CN101603965B - Kit for quantitatively measuring PEG modified medicaments by ELISA competition method - Google Patents
Kit for quantitatively measuring PEG modified medicaments by ELISA competition method Download PDFInfo
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Abstract
The invention relates to a kit for quantitatively measuring PEG modified medicaments by an ELISA competition method. The inside of the kit is provided with an elisa plate, diluent, cleaning solution, substrate solution and stop solution, and the kit also comprises a PEG modified protein standard product and a combined substance of PEG and enzyme, wherein the elisa plate is coated with anti-PEG monoclonal antibody; and the PEG modified protein standard product is prepared by combining the PEG and protein or polypeptide. The kit has the advantages of high detection sensitivity, short detection time and high accuracy, can quantitatively detect the PEG modified medicaments, has wide applicability, and can be used for quantitative detection of different medicaments modified by the PEG and the quantitative detection in vivo and in vitro.
Description
Technical field
The present invention relates to a kind of biomedical product, relate to a kind of kit of ELISA competition law quantitatively measuring PEG modified medicaments particularly, belong to field of medicaments.
Background technology
ELISA is that enzyme connects immunosorbent and measures (Enzyme-Linked Immunosorbnent Assay)) abbreviation; Be to be the basis with the immunological response; The specific reaction of antigen, antibody and enzyme to the very high experimental technique of a kind of susceptibility that the efficient catalytic effect of substrate combines, can be detected the specific antibody or the antigen of trace in the body fluid sensitively.Owing to carry out in the hole that is reflected at a kind of solid phase carrier Zhao polystyrene microtiter plates of antigen, antibody, after a kind of reagent of every adding is hatched, can remove unnecessary free reactant through washing, thus warranty test result's specificity and stability.In practical application, through different designs, concrete method step can have multiple, that is: be used to detect antibody indirect method, be used to detect the double antibody sandwich method of antigen and be used to detect micromolecule antigen or haptenic antigenic competition method or the like.
(polyethylene glycol is the polymkeric substance of modifying protein the most commonly used PEG) to polyglycol, and it is to be polymerized by monoethylene glycol monomer: HO-(CHCHO)-CH-CH-OH.What it was widely used in modifying is former because have the following advantages:
1.PEG have amphiphilic, the both available water that is dissolved in can be dissolved in most organic solvent again.2. nontoxic, immunogenicity is low, is easy in the body remove, and its molecule compatibility is also through the U.S. FDA authentication.3.PEG molecular weight very wide, the choice is big.4. can give the biomolecule after the modification with its many advantageous properties.For various albumen with medicinal potential, the advantage of following three aspects can be arranged after employing PEG modifies: (1) increases stability, prolongs plasma half-life; (2) reduce immunogenicity and antigenicity; (3) reduce toxic and side effect.Can increase albumen biodistribution in vivo in addition.At present, be used for the clinical adenosine deaminase that mainly contains, Intederon Alpha-2a, α-2b, granulocyte colony stimulating factor, the PEG modified medicaments of growth hormone release inhibiting hormone etc., the PEG modified medicaments that also has tens is just at clinical stage.
For the degree of modification that detects the PEG trim and the detection of decorating site; Available following method: AAS, liquid phase chromatography, electrophoresis, mass spectroscopy, nuclear magnetic resonance, light scattering method, fourier transform infrared spectroscopy etc.; But the influence of many aspects such as the often examined sensitivity of these methods at present, professional, equipment implementation condition and cost, and the sample concentration after can not quantitative measurement modifying.At present, the global kit that does not still have ELISA method or other immunization method detection PEG modified medicaments supplies application such as pharmaceutical factory or hospital.
Summary of the invention
The kit that the purpose of this invention is to provide a kind of ELISA competition law quantitatively measuring PEG modified medicaments is used for the detection by quantitative of PEG modified medicaments, solves the not enough problem of detection method of present PEG modified medicaments effectively.
The present invention adopts a kind of brand-new design philosophy, competition law is introduced the design research and development of kit.Competition law is meant: specific antibody and solid phase are connect; Form insolubilized antibody, add sample to be measured (containing corresponding antigens) and corresponding a certain amount of enzyme-labelled antigen, antigen in the sample to be measured and enzyme-labelled antigen competition combine with insolubilized antibody; Antigenic content is high more in the sample to be measured; Then combine also manyly more, make that the chance that enzyme-labelled antigen combines with insolubilized antibody is just few more, even have no chance to combine with insolubilized antibody.It is very shallow not develop the color or develop the color behind the adding substrate, and the dark person that develops the color is negative.
The present invention is to realize through following technical scheme for reaching above purpose:
A kind of kit of ELISA competition law quantitatively measuring PEG modified medicaments includes ELISA Plate, dilution, cleaning fluid, substrate solution, stop buffer in the kit, it is characterized in that also comprising: the protein standard items that PEG modifies, the bond of PEG and enzyme; Described ELISA Plate is the ELISA Plate that is coated with anti-PEG monoclonal antibody; The protein standard items that described PEG modifies are with PEG and protein or polypeptide be combined into; Its method comprises with the PEG of activation and protein or polypeptide directly reacts; Or end is changed into not isoplastic PEG and protein or polypeptide adopts bifunctional group to be connected be combined into, perhaps adopts different arms to be formed by connecting.The bond of PEG of the present invention and enzyme is processed with following method; Comprise with the directly reaction and getting of the PEG of activation and enzyme; Or end is changed into not isoplastic PEG and enzyme and is adopted bifunctional group to be connected be combined into; Perhaps adopt different arms to be formed by connecting, or make it to combine with PEG with the method for first treatment enzyme.The bond of PEG described in the present invention and enzyme is the PEG of enzyme labeling.
The described ELISA Plate that is coated with anti-PEG monoclonal antibody is to process with following method: with KLH-PEG as immunizing antigen immunity New Zealand large ear rabbit; And get its spleen and 240E cell (US5675063) and merge; The ELISA screening enlarges and produces monoclonal antibody purification; After the monoclonal antibody coated elisa plate sealing with purifying, add the sugar solution treatment ELISA Plate.Add the stability that the sugar solution treatment ELISA Plate can prolong antibody on the immobilized enzyme target.
As preferably, the sugar juice of described treatment enzyme target is one or more the WS that contains in sucrose, glucose, trehalose, lactose, the galactose, and wherein the mass concentration of sugar is 0.3～20%.
As preferably, be coated with the ELISA Plate of anti-PEG monoclonal antibody, the concentration that encapsulates of its antibody is 2 μ g/ml.
The proportioning of described dilution is: 0.5-3gBSA, and the Sodium Mercurothiolate of massfraction 0.01-0.02%, 0.1-0.3g Tris, 0.5-0.7g NaCl adds deionized water to 100ml, regulates pH to 7.4.The proportion optimization of dilution is: every 100ml contains 1gBSA, the Sodium Mercurothiolate of 1ml mass concentration 1%, and Tris0.241g, NaCl 0.584g, all the other are deionized water, regulate the low protein adsorption membrane filtration degerming of pH to 7.4 back 0.22 μ m.
The proportioning of described cleaning fluid is: the Sodium Mercurothiolate of mass concentration 0.01-0.02%, and 1-3g Tris, 5-7g NaCl, 1ml Tween-20 adds deionized water to 100ml, regulates pH to 7.4.The proportion optimization of cleaning fluid is: every 100ml contains the Sodium Mercurothiolate of 1ml mass concentration 1%, 2.41gTris, and 5.84gNaCl, 1ml Tween-20, all the other are deionized water, regulate the low protein adsorption membrane filtration degerming of pH to 7.4 back 0.22 μ m.
Described substrate solution is made up of substrate A liquid and substrate B liquid, and wherein the proportioning of substrate A liquid is: the 1-2g sodium hydrogen phosphate, and the 0.5-1g citric acid, 30-50 μ l mass concentration 30% hydrogen peroxide adds deionized water to 100ml; The proportioning of substrate B liquid is: 0.03-0.08gTMB, and the 0.1-0.2g citric acid, 0.01-0.05gEDTA, 20ml glycerine, 3ml DMSO adds deionized water to 100ml.The optimum ratio of substrate A liquid is: every 100ml contains the 1.84g sodium hydrogen phosphate, 0.510g citric acid, 33 μ l mass concentrations, 30% hydrogen peroxide, deionized water 50ml.The optimum ratio of substrate B liquid is: every 100ml contains 0.039gTMB, the 0.105g citric acid, and 0.0186g EDTA, 20ml glycerine, 3ml DMSO, all the other are deionized water.
As preferably, the protein standard items that described PEG modifies and the bond of PEG and enzyme, the molecular weight of its PEG is 1000-40000 dalton.Kit of the present invention is in order to detect the PEG modified medicaments, and the PEG scope relatively more commonly used in the PEG modified medicaments is 1000-40000 at present.As preferably, the protein standard items that described PEG modifies, the reactive group of the PEG of its albumen or polypeptide and activation comprises amino, sulfydryl or carboxyl; The bond of said PEG and enzyme, the PEG reactive group of its enzyme and activation comprises amino, sulfydryl or carboxyl.Protein chemistry is modified and is mainly comprised following several kinds: acylation reaction, oxidation reaction, reduction reaction, alkylated reaction, aromatic rings substitution reaction; Wherein amino acylation reaction, the alkylated reaction of participating in; Sulfydryl is participated in acylation reaction, alkylated reaction, oxidation reaction, reduction reaction, and carboxyl is participated in alkylated reaction.
The bond of said PEG and enzyme, enzyme wherein are horseradish peroxidase, alkaline phosphatase or glucose oxidase.These three kinds of enzymes are enzymes relatively commonly used and that be easy to get, and the mensuration wavelength after the substrate colour developing commonly used of these enzymes is between 400～500nm, and the ELIASA of domestic consumer all is equipped with the optical filter between 400～500nm, so these three kinds of enzymes are reasonable selections.The bond of described PEG and enzyme, its preparation method are enzyme and the PEG reaction after first treatment enzyme will be handled again, and the method for described first treatment enzyme is the oxydasis method.With oxydasis method treatment enzyme, make enzyme have reactive group such as aldehyde radical etc. earlier, the amino of these reactive groups abilities and PEG reacts, and forms the bond of PEG-enzyme, is the PEG of enzyme labeling.
The protein standard substance that described PEG modifies, it adopts the bifunctional group of bifunctional group connection method to comprise carbodiimide and derivant thereof, 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester or glutaraldehyde; The bond of described PEG and enzyme, the bifunctional group of its bifunctional group connection method comprises carbodiimide and derivant thereof, 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester or glutaraldehyde.
The inventor is with the prepared ELISA Plate that is coated with anti-PEG monoclonal antibody (hereinafter to be referred as anti-PEG monoclonal antibody) of said method; The protein standard items that PEG modifies; The bond of PEG and enzyme is a core; And combine mentioned reagent to be assembled into all PEG modified medicaments of the methoxyl that can detect, discern PEG, and this method is verified, thereby realized the present invention.
Advantage of the present invention is:
1. it all is conventional that the different pharmaceutical can the different PEG of quantitative measurement modified of the kit of the ELISA competition law quantitatively measuring PEG modified medicaments set up of the present invention, and equipment needed thereby detects.
2. kit detection sensitivity of the present invention is high, and detection time is short, and accuracy is high, and applicability is wide.
3. kit of the present invention can be used for the research or the experiment in vitro of pharmacokinetics, and range of application is wider.
Description of drawings
Fig. 1 is technology path figure of the present invention.
Fig. 2 examines for the purity of PEG-BSA and dyes figure.The 2nd road is the BSA that modifies with PEG, and the 3rd road is the BSA before modifying.
Fig. 3 is different proteins that PEG modifies and the competitive ELISA response curve of PEG-HRP, with and the curve map of relevant detection sensitivity and sensing range.Wherein, the X axle is the concentration of PEG modified medicaments, and the Y axle is corresponding OD value.
Embodiment
Below kit of the present invention is done explanation in further detail through concrete embodiment.
A kind of immunization method detection kit of quantitatively measuring PEG modified medicaments comprises the ELISA Plate that is coated with anti-PEG monoclonal antibody that is located in the box body, the PEG-BSA standard items; The PEG of horseradish peroxidase-labeled, dilution, cleaning fluid; Substrate A liquid, substrate B liquid and stop buffer.
1.PEG the preparation of the protein standard items PEG-BSA that modifies:
1. the BSA with 50mg is dissolved into 10ml PBS damping fluid.
2. in BSA: the PEG5000 that the ratio of PEG=1: 1-9 takes by weighing activation is dissolved into deionized water, immediately with the two hybrid reaction 1～4 hour.This process is carried out at 4 degree refrigerators.
3. add 0.5M Tris 100 μ l, reaction 0.5～2h.
4. crosslinked good PEG-BSA being respectively charged in the bag filter, is dialysed overnight among the PBS of 0.05mol in pH7.4 concentration, changes liquid 3 times, and this process is carried out at 4 degree refrigerators.
5. the BSA-PEG in the bag filter is moved on to centrifuge tube.
6. measure its protein concentration, and adjust its concentration to 1mg/ml according to the result of gained.
7. it is packed as 1ml/ pipe, it is subsequent use to put into-20 ℃ of preservations.
The purity of PEG-BSA of the present invention and content detection adopt SDS glue to identify and the BCA albuminimetry respectively.Concrete grammar is:
A.SDS glue detects the purity of protein of PEG-BSA
With sample buffer PEG-BSA and BSA are diluted to 0.25 μ g/ml respectively, 100 ℃ of water-bath sex change 5min get 20 μ l samples, ten holes comb, 10%SDS glue, 150V, 50 minutes-1.5 hours.Employing is examined the method for dying and is carried out the albumen position and confirm, and the map analysis of scanning glue, and its result is as shown in Figure 2, PEG-BSA＞90% after crosslinked, and the BSA of crosslinked PEG is higher than theoretical molecular weight, about 150kd.Wherein 1 road is mark, the 2 roads BSA of PEG that has been crosslinked, and 3 roads are the BSA before crosslinked.
The preparation of above-mentioned sample buffer: 62mmol/L Tris-HCl (pH 6.8), 25% glycerine, 2%SDS, 0.01% bromophenol blue, 710mM beta-mercaptoethanol adds the deionized water constant volume to 1L.
B. protein quantification adopts the BCA sizing technique, adopts the kit of pierce company to carry out quantitative measurement.Concrete operation steps sees the product description of pierce company for details.
2. the preparation of horseradish peroxidase-labeled PEG:
A. horseradish peroxidase (Horseradish Peroxidase, HRP) mark PEG:
1. the HRP with 20mg is dissolved in the 1ml ionized water.
2. in HRP: the PEG that the ratio of PEG=1: 1-9 takes by weighing activation is dissolved into the WS of HRP, 4 ℃ of lucifuges reactions 1～4 hour.
3. add 0.5M Tris 100 μ l, 4 ℃ of reaction 0.5～2h.
4. crosslinked good PEG-HRP is respectively charged in the bag filter, the PBS dialysed overnight of pH7.4 0.05mol is changed liquid 3 times, and this process should be carried out at 4 ℃ of refrigerators.
5. it is anticorrosion to add 0.05% Sodium Mercurothiolate, and adds 25% glycerine, packing.
6. it is subsequent use to put into-20 ℃ of preservations.
Confirming of the working concentration of b.PEG-HRP and antibody sandwich concentration:
1. coating buffer: NaHCO
32.93g, Na
2CO
31.59g be settled to 1000ml, pH9.6.
2. with coating buffer with antibody dilution to different concentration, 8,4,2,1,0.5,0.25,0.125,0.0625 μ g/ml adds, 4 ℃ are spent the night.
3. after the sealing, crosslinked good HRP-PEG is done 1: 500,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000, the dilution back added in 1: 64000, reacted 45 minutes,
4. after cleaning 3 times with cleaning fluid, add substrate colour developing 15min.
5. add the reaction of stop buffer color development stopping.
6. confirm that according to this experimental result its working concentration is 1: 12000, the concentration that encapsulates of antibody is 2 μ g/ml.
C. pack
When this kit of assembling, HRP-PEG is used the HRP protective agent---the dilution of pierce reagent, filter packing, the term of validity 1 year.Each kit 150 μ l, during use with 60 times of diluted.4 ℃ of preservations are valid for one year.
3. be coated with the preparation of the ELISA Plate of anti-PEG monoclonal antibody:
A. Antibody Preparation
Get New Zealand's large ear rabbit of healthy adult, with KLH-PEG as immunizing antigen immunity (preparation method of KLH-PEG is identical with BSA-PEG), add the emulsification of Fu Shi Freund's complete adjuvant with KLH-PEG for the first time after, the subcutaneous five-spot that rabbits is immune.Add the fully emulsified back of freund 's incomplete adjuvant immune rabbit to the 5th time with KLH-PEG for the second time.Be 2 weeks each immune interval time.Immunizing dose 0.5mg/ time.In merging first three day, ear vein is injected at last, reinforced immunological, and immunizing dose doubles, and does not add adjuvant.Get its spleen, and separating Morr. cell, merge with 240E cell (US5675063).(" antibody molecule and the tumour " that fusion method is write with reference to Chen Zhinan.) utilize the ELISA method to filter out positive colony; With positive colony with limiting dilution assay (with reference to " principle of in vitro culture " of Xue Qingshan) monoclonalization; Obtain the monoclonal cell strain that secretory antibody tires the highest and carry out the high density enlarged culture; And the collection culture supernatant, with protein A affinity column purifying.IgG is quantitative afterwards, and measures its specificity.
B. antibody sandwich concentration is confirmed
Antibody sandwich concentration is definite in above-mentioned 2.b step, and the result shows that the best concentration that encapsulates is 2 μ g/ml.
C. the preparation of ELISA Plate is specially:
1. the concentration of with coating buffer antibody dilution extremely being confirmed, 100 μ l/ holes.4 ℃ are spent the night.
2. every hole adds 1% the BSA sealing of 100 μ l, seals 1 hour.
3. clean 1 time.
4. 10% the aqueous trehalose that adds 100 μ l/ holes was handled sheet material 1 hour.
5. clap and do.
6. use the aluminum plastic film vacuum packet by sheet material, each packing must add drying agent.
4. the prescription of other main material in this kit:
1) component of dilution is: every 100ml contains 1gBSA, the Sodium Mercurothiolate of 1ml mass concentration 1%, and Tris 0.241g, NaCl0.584g, all the other are deionized water, behind the adjusting pH to 7.4,0.22 μ m hangs down the degerming of protein adsorption membrane filtration.
2) component of cleaning fluid is: every 100ml contains the Sodium Mercurothiolate of 1ml mass concentration 1%, 2.41gTris, and 5.84g NaCl, 1ml Tween-20, all the other are deionized water, behind the adjusting pH to 7.4,0.22 μ m membrane filtration degerming.
3) component of substrate A is: every 100ml contains the 1.84g sodium hydrogen phosphate, the 0.510g citric acid, and 33 μ l mass concentrations, 30% hydrogen peroxide, all the other are deionized water,, 0.22 μ m membrane filtration degerming.
4) component of substrate B is: every 100ml contains 0.039gTMB, the 0.105g citric acid, and 0.0186g EDTA, 20ml glycerine, 3ml DMSO, all the other are deionized water, 0.22 μ m membrane filtration degerming.
A kind of immunization method detection kit of quantitatively measuring PEG modified medicaments comprises the ELISA Plate that is coated with anti-PEG monoclonal antibody that is located in the box body, PEG-BSA standard items, the PEG of alkali phosphatase enzyme mark, dilution, cleaning fluid, substrate A liquid, substrate B liquid and stop buffer.
1.PEG the preparation of the protein standard items PEG-BSA that modifies:
Concrete steps are with embodiment 1.1, but wherein step 2. in used PEG be PEG40000.
2. the preparation of alkali phosphatase enzyme mark PEG:
The cross-linking method of alkaline phosphatase-PEG bond is with 2 among the embodiment 1.Different is, 5., it is anticorrosion to add 0.01% Sodium Mercurothiolate among the step a, and adds 5% glycerine.
3. be coated with the preparation of the ELISA Plate of anti-PEG monoclonal antibody:
With embodiment 1.3, different is, the sucrose and the glucose mixed solution that 4. add the mass concentration 20% in 100 μ l/ holes in the c. step are handled sheet material, and wherein the weight ratio of sucrose and glucose is 1: 4.
4. the prescription of other main material in this kit:
1) component of dilution is: every 100ml contains 0.5gBSA, the Sodium Mercurothiolate of 1ml mass concentration 2%, and Tris 0.1g, NaCl 0.5g, all the other are deionized water, behind the adjusting pH to 7.4,0.22 μ m hangs down the degerming of protein adsorption membrane filtration.
2) component of cleaning fluid is: every 100ml contains the Sodium Mercurothiolate of 1ml mass concentration 2%, 1g Tris, and 5g NaCl, 1mlTween-20, all the other are deionized water, behind the adjusting pH to 7.4,0.22 μ m membrane filtration degerming.
3) component of substrate A is: every 100ml contains the 1g sodium hydrogen phosphate, the 0.5g citric acid, and 30 μ l mass concentrations, 30% hydrogen peroxide, all the other are deionized water, 0.22 μ m membrane filtration degerming.
4) component of substrate B is: every 100ml contains 0.03g TMB, the 0.1g citric acid, and 0.01g EDTA, 20ml glycerine, 3mlDMSO, all the other are deionized water, 0.22 μ m membrane filtration degerming.
Embodiment 3
A kind of immunization method detection kit of quantitatively measuring PEG modified medicaments comprises the ELISA Plate that is coated with anti-PEG monoclonal antibody that is located in the box body, PEG-BSA standard items, the PEG of glucose oxidase enzyme labeling, dilution, cleaning fluid, substrate A liquid, substrate B liquid and stop buffer.
1.PEG the preparation of the protein standard items PEG-BSA that modifies:
Concrete steps are with embodiment 1.1, but wherein step 2. in used PEG be PEG1000.
2. the preparation of glucose oxidase enzyme labeling PEG:
The cross-linking method of glucose oxidase-PEG bond is with 2 among the embodiment 1.Different is, 5., it is anticorrosion to add 0.1% Sodium Mercurothiolate among the step a, and adds 50% glycerine.
3. be coated with the preparation of the ELISA Plate of anti-PEG monoclonal antibody:
With embodiment 1.3, different is that the lactose solution that 4. adds the mass concentration 0.3% in 100 μ l/ holes in the c. step is handled sheet material.
4. the prescription of other main material in this kit:
1) component of dilution is: every 100ml contains 3gBSA, the Sodium Mercurothiolate of 1ml mass concentration 1.5%, and Tris 0.3g, NaCl0.7g, all the other are deionized water, regulate the low protein adsorption membrane filtration degerming of pH to 7.4 back 0.22 μ m.
2) component of cleaning fluid is: every 100ml contains the Sodium Mercurothiolate of 1ml mass concentration 1.5%, 3g Tris, and 7g NaCl, 1mlTween-20, all the other are deionized water, regulate pH to 7.4 back 0.22 μ m membrane filtration degerming.
3) component of substrate A is: every 100ml contains the 2g sodium hydrogen phosphate, the 1g citric acid, and 50 μ l mass concentrations, 30% hydrogen peroxide, all the other are deionized water,, 0.22 μ m membrane filtration degerming.
4) component of substrate B is: every 100ml contains 0.08g TMB, the 0.2g citric acid, and 0.05g EDTA, 20ml glycerine, 3mlDMSO, all the other are deionized water, 0.22 μ m membrane filtration degerming.
The mensuration program of embodiment 4 kits of the present invention
1. application of sample
1. blood serum sample to be measured or other sample are done dilution in 1: 5 with dilution.
2. get the standard items dissolving of 500 μ l deionized waters with freeze-drying, this moment, the concentration of standard items was 200ng/ml.
3. use dilution that standard items are diluted to gradient to be: 150,100,70,40,20,10,2,0ng/ml.
4. calculate the amount of the required HRP-PEG of this experiment, with 60 times of dilutions of dilution HRP-PEG.
5. get 1. and the 4. liquid of step gained abundant mixing in transfer blade of equivalent respectively, get respectively simultaneously equivalent 3. and 4. the liquid of step gained in transfer blade, fully get 50 μ l respectively after the mixing and be added in the ELISA Plate that is coated with anti-PEG monoclonal antibody.30 ℃ of constant temperature were hatched 45 minutes.
2. clean
1. with deionized water with 10 times of dilutions of cleaning fluid.
2. pour out the reaction liquid in the ELISA Plate, every hole adds the cleaning fluid of 200 μ l, cleans 3 times, and claps and do.
3. add substrate
1. substrate A and substrate B are done the equal-volume mixing.
2. every hole adds 100 μ l, 37 degree colour developings 1 hour.
4. stop: every hole adds 100 μ l stop buffer cessation reactions.
5. measure: the OD value of measuring every hole with ELIASA at the 450nm place.
6. the result judges: the OD value of removing 0ng/ml.The result is put into the excel form.With standard items concentration is horizontal ordinate, and the OD value is an ordinate, selects suitable curve, and draws linear equation.With the OD value substitution linear equation of sample, can by formula calculate the PEG content of sample.
The detection sensitivity of embodiment 5 kits of the present invention and the mensuration of sensing range
Sensitivity uses detectability (LOD) expression, LOD to refer to make the required reference material Cmin of remarkable change on the level generation statistical significance that combines of titer and antibody usually, generally gets B
0The mean value (X) that (zero-dose standard point) measured the result for 10 times deducts its 2 times of standard deviations (SD); Promptly calculate according to following formula: Y=[(X-2SD)/X] * 100%; Y value substitution typical curve is obtained corresponding mass concentration; Be the theoretic detection lower limit of this kit (LOD), the concrete steps of this experiment are following:
1. application of sample
And prepare the BSA-PEG of 2 parts of 0ng/ml 1. 2., with the step of embodiment 4.1.
2. 3. with the step of embodiment 4.1.
3. 4. with the step of embodiment 4.1.
Get BSA-PEG and the 3. step gained solution abundant mixing in transfer blade of the 0ng/ml of 2 parts of equivalent respectively, get respectively simultaneously equivalent 2. and 3. step gained solution in transfer blade, fully get 50 μ l respectively after the mixing and be added in the ELISA Plate that is coated with anti-PEG monoclonal antibody.30 ℃ of constant temperature were hatched 45 minutes.
2,3,4,5,6 steps are with 2,3 of embodiment 4,4,5,6 steps.
7. repeat said determination 10 times.
8. press Y=[(X-2SD)/X] * 100%, calculate the y value.Wherein X is 10 mensuration results' a mean value, and SD is a standard deviation.
9. y value substitution typical curve is obtained corresponding mass concentration, be the theoretic detection lower limit of this kit (LOD), i.e. sensitivity.The theoretical value that determines is 0.36ng/ml, but considers the needs of real work, and the detection sensitivity of this kit is finally confirmed as 1ng/ml.
Use dilution that the standard items dilution is series concentration; Accomplish test according to the 2-6 step among the embodiment 4; And make the canonical plotting of the corresponding with it OD of concentration, it is better to draw when 0ng～75ng/ml curve, therefore sensing range is decided to be: 0ng～75ng/ml; In this scope, can obtain testing result accurately, typical curve as shown in Figure 3.
The widespread popularity experiment of 6 kits of embodiment
A) with BSA-PEG, it is 200,150,70,40,20,10,2 that ADI-PEG, Mouse IgG use dilution to dilute respectively, 0ng/ml.
B) calculate the amount that this tests required HRP-PEG, with 60 times of dilutions of dilution HRP-PEG.
C) get respectively equivalent a) and b) solution of step gained abundant mixing in transfer blade, get 50 μ l afterwards respectively and be added in the ELISA Plate that is coated with anti-PEG monoclonal antibody.
D) with embodiment 4 said 2-6 procedure.
The result is as shown in Figure 3; This kit of experiment proof can be used for the detection of above-mentioned 3 kinds of PEG trims; The single-minded identification of the PEG antibody capable of developing among the present invention end is the PEG of methoxy group, and kit of the present invention in theory can all ends of detection by quantitative contains the PEG of methoxy group, no matter promptly which kind of pharmaceutical protein PEG modifies; As long as the PEG end in the PEG modified medicaments still contains the methoxy group, just can be gone out by kit detection by quantitative of the present invention.Through using BSA-PEG, ADI-PEG, Mouse IgG-PEG test and can find out, kit of the present invention is these three kinds of PEG trims of ability detection by quantitative all, so kit of the present invention can be used for the detection by quantitative of most of PEG modified medicaments.
Precision, accuracy, the repeated experiment of 7 kits of embodiment
1. the mensuration of precision:
Standard items are diluted 3 different concentration with cell culture fluid and human plasma respectively, 3 multiple holes of each concentration.According to competition law ELISA replication 20 times, the regression equation of application standard curve calculates the measured value of each concentration, in calculating batch and batch between the coefficient of variation, the result sees table 1 and table 2.
Table 1 cell culture fluid
Table 2 human plasma
Precision is meant same sample with the method repeated sampling analysis of setting up, the size of observing coefficient of variation Cv=standard deviation (the S)/average (x) * 100% between each time result, and the coefficient of variation is more little, shows that repeatability is good more, and precision is high more.Conclusion: the precision of kit is good, and repeatability is strong, and a batch differences meets the requirements.
2. determination of recovery rates:
PEG-BSA is diluted three gradients respectively with cell culture fluid and human plasma respectively, 6 repetitions of each concentration, replication 3 times, and calculate by following formula:
The recovery (100%)=measured concentration/interpolation concentration * 100%
The result sees table 3:
Table 3
| The dilution type | Average recovery rate (%) | Scope |
| Human serum (n=6) | 113 | 90-122％ |
| Human plasma (n=6) | 100 | 85-114％ |
| Nutrient solution (n=6) | 106 | 85-117％ |
Conclusion: no matter be that sample to be detected is a serum, blood plasma or nutrient solution can both obtain the content of PEG trim comparatively accurately through the mensuration of this kit.
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.The sugar juice of the treatment enzyme target described in embodiment 1 can be one or more the WS that contains in sucrose, glucose, trehalose, lactose, the galactose, and wherein the mass concentration of sugar is 0.3～20%.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, the some simple deduction or replace of making all should be regarded as belonging to the definite scope of patent protection of claims that the present invention submits to.
Claims (9)
1. the kit of an ELISA competition law quantitatively measuring PEG modified medicaments; Include ELISA Plate, dilution, cleaning fluid, substrate solution, stop buffer in the kit; It is characterized in that also comprising: the protein standard items that PEG modifies; The bond of PEG and enzyme, described ELISA Plate are the ELISA Plates that is coated with anti-PEG monoclonal antibody, and the protein standard items that described PEG modifies are with PEG and protein or polypeptide be combined into; The described ELISA Plate that is coated with anti-PEG monoclonal antibody is to process with following method: with KLH-PEG as immunizing antigen immunity New Zealand large ear rabbit; And get its spleen and 240E Fusion of Cells; The ELISA screening enlarges and produces monoclonal antibody purification; After the monoclonal antibody coated elisa plate sealing with purifying, use the sugar solution treatment ELISA Plate; The single-minded identification of described anti-PEG monoclonal antibody end is the PEG of methoxy group.
2. kit according to claim 1 is characterized in that: the sugar juice of described treatment enzyme target is one or more the WS that contains in sucrose, glucose, trehalose, lactose, the galactose, and wherein the concentration of sugar is 0.3～20%.
3. kit according to claim 1 and 2 is characterized in that: be coated with the ELISA Plate of anti-PEG monoclonal antibody, the concentration that encapsulates of its antibody is 2 μ g/ml.
4. kit according to claim 1 and 2 is characterized in that: the bond of described PEG and enzyme, its preparation method are enzyme and the PEG reaction after first treatment enzyme will be handled again, and the method for described first treatment enzyme is the oxydasis method.
5. kit according to claim 3 is characterized in that: the bond of described PEG and enzyme, its preparation method are enzyme and the PEG reaction after first treatment enzyme will be handled again, and the method for described first treatment enzyme is the oxydasis method.
6. kit according to claim 1 and 2 is characterized in that: the protein standard items that described PEG modifies and the bond of PEG and enzyme, and the molecular weight of its PEG is 1000-40000 dalton; The protein standard items that described PEG modifies, the reactive group of the PEG of its albumen or polypeptide and activation comprises amino, sulfydryl or carboxyl; The bond of said PEG and enzyme, the PEG reactive group of its enzyme and activation comprises amino, sulfydryl or carboxyl.
7. kit according to claim 1 and 2 is characterized in that: the bond of said PEG and enzyme, enzyme wherein are horseradish peroxidase, alkaline phosphatase or glucose oxidase.
8. kit according to claim 1 and 2; It is characterized in that: the protein standard substance that described PEG modifies, it adopts the bifunctional group of bifunctional group connection method to comprise carbodiimide and derivant thereof, 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester or glutaraldehyde; The bond of described PEG and enzyme, the bifunctional group of its bifunctional group connection method comprises carbodiimide and derivant thereof, 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester or glutaraldehyde.
9. kit according to claim 1 is characterized in that:
The proportioning of described dilution is: 0.5-3g BSA, and the Sodium Mercurothiolate of massfraction 0.01-0.02%, 0.1-0.3g Tris, 0.5-0.7g NaCl adds deionized water to 100ml, regulates pH to 7.4;
The proportioning of cleaning fluid is: the Sodium Mercurothiolate of massfraction 0.01-0.02%, and 1-3g Tris, 5-7g NaCl, 1 ml Tween-20 adds deionized water to 100ml, regulates pH to 7.4;
Substrate solution is made up of substrate A liquid and substrate B liquid, and wherein the proportioning of substrate A liquid is: the 1-2g sodium hydrogen phosphate, and the 0.5-1g citric acid, 30-50 μ l mass concentration 30% hydrogen peroxide adds deionized water to 100ml;
The proportioning of substrate B liquid is: 0.03-0.08g TMB, and the 0.1-0.2g citric acid, 0.01-0.05g EDTA, 20ml glycerine, 3ml DMSO adds deionized water to 100ml.
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| CN102818896B (en) * | 2011-06-09 | 2014-12-17 | 北京华大蛋白质研发中心有限公司 | Detection method of nitration modification sites based on specific antibodies and antibody capable of specifically recognizing succinyl-CoA: 3-oxoacid CoA transferase (SCOT) nitration sites |
| CN103926411B (en) * | 2014-04-22 | 2015-11-18 | 王少雄 | For the screening technique that the ELISA detection specific antibody of protein drug is right |
| CN104459110B (en) * | 2014-11-25 | 2016-01-13 | 成都威尔诺生物科技有限公司 | A kind of one-component TMB nitrite ion |
| CN104459114B (en) * | 2014-11-25 | 2016-03-30 | 成都威尔诺生物科技有限公司 | A kind of anti-interference one-component chromogenic enzyme substrate reagent |
| CN104459109B (en) * | 2014-11-25 | 2016-03-23 | 成都威尔诺生物科技有限公司 | A kind of one-component TMB nitrite ion for enzyme linked immunoassay |
| CN109696554A (en) * | 2019-03-08 | 2019-04-30 | 重庆派金生物科技有限公司 | The detection method of Pegylation protein drug metabolite in a kind of quantitative analysis body |
| CN115340598A (en) * | 2021-05-12 | 2022-11-15 | 复旦大学 | Application of PEG-scFv antibody in separation of free drug and PEG carrier drug and separation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1179469A (en) * | 1996-10-11 | 1998-04-22 | 财团法人生物技术开发中心 | Monoclonal antibody of p-methyl benzedrine with specificity, hybridoma producing same and kit containing monoclonal antibody and its use |
| WO2002094853A2 (en) * | 2001-05-21 | 2002-11-28 | Nektar Therapeutics Al, Corporation | Antibodies specific for poly(ethylene glycol) |
| WO2008063663A2 (en) * | 2006-11-21 | 2008-05-29 | University Of Southern California | Poly (ethylene glycol) anti-body detection assays and kits for performing thereof |
-
2009
- 2009-04-08 CN CN 200910301405 patent/CN101603965B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1179469A (en) * | 1996-10-11 | 1998-04-22 | 财团法人生物技术开发中心 | Monoclonal antibody of p-methyl benzedrine with specificity, hybridoma producing same and kit containing monoclonal antibody and its use |
| WO2002094853A2 (en) * | 2001-05-21 | 2002-11-28 | Nektar Therapeutics Al, Corporation | Antibodies specific for poly(ethylene glycol) |
| WO2008063663A2 (en) * | 2006-11-21 | 2008-05-29 | University Of Southern California | Poly (ethylene glycol) anti-body detection assays and kits for performing thereof |
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