CN102207501A - Biotinylation bovine serum albumin and streptavidin enzyme-labeled reaction plate and preparation method thereof - Google Patents
Biotinylation bovine serum albumin and streptavidin enzyme-labeled reaction plate and preparation method thereof Download PDFInfo
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- CN102207501A CN102207501A CN2011100687829A CN201110068782A CN102207501A CN 102207501 A CN102207501 A CN 102207501A CN 2011100687829 A CN2011100687829 A CN 2011100687829A CN 201110068782 A CN201110068782 A CN 201110068782A CN 102207501 A CN102207501 A CN 102207501A
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- 108091003079 Bovine Serum Albumin Proteins 0.000 title claims abstract description 50
- 229940098773 bovine serum albumin Drugs 0.000 title claims abstract description 50
- 230000006287 biotinylation Effects 0.000 title claims abstract description 49
- 238000007413 biotinylation Methods 0.000 title claims abstract description 49
- 108010090804 Streptavidin Proteins 0.000 title claims abstract description 45
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 title abstract description 29
- 108090000790 Enzymes Proteins 0.000 title abstract description 29
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 31
- 229960002685 biotin Drugs 0.000 claims abstract description 16
- 235000020958 biotin Nutrition 0.000 claims abstract description 16
- 239000011616 biotin Substances 0.000 claims abstract description 16
- 239000007791 liquid phase Substances 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 229920002223 polystyrene Polymers 0.000 claims abstract description 10
- 239000004793 Polystyrene Substances 0.000 claims abstract description 9
- 238000010790 dilution Methods 0.000 claims description 52
- 239000012895 dilution Substances 0.000 claims description 52
- 238000006911 enzymatic reaction Methods 0.000 claims description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 238000002965 ELISA Methods 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000012153 distilled water Substances 0.000 claims description 17
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 16
- 239000002953 phosphate buffered saline Substances 0.000 claims description 16
- 238000012546 transfer Methods 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 10
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 10
- 235000019800 disodium phosphate Nutrition 0.000 claims description 10
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 10
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 10
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- BMVXCPBXGZKUPN-UHFFFAOYSA-N 1-hexanamine Chemical compound CCCCCCN BMVXCPBXGZKUPN-UHFFFAOYSA-N 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 229960004799 tryptophan Drugs 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 2
- 239000000427 antigen Substances 0.000 abstract description 18
- 102000036639 antigens Human genes 0.000 abstract description 18
- 108091007433 antigens Proteins 0.000 abstract description 18
- 238000008416 Ferritin Methods 0.000 abstract description 15
- 102000008857 Ferritin Human genes 0.000 abstract description 11
- 108050000784 Ferritin Proteins 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 10
- 239000000463 material Substances 0.000 abstract description 8
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- 239000003814 drug Substances 0.000 abstract 1
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- 230000035484 reaction time Effects 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 6
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- 108090001008 Avidin Proteins 0.000 description 3
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- 238000010998 test method Methods 0.000 description 3
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- 238000007865 diluting Methods 0.000 description 2
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- 238000004321 preservation Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
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- PYXDZWUIPFCTJP-XFNAGHOKSA-N (2s)-2-azanyl-3-(1h-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1.C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 PYXDZWUIPFCTJP-XFNAGHOKSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- WZELXJBMMZFDDU-UHFFFAOYSA-N Imidazol-2-one Chemical group O=C1N=CC=N1 WZELXJBMMZFDDU-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a biotinylation bovine serum albumin and streptavidin enzyme-labeled reaction plate and a preparation method thereof, belonging to the field of labeling immunoassay in laboratory medicine. The reaction plate comprises a polystyrene enzyme-labeled plate; each micropore is respectively coated with the biotinylation bovine serum albumin and the streptavidin; and a biotinylation antibody is stored in a liquid phase to serve as a reaction reagent for later use. When in detection, the biotinylation antibody and an antigen to be detected are simultaneously added on the enzyme-labeled reaction plate; the biotinylation antibody and the antigen to be detected are simultaneously reacted; and biotin molecules and the streptavidin are combined and fixed on the surface of solid phase materials. According to the invention, the antigen to be detected and the biotinylation antibody are placed in the liquid phase so as to improve reaction rate, widen detection range of ferritin, and reduce hook effects in double antibody sandwich experiment. Indexes such as reaction sensitivity, accuracy, precision and the like of the enzyme label satisfy the technical requirements; the usage ratio of the coated antibody is improved; the usage amount of the antibody is reduced; and the cost of a reagent box is greatly reduced.
Description
Technical field
The present invention relates to the enzyme-linked immuno assay technology, specifically is that a kind of bag is by enzyme reaction plate of biotinylation bovine serum albumin(BSA) and Streptavidin and preparation method thereof.
Background technology
Enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) be to be main agents with enzymic-labelled antibody (or antigen) and the enzyme reaction plate that is coated with antibody (antigen), a kind of labelled immune analytical technology that the specificity and the substrate for enzymatic activity high efficiency of antigen-antibody reaction combined.ELISA major technique type comprises: double antibody sandwich method, indirect method, competition law, prize law etc.Double antibody sandwich method is one of common technology, can be used for micro substance in the detection bodies.
Enzyme linked immunosorbent assay belongs to heterogeneous immunoassay, needs separating and combining label and free label during mensuration, just can make detection signal relevant with antigen-antibody reaction intensity.The general solid phase adsorption mode that adopts realizes that free label separates, and measures binding label.Be using polystyrol as solid phase material (microwell plate, test tube, microballoon), connect specific antibody by the non-specific adsorption mode.As containing corresponding antigens in the sample, will combine with specific antibody, form immune complex and be fixed on the solid phase material surface equally, bound substances is not free in the liquid phase, can remove by mode of washing.
The double-antibody sandwich enzymoimmunoassay need combine capture antibody with enzyme reaction plate (96 hole micro-reaction plate), form the enzyme reaction plate of coated antibody in advance, and this process is called bag (promptly being prepared enzyme reaction plate).After adding determined antigen and enzyme labelled antibody, the immune complex that forms is fixed on the reaction plate surface by the capture antibody on the enzyme reaction plate, superfluous enzyme labelled antibody (and other non-material-specifics) is present in the liquid phase, reject reactant liquor and washing reaction plate just can be removed the label (enzyme labelled antibody) that does not have combination.Enzyme reaction plate is the enzyme-linked immuno assay important materials, and therefore, the preparation enzyme reaction plate is the enzyme linked immunosorbent assay key link.
The non-specific adsorption technology is adopted in the preparation of tradition enzyme reaction plate.The preparation method be with specific antibody with bag be cushioned liquid (phosphate or carbonate) be diluted to finite concentration (3 ~ 10ng/L), add in each micropore of enzyme reaction plate.Because polystyrene has the adhesion protein characteristic, can cross physics (electric charge) absorption antibody molecule.After after a while, antibody promptly is fixed in the solid phase material surface, keeps original antibody activity simultaneously.Because protein concentration is very low in the coating buffer, antibody molecule can not cover all sites fully, and non-specific adsorption takes place when preventing subsequent reactions, need add high concentration bovine serum albumin(BSA) (bovine serum again, albumin, BSA) (1% ~ 2%) reaches the blank site of sealing purpose.At last, the reject lock solution gets final product through the vacuum drying packing.
The enzyme reaction plate of classical way preparation be with the antibody molecule direct coated in the enzyme reaction plate surface, be commonly referred to as the direct coated pattern.Advantages such as that the direct coated pattern has is simple to operate, technical maturity.But there are many shortcomings in the direct coated pattern, mainly shows following aspect:
One, antibody molecule are attracted to the solid phase material surface, because antigen-antibody reaction depends on space conformation, the conformation change that the antibody immobilization is caused certainly will influence the utilization ratio of antibody, causes the attenuating of affinity between the antigen-antibody.
Two, the surface area in the micro-ELISA Plate micropore is less, cause the coated antibody molecular amounts limited, add the reduction of utilization factor that immobilization causes, all can make antibody molecule quantity can not satisfy the demand of determined antigen, influence the sensing range and the detection time of test method.
Three, the immobilization antibody molecule remains static, and causes on the solid phase that the probability that collides of antigen will inevitably increase antigen-antibody reaction and reach the required time of balance forever less than liquid phase reactor in the antibody and liquid phase.When single stage method, need add determined antigen and enzyme labelled antibody simultaneously simultaneously, the reaction rate of determined antigen and capture antibody causes " ditch shape effect " to take place less than the reaction rate of determined antigen and enzyme labelled antibody, false negative result occurs.
(biotin, B) molecular weight is 244. 31 to biotin, now can manually synthesize, and is easy to prepare.The basic structure of biotin is that twin nuclei: I ring is the imidazolone ring, is the position that combines with Avidin; II is a thiphene ring, contains a valeric acid side chain, and its terminal carboxyl group can be connected with biomacromolecule, forms biotin labeling antigen, antibody, enzyme etc.Biotin with do not influence biomolecule and the original biologically active of biotin after biomacromolecule combines, promptly biotin labeling antibody possesses the characteristic that combines with Avidin and corresponding antigens simultaneously.
Strepto-affinity element (streptavidin, SA), molecular weight 65KD is made up of four identical peptide chains, and the plain molecular energy of strepto-affinity is in conjunction with four biotin molecules.Compare with Avidin, Streptavidin has high affinity to biotin, and (affinity costant is 10
15/ mol), combine the back with biotin and form highly stable compound, be difficult to dissociate.
Summary of the invention
The present invention is exactly in order to solve classical directly antibody sandwich pattern inherent shortcoming, providing a kind of is primary raw material with biotinylation bovine serum albumin(BSA), Streptavidin, the characteristic of utilizing biotin to combine with Streptavidin, and a kind of liquid phase antigen and antibody response mode are provided, shorten biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate that reaction reaches the balance required time.
The present invention realizes by following technical scheme.
A kind of biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, comprising the polystyrene ELISA Plate, is the Streptavidin of 1:3800-4200 and be coated with biotinylation bovine serum albumin(BSA) and the dilution ratio that dilution ratio is 1:2850-3150 respectively in each micropore of ELISA Plate; Preserve 4 ℃ of liquid phases of biotinylated antibody 0.1M phosphate buffered saline (PBS) standby in addition as reaction reagent.
Described a kind of biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, the biotinylation bovine serum albumin(BSA) and the dilution ratio that are coated with dilution ratio in each micropore of its ELISA Plate respectively and are 1:3000 are the Streptavidin of 1:4000; Preserve 4 ℃ of liquid phases of biotinylated antibody 0.1M phosphate buffered saline (PBS) standby in addition as reaction reagent.
The preparation method of described a kind of biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, comprise the polystyrene ELISA Plate, be cushioned liquid 150 μ l and add biotinylation bovine serum albumin(BSA) and dilution I in the every hole of polystyrene ELISA Plate by the biotinylation bovine serum albumin(BSA) bag of 1:2850-3150 dilution proportion, the room temperature temperature is bathed 16～18h, get rid of clean bag and be cushioned liquid, wash ELISA Plate 2 times with the dilution I, need leave standstill 10min at every turn, pat dry;
Every hole adds Streptavidin and dilution II again and is cushioned liquid 150 μ l by the Streptavidin bag of 1:3800-4200 dilution proportion, the normal temperature temperature is bathed 2h, get rid of clean bag and be cushioned liquid, wash ELISA Plate 2 times, need leave standstill 10min at every turn with the dilution II, get rid of clean dilution II, last every hole adds 250 μ l lock solution, and 4 ℃ of temperature are bathed and spent the night, and get rid of clean lock solution, vacuum drying is 6h at least, and is vacuum-packed standby;
With biotinylated antibody 0.1M phosphate buffered saline (PBS), 4 ℃ of liquid phases are preserved standby as reaction reagent in addition.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its dilution I be by following feedstock production,
Trishydroxymethylaminomethane 5.76-6.36g
Sodium chloride 8.33-9.21g
Proclin?300 0.5ml
Distilled water 950 ml
It is fixed molten to 1000 ml to transfer pH=8.0 to add 0.5 ml Procilin 300.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its dilution II be by following feedstock production,
Sodium dihydrogen phosphate 4.45-4.91g
Sodium hydrogen phosphate 6.8-7.52g
L-tryptophane 2.09-2.1g
Sodium chloride 8.33-9.21 g
Distilled water 950 ml
Transfer pH=8.0 fixed molten to 1000 ml.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its lock solution be by following feedstock production,
Sodium dihydrogen phosphate 0.138-0.152g
Sodium hydrogen phosphate 1.38-1.52g
6-aminohexane 1.43-1.57g
Sodium chloride 4.16-4.6g
Distilled water 450 ml transfer pH=7.3 ~ 7.5
Bovine serum albumin(BSA) 1.9-2.1g
Sucrose 16.63-18.37g
Procilin?300 0.25?ml
Adding distil water is fixed molten to 500 ml.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its biotinylated antibody is to be that the ratio of 20:1 is mixed with the mol ratio with antibody and biotin, stirring at room 1h, with reacted liquid after 0.1M phosphate buffered saline (PBS) dialysis 4
oC preserves standby.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its dilution I be by following feedstock production,
Trishydroxymethylaminomethane 6.06 g
Sodium chloride 8.77 g
Proclin?300 0.5ml
Distilled water 950 ml
It is fixed molten to 1000 ml to transfer pH=8.0 to add 0.5 ml Procilin 300.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its dilution II be by following feedstock production,
Sodium dihydrogen phosphate 4.68 g
Sodium hydrogen phosphate 7.16 g
L-tryptophane 0.2 g
Sodium chloride 8.77 g
Distilled water 950 ml
Transfer pH=8.0 fixed molten to 1000 ml.
The preparation method of described biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, its lock solution be by following feedstock production,
Sodium dihydrogen phosphate 0.145 g
Sodium hydrogen phosphate 1.45 g
6-aminohexane 1.5 g
Sodium chloride 4.38 g
Distilled water 450 ml transfer pH=7.3 ~ 7.5
Bovine serum albumin(BSA) 2.0 g
Sucrose 17.5 g
Procilin?300 0.25?ml
Adding distil water is fixed molten to 500 ml.
She Ji the present invention like this owing to have higher affinity between Streptavidin and the biotin, guarantees that enzyme reaction plate has fine stability.Bovine serum albumin(BSA) molecule source is abundant, cheap, and is easy to the bag quilt in enzyme reaction plate, utilizes bovine serum albumin(BSA) molecule space effect, can increase the solid phase material reaction area, improves binding antibody molecule number, widens sensing range.
Biotinylated antibody and determined antigen are in the liquid phase simultaneously, are beneficial to antigen-antibody reaction, make it to reach rapidly balance.During use, add corresponding biotinylation capture antibody as required again, flexible; Antibody molecule places liquid phase, can keep the original space conformation of antibody molecule, effectively improves the antibody utilization ratio, reduces the antibody consumption, reduces the kit cost to greatest extent.
Description of drawings
Fig. 1 is that serum ferritin wraps indirectly by biotinylated antibody and enzyme labelled antibody dilute concentration one suite line chart;
Fig. 2 is that serum ferritin wraps indirectly by biotinylated antibody and enzyme labelled antibody dilute concentration two suite line charts;
Fig. 3 is serum ferritin control group direct coated capture antibody and enzyme labelled antibody dilute concentration one suite line chart;
Fig. 4 is serum ferritin control group direct coated capture antibody and enzyme labelled antibody dilute concentration two suite line charts;
Fig. 5 is that the serum prostate specific antigen wraps indirectly by biotinylated antibody and enzyme labelled antibody dilute concentration one suite line chart;
Fig. 6 is that the serum prostate specific antigen wraps indirectly by biotinylated antibody and enzyme labelled antibody dilute concentration two suite line charts;
Fig. 7 is serum prostate specific antigen control group direct coated capture antibody and enzyme labelled antibody dilute concentration one suite line chart;
Fig. 8 is serum prostate specific antigen control group direct coated capture antibody and enzyme labelled antibody dilute concentration two suite line charts.
Embodiment
The present invention will be described in detail below in conjunction with drawings and Examples.
One. material:
1. polystyrene ELISA Plate: commercially available U.S. COSTAR product, 96 holes are detachable, and enzyme linked immunosorbent assay is mutatis mutandis.
2. biotinylation bovine serum albumin(BSA): Sigma company product is bovine serum albumin(BSA) and biotin bond.
3. Streptavidin: Sigma company product.
4. bovine serum albumin(BSA): Sigma company product.
Two. the preparation method
1. component and proportioning
1. dilution I
Trishydroxymethylaminomethane (Tris) 6.06 g
Sodium chloride (NaCl) 8.77 g
Proclin?300 0.5ml
Distilled water (D H
2O) it is fixed molten to 1000 ml that 950 ml accent pH=8.0 adds 0.5 ml Procilin 300.
2. dilution II
Sodium dihydrogen phosphate (NaH
2PO
42H
2O) 4.68 g
Sodium hydrogen phosphate (Na
2HPO
412H
2O) 7.16 g
L-tryptophane (L-Tryptophan) 0.2 g
Sodium chloride (NaCl) 8.77 g
Distilled water (D H
2O) 950 ml transfer pH=8.0 fixed molten to 1000 ml.
3. biotinylation bovine serum albumin(BSA) bag is cushioned liquid
Biotinylation bovine serum albumin(BSA) and dilution I are pressed the 1:2850-3150 dilution proportion, and final concentration is 5 ± 0.25 μ g/ml, fully stirring and evenly mixing.
4. the Streptavidin bag is cushioned liquid
Streptavidin and dilution II are pressed the 1:3800-4200 dilution proportion, and final concentration is 3 ± 0.15 μ g/ml, fully stirring and evenly mixing.
5. lock solution
Sodium dihydrogen phosphate (NaH
2PO
42H
2O) 0.145 g
Sodium hydrogen phosphate (Na
2HPO
412H
2O) 1.45 g
6-aminohexane (ACA) 1.5 g
Sodium chloride (NaCl) 4.38 g
Distilled water (D H
2O) 450 ml transfer pH=7.3 ~ 7.5
Bovine serum albumin(BSA) (BSA) 2.0 g
Sucrose 17.5 g
Procilin?300 0.25?ml
Adding distil water (D H
2O) fixed molten to 500 ml.
2. concrete steps:
1. every hole add biotinylation bovine serum albumin(BSA) and dilution I by the 1:3000 dilution proportion biotinylation bovine serum albumin(BSA) bag be cushioned liquid, volume 150 μ l are placed on microwell plate in the container of sealing, 18 ~ 25 ℃, temperature bath 16 ~ 18h.
2. get rid of clean bag and be cushioned liquid, wash ELISA Plate 2 times with the dilution I, 300 μ l/well need leave standstill 10min at every turn.
3. get rid of clean dilution, and on clean towel, pat dry.
4. every hole adding Streptavidin and dilution II are cushioned liquid by the Streptavidin bag of 1:4000 dilution proportion, and volume 150 μ l are placed on microwell plate in the container of sealing, and 18 ~ 25 ℃, temperature is bathed 2h.
5. get rid of clean bag and be cushioned liquid, wash ELISA Plate 2 times with the dilution II, 300 μ l/well need leave standstill 10min at every turn.
6. repeat 3. to get rid of clean dilution, and on clean towel, pat dry.
7. every hole adds lock solution, and volume 250 μ l are placed on microwell plate in the container of sealing, and 4 ℃ of temperature are bathed and spent the night.
8. get rid of clean confining liquid, and on clean towel, pat dry.With microwell plate vacuum drying 6h at least.It is in time vacuum-packed to be positioned over hothouse after the taking-up.
Three. the clinical practice situation
Measure serum ferritin (Ferr):
(1) reagent is formed
1. enzyme reaction plate of the present invention
2. the anti-ferritin monoclonal antibody of biotinylation (capture antibody)
3. the anti-ferritin polyclonal antibody of enzyme labeling (detection antibody)
4. ferritin standard items (antigen) and quality controlled serum
5. lavation buffer solution
6. liquid A and colour developing liquid B(zymolyte develop the color)
7. stop buffer (0.2N H
2SO
4)
Above reagent is dezymotized outside the mark reaction plate, and tool is provided by the kit itself that Watson ﹠ Crick (Tianjin) Biotechnology Co., Ltd. produces.
(2) detection method is strict with Watson ﹠ Crick's kit instructions operation.
1. prepare:
1. before the test reagent constituents is placed room temperature (18-25 ℃) balance 30 minutes, should put back to 2-8 ℃ of preservation immediately after the test.
2. lavation buffer solution is 10 times and concentrates, with preceding with distilled water diluting to 200 ml.
If 3. ferritin concentration may be higher than 1000ng/ml, the dilution back is detected.
2. reaction:
1. take out enzyme reaction plate of the present invention.
2. add 50 μ l ferritin standard items, quality controlled serum, sample respectively in the hole, add the anti-ferritin monoclonal antibody of 50 μ l biotinylations more respectively, vibrations 10-20 mixing second was hatched 30 minutes for 37 ℃.
3. deduct liquid in the hole,, on clean paper handkerchief or towel, pat dry, remove residual liquid as far as possible with dilution back lavation buffer solution washing 5 times.
4. add the anti-ferritin polyclonal antibody of 100 μ l enzyme labelings in every hole, vibrations 10-20 mixing second was hatched 30 minutes for 37 ℃.
5. repeating step 3..
6. every hole adds 1 colour developing zymolyte A and B, places room temperature (18-25 ℃) lucifuge colour developing 15 minutes.
7. every hole adds 1 stop buffer, shakes mixing several times gently.
8. in 15 minutes, as the reference wavelength, measure the absorbance (A450) at 450nm place with 630nm.
2. calculate:
1. the absorbance with standard items is a Y-axis, and concentration is the X-axis mapping.
2. according to the absorbance of quality controlled serum and sample, the concentration of inverse ferritin from the typical curve.
If 3. sample is through dilution, when calculating its concentration, should multiply by corresponding extension rate.
3. experimental result
1. different bags are by the resultant typical curve of pattern, with reference to Fig. 1-4.
Table 1 the present invention wraps indirectly by biotinylated antibody and labelled antibody optium concentration
Table 2 direct coated pattern capture antibody and labelled antibody optium concentration
2. precision
Table 3 precision measuring result
The coefficient of variation of low concentration testing result is not more than 10%, and the coefficient of variation of high concentration and middle concentration testing result illustrates that less than 5% test method has good repeatability.
3. sensing range experiment
With high value serum doubling dilution, with accurately recording the high value serum-concentration of an inverse, the maximum detected value of hook effect point before as this detection method will appear again.This method sensing range is 0 ~ 1000ng/ml after measured.
4. reaction time experiment
Table 4 bag was indirectly determined by the pattern reaction time
The table 5 direct coated pattern reaction time is determined
4. effect
1.. the capture antibody consumption: by experimental result as can be known, and under the direct coated pattern, capture antibody consumption 1:1000 (8 mcg/ml) enzyme labelled antibody consumption 1:2000(2 mcg/ml); And when adopting the indirect coating technique of the present invention, capture antibody consumption 1:4000 (2 mcg/ml) enzyme labelled antibody consumption 1:8000(0.5 mcg/ml), greatly reduce the consumption of capture antibody and enzyme labelled antibody, saved cost.With reference to table 1, table 2.
2.. sensing range: by the result as can be known, the sensing range under the direct coated pattern is 0 ~ 1000ng/ml, adopts the indirect coating technique of the present invention to reduce the generation of hook effect, and sensing range still is 0 ~ 1000ng/ml under the prerequisite of saving cost.
Measure serum prostate specific antigen (PSA):
(1) reagent is formed
1. enzyme reaction plate of the present invention
2. the anti-PSA monoclonal antibody of biotinylation (capture antibody)
3. the anti-PSA polyclonal antibody of enzyme labeling (detection antibody)
4.PSA standard items (antigen) and quality controlled serum
5. washing lotion (10 times concentrate)
6. liquid A and colour developing liquid B(zymolyte develop the color)
7. stop buffer (0.2N H
2SO
4)
(2) detection method is strict with Watson ﹠ Crick's kit instructions operation
1. prepare:
1. before the test reagent constituents is placed room temperature (18-25 ℃) balance 30 minutes, should put back to 2-8 ℃ of preservation immediately after the test.
2. lavation buffer solution is 10 times and concentrates, with before using distilled water diluting.
If 3. PSA concentration may be higher than 100ng/ml, the dilution back is detected.
2. reaction:
1. take out enzyme reaction plate of the present invention.
2. add 50 μ l ferritin standard items, quality controlled serum, sample respectively in the hole, add the anti-PSA monoclonal antibody of 50 μ l biotinylations more respectively, vibrations 10-20 mixing second was hatched 30 minutes for 37 ℃.
3. deduct liquid in the hole,, on clean paper handkerchief or towel, pat dry, remove residual liquid as far as possible with dilution back lavation buffer solution washing 5 times.
4. add the anti-PSA polyclonal antibody of 100 μ l enzyme labelings in every hole, vibrations 10-20 mixing second was hatched 30 minutes for 37 ℃.
5. repeating step 3..
6. every hole adds 1 colour developing zymolyte A and B, places room temperature (18-25 ℃) lucifuge colour developing 15 minutes.
7. every hole adds 1 stop buffer, shakes mixing several times gently.
8. in 15 minutes, as the reference wavelength, measure the absorbance (A450) at 450nm place with 630nm.
2. calculate:
1. the absorbance with standard items is a Y-axis, and concentration is the X-axis mapping.
2. according to the absorbance of quality controlled serum and sample, the concentration of inverse PSA from the typical curve.
3., when calculating its concentration, should multiply by corresponding extension rate if sample is through dilution.
3. experimental result:
1. different bags are by the resultant typical curve of pattern, with reference to Fig. 5-8.
Table 6 wraps indirectly by biotinylated antibody and labelled antibody optium concentration
Table 7 direct coated pattern capture antibody and labelled antibody optium concentration
2. precision
Table 8 precision measuring result
The coefficient of variation of low concentration testing result is not more than 10%, and the coefficient of variation of high concentration and middle concentration testing result illustrates that less than 5% test method has good repeatability.
3. the sensing range experiment with accurately recording the high value serum-concentration of an inverse, will occur the maximum detected value of hook effect point before as this detection method with high value serum doubling dilution again.This method sensing range is 0 ~ 100ng/ml after measured.
4. reaction time experiment
Table 9 bag was indirectly determined by the pattern reaction time
The table 10 direct coated pattern reaction time is determined
Above result shows that direct coated pattern and indirect bag are 30min by the pattern first step and second reaction time in step.
4. effect
1. the capture antibody consumption by experimental result as can be known, under common direct coated pattern, capture antibody consumption 1:1000, enzyme labelled antibody consumption 1:3000; And when adopting the indirect coating technique of the present invention, capture antibody consumption 1:4000, enzyme labelled antibody consumption 1:5000 greatly reduces the consumption of capture antibody and enzyme labelled antibody, has saved cost.
2. sensing range by the result as can be known, the sensing range under the common direct coated pattern is 0 ~ 100ng/ml; Adopt the indirect coating technique of the present invention to reduce the generation of hook effect, sensing range still is 0 ~ 100ng/ml under the prerequisite of saving cost.
Claims (10)
1. biotinylation bovine serum albumin(BSA) and Streptavidin enzyme reaction plate, comprise the polystyrene ELISA Plate, it is characterized in that: the biotinylation bovine serum albumin(BSA) and the dilution ratio that are coated with dilution ratio in each micropore of ELISA Plate respectively and are 1:2850-3150 are the Streptavidin of 1:3800-4200; In addition 4 ℃ of liquid phases of biotinylated antibody 0.1M phosphate buffered saline (PBS) are preserved, standby as reaction reagent.
2. according to described a kind of biotinylation bovine serum albumin(BSA) of claim 1 and Streptavidin enzyme reaction plate, it is characterized in that: the biotinylation bovine serum albumin(BSA) and the dilution ratio that are coated with dilution ratio in each micropore of ELISA Plate respectively and are 1:3000 are the Streptavidin of 1:4000; In addition biotinylated antibody, 4 ℃ of liquid phases of 0.1M phosphate buffered saline (PBS) are preserved, standby as reaction reagent.
3. according to the preparation method of described a kind of biotinylation bovine serum albumin(BSA) of claim 1 and Streptavidin enzyme reaction plate, comprise the polystyrene ELISA Plate, it is characterized in that: polystyrene ELISA Plate every hole adding biotinylation bovine serum albumin(BSA) and dilution I are cushioned liquid 150 μ l by the biotinylation bovine serum albumin(BSA) bag of 1:2850-3150 dilution proportion, the room temperature temperature is bathed 16h～18h, get rid of clean bag and be cushioned liquid, wash ELISA Plate 2 times with the dilution I, need leave standstill 10min at every turn, pat dry;
Every hole adds Streptavidin and dilution II again and is cushioned liquid 150 μ l by the Streptavidin bag of 1:3800-4200 dilution proportion, the normal temperature temperature is bathed the clean bag of 2h and is cushioned liquid, wash ELISA Plate 2 times with the dilution II, need leave standstill 10min at every turn, get rid of clean dilution II, last every hole adds 250 μ l lock solution, 4 ℃ of temperature are bathed and are spent the night, get rid of clean lock solution, vacuum drying is 6h at least, and is vacuum-packed standby;
With biotinylated antibody 0.1M phosphate buffered saline (PBS), 4 ℃ of liquid phases are preserved standby as reaction reagent in addition.
4. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 3 and Streptavidin enzyme reaction plate, it is characterized in that: the dilution I is by following feedstock production,
Trishydroxymethylaminomethane 5.76-6.36g
Sodium chloride 8.33-9.21g
Proclin?300 0.5ml
Distilled water 950 ml
It is fixed molten to 1000 ml to transfer pH=8.0 to add 0.5 ml Procilin 300.
5. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 3 and Streptavidin enzyme reaction plate, it is characterized in that: the dilution II is by following feedstock production,
Sodium dihydrogen phosphate 4.45-4.91g
Sodium hydrogen phosphate 6.8-7.52g
L-tryptophane 2.09-2.1g
Sodium chloride 8.33-9.21 g
Distilled water 950 ml
Transfer pH=8.0 fixed molten to 1000 ml.
6. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 3 and Streptavidin enzyme reaction plate, it is characterized in that: lock solution is by following feedstock production,
Sodium dihydrogen phosphate 0.138-0.152g
Sodium hydrogen phosphate 1.38-1.52g
6-aminohexane 1.43-1.57g
Sodium chloride 4.16-4.6g
Distilled water 450 ml accent pH=7.3 ~ 7.5,
Bovine serum albumin(BSA) 1.9-2.1g
Sucrose 16.63-18.37g
Procilin?300 0.25?ml
Adding distil water is fixed molten to 500 ml.
7. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 3 and Streptavidin enzyme reaction plate, it is characterized in that: biotinylated antibody is to be that the ratio of 20:1 is mixed with the mol ratio with antibody and biotin, stirring at room 1h, with reacted liquid after 0.1M phosphate buffered saline (PBS) dialysis 4
oC preserves standby.
8. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 4 and Streptavidin enzyme reaction plate, it is characterized in that: the dilution I is by following feedstock production,
Trishydroxymethylaminomethane 6.06 g
Sodium chloride 8.77 g
Proclin?300 0.5ml
Distilled water 950 ml
It is fixed molten to 1000 ml to transfer pH=8.0 to add 0.5 ml Procilin 300.
9. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 5 and Streptavidin enzyme reaction plate, it is characterized in that: the dilution II is by following feedstock production,
Sodium dihydrogen phosphate 4.68 g
Sodium hydrogen phosphate 7.16 g
L-tryptophane 0.2 g
Sodium chloride 8.77 g
Distilled water 950 ml
Transfer pH=8.0 fixed molten to 1000 ml.
10. according to the preparation method of described biotinylation bovine serum albumin(BSA) of claim 6 and Streptavidin enzyme reaction plate, it is characterized in that: lock solution is by following feedstock production,
Sodium dihydrogen phosphate 0.145 g
Sodium hydrogen phosphate 1.45 g
6-aminohexane 1.5 g
Sodium chloride 4.38 g
Distilled water 450 ml transfer pH=7.3 ~ 7.5
Bovine serum albumin(BSA) 2.0 g
Sucrose 17.5 g
Procilin?300 0.25?ml
Adding distil water is fixed molten to 500 ml.
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