CN105527434A - A kit used for detecting N1,N<12>-diacetylspermine (DAS) - Google Patents

A kit used for detecting N1,N<12>-diacetylspermine (DAS) Download PDF

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CN105527434A
CN105527434A CN201511020936.1A CN201511020936A CN105527434A CN 105527434 A CN105527434 A CN 105527434A CN 201511020936 A CN201511020936 A CN 201511020936A CN 105527434 A CN105527434 A CN 105527434A
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das
spermine
reagent
diacetyl
room temperature
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CN105527434B (en
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李文欣
朱明光
刘峰
宫晓丽
王帅
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Liaoning Maidi biological Polytron Technologies Inc
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LIAONING MAIDI BIOLOGIC TECHNOLOGY Co Ltd
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney

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Abstract

The invention relates to a method of detecting the content of N1,N<12>-diacetylspermine (called as DAS for short), and particularly relates to a kit used for detecting the content of the N1,N<12>-diacetylspermine (DAS), preparing methods of an antigen used in the kit and an antibody used in the kit, a developing method of the kit, and a preparing scheme of the kit. The kit optimizes detecting methods of the content of the DAS. Compared with the prior art, the kit is advantaged by simple and convenient operation, a high detection speed, high precision, good repeatability, capability of allowing detection results to be not influenced by operation, capability of being used for large-scale sample testing, and the like.

Description

A kind of kit being used for diacetyl spermine (DAS) and detecting
Technical field
The present invention relates to and detect diacetyl spermine (N1, N 12-Diacetylspermine, is called for short DAS) method of content, specifically a kind of kit being used for diacetyl spermine (DAS) and detecting.
Background technology
Colorectal cancer comprises colon cancer and the carcinoma of the rectum, colorectal cancer comes the 3rd at the global incidence of disease in malignant tumour, comes the 5th at the incidence of disease of China, and the intestinal cancer incidence of disease of China male sex is 16/100000ths, women is 14/100000ths, is one of modal tumour.Westerner is mainly based on colon, and 70% to 80% patient's trouble is colon cancer, and rectal cancer patient accounts for 20% to 30%.China is contrary, and rectal cancer patient accounts for 60% to 70%, and colon cancer accounts for 30% to 40%.In recent years research and analyse discovery, the carcinoma of the rectum incidence of disease of China remains unchanged substantially, colon cancer disease rates but rising.Although chemotherapy is effective to metastatic colorectal cancer patients, prognosis is poor.Therefore, the kit studied for the preparation of detecting colorectal cancer patients tumor markers content has important social effect and scientific value.
Polyamines comprises putrescine, cadaverine, spermidine and spermine, is the general name that a class has the nitrogenous low molecular weight organic compound of biologically active.The material that in amino molecule, 1-3 hydrogen atom generates after being replaced by alkyl or aryl can be regarded as.Can be produced by protokaryon and eukaryotic.The tissue of quick growth has active polyamines synthesis system, usually containing a large amount of polyamines.When there is such organizing in body, in urine, the synthesis of polyamines can increase.Previous research shows compared with Healthy People, and in cancer patient's urine, the content of polyamines increases.But in follow-up research display urine, total and free polyamine content is owing to creating a large amount of false positives and negative findings, thus can not as reliable tumor markers.
DAS molecular formula is C 14h 30n 4o 2, molecular weight 286.41 is diacetyl derivative of a polyamine species, is generated in vivo by ornithine through ornithine decarboxylase effect.Diacetyl spermine is the metabolic product of tumour cell, is excreted in urine.After cell carcinogenesis, the secretion of acetyl group polyamines increases, and causes diacetyl spermine concentration in tumor patient urine obviously to raise.The early detection of DAS is completed by high performance liquid chromatography (HPLC), establishes the Enzyme-linked Immunosorbent Assay system being applicable to DAS content detection in urine afterwards.Have research display, in urine, the detection of DAS expression can as the effective novel tumor markers of colon cancer, breast cancer and liver cancer.So far, clinical research is widely obtained for the ELISA detection method of DAS content detection in urine.But there is complicated operation equally in ELISA detection method, can not the problem such as batch detection.The automatic clinical chemistry analyzer that utilizes of the present invention's exploitation detects the turbidimetry detection kit of DAS content in clinical samples, a kind of, efficiently detection technique more more convenient than ELISA detection method, can once detect great amount of samples, and it is highly sensitive, simple to operate.
Summary of the invention:
The object of the invention is to provide a kind of diacetyl spermine (DAS) immunoturbidimetry vitro detection kit.
For achieving the above object, the technical solution used in the present invention is:
A kind of diacetyl spermine (DAS) immunoturbidimetry vitro detection kit, comprise R1 reagent, R2 reagent, standard items and quality-control product, described R1 reagent is by diacetyl spermine antibody coupling on nano particle, the reagent preserved with the solid form after liquid form or freeze-drying after adding protection liquid; Described R2 reagent for acetyl spermine (DAS) molecule is coupled on carrier protein by coupling reagent, with after liquid form or freeze-drying solid forms preserve reagent.
Described diacetyl spermine antibody is coupled at the product of gained on carrier protein as immunogene using acetyl spermine (DAS) molecule by coupling reagent, then immune animal obtains polyclonal antibody or obtains monoclonal antibody through screening.
Liquid is protected to be by weight percentage in described R1: the PEG6000 of the sucrose of PEG20000,2-5% of BSA, 0.1-0.5% of PVP40,0.5-1% of Tris, 0.5-2% of 0.1-0.5%, the Sodium azide of TWEEN20,0.02-0.1% of 0.01-0.8%, NaCl, 1-4% of 0.85-1.5%, surplus is water.
Described sample is including but not limited to serum, blood plasma, urine etc.Described nano particle includes but not limited to nm of gold, microballoon etc.Described carrier protein includes but not limited to seralbumin (BSA), poly-D-lysine, ovalbumin etc.
Described coupling reagent is the sub-amide-type of carbon two or maleimide; Wherein, the sub-amide-type of carbon two is EDC, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide or N-hydroxy-succinamide ester class (NHS ester); Maleimide is 4-maleimidobutyric acid-N-succinimide ester (GMBS) or 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC).
Described diacetyl spermine (DAS) molecular conjugate vectors albumen is: using carbodiimide/N-hydroxy-succinamide (EDC/NHS) as coupling reagent, carrier protein after activation process is added in the PBS of the pH=6.5-8.0 of excessive 0.01M-0.1M, add water miscible carbodiimide (EDC) and N-hydroxy-succinamide (NHS) again, mixing is extremely as clear as crystal, then left at room temperature 15-30 minute; Add excessive acetyl spermine after leaving standstill, mixing is to as clear as crystal, and regulation system pH value, to 10-12, reacts 4-6 hour under room temperature; Through dialysis products therefrom after reaction, obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
Be specially: described diacetyl spermine (DAS) molecular conjugate vectors albumen is using carbodiimide/N-hydroxy-succinamide (EDC/NHS) as coupling reagent, 6-15mg carrier protein after activation process is added in the pH=6.5-8.0PBS3-8mL of 0.01M-0.1M, add water miscible carbodiimide (EDC) 7-10mg and N-hydroxy-succinamide (NHS) 3-5mg again, mixing is extremely as clear as crystal, then left at room temperature 15-30 minute; Add acetyl spermine after leaving standstill, mixing is to as clear as crystal, and regulation system pH value is adjusted to 10-12, reacts 4-6 hour under room temperature; Through dialysis products therefrom after reaction, obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
Described diacetyl spermine (DAS) molecular conjugate vectors albumen is: using carbodiimide/N-hydroxy-succinamide (EDC/NHS) as coupling reagent, carrier protein after activation process is added in the PBS of the pH=6.5-8.0 of excessive 0.01M-0.1M, add water miscible carbodiimide (EDC) and N-hydroxy-succinamide (NHS) again, mixing is extremely as clear as crystal, then left at room temperature 15-30 minute; Add spermine after leaving standstill, mixing is to as clear as crystal, and regulation system pH value is adjusted to 10-12, reacts 4-6 hour under room temperature; After reaction through dialysis products therefrom in 2-8 DEG C, the acylating agent added in reaction system, then reacts 1-3 hour again at 2-8 DEG C, and reactant is through dialysing and obtaining diacetyl spermine (DAS) molecular conjugate vectors albumen.
Be specially: described diacetyl spermine (DAS) molecular conjugate vectors albumen is: during using carbodiimide/N-hydroxy-succinamide (EDC/NHS) as coupling reagent, 6-15mg carrier protein after activation process is added in the pH=6.5-8.0PBS3-8mL of 0.01M-0.1M, add water miscible carbodiimide (EDC) 7-10mg and N-hydroxy-succinamide (NHS) 3-5mg again, mixing is extremely as clear as crystal, then left at room temperature 15-30 minute; Add spermine after leaving standstill, mixing is to as clear as crystal, and regulation system pH value is adjusted to 10-12, reacts 4-6 hour under room temperature; After reaction through dialysis products therefrom in 2-8 DEG C, in 3-10mL reaction system, add the acylating agent of 40 μ L-80 μ L, then at 2-8 DEG C, react 1-3 hour again, reactant is through dialysing and obtaining diacetyl spermine (DAS) molecular conjugate vectors albumen.
Described diacetyl spermine (DAS) molecular conjugate vectors albumen is using N-[γ-maleimide butyryl oxygen] succinimide ester (GMBS) as coupling reagent, acylating agent (acetic anhydride or anhydrous acetic acid or acetyl chloride) and spermine are reacted 60-90min at 2-8 DEG C, mix in the PBS of the pH=6.5-8.0 of 0.01M-0.1M with coupling agent (GMBS) after reaction, react 3-4 hour under room temperature, obtain acetyl spermine-GMBS coupled product; By carrier protein with acetyl mercapto succinic acid mix in the PBS of the pH=6.5-8.0 of 0.01M-0.1M, react 3-5 hour under room temperature; Add 0.1M azanol after reaction, react 30-35 minute under room temperature except deacetylate; Dialyse in the PBS of the pH=6.5-8.0 of 0.01M-0.1M, obtain the BSA that sulfhydrylation is modified; Acetyl spermine-GMBS coupled product is mixed with the BSA that above-mentioned sulfhydrylation is modified, under room temperature, reacts 4-6 hour; To dialyse in the PBS of the pH=6.5-8.0 of 0.01M-0.1M to obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
Be specially: described diacetyl spermine (DAS) molecular conjugate vectors albumen is: using N-[γ-maleimide butyryl oxygen] succinimide ester (GMBS) as coupling reagent, the acylating agent of 10-15 μ L and 15-25mg spermine are reacted 60-90min at 2-8 DEG C, mix in the pH=6.5-8.0PBS3-5mL buffer solution of 0.01M-0.1M with 2-4mg coupling agent (GMBS) after reaction, react 3-4 hour under room temperature, obtain acetyl spermine-GMBS coupled product; 8-15mg carrier protein is mixed with the acetyl mercapto succinic acid of 3-7mg in the pH=6.5-8.0PBS3-5mL buffer solution of 0.01M-0.1M, under room temperature, reacts 3-5 hour; Add 0.1M azanol after reaction, react 30-35 minute under room temperature except deacetylate; Dialyse in the pH=6.5-8.0PBS damping fluid of 0.01M-0.1M, obtain the BSA that sulfhydrylation is modified; Acetyl spermine-GMBS coupled product is mixed with the BSA that above-mentioned sulfhydrylation is modified, under room temperature, reacts 4-6 hour; To dialyse in the pH=6.5-8.0PBS damping fluid of 0.01M-0.1M to obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
Described carrier protein activation process is by carrier protein 0.01M-0.1MpH=6.5-8.0PBS buffer solution, adds MES after dissolving, is placed in 2-8 DEG C of environment and reacts half an hour.
。Described acylating agent comprises acetic anhydride, anhydrous acetic acid or acetyl chloride.
Described kit is utilized to detect the diacetyl spermine in sample by immunoturbidimetry A competitive inhibition method.
Specifically, diacetyl spermine (N1, N 12-Diacetylspermine, is called for short DAS) molecular formula is C 14h 30n 4o 2, molecular weight 286.41 is diacetyl derivative of a polyamine species, is generated in vivo by ornithine through ornithine decarboxylase effect.DAS is originally as small-molecule substance, it is haptens, do not possess immunogenicity, only there is reactionogenicity, DAS itself cannot be used to prepare DAS antibody as immunogene, DAS need be prepared into complete antigen by coupling agent and carrier protein couplet, just may obtain corresponding DAS antibody by comlete antigen immune animal.
Its detection method principle is: the DAS antigen of carrier protein couplet can carry out specific binding with the anti-DAS monoclonal antibody on nano particle and form immune complex, occurs turbidity; The DAS competitive binding DAS monoclonal antibody of the DAS in urine and carrier protein couplet.Solution turbidity is measured by Biochemical Analyzer.In turbidity and urine, the content of DAS is inversely proportional to.By bioassay standard product, set up the typical curve of a degree of reaction corresponding concentration, DAS concentration value in urine sample can be measured.
The desired use of DAS kit is the content that Quantitative in vitro detects diacetyl spermine (N1, N12-Diacetylspermine, DAS) in human urine.
The advantage that the present invention has:
The content of vitro detection kit of the present invention comprises and measures required reagent for the monoclonal antibody specific of DAS and nanoparticle label, combine with novel nano particle immunologic detection method, for the vitro detection of the DAS content of human body biological sample, open the new method of DAS content detection, compared with ELISA method, have easy and simple to handle, detection speed is fast, precision is high, reproducible, testing result is by advantages such as operating influences.
Accompanying drawing explanation
The typical curve that Fig. 1 provides for the embodiment of the present invention.
The employing immunoturbidimetry of the present invention that Fig. 2 provides for the embodiment of the present invention and ELISA detect sample DAS content balance figure.
The employing immunoturbidimetry of the present invention that Fig. 3 provides for the embodiment of the present invention and ELISA detect sample DAS content correlativity.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but the invention is not restricted to following specific embodiment.
Embodiment 1
The preparation of the DAS antigen of BSA coupling:
The activation process of 1.BSA: precise bovine serum albumin(BSA) (BSA) 66mg, dissolves with 0.01MpH=7.2PBS damping fluid 5ml; Take 10mgMES, add in above-mentioned lysate, be placed in 2-8 DEG C of environment and react half an hour;
2. measure activated b SA product 10mg, be placed in a clean 10ml test tube, add 0.01MpH=7.2PBS to volume 4ml, add EDC/NHS (7mg/3mg), mixing to as clear as crystal, left at room temperature 25 minutes;
3. accurately measure acetyl spermine 5mg, add above-mentioned reaction system, mixing is to as clear as crystal, and pH value is adjusted to 10, reacts 5 hours under room temperature;
4. reaction product is dialysed 48 hours in 0.01MpH=7.2PBS dislysate 1000ml, and liquid is changed 4 times in centre;
5. adjust final concentration to 2mg/ml with 0.01MpH=7.2PBS;
6. be sub-packed in (0.5ml/ pipe) in clean EP pipe, save backup in-20 DEG C of refrigerators.
Embodiment 2
The activation process of 1.BSA: precise bovine serum albumin(BSA) (BSA) 66mg, dissolves with 0.05MpH=8.0PBS damping fluid 5ml; Take 10mgMES, add in above-mentioned lysate, be placed in 2-8 DEG C of environment and react half an hour;
2. measure activated b SA product 10mg, be placed in clean 10ml test tube, add 0.05MpH=8.0PBS to volume 4ml, add EDC/NHS (10mg/5mg), mixing is extremely as clear as crystal, left at room temperature 15 minutes;
3. measure spermine 10mg, be added to above-mentioned reaction system, mixing is to as clear as crystal, and pH value modulation 11.2, reacts 4 hours under room temperature;
4. reaction product is dialysed 48 hours in 0.05MpH=8.0PBS dislysate 1000ml, and liquid is changed 4 times in centre;
5. in 2-8 DEG C of environment, in reaction system, add 60 μ l acetic anhydrides, react 1 hour under 2-8 DEG C of environment;
6. reaction product is dialysed 48 hours in 0.05MpH=8.0PBS dislysate 1000ml, and liquid is changed 4 times in centre;
7. adjust final concentration to 2mg/ml with 0.05MpH=8.0PBS, be sub-packed in (0.5ml/ pipe) in clean EP pipe, save backup in-20 DEG C of refrigerators.
Embodiment 3
1. the synthesis of acetyl spermine: with 20mg spermine and 10 μ L anhydrous acetic acids for synthesis material, add in the 0.1MpH=7.2PBS of 1mL, reacts 60 minutes, does not stop to stir and evenly mix between the reaction period in 2-8 DEG C of environment;
2. above-mentioned obtained acetyl spermine is mixed with coupling agent 4mgGMBS in 2mL0.1MpH=7.2PBS buffer solution, react 3 hours under room temperature;
3. 10mgBSA and 4mg acetyl mercapto succinic acid is mixed in 2mL0.1MPBS buffer solution, reflect 3 hours under room temperature; Add 10 μ L0.1M azanols, react 30 minutes under room temperature except deacetylate; Dialyse 4 hours in 0.1MpH=7.2PBS damping fluid, removing reaction impurities;
4. acetyl spermine-GMBS coupled product is mixed with the BSA that above-mentioned sulfhydrylation is modified, react 4 hours under room temperature; Dialyse 24 hours in the 0.1MpH=7.2PBS damping fluid of 1000mL ,-20 DEG C save backup.
Embodiment 4
1. the synthesis of acetyl spermine: with 20mg spermine and 10 μ L acetic anhydrides or acetic acid for synthesis material, the 0.01MpH=7.2PBS joining 1mL in 2-8 DEG C of environment reacts 60 minutes, does not stop to stir and evenly mix between the reaction period;
2. get activated b SA product 10mg, be placed in clean 10ml test tube, add in 4mL0.01MpH=7.2PBS, then add EDC/NHS (8mg/4mg), mixing is extremely as clear as crystal, left at room temperature 45 minutes;
3. get the acetyl spermine of said method synthesis, add reaction system, mixing is to as clear as crystal, and pH value is adjusted to 11, reacts more than 4 hours under room temperature;
4. reaction product is dialysed 48 hours in 0.01MpH=7.2PBS dislysate 1000ml, and liquid is changed 4 times in centre;
5. adjust final concentration to 2mg/ml with 0.01MpH=7.2PBS;
6. be sub-packed in (0.5ml/ pipe) in clean EP pipe, save backup in-20 DEG C of refrigerators.
Embodiment 5
1. take spermine 20mg, it is mixed with 4mg coupling agent GMBS in 1mL0.01MpH=6.5PBS buffer solution, react 1.5 hours under room temperature;
2. activated b SA10mg is mixed with 4mg acetyl mercapto succinic acid in 2mL0.01MpH=6.5PBS buffer solution, react 3 hours under room temperature; Add 10 μ L0.1M azanols, react 30 minutes under room temperature except deacetylate; Dialyse 4 hours in 0.01MMPBS damping fluid 1000mL, removing reaction impurities;
3. the BSA that spermine-GMBS coupled product and above-mentioned sulfhydrylation are modified is mixed, react 4 hours under room temperature;
4.0.01MpH=6.5PBS dialyse 24 hours in damping fluid 1,000ml, liquid is changed 2 times in centre;
5. in 2-8 DEG C of environment, add 60 μ l acetic anhydrides in reaction system, 2-8 DEG C is reacted 1 hour;
6. reaction product is dialysed 48 hours in 0.01MpH=6.5PBS dislysate 1000ml, and liquid is changed 4 times in centre;
7. adjust final concentration to 2mg/ml with 0.01MpH=6.5PBS, be sub-packed in (0.5ml/ pipe) in clean EP pipe, save backup in-20 DEG C of refrigerators.
Embodiment 6
Polyclonal antibody or the monoclonal antibody through screening gained.
Preparation method for antibody process is as follows:
1. use above-mentioned prepared antigen as immunogene, the animals such as immune sheep, rabbit, mouse, after repeatedly immunity, obtain the plasma/serum containing anti-DAS antibody or the effect B cell of anti-DAS antibody can be expressed;
2., after obtaining plasma/serum, obtain polyclonal antibody needed for kit developing by Protein G column chromatography;
3. obtain after can expressing the effect B cell of anti-DAS antibody, B cell and myeloma cell are merged, screening, obtain the hybridoma cell strain having simultaneously and express anti-DAS antibody and infinite multiplication function, after carrying out expansion cultivation, enrichment of cell supernatant, obtains monoclonal antibody by protein A chromatography purifying.
Embodiment 7
The preparation of anti-DAS antibody
One, immunogene is the product (concrete operations detailed in Example 1-embodiment 5) that acetyl spermine molecule is coupled at gained on carrier protein BSA
Concentration: 2mg/ml
Two, animal is selected and immunity
(1), animal is selected
Select and the Balb/c Healthy female mouse of myeloma cell's homology used, mouse age was at 6 ~ 10 weeks.For avoiding mouse to react dead in not good or immunologic process, immunity 5 mouse simultaneously, No. 1-5, numbering.
(2), immunization protocol
1, initial immunity
Be dissolved in PBS by 300ug antigen, add isopyknic Freund's complete adjuvant (CFA), be emulsified into Water-In-Oil shape, adopt abdominal cavity and dorsal sc multi-point injection, 0.5ml/ only, carries out second time immunity after three weeks;
2, second time immunity
Be dissolved in PBS by 300ug antigen, add isopyknic freund 's incomplete adjuvant (IFA), be emulsified into Water-In-Oil shape, adopt abdominal cavity and dorsal sc multi-point injection, 0.5ml/ only, carries out third time immunity after three weeks;
3, third time immunity
Be dissolved in PBS by 300 μ g antigens, add isopyknic freund 's incomplete adjuvant (IFA), be emulsified into Water-In-Oil shape, adopt abdominal cavity and dorsal sc multi-point injection, only, after one week, blood sampling detection antiserum is active for 0.5ml/;
4, antiserum Activity determination (it is active that ELISA method detects antiserum)
4.1 experimental procedure
4.1.1 antigen coated
Pure antigen concentration 2mg/ml, gets 100 μ l add in each hole of polystyrene enzyme joint inspection drafting board with after coating buffer dilution, 4 DEG C spend the night after, wash liquid 3 times.
4.1.2 close
Every hole adds 200 μ l confining liquids, and 4 DEG C are spent the night or 37 DEG C after two hours, wash 3 times, pat dry.Put 4 DEG C of Refrigerator stores for subsequent use.
4.1.3ELISA detect
1), when detecting serum titer, doubling dilution is done to serum, 100 μ l/ holes, take Normal Mouse Serum as negative control, hatch 30min for 37 DEG C, wash 3 times, pat dry.
2) add sheep anti mouse-HRP, tiring is 105,100 μ l/ holes, hatches 30min for 37 DEG C, washs 3 times, pats dry.
3) develop the color: add each 50 μ l/ holes of A, B liquid, 37 DEG C of colour developing 15min.
4) stop: add stop buffer 50 μ l/ hole.
5) reading: measure each hole OD value with 450nm, 630nm dual wavelength, is limited to be greater than 2.1 with the ratio (P/N) of negative control hole OD value, as being judged as the positive or determining the critical point of tiring.
After testing, 5 mouse activity intensities are: 4#>5#>1#>3#Gre atT.GreaT.GT2#, booster immunization 4# mouse after two weeks.
5, booster immunization
Mixed with 0.5mlPBS by 50ug antigen, tail vein injects in 4# Mice Body, carries out Fusion of Cells after three days.
Three, Fusion of Cells
(1), feeder cells preparation
1, reagent consumptive material:
1.1RPMI-1640 complete culture solution;
The incomplete nutrient solution of 1.2RPMI-1640;
1.3HAT nutrient solution;
1.4 hydro-extractor;
1.5 other materials: cell diluent, blood cell counting plate, cover glass, sterile centrifugation tube, 96 porocyte culture plates, eye scissors, tweezers, large tweezers, plate, Dispensable 1 ml syringe, disposable 10ml syringe.
2, animal used as test
Balb/c or Kunming healthy mice (male and female) 1,6 ~ 10 week age, body weight 18 ~ 22 grams.
3, experimental technique
Balb/c or kunming mice draw neck to dislocate by 3.1 to be put to death, and be soaked in 75% alcohol 5 minutes, put into superclean bench immediately, belly is put in plate upward;
3.2 play mouse part skin with ophthalmic tweezers sub-folder, cut an osculum with scissors, note being sure not to break peritonaeum, in order to avoid peritoneal fluid outflow, then do blunt separation in both sides up and down with scissors, fully expose peritonaeum, sterilize with cotton ball soaked in alcohol wiping peritonaeum;
3.3 draw incomplete nutrient solution with syringe, inject mouse peritoneal, and syringe stops motionless, rocks mouse or repeatedly aspirates several times, draw back abdomen intracavity liquid by original annotation emitter, inject centrifuge tube;
3.41000rpm centrifugal 10min, abandons supernatant;
3.5, with 10ml complete culture solution re-suspended cell, count, and adjustment cell concentration is 200,000 every milliliter, and 100 μ l/ holes are added drop-wise to 96 porocyte culture plates, put 37 DEG C, cultivate for subsequent use in 5%CO2 incubator;
The growth conditions of 3.6 observation feeder cells, general well-grown feeder cells and macrophage are fusiformis or polygon, cell is bright, refractivity is strong.
(2), Fusion of Cells
1, reagent consumptive material
1.1 ophthalmology tweezers, scissors number handle, fixed head
1.237 DEG C warm water
1.3 spirit lamps, centrifuge tube, hydro-extractor, plate
1.4 fusion agent: 50%PEG, W=4000 (37 DEG C of preheatings)
1.5RPMI-1640 not exclusively nutrient solution (37 DEG C of preheatings)
1.6RPMI-1640 complete culture solution (37 DEG C of preheatings)
1.7HAT nutrient solution adjuvant (37 DEG C of preheatings)
2, splenocyte suspension is prepared
Get booster immunization 4# mouse, dislocate after eye socket blood sampling execution, takes out spleen, make splenocyte suspension in 75% alcohol after invading bubble sterilization under aseptic condition, centrifugal, 1000rpm, 10min, and incomplete nutrient solution washes twice, counting, stand-by;
3, myeloma cell's suspension is prepared
Get (viable count >95%) myeloma cell that 3 bottles of growth conditions are good, aseptic dropper is used to carry out pressure-vaccum to culture flask inner cell, then cell suspension is transferred in 50ml centrifuge tube, centrifugal, 1000rpm, 10min, the full nutrient solution that cannots be used up washes twice, counting, stand-by;
4, Fusion of Cells
Myeloma cell and splenocyte mix by the quantitative proportion of 1: 10 or 1: 5 by 4.1, and use 50ml centrifuge tube centrifugal, the full nutrient solution that cannots be used up washes 1 time, 1000rpm, 10min;
4.2 abandon supernatant, to exhaust residual liquid, in order to avoid affect the concentration of PEG with dropper;
4.3 gently at the bottom of attack centrifuge tube, make cell precipitation loosening slightly;
4.4 at room temperature merge
1. add the 1ml50%PEG (molecular weight 4000) of preheating in 1 minute, limit edged rocks;
2. acted on for 90 seconds;
3. add the incomplete nutrient solution of RPMI-1640 of preheating, stop PEG effect, added 1ml respectively every 2 minutes, the incomplete nutrient solution of RPMI-1640 of 2ml, 3ml, 4ml, 5ml and 10ml preheating;
4.5 is centrifugal, 1000rpm, 10min;
4.6 abandon supernatant, first with the RPMI1640 complete culture solution gently suspendible cell of about 6ml containing 20% calf serum, make sure to keep in mind firmly to blow and beat, in order to avoid make the cell merged scatter;
4.7 according to the quantity of 96 well culture plates used, adds complete culture solution (RPMI-1640 complete culture solution adds 1mlHAT nutrient solution adjuvant in every 50ml complete culture solution);
Cell suspension after fusion is added 96 orifice plates containing feeder cells, 100 μ l/ holes by 4.8,37 DEG C, the cultivation of 5%CO2 incubator;
Four, filtering hybridoma
(1), HAT selects hybridoma
HAT Selective agar medium contains hypoxanthine, aminopterin-induced syndrome and thymine, the main path of wherein aminopterin-induced syndrome DNA synthesis capable of blocking.After main path blocks, by complying with emergent approach namely under HGPRT (hypoxanthine guanine phosphoribosyltransferase) and TK (thymidine kinase) effect, utilize thymine and hypoxanthine synthetic DNA, lack wherein a kind of, DNA synthesis can not occur.For the myeloma cell line of hybridizing by forming the auxotrophic cell selecting to produce through toxic medicament induction, all without TK or HGPRT in cell, thus single or fused bone myeloma cells in HAT nutrient solution by death.Although B cell has HGPRT and TK, in vitro usually under condition of culture, especially under individual cells environment, be difficult to long-term surviving and propagation goes down to posterity, therefore only have hybridoma could in HAT nutrient solution growth and breeding.
(2), cell chulture, change liquid
After Fusion of Cells, first day starts, and examines cell, has recorded the growth conditions of cell, nutrient solution has situation that is pollution-free, feeder cells.Cultivate 3 ~ 5 days HAT nutrient solutions and change liquid once, within 10 days, change HT nutrient solution and be cultured to 20 days, change 1640 complete culture solutions.
(3) hybridoma (with wherein one piece of culture plate accompanying drawing) of secretory antibody, is screened
When hybridoma colonies grows into a certain size after fusion, nutrient solution starts to turn yellow, and just can start screening antibodies activity.General conventional having: immunofluorescence, radio-immunity (RIA), histochemical method and enzyme-linked immuno assay (ELISA) method etc.Through common method be ELISA method.When collecting supernatant, should carry out, so that antibody accumulation for 3-4 days after changing liquid last time.Supernatant can survey antibody activity again without after dilution or 1:5-1:10 dilution, and can screen out p+ antibody when screening with dilution antibody, also can remove the false positive because high background causes, detection method is as follows:
1, antiserum Activity determination (it is active that ELISA method detects antiserum)
1.1 antigen coated
Pure antigen concentration 2mg/ml, gets 100 μ l add in each hole of polystyrene enzyme joint inspection drafting board with after coating buffer dilution, 4 DEG C spend the night after, wash liquid 3 times.
1.2 close
Every hole adds 200 μ l confining liquids, and 4 DEG C are spent the night or 37 DEG C after two hours, wash 3 times, pat dry.Put 4 DEG C of Refrigerator stores for subsequent use.
1.3ELISA detect
1) draw the cell conditioned medium of culture hole to be detected, 100 μ l/ holes, with not celliferous nutrient culture media for negative control, using positive serum as positive control, hatch 30min for 37 DEG C, wash 3 times, pat dry.
2) add sheep anti mouse-HRP, tiring is 105,100 μ l/ holes, hatches 30min for 37 DEG C, washs 3 times, pats dry.
3) develop the color: add each 50 μ l/ holes of A, B liquid, 37 DEG C of colour developing 15min.
4) stop: add stop buffer 50 μ l/ hole.
5) reading: measure each hole OD value with 450nm, 630nm dual wavelength, is limited (see table 1) to be greater than 2.1 with the ratio (P/N) of negative control hole OD value.
Table 1
1 2 3 4 5 6 7 8 9 10 11 12
A 0.010 0.020 0.015 0.020 0.040 0.020 0.060 0.020 0.973 1.132 0.020 0.006
B 0.008 0.020 0.010 0.008 0.007 0.008 0.041 0.008 0.079 0.080 0.020 0.008
C 0.050 0.035 0.030 0.044 0.063 0.020 0.004 0.023 0.050 0.014 1.257 0.891
D 0.024 0.010 0.016 1.453 0.010 0.023 0.030 0.010 0.010 0.055 0.018 0.019
E 0.017 0.019 0.022 0.010 0.060 0.010 0.050 0.010 0.041 0.007 0.025 0.023
F 0.017 0.011 0.015 0.006 0.007 0.011 0.050 0.031 0.020 0.062 0.010 0.010
G 0.050 0.050 0.008 0.035 0.050 0.886 0.008 0.984 0.062 0.042 0.009 0.006
H 0.046 0.008 0.005 0.008 1.266 0.008 0.030 0.052 0.006 1.102 0.005 0.008
2, antiserum Activity determination (colloidal gold strip detection)
2.1 test strips make:
Mark gold pad: 525nm collaurum, add K2CO3 and sheep anti-mouse igg by 6/25 volume ratio, shaking table shakes 10min, adds 10% casein, closes 10min, centrifugal, redissolves, and paving gold, drains;
Bag quilt: ASP-PLL, 12mg/mL, dilutes 5 times with 1*PBS, wraps by 30uL/ plate, draws a line;
Assembling: PVC board is followed successively by from top to bottom: thieving paper, NC film, gold pad (2 layers of 6mm), application of sample pad;
Slitting: 3mm/ bar slitting;
2.2 detect:
Application of sample 50-100ul cell conditioned medium liquid, static 20min, the colour developing of contrast chromogenic line is strong and weak, and judge that in cell culture medium, antibody activity is strong and weak, develop the color stronger, antibody activity is higher.
By above-mentioned, the testing result of two kinds of methods is through comparing discovery, and it is high that numerical value is surveyed in enzyme joint inspection, and the colour developing of colloidal gold strip detection line is dark, and testing result is consistent, therefore later antibody screening adopts colloidal gold strip method.
Five, the cloning (limiting dilution assay) of hybridoma
Fused cell is after antibody activity screening, and carry out subclone, pick out monoclonal cell strain, step is as follows:
1, in 96 porocyte culture plates, every hole adds 100 μ l feeder cells suspensions (about containing 2.0 × 104 abdominal cavity cells) successively;
2, prepare hybridoma suspension to be cloned, be diluted to 10 cell/ml with the HT nutrient culture media containing 10% serum, every hole inoculum concentration is 0.1ml cell suspension, and namely every hole is containing 1 cell;
3,37 DEG C, 5%CO2 cultivate 7 ~ 10 days, there is macroscopic clone or hybridoma can be observed under inverted microscope being covered with 1/3 area and detectable antibody at the bottom of hole; Mark the hole only having single clonal growth, get supernatant and do antibody test,
4, get antibody test positive hole isolated cell, and transferred species expands cultivation in 24 orifice plates, and frozen;
5, repeat appeal 1-4 step 2 time, choose all monoclonal cell expansion cultivations being the positive containing cell hole after cloning, conservation freeze-stored cell.
The finished product cell line numbering finally filtered out: 5D4A7A10
Six, the production of monoclonal antibody
Cell after conservation is continued expand again and cultivate, carry out a large amount of preparations of monoclonal antibody.
1, adult Balb/c mouse (3 monthly age) intraperitoneal inoculation whiteruss, every mouse 0.3 ~ 0.5ml, used after 7 days;
2, the hybridoma of exponential phase is collected, with the centrifugal 10min of brine twice, 1500r/min;
3, sample, with Trypan Blue, living cell counting, again become the suspension of 1 × 106 cell/ml with normal saline
4, the hybridoma of exponential phase is in every mouse peritoneal injection 1ml;
5, interval is after 5 days, observes the production of mouse ascites every day, and as belly obviously expands, when touching with hand, skin has tension, puts to death mouse, gathers ascites;
6, the centrifugal ascites 15min of 4000r/min, removing cell component and other sediment, collect supernatant, packing ,-70 DEG C are frozen for subsequent use;
7, will the ascites of 5D4A7A10 be numbered, amount to 10ml, and use protein A chromatography purifying, and carry out desalting processing, after purifying, be anti-DAS antibody.
Embodiment 8
Kit, comprises R1 reagent, R2 reagent, standard items and quality-control product, described R1 reagent be by liquid form or freeze-drying after adding protection liquid after diacetyl spermine antibody coupling is on nano particle after the reagent preserved of solid form.By the reagent that acetyl spermine (DAS) molecule is preserved by the solid forms after coupling reagent is coupled at liquid form after on carrier protein or freeze-drying during described R2 reagent, prepared by above-described embodiment 1-5.Described diacetyl spermine antibody is coupled at the product of gained on carrier protein as immunogene by acetyl spermine (DAS) molecule by coupling reagent, immune animal gained polyclonal antibody or the monoclonal antibody (being prepared by above-described embodiment 7) through screening gained.
Described sample is including but not limited to serum, blood plasma, urine.Described nano particle includes but not limited to nm of gold, microballoon.
Described protection liquid is by weight percentage: the PEG6000 of the sucrose of PEG20000,2-5% of BSA, 0.1-0.5% of PVP40,0.5-1% of Tris, 0.5-2% of 0.1-0.5%, the Sodium azide of TWEEN20,0.02-0.1% of 0.01-0.8%, NaCl, 1-4% of 0.85-1.5%, and surplus is water.
The preparation of described R1 reagent:
1. prepare the MES damping fluid of 50mMpH6.0, be used for activating nano particle, make its final concentration be 1%w/v.
2. every milliliter of nano particle suspension adds the EDC of 20mg, incubated at room 20min.
The centrifugal 30min of 3.12000rpm, is buffered liquid (100mMHepespH7.0 is containing DAS antibody 100 μ g/mL) with isopyknic bag and suspends, incubated at room 2-5 hour.
4. add 2.5 μ L monoethanolamines in every milliliter of reaction solution, Keep agitation incubated at room 10min.
The centrifugal 30min of 5.12000rpm, suspends with protection liquid.
R2 reagent is the DAS antigen of 800ng/mLBSA coupling;
Standard items are the DAS standard items through water gradient dilution, and gradient dilution concentration is 0nM, 250nM, 500nM, 750nM, 1000nM5 concentration.
The volume ratio of R1 and R2 is=150:40
Detect
Adopt the vitro detection kit of above-described embodiment to carry out vitro detection, and contrast with ELISA detection method:
(1) gather 40 routine biological samples (i.e. human urine) immediately in 30 minutes, under 4 DEG C of conditions, centrifuging and taking supernatant is as measuring samples, to be placed in ice chest 1 hour use or-20 DEG C save backup;
(2) take out reagent R1, R2 and place room temperature down to equalized temperature;
(3) R1, R2 reagent and DAS standard items are placed on the corresponding position of Biochemical Analyzer, formulate typical curve (table 2 and see Fig. 1);
Table 2
Typical curve 1 2 3 4 5
Concentration (nM) 0 250 500 750 1000
Degree of reaction 0.4625 0.3545 0.2139 0.1280 0.0844
(4) DAS quality-control product is detected by automatic clinical chemistry analyzer, setting adds R1 reagent 150 μ L, R2 reagent 40 μ L, add sample 5 μ L, within 22nd o'clock, detected as first o'clock, the 45th is second point detection, with 2 differences for detected value, returned by typical curve, determine testing result validity (see table 3);
Table 3
(5) detect DAS sample with automatic clinical chemistry analyzer, arrange and add R1 reagent 150 μ L, R2 reagent 40 μ L, add sample 5 μ L, within the 22nd o'clock, detected as first o'clock, the 45th is second point detection, with 2 differences for detected value, returned by typical curve and draw net result.
(6) adopt mode of the present invention and ELISA method to detect DAS pattern detection result see table 4, Fig. 2, be horizontal ordinate by ELISA method measured value, this law measured value is ordinate, utilizes related software to do straight line as shown in Figure 3.
Table 4 two kinds of methods detect DAS content results
As can be seen from table 4 and Fig. 2 as seen by ratio, it is consistent that two kinds of methods detect DAS content trend, and as seen from Figure 3, linear equation is y=0.5714x-68.630, r 2=0.9272 correlation coefficient r=0.9629 (shown in Fig. 3), result shows, the DAS value of two kinds of method mensuration is closely related.And the inventive method detects DAS biochemical reagents box compared with ELISA method, detection time is only 10-15min, saves a large amount of time, easy and simple to handle, quick.
Embodiment 9
Kit comprises R1 reagent, R2 reagent, standard items and quality-control product.
The preparation of described R1 reagent, gets the 0.2MK that 1mL nm of gold adds 7 μ L 2cO 3; Add 15 μ gDAS antibody after mixing, mixing leaves standstill 10min; Add 10 μ L confining liquids again to close, mixing leaves standstill 10min again; Centrifugal 10min, rotating speed is 8000r/min, abandons supernatant, precipitates and protects liquid to redissolve with 1mLR1.
R1 protect liquid be by weight percentage: the Tris of 0.3%, the PVP40 of 2%, 1% BSA, the PEG20000 of 0.1%, sucrose, 0.1%TWEEN20,0.04% Sodium azide, the NaCl of 1%, the PEG6000 of 4% of 5%, surplus is water.
R2 reagent is the DAS antigen of 800ng/mLBSA coupling;
Standard items are the DAS standard items through water gradient dilution, and gradient dilution concentration is 0nM, 250nM, 500nM, 750nM, 1000nM5 concentration.
The volume ratio of R1 and R2 is=150:40
Embodiment 10
Kit comprises R1 reagent, R2 reagent, redissolution liquid, standard items and quality-control product.
Described R1 reagent preparation: be prepare rear packing according to R1 in embodiment 8, put into freeze drier and carry out freeze-drying, redissolves R1 reagent redissolution liquid during use again.
Described redissolution liquid is: R1 protect liquid be by weight percentage: the Tris of 0.3%, the PVP40 of 2%, 1% BSA, the PEG20000 of 0.1%, sucrose, 0.1%TWEEN20,0.04% Sodium azide, the NaCl of 1%, the PEG6000 of 4% of 5%, surplus is water.
Other compositions described are identical with embodiment 8.
It is as shown in table 5 below that the inventive method and comparative example detect DAS pattern detection result.
Table 5
Shown by the above results, R1 reagent detects DAS with lyophilized form in liquid form and is more or less the same containing numerical quantity, all meets product request for utilization.

Claims (8)

1. diacetyl spermine (DAS) immunoturbidimetry vitro detection kit, comprise R1 reagent, R2 reagent, standard items and quality-control product, it is characterized in that: described R1 reagent for by diacetyl spermine antibody coupling on nano particle, and after adding protection liquid, the reagent preserved with the solid form after liquid form or freeze-drying; Described R2 reagent for acetyl spermine (DAS) molecule is coupled on carrier protein by coupling reagent, with after liquid form or freeze-drying solid forms preserve reagent.
2. by diacetyl spermine (DAS) immunoturbidimetry vitro detection kit according to claim 1, it is characterized in that: described diacetyl spermine antibody is coupled at the product of gained on carrier protein as immunogene using acetyl spermine (DAS) molecule by coupling reagent, immune animal obtains polyclonal antibody or through screening to obtain monoclonal antibody.
3. by diacetyl spermine (DAS) immunoturbidimetry vitro detection kit according to claim 1; it is characterized in that: in described R1, protect liquid to be by weight percentage: the PEG6000 of the sucrose of PEG20000,2-5% of BSA, 0.1-0.5% of PVP40,0.5-1% of Tris, 0.5-2% of 0.1-0.5%, the Sodium azide of TWEEN20,0.02-0.1% of 0.01-0.8%, NaCl, 1-4% of 0.85-1.5%, surplus is water.
4., by diacetyl spermine (DAS) immunoturbidimetry vitro detection kit according to claim 1, it is characterized in that: described coupling reagent is the sub-amide-type of carbon two or maleimide; Wherein, the sub-amide-type of carbon two is EDC, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide or N-hydroxy-succinamide ester class (NHS ester); Maleimide is 4-maleimidobutyric acid-N-succinimide ester (GMBS) or 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC).
5. by diacetyl spermine (DAS) the immunoturbidimetry vitro detection kit described in claim 2 or 4, it is characterized in that: described diacetyl spermine (DAS) molecular conjugate vectors albumen is using carbodiimide/N-hydroxy-succinamide (EDC/NHS) as coupling reagent, carrier protein after activation process is added in the PBS of the pH=6.5-8.0 of excessive 0.01M-0.1M, add water miscible carbodiimide (EDC) and N-hydroxy-succinamide (NHS) again, mixing is extremely as clear as crystal, then left at room temperature 15-30 minute; Add excessive acetyl spermine after leaving standstill, mixing is to as clear as crystal, and regulation system pH value is adjusted to 10-12, reacts 4-6 hour under room temperature; Through dialysis products therefrom after reaction, obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
6. by diacetyl spermine (DAS) the immunoturbidimetry vitro detection kit described in claim 2 or 4, it is characterized in that: described diacetyl spermine (DAS) molecular conjugate vectors albumen is using carbodiimide/N-hydroxy-succinamide (EDC/NHS) as coupling reagent, carrier protein after activation process is added in the PBS of the pH=6.5-8.0 of excessive 0.01M-0.1M, add water miscible carbodiimide (EDC) and N-hydroxy-succinamide (NHS) again, mixing is extremely as clear as crystal, then left at room temperature 15-30 minute; Add spermine after leaving standstill, mixing is to as clear as crystal, and regulation system pH value is adjusted to 10-12, reacts 4-6 hour under room temperature; Through dialysing products therefrom in 2-8 DEG C after reaction, in reaction system, add acylating agent, then at 2-8 DEG C, react 1-3 hour again, reactant is through dialysing and obtaining diacetyl spermine (DAS) molecular conjugate vectors albumen.
7. by diacetyl spermine (DAS) the immunoturbidimetry vitro detection kit described in claim 2 or 4, it is characterized in that: described diacetyl spermine (DAS) molecular conjugate vectors albumen is using N-[γ-maleimide butyryl oxygen] succinimide ester (GMBS) as coupling reagent, acylating agent (acetic anhydride or anhydrous acetic acid or acetyl chloride) and spermine are reacted 60-90min at 2-8 DEG C, mix in the PBS of the pH=6.5-8.0 of 0.01M-0.1M with coupling agent (GMBS) after reaction, 3-4 hour is reacted under room temperature, obtain acetyl spermine-GMBS coupled product, carrier protein is mixed with acetyl mercapto succinic acid in the PBS of the pH=6.5-8.0 of 0.01M-0.1M, under room temperature, reacts 3-5 hour, add 0.1M azanol after reaction, react 30-35 minute under room temperature except deacetylate, dialyse in the PBS of the pH=6.5-8.0 of 0.01M-0.1M, obtain the BSA that sulfhydrylation is modified, acetyl spermine-GMBS coupled product is mixed with the BSA that above-mentioned sulfhydrylation is modified, under room temperature, reacts 4-6 hour, to dialyse in the PBS of the pH=6.5-8.0 of 0.01M-0.1M to obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
8. by diacetyl spermine (DAS) immunoturbidimetry vitro detection kit according to claim 1, it is characterized in that: utilize described kit to detect the diacetyl spermine in sample by immunoturbidimetry A competitive inhibition method.
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