CN101900725A - Method for detecting trace protein or polypeptide by utilizing positive-feedback enzyme reaction amplification system - Google Patents
Method for detecting trace protein or polypeptide by utilizing positive-feedback enzyme reaction amplification system Download PDFInfo
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Abstract
The invention belongs to the field of protein or polypeptide detection and discloses a method for detecting trace protein or polypeptide by utilizing a positive-feedback enzyme reaction amplification system. The method adopts an enzyme-linked immunosorbent assay method combining a positive-feedback enzymatic reaction amplification system and an enzyme reaction kinetic analysis method to detect the protein or polypeptide to be detected. The method has the advantages that the detection sensitivity can reach 1-50 fmol/L and is improved by 4-5 orders of magnitude compared with the conventional ELISA sensitivity.
Description
Technical field
The invention belongs to protein or polypeptide detection range, relate to a kind of highly sensitive positive-feedback enzyme reaction amplification system that utilizes and detect the method for trace amount of protein or polypeptide.
Background technology
Since Engvall in 1971 etc.
1Since the invention ELISA, this technology has been widely used in fundamental research and clinical diagnosis, be characterized in high specificity, good reproducibility, easy and simple to handle, do not need large-scale specific apparatus.
Since the eighties, scientist just explores the method that improves the ELISA detection sensitivity from many aspects, and wherein the introducing effect of amplification system is the most obvious, such as alkaline phosphatase and coenzyme I amplification system
2-4, clotting factor amplification system (ELCA)
5-9, the biotin-avidin amplification system
10, all improved the detection sensitivity of common ELISA greatly.
Along with the development of proteomics and clinical diagnosis, the requirement of ELISA detection sensitivity is improved constantly.The concentration change of some trace amount of protein or polypeptide is to the prediction of disease and diagnose significant in the blood plasma.Concentration such as the short natruresis peptide (BNP) of Type B in the human plasma is all significant for (HF) patient's in heart failure diagnosis, classification, clinical detection and prognosis etc.
11-14, and the concentration of BNP in blood plasma very low (10～100pg/ml), very high to the requirement of detection sensitivity.Up-to-date human plasma protein fraction matter group research
15Confirmed to have 4590 kinds of albumen in the blood plasma, shown to exist the huge utmost point low-abundance protein of number in the human plasma, further study the function of these utmost point low-abundance proteins and just must set up the very sensitive detection method of a cover.But still there is certain difficulty in the trace detection of protein, and most important reason is to lack technology highly sensitive the PCR of picture detection nucleic acid, that can increase to target protein.At present, Shang Weiyou adopts positive-feedback enzyme reaction amplification system desmoenzyme reaction kinetics analytical approach to detect the ELISA method of trace amount of protein or polypeptide.
Summary of the invention
Purpose of the present invention provides a kind of highly sensitive positive-feedback enzyme reaction amplification system desmoenzyme reaction kinetics analytical approach of utilizing to detect the ELISA method of trace amount of protein or polypeptide.
Another object of the present invention provides a kind of positive-feedback enzyme reaction amplification system.
The objective of the invention is to realize by following technical measures:
A kind of method that detects trace amount of protein or polypeptide, this method is for adopting the enzyme-linked immunosorbent assay method that combines positive feedback enzymatic reaction amplification system and enzyme kinetics analytical approach to trace amount of protein to be measured or polypeptide.
The method of described detection trace amount of protein or polypeptide is wherein carried out the ELISA detection to trace antigen protein to be measured or polypeptide; Be used to detect the detection antibody urokinase mark of antigen protein or polypeptide; Set up one by plasminogen and prourokinase or urokinase zymogen mutant M5, and the positive feedback enzymatic reaction amplification system of the urokinase of marker detection antibody composition; By the enzyme kinetics analytical approach, use the synthetic chromophoric substrate of fibrinolysin or the amount that the fluorescent small molecule substrate is measured the fibrinolysin that produces in the positive feedback enzymatic reaction amplification system, reflect the amount of the urokinase of marker detection antibody, thereby draw the content of testing protein or polypeptide.
The method of described detection trace amount of protein or polypeptide, wherein positive-feedback enzyme reaction amplification system is that the potpourri of plasminogen and prourokinase or urokinase zymogen mutant M5 is under the catalysis of the urokinase of marker detection antibody, by the constantly positive feedback enzymatic reaction of catalysis mutually, produce the detected fibrinolysin of available enzyme dynamic method.
The method of described detection trace amount of protein or polypeptide wherein is to use the amino of the covalently bound detection antibody of SMPT and the interchain sulfydryl of urokinase to detecting antibody with the urokinase mark, obtains detecting the covalent coupling thing of antibody and urokinase.
The method of described detection trace amount of protein or polypeptide, the beta-mercaptoethanol that wherein uses 0.2mmol/L is the prourokinase interchain disulfide bond also, the interchain sulfydryl that generation can be reacted, and do not influence the intrachain disulfide bond relevant with urokinase activity.
The method of described detection trace amount of protein or polypeptide, wherein the method for purification assays antibody and urokinase covalent coupling thing is to contain Protein G affinity chromatographic column on the solution that detects antibody and urokinase covalent coupling thing, use 0.1mol/L, the detection antibody of not coupling is removed in the glycine solution washing of pH2.8, use contains the 0.1mol/L of 0.5mol/L sodium chloride, and the glycine solution wash-out of pH2.8 obtains detecting the covalent coupling thing of antibody and urokinase.
The method of described detection trace amount of protein or polypeptide, wherein the synthetic chromophoric substrate of fibrinolysin is chromophoric substrate S2251; The fluorescent small molecule substrate is H-D-Val-Leu-Lys-AMC.
A kind of urokinase zymogen mutant M5, this mutant are that the 300th lysine is replaced by histidine in the former amino acid sequence of natural urokinase.
The method of described detection trace amount of protein or polypeptide, wherein the preparation method of plasminogen is to use lysine affinity chromatographic column separation and purification plasminogen from people's fresh plasma, adds phenylmethylsulfonyl fluoride in all solution of chromatography process and blood plasma.
A kind of positive feedback enzymatic reaction amplification system, this system are by plasminogen and prourokinase or urokinase zymogen mutant M5, and urokinase is formed; Plasminogen is converted into fibrinolysin under the catalysis of urokinase, the fibrinolysin that produces makes prourokinase or urokinase zymogen mutant M5 in the system be converted into urokinase, urokinase continues the catalysis plasminogen again and is converted into fibrinolysin, thereby produce more fibrinolysin, form the enzymatic reaction system that a positive feedback is constantly amplified.
Technological core of the present invention is a kind of positive-feedback enzyme reaction amplification system of being made up of plasminogen, prourokinase or its mutant, urokinase (as shown in Figure 1), this system has by a pair of that the interactional proteinase of positive feedback---plasminogen and prourokinase or its mutant are formed, and urokinase is the triggering agent as this system.Usually, be not higher than the plasminogen of 2 μ mol/L and be not higher than the prourokinase of 40pmol/L or the mixed system of its mutant is a metastable enzyme system, enter this system, can quicken whole enzymatic reaction process as the trace urokinase.Be that the trace urokinase can make plasminogen be converted into fibrinolysin, the fibrinolysin that produces makes the prourokinase in the system be converted into double chain urokinase, double chain urokinase can continue the catalysis plasminogen again and be converted into fibrinolysin, thereby produce more fibrinolysin, therefore form the process that a positive feedback is constantly amplified.Use sensitive enzyme kinetic analysis method to come the fibrinolysin that produces in the detection architecture, thereby reflect the amount of urokinase.When being used for ELISA when reaction, by the bag antibody that is hunted down, seal non-specific site, after the washing, add antigenic solution to be detected and hatch.Behind the flush away antigenic solution, add the detection antibody that is marked with urokinase, fully washing, add above-mentioned positive-feedback enzyme reaction amplification system again, the final amount of using fibrinolysin in the enzyme kinetics analytical approach mensuration system, thereby reflect that coupling has the amount of the detection antibody of urokinase, therefore can calculate the concentration of antigen to be detected.
Characteristics of the present invention also are the detection antibody among the ELISA and the covalent coupling of urokinase, by the beta-mercaptoethanol that uses 0.2mmol/L urokinase is carried out partial reduction, promptly only go back the interchain disulfide bond of prourokinase, the interchain sulfydryl that generation can be reacted, and do not influence active closely-related intrachain disulfide bond with it, therefore have the urokinase catalyst activated centre the light chain part can by sulfydryl with through SMPT (4-succinimide oxygen carbonyl-methyl-a[2-pyridine radicals two sulphur] for toluene, the detection antibodies that 4-succinimidyloxycarbonyl-methyl-a-(2-pyridyldithio)-toluene) modifies.
The present invention has adopted a kind of urokinase zymogen mutant (M5) in positive-feedback enzyme reaction amplification system, it has the former amino acid sequence of natural urokinase, but its lysine of the 300th is suddenlyd change, become histidine, this sudden change can make its inherent activity ratio's natural urokinase former low more than 4 times, and to make its double chain activity be 2 times of urokinase.When this mutant is used for positive-feedback enzyme reaction amplification system, its lower inherent enzymatic activity has guaranteed the relative stability of itself and plasminogen mixed system, after it is converted to double chain urokinase, the catalysis plasminogen is converted into fibrinolysin at faster speed again, further improves the efficient of whole positive-feedback enzyme reaction amplification system.
Effective effect of the present invention:
The present invention is in order to improve the sensitivity that trace amount of protein or polypeptide detect, the method that a kind of use in conjunction ELISA, positive feedback enzymatic reaction amplification system and enzyme kinetics analysis improve detection sensitivity is proposed, its detection sensitivity reaches 1～50fmol/L, than 4～5 orders of magnitude of sensitivity raising of conventional ELISA.The positive-feedback enzyme reaction amplification system of the present invention's design and present any reported method are all inequality.
Description of drawings
Fig. 1 is the synoptic diagram of ELISA, positive-feedback enzyme reaction system.The positive-feedback enzyme reaction amplification system that expression is made up of plasminogen, prourokinase, urokinase in the square frame.
Fig. 2 is 26E2-UK SDS-PAGE electrophoretogram (8% a non-reduced gel).Molecular weight is 190kD, indirect proof detection antibody and urokinase activity chain part to form the covalent coupling thing at 1: 1.
Fig. 3 is plasminogen SDS-PAGE electrophoretogram (10% goes back virgin rubber).Gel density gradient scanning analysis shows purity greater than 90%, and does not have fibrinolysin through the S2251 enzyme kinetic analysis and pollute, and meets the requirement of positive-feedback enzyme reaction amplification system.
Fig. 4 is the activation experiment of plasminogen.Solid line is represented to be activated by prourokinase, and dotted line is represented to be activated by M5.Wherein plasminogen (Glu-plasminogen) concentration is 2 μ mol/L, and S2251 concentration is 1.5mmol/L, and the concentration of prourokinase or M5 is 0.075nmol/L.By curve among the figure as can be known M5 compare with natural prourokinase, long lag period is arranged, but just rises rapidly afterwards, reflect its higher double chain activity.
Fig. 5 is that ELISA associating positive-feedback enzyme reaction amplification system and enzyme kinetic analysis detect BNP.(a) use common chromophoric substrate S2251; (b) use fluorogenic substrate H-D-Val-Leu-Lys-AMC.Wherein * represents this BNP concentration group (comprising all BNP concentration groups that are higher than this concentration) there were significant differences (P＜0.01) with blank group.
Fig. 6 is that ELISA associating positive-feedback enzyme reaction amplification system and enzyme kinetic analysis detect rhEPO.(a) use common chromophoric substrate S2251; (b) use fluorogenic substrate H-D-Val-Leu-Lys-AMC.Wherein * represents this rhEPO concentration group (comprising all rhEPO concentration groups that are higher than this concentration) there were significant differences (P＜0.01) with blank group.
Fig. 7 is that ELISA associating positive-feedback enzyme reaction amplification system and enzyme kinetic analysis detect rhGH.(a) use common chromophoric substrate S2251; (b) use fluorogenic substrate H-D-Val-Leu-Lys-AMC.Wherein * represents this rhGH concentration group (comprising all rhGH concentration groups that are higher than this concentration) there were significant differences (P＜0.01) with blank group.
Fig. 8 is that ELISA associating positive-feedback enzyme reaction amplification system (prourokinase) and enzyme kinetic analysis detect BNP.(a) use common chromophoric substrate S2251; (b) use fluorogenic substrate H-D-Val-Leu-Lys-AMC.Wherein * represents this BNP concentration group (comprising all BNP concentration groups that are higher than this concentration) there were significant differences (P＜0.01) with blank group.
Embodiment
The invention will be further elaborated by the following examples.
1. material of the present invention:
1. main agents: urokinase and BNP are provided by Suzhou Su Lan biological medicine scientific and technological development company limited.Pairing monoclonal antibody 26E2 (detection antibody) that BNP detects and 50E1 (capture antibody) are available from Abcam company.4-succinimidyloxycarbonyl-methyl-a-(2-pyridyldithio)-toluene (SMPT) is available from Pierce company.Protein G affinity column is available from Pierce company.Human plasma is available from Nanjing HongShiZi blood Center.Micromolecule chromophoric substrate S2444 (Glu-Gly-Arg-pNAHCl) (urokinase substrate), S2251 (H-D-Val-Leu-Lys-pNA2HCl) (fibrinolysin substrate) are available from Chromogenix company.Fibrinolysin micromolecule fluorogenic substrate (H-D-Val-Leu-Lys-AMC) is available from Bachem company.Lysine-Sepharose 4B is available from GE (Amersham) company.
RhEPO is available from AMGEN company, and anti-hEPO monoclonal antibody (MAB287) and anti-rhEPO polyclonal antibody (AB-286-NA) are available from R ﹠amp; D SYSTEMS company.
RhGH is available from Genentech company, and anti-hGH monoclonal antibody (MAB1067) and anti-rhGH polyclonal antibody (AF1067) are available from R ﹠amp; D SYSTEMS company.
2. key instrument and consumptive material: the long fluorescence microplate reader of Synergy HT all-wave is available from Bio-Tek company.
96 hole ELISA Plate are available from Corning company.
1. detect the covalent coupling of antibody and urokinase and the purifying of conjugate: the detection antibody 26E2 of BNP is dissolved in the PBS solution with the concentration of 2mg/ml, adds excessive SMPT, SMPT is by the amino and the antibody covalent cross-linking of antibody.Remove free unreacted SMPT by dialysis, thereby obtain the 26E2 antibody (SMPT-26E2) that SMPT modifies.Urokinase is dissolved in (pH 7.8) in phosphate-EDTA solution, and the beta-mercaptoethanol that uses 0.2mmol/L is with the restricted interchain sulfydryl that is reduced to of the interchain disulfide bond of urokinase.Light chain with urokinase catalyst activated centre forms the covalent coupling thing 26E2-UK that detects antibody and urokinase by interchain sulfydryl and equimolar SMPT-26E2 coupling.Use the Protein G affinity chromatographic column above-mentioned covalent coupling thing of purifying (26E2-UK), use 0.1mol/L glycine solution (pH2.8) washing to remove free antibodies, use 0.1mol/L glycine solution (pH2.8) wash-out that contains 0.5mol/L sodium chloride to obtain 26E2-UK.Use the S2444 enzyme kinetic analysis to identify the enzyme activity of 26E2-UK.
2. the preparation of plasminogen: 25ml Freshman blood plasma dilutes with 20ml sodium phosphate buffer (pH7.5).With 0.1mol/L sodium phosphate buffer (pH7.5) balance Lysine-Sepharose 4B, go up sample subsequently.Last sample finishes, with containing sodium phosphate buffer (pH7.5) the washing column bed of 0.5mol/L sodium chloride, until 280nm absorbance value (OD 280nm) to baseline values.With 0.2mol/L EACA (ε-amino-n-caproic acid, SIGMA) wash-out Lysine-Sepharose4B, the OD 280nm of mensuration eluent.All add PMSF and EDTA in above-mentioned all solution and the blood plasma, make its final concentration be respectively 1mmol/L and 3mmol/L.Wash-out is collected liquid the solution of 50mmol/L Tris, 100mmol/L NaCl (pH7.5) is dialysed, to remove EACA.SDS-PAGE identifies its purity, uses the S2251 enzyme kinetic analysis to measure and has or not fibrinolysin to pollute.
3. the preparation of urokinase zymogen mutant M5
16: the pFC16 plasmid that contains the former full-length cDNA of natural urokinase with the HindIII-BamHI double digestion
17The endonuclease bamhi subclone is to M13 carrier (mp18), the former amino acid sequence mutational site of one section of the synthetic coding natural urokinase (oligonucleotides of Lys300 → His), make itself and the hybridization of reorganization M13 carrier, through after amplification, connecting and transform, (positive colony of Lys300 → His) carries out dna sequencing to select sudden change.Use the HindIII-BamHI double digestion once more, make sudden change back total length (M5) sequence insert the pFC16 plasmid vector again, Transformed E scherichia coli type B bacterial strain.Through the ultrasonic processes such as bacterium, change renaturation, prourokinase antibody affinity chromatography, G25 gel chromatography of splitting, obtain M5.Wherein (DFP diisopropylfluorophosphate) handles and thoroughly removes the double-stranded M5 of trace, obtains pure product M5 by benzamidine-Sepharose chromatographic column and diisopropyl fluorophosphate.
4. enzyme linked immunological adsorption reaction (ELISA): use the concentration bag quilt of the capture antibody 50E1 of BNP with 5 μ g/ml
The hole ELISA Plate, 4 ℃ spend the night or 37 ℃ hatched 1 hour.Use PBS-T (adding 0.1% Tween-20 in the PBS damping fluid) detersive enzyme target 3 times.Use the PBS-T solution of 1.5%BSA to seal non-specific site.After PBS-T washing 3 times, add BNP to be measured (every hole 100 μ L), hatched 1 hour for 37 ℃.Use PBS-T detersive enzyme target 3 times.The 26E2-UK solution that adds 0.2 μ g/mL was hatched 1 hour for 37 ℃.Thoroughly flush away is not in conjunction with after the compound, the potpourri that adds plasminogen (1 μ mol/L) and urokinase zymogen mutant M5 (5pmol/L), 37 ℃ of joltings were reacted 2 hours, finally added fibrinolysin fluorogenic substrate (H-D-Val-Leu-Lys-AMC) or common chromophoric substrate S2251 colour developing.In fluorescence microplate reader (Synergy HT, Bio-Tek) carrying out enzyme kinetics on measures, read and calculate Δ F/min respectively (for fluorogenic substrate, exciting light 310nm, emission light 460nm) and Δ mOD/min (for chromophoric substrate, wavelength 405nm), Δ F/min and Δ mOD/min and BNP concentration are proportional.
The result:
1. the preparation of 26E2-UK and evaluation: use Protein G purifying conjugate, (the 0.1mol/L glycine solution antibody of coupling pH2.8) can only wash-out take place, and conjugate is combined in still on the Protein G conventional elution requirement.Using the 0.1mol/L glycocoll to contain the solution (pH2.8) of 0.5mol/L sodium chloride just can wash-out conjugate 26E2-UK.Show that through irreducibility SDS-PAGE (SDS-polyacrylamide gel electrophoresis) the 26E2-UK molecular weight is 190kDa (Fig. 2).Use the S2444 enzyme kinetic analysis to measure the enzyme activity of 26E2-UK, be about 86% of initial adding urokinase vigor.Further the beta-mercaptoethanol of proof 0.2mmol/L has only reduced the interchain disulfide bond of urokinase, and has guaranteed that its intrachain disulfide bond is unaffected.The amount of measuring 26E2-UK by BCA (Pierce) protein quantification is 18.1mg, and the urokinase activity that enzyme kinetic analysis is measured among the 26E2-UK is equivalent to 5.2mg urokinase albumen, can calculate detection antibody and 1: 1 covalent complex of urokinase activity chain part formation that SMPT modifies.
2. the preparation of plasminogen and evaluation: the separable 4mg plasminogen that obtains of 25ml blood plasma.SDS-PAGE shows that its purity is greater than 90% (Fig. 3).Measure through the S2251 enzyme kinetic analysis, no fibrinolysin pollutes.
3. the preparation of urokinase zymogen mutant M5 and evaluation:, shown the low inherent enzymatic activity of M5 and higher SK-SD activity by to a series of enzyme kinetic analysis of M5 and comparison (table 1, Fig. 4) former with natural urokinase, urokinase thereof.
Table 1: the enzyme kinetic analysis of prourokinase, M5, urokinase, double-stranded M5 hydrolysis S2444.
4. ELISA associating positive-feedback enzyme reaction amplification system and enzyme kinetic analysis detect BNP: shown in Fig. 5 a, use the common chromophoric substrate of fibrinolysin can detect the BNP that is low to moderate 50fmol/L (150fg/ml), and use fibrinolysin fluorogenic substrate (Fig. 5 b) can detect the BNP that is low to moderate 1fmol/L (3fg/ml).
Embodiment 2---the detection of recombined human erythropoietin (rhEPO)
EPO is a kind of glycoprotein that is mainly produced by kidney, and its major function is to promote erythropoiesis, improves the concentration of haemoglobin in the blood.Be mainly used in the treatment of renal anemia clinically.Because EPO to the raising effect of oxygen carrying capacity, often is used as excitant by some sportsmen that participate in endurance events.EPO content of (blood, urine etc.) in body fluid is lower, and this requires detection method that enough sensitivity will be arranged.
The covalent coupling of 1. anti-rhEPO monoclonal antibody (MAB287) and urokinase: coupling method, condition are with the coupling of monoclonal antibody 26E2 and urokinase among the embodiment 1, and the polyclone for preparing the urokinase mark thus detects antibody MAB287-UK.
2. the enzyme linked immunological adsorption reaction of amplifying detects rhEPO: use the concentration bag quilt of anti-rhEPO polyclonal antibody AB-286-NA with 5 μ g/ml
96 hole ELISA Plate, 4 ℃ spend the night or 37 ℃ hatched 1 hour.Use PBS-T detersive enzyme target 3 times.Use the PBS-T solution of 1.5%BSA to seal non-specific site.After the PBS-T washing 3 times, add rhEPO to be measured, hatched 1 hour for 37 ℃.Use PBS-T detersive enzyme target 3 times.The MAB287-UK solution that adds 0.2 μ g/mL was hatched 1 hour for 37 ℃.Thoroughly flush away is not after the binding antibody, the potpourri that adds plasminogen (1 μ mol/L) and urokinase zymogen mutant M5 (5pmol/L), 37 ℃ of joltings were reacted 2 hours, finally added fibrinolysin fluorescent small molecule substrate (H-D-Val-Leu-Lys-AMC) or common chromophoric substrate S2251 colour developing.(Synergy HT carries out enzyme kinetics on Bio-Tek) and measures, and reads and calculate Δ F/min and Δ mOD/min respectively, and Δ F/min and Δ mOD/min and rhEPO concentration are proportional at fluorescence microplate reader.
The result:
ELISA associating positive-feedback enzyme reaction amplification system and enzyme kinetic analysis detect rhEPO: shown in Fig. 6 a, use the common chromophoric substrate of fibrinolysin can detect the rhEPO that is low to moderate 30fmol/L (1pg/ml); Shown in Fig. 6 b, use the fibrinolysin fluorogenic substrate can detect the rhEPO that is low to moderate 0.5fmol/L (17fg/ml).
Embodiment 3---human growth hormone recombinant's (rhGH) detection
Human growth hormone (HGH) (hGH) is synthetic by anteriorpituitary, and it is a pleiotropism cell factor, by with the transmembrane receptor of cell surface in conjunction with the performance biological effect, its topmost function is to promote protein synthesis and body growth metabolism.It is of short and small stature to be mainly used in treatment clinically.Because it promotes the physiological action of growth to lure that some sportsman makes a desperate move into it is used as excitant.Therefore the high-sensitivity detection of rhGH is very necessary for clinical diagnosis and drug-testing.
The covalent coupling of 1. anti-rhGH monoclonal antibody (MAB1067) and urokinase: coupling method, condition are with the coupling of monoclonal antibody 26E2 and urokinase among the embodiment 1, and the polyclone for preparing the urokinase mark thus detects antibody MAB1067-UK.
2. the enzyme linked immunological adsorption reaction of amplifying detects rhGH: use the concentration bag quilt of anti-rhGH polyclonal antibody AF1067 with 5 μ g/ml
96 hole ELISA Plate, 4 ℃ spend the night or 37 ℃ hatched 1 hour.Use PBS-T detersive enzyme target 3 times.Use the PBS-T solution of 1.5%BSA to seal non-specific site.After PBS-T washing 3 times, add rhGH to be measured, hatched 1 hour for 37 ℃.Use PBS-T detersive enzyme target 3 times.The MAB1067-UK solution that adds 0.2 μ g/mL was hatched 1 hour for 37 ℃.Thoroughly flush away is not after the binding antibody, the potpourri that adds plasminogen (1 μ mol/L) and urokinase zymogen mutant M5 (5pmol/L), 37 ℃ of joltings were reacted 2 hours, finally added fibrinolysin fluorescent small molecule substrate (H-D-Val-Leu-Lys-AMC) or common chromophoric substrate S2251 colour developing.(Synergy HT carries out enzyme kinetics on Bio-Tek) and measures, and reads and calculate Δ F/min and Δ mOD/min respectively, and Δ F/min and Δ mOD/min and rhGH concentration are proportional at fluorescence microplate reader.
The result:
ELISA associating positive-feedback enzyme reaction amplification system and enzyme kinetic analysis detect rhGH: shown in Fig. 7 a, use the common chromophoric substrate of fibrinolysin can detect the rhGH that is low to moderate 60fmol/L (1.3pg/ml); Shown in Fig. 7 b, use the fibrinolysin fluorogenic substrate can detect the rhGH that is low to moderate 1fmol/L (22fg/ml).
Embodiment 4---the detection (the enzyme amplification system uses prourokinase) of BNP
The enzyme linked immunological adsorption reaction of amplifying detects BNP: use the concentration bag quilt of the capture antibody 50E1 of BNP with 5 μ g/ml
96 hole ELISA Plate, 4 ℃ spend the night or 37 ℃ hatched 1 hour.Use PBS-T detersive enzyme target 3 times.Use the PBS-T solution of 1.5%BSA to seal non-specific site.After PBS-T washing 3 times, add BNP to be measured, hatched 1 hour for 37 ℃.Use PBS-T detersive enzyme target 3 times.The 26E2-UK solution that adds 0.2 μ g/mL was hatched 1 hour for 37 ℃.Thoroughly flush away is not in conjunction with after the compound, the potpourri that adds plasminogen (1 μ mol/L) and prourokinase (5pmol/L), 37 ℃ of joltings were reacted 4 hours, finally added fibrinolysin fluorogenic substrate (H-D-Val-Leu-Lys-AMC) or common chromophoric substrate S2251 colour developing.In fluorescence microplate reader (Synergy HT, Bio-Tek) carrying out enzyme kinetics on measures, read and calculate Δ F/min respectively (for fluorogenic substrate, exciting light 310nm, emission light 460nm) and Δ mOD/min (for chromophoric substrate, wavelength 405nm), Δ F/min and Δ mOD/min and BNP concentration are proportional.
The result:
ELISA associating positive-feedback enzyme reaction amplification system (prourokinase) and enzyme kinetic analysis detect BNP: shown in Fig. 8 a, use the common chromophoric substrate of fibrinolysin can detect the BNP that is low to moderate 50fmol/L (150fg/ml), and use fibrinolysin fluorogenic substrate (Fig. 8 b) can detect the BNP that is low to moderate 5fmol/L (15fg/ml).
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Claims (10)
1. a method that detects trace amount of protein or polypeptide is characterized in that this method is for adopting the enzyme-linked immunosorbent assay method that combines positive feedback enzymatic reaction amplification system and enzyme kinetics analytical approach to trace amount of protein to be measured or polypeptide.
2. the method for detection trace amount of protein according to claim 1 or polypeptide is characterized in that trace antigen protein to be measured or polypeptide are carried out the ELISA detection; Be used to detect the detection antibody urokinase mark of antigen protein or polypeptide; Set up one by plasminogen and prourokinase or urokinase zymogen mutant M5, and the positive feedback enzymatic reaction amplification system of the urokinase of marker detection antibody composition; By the enzyme kinetics analytical approach, use the synthetic chromophoric substrate of fibrinolysin or the amount that the fluorescent small molecule substrate is measured the fibrinolysin that produces in the positive feedback enzymatic reaction amplification system, reflect the amount of the urokinase of marker detection antibody, thereby draw the content of testing protein or polypeptide.
3. the method for detection trace amount of protein according to claim 2 or polypeptide, it is characterized in that positive-feedback enzyme reaction amplification system is that the potpourri of plasminogen and prourokinase or urokinase zymogen mutant M5 is under the catalysis of the urokinase of marker detection antibody, by the constantly positive feedback enzymatic reaction of catalysis mutually, produce the detected fibrinolysin of available enzyme dynamic method.
4. the method for detection trace amount of protein according to claim 2 or polypeptide, it is characterized in that being to use the amino of the covalently bound detection antibody of SMPT and the interchain sulfydryl of urokinase with the urokinase mark, obtain detecting the covalent coupling thing of antibody and urokinase detecting antibody.
5. the method for detection trace amount of protein according to claim 4 or polypeptide, the beta-mercaptoethanol that it is characterized in that using 0.2mmol/L is the prourokinase interchain disulfide bond also, the interchain sulfydryl that generation can be reacted, and do not influence the intrachain disulfide bond relevant with urokinase activity.
6. the method for detection trace amount of protein according to claim 4 or polypeptide, the method that it is characterized in that purification assays antibody and urokinase covalent coupling thing is to contain ProteinG affinity chromatographic column on the solution that detects antibody and urokinase covalent coupling thing, use 0.1mol/L, the detection antibody of not coupling is removed in the glycine solution washing of pH2.8, use contains the 0.1mol/L of 0.5mol/L sodium chloride, and the glycine solution wash-out of pH2.8 obtains detecting the covalent coupling thing of antibody and urokinase.
7. the method for detection trace amount of protein according to claim 2 or polypeptide, the synthetic chromophoric substrate that it is characterized in that fibrinolysin is chromophoric substrate S2251; The fluorescent small molecule substrate is H-D-Val-Leu-Lys-AMC.
8. a urokinase zymogen mutant M5 is characterized in that this mutant is that the 300th lysine is replaced by histidine in the former amino acid sequence of natural urokinase.
9. according to the method for claim 2 or 3 described detection trace amount of protein or polypeptide, the preparation method who it is characterized in that plasminogen is to use lysine affinity chromatographic column separation and purification plasminogen from people's fresh plasma, adds phenylmethylsulfonyl fluoride in all solution of chromatography process and blood plasma.
10. a positive feedback enzymatic reaction amplification system is characterized in that this system by plasminogen and prourokinase or urokinase zymogen mutant M5, and urokinase is formed; Plasminogen is converted into fibrinolysin under the catalysis of urokinase, the fibrinolysin that produces makes prourokinase or urokinase zymogen mutant M5 in the system be converted into urokinase, urokinase continues the catalysis plasminogen again and is converted into fibrinolysin, thereby produce more fibrinolysin, form the enzymatic reaction system that a positive feedback is constantly amplified.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0268465A2 (en) * | 1986-11-19 | 1988-05-25 | Bioclones (Proprietary) Limited | Method for detecting the presence of a plant antigen in a plant |
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|---|---|---|---|---|
| EP0268465A2 (en) * | 1986-11-19 | 1988-05-25 | Bioclones (Proprietary) Limited | Method for detecting the presence of a plant antigen in a plant |
Non-Patent Citations (3)
| Title |
|---|
| 兰风华; 朱忠勇: "放大与ELISA", 《国际免疫学杂志》 * |
| 杨永钦: "ELISA的放大系统", 《畜牧与兽医》 * |
| 程伟华综述 黎才海审校: "uPA系统与肿瘤侵袭转移的关系", 《实用癌症杂志》 * |
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