetic bead diluted, is configured to the kit magnetic bead of suitable concentration Working solution, magnetic bead working solution concentration are 1mg/ml.The specific component of magnetic bead dilution is：HEPES, 250mmol/LNaCl of 30mM, 1%BSA, 1% sucrose, 1% gelatin, 0.1% polysorbas20,1%PVP360 and 3% preservative, pH 8.0；Preservative is preferably Proclin-300。
5. substrate solution
Accurately weigh 3- (the spiral adamantane of 2-) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxanes two Sodium salt (AMPPD) 0.5g, trishydroxymethylaminomethane 18g, NaCl 100g, hexadecyltrimethylammonium chloride 0.02g, water are fixed It is 9.4 ± 0.05 to hold to 1000mL, adjustment chemical luminous substrate pH value of solution；This chemical luminous substrate is for alkaline phosphatase Epidioxy ethane substrate, it is necessary to be kept in dark place under the conditions of 2~8 DEG C, the used time slowly mixes again.
6. cleaning solution
Trishydroxymethylaminomethane 1211.4mg, NaCl 9g, polysorbas20 1g, water 900mL are added into container successively, is mixed It is even to be dissolved to each component, pH value of solution is adjusted to 8.0 ± 0.5 with sodium hydroxide solution, is added water and is settled to 1000mL, mixes 30 points Clock, 0.22 μm of filtering obtain cleaning solution.
Embodiment 8
The step of the present embodiment provides the injury of kidney factor 1 in urine is detected using any kits of embodiment 5-7：
1. respectively plus 25 μ lKIM-1 calibration objects, KIM-1 quality-control products and urine specimen are into corresponding test tube；
2. the mixing of 25 μ l pre-treatment reagents is added in each test tube；
Mixed after 3. 50 μ l enzyme conjugates working solutions and 50 μ l magnetic bead working solutions are added in each test tube；42 DEG C of reactions 5min, obtains the first reaction solution；
4. above-mentioned first reaction solution is carried out Magneto separate, magnetic bead is collected；300 μ l cleaning solutions cleaning magnetic is added in each test tube Pearl, repeats 3-5 times, removes cleaning solution；
5. adding 100 μ l substrate solutions in each test tube, luminous value is detected after mixing.
Embodiment 9
The step of the present embodiment provides the injury of kidney factor 1 in urine is detected using any kits of embodiment 5-7：
1. respectively plus 25 μ lKIM-1 calibration objects, KIM-1 quality-control products and urine specimen are into corresponding test tube；
2. the mixing of 25 μ l pre-treatment reagents is added in each test tube；
Mixed after 3. 50 μ l enzyme conjugates working solutions and 50 μ l magnetic bead working solutions are added in each test tube；37 DEG C of reactions 10min, obtains the first reaction solution；
4. above-mentioned first reaction solution is carried out Magneto separate, magnetic bead is collected；300 μ l cleaning solutions cleaning magnetic is added in each test tube Pearl, repeats 3-5 times, removes cleaning solution；
5. adding 100 μ l substrate solutions in each test tube, luminous value is detected after mixing.
1 linear verification of experimental example
Kit Plays product or quality-control product are taken, measure 3 times, is averaged, and is as a result returned with expected concentration at every Straight line, is calculated regression coefficient r ＞ 0.99, illustrates the dilution good linearity of kit provided by the invention.
2 precision of experimental example is verified
The urine high level quality-control product with traceability, each portion of low value quality-control product are taken, 10 inspections are carried out to every part of quality-control product Survey, totally 10 testing results will calculate average value, standard value.According to coefficient of variation CV=(standard deviation/average value) × 100%, it is calculated：CV1(100ng/mL)=3%, CV2(20ng/mL)=3%.It follows that reagent provided by the invention Box has higher precision.
3 sensitivity of experimental example verification,
Take the quality-control product with traceability to be diluted to detection range lower limit (0.1ng/mL) to be nearby measured, replication 3 times, average value is calculated, is compareed afterwards with quality-control product target value.The results show that the kit detected value of the present invention connects with target value Closely, illustrate that kit provided by the invention has higher sensitivity.
4 detection range of experimental example is verified
The standard items with traceability are taken, luminescence phenomenon has been detected whether respectively after diluting different multiples, the results show that In the range of concentration is 0.1-1000ng/ml, there is luminescence phenomenon, the kit detection range for showing the present invention is 0.1- 1000ng/ml。
Obviously, the above embodiments are merely examples for clarifying the description, and the restriction not to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or Among changing still in the protection domain of the invention.

Claims (11)

  1. A kind of 1. 1 detection kit of the injury of kidney factor, it is characterised in that including
    First liquid storage component, for storing KIM-1 calibration objects；
    Second liquid storage component, for storing enzyme conjugates working solution；
    3rd liquid storage component, for storing magnetic bead working solution；
    4th liquid storage component, for storing cleaning solution；
    5th liquid storage component, for storing substrate solution；
    6th liquid storage component, for storing pre-treatment reagent；
    7th liquid storage component, for storing KIM-1 quality-control products.
  2. 2. 1 detection kit of the injury of kidney factor according to claim 1, it is characterised in that the magnetic bead working solution includes It is marked with the carboxyl magnetic bead of KIM-1 antibody；The particle diameter of the magnetic bead is 1-5 μm.
  3. 3. 1 detection kit of the injury of kidney factor according to claim 2, it is characterised in that the mark has antibody The concentration of carboxyl magnetic bead be 0.1-1mg/ml.
  4. 4. 1 detection kit of the injury of kidney factor according to claim 1, it is characterised in that the enzyme conjugates working solution Anti- KIM-1 antibody including alkali phosphatase enzyme mark.
  5. 5. 1 detection kit of the injury of kidney factor according to claim 4, it is characterised in that the alkali phosphatase enzyme mark The concentration of anti-KIM-1 antibody be 0.5~2 μ g/ml.
  6. 6. 1 detection kit of the injury of kidney factor according to claim 1, it is characterised in that the KIM-1 calibration objects and institute It is the HEPES solution containing KIM-1 antigens to state KIM-1 quality-control products.
  7. 7. 1 detection kit of the injury of kidney factor according to claim 1, it is characterised in that the substrate solution is enzymatic Chemical luminous substrate solution.
  8. 8. 1 detection kit of the injury of kidney factor according to claim 1, it is characterised in that the pre-treatment reagent is bag Include the buffer solution of NaCl, sucrose, glycerine, polysorbas20 and preservative.
  9. A kind of 9. method for detecting the injury of kidney factor 1, it is characterised in that usage right requires the detection examination of 1-8 any one of them Agent box detects, and includes the following steps：
    (1) sample to be tested, KIM-1 calibration objects and KIM-1 quality-control products are added separately in pre-treatment reagent, are added after mixing Magnetic bead working solution, enzyme conjugates working solution, 37 DEG C of -42 DEG C of reaction 5min-10min, obtain the first reaction solution after mixing；
    (2) first reaction solution is subjected to Magneto separate, collects magnetic bead；The magnetic bead is cleaned by cleaning solution；
    (3) substrate solution is added in the magnetic bead after cleaning, obtains mixed liquor；Detect the luminous intensity of the mixed liquor.
  10. 10. according to the method described in claim 9, it is characterized in that, the sample to be tested, the KIM-1 calibration objects or described The ratio of KIM-1 quality-control products and the pre-treatment reagent is 1:1.
  11. 11. according to the method described in claim 9, it is characterized in that, the magnetic bead working solution, the enzyme conjugates working solution with The ratio of the substrate solution is 1:1:2.
CN201711145688.2A 2017-11-17 2017-11-17 1 detection kit of the injury of kidney factor Pending CN107942072A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711145688.2A CN107942072A (en) 2017-11-17 2017-11-17 1 detection kit of the injury of kidney factor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711145688.2A CN107942072A (en) 2017-11-17 2017-11-17 1 detection kit of the injury of kidney factor

Publications (1)

Publication Number Publication Date
CN107942072A true CN107942072A (en) 2018-04-20

Family

ID=61932830

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711145688.2A Pending CN107942072A (en) 2017-11-17 2017-11-17 1 detection kit of the injury of kidney factor

Country Status (1)

Country Link
CN (1) CN107942072A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111044724A (en) * 2019-12-22 2020-04-21 上海复星长征医学科学有限公司 Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof
CN112129951A (en) * 2020-09-16 2020-12-25 武汉生之源生物科技股份有限公司 Enzyme-labeled diluent for SAA chemiluminescence enzyme immunoassay and application thereof
CN113702647A (en) * 2021-08-31 2021-11-26 普十生物科技(北京)有限公司 Human growth differentiation factor 15 instant detection kit, preparation method and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102353782A (en) * 2010-12-31 2012-02-15 上海华谷生物技术有限公司 In vitro diagnostic kit of antihuman kidney injury molecule-1 and its detection method
CN104730250A (en) * 2013-12-20 2015-06-24 北京义翘神州生物技术有限公司 Enzyme linked immunosorbent assay kit for detecting human kidney injury molecule-1
CN106370864A (en) * 2016-08-31 2017-02-01 镇江华测金太医学检验所有限公司 Chemiluminiscence box of neutrophile granulocyte gelatinase related lipid transport protein
CN106501504A (en) * 2016-09-26 2017-03-15 王键 Laminin，LN test kit and preparation method thereof
CN106771133A (en) * 2016-11-30 2017-05-31 三诺生物传感股份有限公司 A kind of composition and its application as enzyme mark compound preservation liquid

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102353782A (en) * 2010-12-31 2012-02-15 上海华谷生物技术有限公司 In vitro diagnostic kit of antihuman kidney injury molecule-1 and its detection method
CN104730250A (en) * 2013-12-20 2015-06-24 北京义翘神州生物技术有限公司 Enzyme linked immunosorbent assay kit for detecting human kidney injury molecule-1
CN106370864A (en) * 2016-08-31 2017-02-01 镇江华测金太医学检验所有限公司 Chemiluminiscence box of neutrophile granulocyte gelatinase related lipid transport protein
CN106501504A (en) * 2016-09-26 2017-03-15 王键 Laminin，LN test kit and preparation method thereof
CN106771133A (en) * 2016-11-30 2017-05-31 三诺生物传感股份有限公司 A kind of composition and its application as enzyme mark compound preservation liquid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HTTP://WWW.CAIGOU.COM.CN/NEWS/2012031215.SHTML: "人肾损伤分子1(Kim-1)酶联免疫分析（ELISA）说明书", 《中国教育装备采购网》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111044724A (en) * 2019-12-22 2020-04-21 上海复星长征医学科学有限公司 Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof
CN111044724B (en) * 2019-12-22 2023-11-10 复星诊断科技(上海)有限公司 Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof
CN112129951A (en) * 2020-09-16 2020-12-25 武汉生之源生物科技股份有限公司 Enzyme-labeled diluent for SAA chemiluminescence enzyme immunoassay and application thereof
CN113702647A (en) * 2021-08-31 2021-11-26 普十生物科技(北京)有限公司 Human growth differentiation factor 15 instant detection kit, preparation method and application thereof
CN113702647B (en) * 2021-08-31 2024-03-15 普十生物科技(北京)有限公司 Human growth differentiation factor 15 instant detection kit, preparation method and application thereof

Similar Documents

Publication Publication Date Title
CN104198732B (en) A kind of neutrophil gelatinase-associated lipocalin reagent box for detecting content
CN104849469A (en) Kit for detecting NGAL content and preparation method thereof
JP5554247B2 (en) Method for measuring cystatin C in human body fluid
CN107942069A (en) A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof
CN107656071A (en) A kind of NT ProBNP detection kits and its application method
CN106959367B (en) For measuring the method and kit of the ingredient to be determined in sample
CN101699287B (en) Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof
CN102944679A (en) Kit for performing retinol binding protein detection by using latex turbidimetry
CN107942047A (en) Interleukin-18 detection kit
CN105866401A (en) Method of immunoassaying a component to be measured
CN107942072A (en) 1 detection kit of the injury of kidney factor
CN107918022A (en) A kind of cTnI detection kits and its application method
CN107957495A (en) A kind of CK-MB detection kits and its application method
CN108445222A (en) A kind of kit and preparation method quantitatively detecting cardic fatty acid binding protein
CN102539784B (en) Method for detecting cardiac troponin I and application thereof
CN104535770A (en) Myoglobin determination kit of compound antibody
CN107907691A (en) A kind of myoglobin assay kit and its application method
CN107942051A (en) A kind of D dimer detection kits and its application method
US20090111091A1 (en) Specimen pretreatment liquid, kit for measuring virus, and method for detecting virus
CN108508001A (en) Chemiluminescence detection kit
CN103954776A (en) NGAL (Neutrophil Gelatinase Associated Lipocalin) optical excitation chemiluminescence detection kit and preparation and use methods of kit
CN107656072A (en) Liver fatty acid binding protein detection kit
CN107918021A (en) Neutrophil gelatinase-associated lipocalin detection kit
CN104483477B (en) It is coupled the preparation method of the troponin diagnosis test paper of immunomagnetic beads
CN105181969B (en) A kind of neutrophil gelatinase-associated lipocalin reagent box for detecting content

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20180420