CN102539761A - Preparation method of TSH (thyroid stimulating hormone) quantitative detection kit based on time-resolved fluoroimmunoassay - Google Patents
Preparation method of TSH (thyroid stimulating hormone) quantitative detection kit based on time-resolved fluoroimmunoassay Download PDFInfo
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- CN102539761A CN102539761A CN2010105781574A CN201010578157A CN102539761A CN 102539761 A CN102539761 A CN 102539761A CN 2010105781574 A CN2010105781574 A CN 2010105781574A CN 201010578157 A CN201010578157 A CN 201010578157A CN 102539761 A CN102539761 A CN 102539761A
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- resolved fluoroimmunoassay
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Abstract
The invention provides a TSH (thyroid stimulating hormone) quantitative detection kit based on time-resolved fluoroimmunoassay, applicable to clinical screening of neonatal hypothyroidism. The preparation method of the kit is characterized by comprising the following steps of: 1, separating an antibody conjugate (Eu-Anti-TSH) from free rare earth ions (Eu<3+>) by using a specific chromatograph column; (2) using a bifunctional chelator (DTTA) for marking, wherein one end of the bifunctional chelator is chelated with rare earth ions (Eu) and the other end of the bifunctional chelator is connected with -NH2 of protein; adding beta-NTA to reinforcing liquid and regulating the concentration so that the fluorescence intensity of a finally obtained reagent is improved; and (4) adding a substitute Proclin300 for sodium azide to an analysis buffer system. Based on the characteristics of the steps in the preparation method, the TSH quantitative detection kit based on time-resolved fluoroimmunoassay can be produced and other in vitro diagnosis detection reagents can be developed.
Description
Technical field
The present invention relates to the Application in Immunodiagnosis technical field; Particularly; The invention provides a kind of time-resolved fluoroimmunoassay method of utilizing; " double-antibody sandwich " technology of different loci on the antibody of employing Eu mark and the antibodies antigen on the solid phase carrier, detection by quantitative is captured in the concentration of thyroid-stimulating hormone (TSH) in the neonate's whole blood sample on the 903# filter paper, is mainly used in the examination of neonate's hypothyroidism.
Technical background
" time resolved fluoro-immunoassay " theory is that the Soini and the Hemmila of Finland proposing in 1979 first, and 1984 cars, Hemmila have been confirmed time resolution immuno analytical method scheme.
At present domestic be used for detecting blood of neonate thyroid-stimulating hormone concentration the most widely experimental technique be enzyme process and fluorescence method.Though enzyme is a kind of desirable label, be difficult to the high concentration determinand directly accurately quantitative for measuring add lustre to its sensitivity of enzyme process of product OD value of enzyme reaction.The conventional fluorescent method that conventional fluorescence molecule is a label is owing to receiving scattered light, and the impact analysis sensitivity of factors such as the background fluorescence of separate sources and fluorescent quenching is generally 10
-8～10
-11Mol/L is only limited to and measures some to sensitivity less demanding determinand and some micromolecule.The time-resolved fluoroimmunoassay method integrates the advantage of enzyme labeling technology and isotope labelling techniques, and its reaction product stability is high, has the height detectability, and high analyte sensitivity is generally 10
-18Mol/L, wide measurement range, good analysis accuracy, the reagent life-span is longer, and easy and simple to handle is the detection method of at present tool advantage.
Summary of the invention
The object of the present invention is to provide a kind of employing time-resolved fluoroimmunoassay method, the kit of detection by quantitative thyrotropic hormone concentration.
The present invention adopts the time-resolved fluoroimmunoassay method, and prepared thyrotropic hormone detection by quantitative kit detection sensitivity is high, and sensing range is wide.
The further purpose of the present invention provides the screening method that more is applicable to neonate's thyroid hypofunction.
Employing time-resolved fluoroimmunoassay method provided by the invention, it comprises following step:
(1) the TSH monoclonal antibody is encapsulated on 96 orifice plates;
(2) with rare earth ion Eu mark on the TSH monoclonal antibody;
(3) through specific chromatogram column separating purification Eu-Anti-TSH bond and free Eu
3+
(4) preparation of enhancing liquid;
(5) in analyzing buffer system, add Sodium azide substitute Proclin300.
The present invention provides a kind of thyrotropic hormone detection by quantitative kit, is applicable to the clinical examination that neonate's thyroxine is low.Concrete detection method derives from time-resolved fluoroimmunoassay (TRFIA) technology.
The present invention also provides a kind of method of labelled antibody.The difunctional chelate mark that will have rare earth ion is to TSH antibody, and under neutral or approaching neutral condition, antibody conjugates has sufficiently high complex stability.
The present invention also provides a kind of method of antibody separation and purification.In order to obtain high activity and highly purified antibody, the present invention is through Sephadex G-50 chromatographic column separation antibody bond and free rare earth ion.
According to the present invention, the above-mentioned method of purification that is used for the required antibody conjugates of preparation time resolution fluorescence immunoassay reagent can be used Sephadex G-50 chromatographic column separation antibody bond and free rare earth ion.
The present invention develops the reagent that is used for TSH time-resolved fluoroimmunoassay method with known method, through rare earth ion on the difunctional chelate mark, uses chromatographic column separation antibody bond and free rare earth ion again.Prepare antibody conjugates.
The method of mark separation antibody is applied to the preparation of reagent in the TSH kit (time-resolved fluoroimmunoassay method), also comprises any test item that can be used for double antibodies sandwich method principle.HCG (short sexual gland chorionic hormone) for example, AFP (alpha-fetoprotein), the labelled antibody of can purifying as required.
Strengthen the preparation method of liquid.Can be used for the raising of the enhancing liquid usefulness in the various time-resolved fluoroimmunoassay method test items, HCG for example, AFP.
According to the present invention, with the reagent in the known method research and development TSH detection kit (time-resolved fluoroimmunoassay method).For example, the difunctional chelate mark that will have rare earth ion uses chromatographic column separation antibody bond and free rare earth ion again to antibody, prepare Eu-antibody.Strengthen the preparation of liquid.The antibody sandwich culture plate.
Encapsulate 96 well culture plates with the TSH monoclonal antibody, thereby the TSH for preparing in the time-resolved fluoroimmunoassay standard measure detection kit encapsulates plate.
In order to improve the effect that strengthens liquid, in strengthening liquid, add β-NTA, make the fluorescence intensity of final reagent be greatly improved through adjusting its concentration, also improved the reaction sensitivity of reagent simultaneously.Thereby prepare the enhancing liquid reagent in the time-resolved fluoroimmunoassay standard measure detection kit.
Eu-Antibody Preparation scheme in the thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method); Can be used for producing thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be used to develop new time-resolved fluoroimmunoassay standard measure detection kit.
Enhancing liquid in the thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method) prepares scheme; Can be used for producing thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be applicable to other time-resolved fluoroimmunoassay standard measure detection kit.
Add Sodium azide substitute Proclin300 in the analysis buffer system in the thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method); Can be used for producing thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be applicable to the time-resolved fluoroimmunoassay standard measure detection kit of other test item.
Description of drawings
Fig. 1: the theoretical detection sensitivity diagram of immuno-labelling technique.
Fig. 2: antibody labeling technology.
Fig. 3: double antibodies sandwich method synoptic diagram.
Fig. 4: TRFIA aglucon label β-NTA.
Embodiment
For making content of the present invention more clear and definite, below be the emphasis preparation manipulation process of reagent in the thyrotropic hormone detection by quantitative kit (time-resolved fluoroimmunoassay method).
1, the flow process of Eu-Anti-TSH mark and separation antibody
(1) dissolves Eu with distilled water
3+Chelate;
(2) wait to dissolve after, get 1mg Eu
3+Chelate solution joins in the 1mg TSH monoclonal anti liquid solution, room temperature reaction 48h;
(3) separate Eu-Anti-TSH bond and free Eu through Sephadex G-50 chromatographic column
3+
(4) collect fluorescence and count eluent, be the Eu-Anti-TSH bond greater than 1,000,000.
2, preparation strengthens liquid
Primary raw material title glacial acetic acid, Triton X-100 (triton x-100, Triton X-100); 0.1M Potassium Hydrogen Phthalate (KHP), β-NTA (β-naphthoyltrifluoroacetone), TOPO (trioctylphosphine oxide); Proclin300, collocation method sees the following form.
Strengthen formula of liquid
| Main supplementary material title | Dosage |
| Glacial acetic acid | 5.86mL |
| Triton?X-100 | 1ml |
| 0.1M Potassium Hydrogen Phthalate | Adjust pH |
| β-NTA | 0.04g |
| topO | 0.386g |
| Proclin300 | 0.5ml |
| Deionized water | Constant volume 1000ml |
3, preparation TSH encapsulates plate
(PH7.4) preparation waits to encapsulate TSH monoclonal anti liquid solution for PBS, 0.02M, and concentration is 5.0 μ g/mL with phosphate buffer.In each micropore of microwell plate, add 200 μ L coating buffers (table 1 is seen in the preparation of coating buffer); Place room temperature (18-30 ℃) spend the night (16-24 hour); Abandon and add confining liquid (table 2 is seen in the preparation of confining liquid) 300 μ L behind the coating buffer and place ambient temperature overnight (16-24 hour), the deblocking liquid that inclines is put 20-25 ℃ of drying at room temperature; The plate that encapsulates of drying is contained in the aluminium foil bag, and the room temperature vacuum is drained sealing.
Table 1 coating buffer prescription
| Main supplementary material title | Dosage |
| Proclin300 | 0.5ml |
| Encapsulate with TSH monoclonal antibody (1.06mg/ml) | 4.7ml |
| 0.02M?PB(PH＝7.4) | Constant volume 1000ml |
Table 2 confining liquid prescription
| Main supplementary material title | Dosage |
| Sucrose | 50g |
| Bovine serum albumin(BSA) | 2g |
| 0.02M?PB(PH＝7.4) | Constant volume 1000ml |
3, preparation experiment damping fluid
Primary raw material title NaCl (sodium chloride), BSA (bovine serum albumin(BSA)), PEG6000 (Macrogol 6000); Proclin300; Tween-40 (Tween-40), EDTA (ethylenediamine tetraacetic acid), eosin (eosin disodium); Tris (trishydroxymethylaminomethane)-HCl (hydrochloric acid), collocation method sees the following form.
Table 5 experiment buffer formulation
| Main supplementary material title | Dosage |
| NaCl | 9g |
| BSA | 10g |
| PEG6000 | 15g |
| 0.5％Tween-40 | 20ml |
| 1mM?EDTA | 25 |
| Proclin300 | 0.5ml |
| 1% eosin | 0.5ml |
| 0.05M?Tris-HCl | Constant volume 1000ml |
4, kit operating process
1. experimental temperature 18-25 ℃, kit and sample return to room temperature from 2-8 ℃ of taking-up;
2. concentrate the dilution in 1: 25 of washing lotion and deionized water, the dilution back can be preserved 1 month for 2～8 ℃;
3.Eu-1: 150 times of dilution of antibody and experiment damping fluid;
4. special-purpose perforating plier is laid diameter 3mm standard items, quality-control product and sample blood sheet and is put into corresponding micropore;
5. every hole adds the Eu-antibody diluent of 200 μ l, room temperature vibration 5 hours;
6. wash plate: reaction discards reaction mixture after finishing, and every hole adds washing lotion 400 μ l, discards, and washs altogether 6 times.
7. every hole adds 200 μ l and strengthens liquid, room temperature vibration 11 minutes;
8. in 20 minutes, read fluorescent value, time resolution immunofluorescence analysis appearance excitation wavelength 340nm, emission wavelength 614nm.
9. utilize the time-resolved fluorescence appearance with software, automatic calculation sample concentration value.
Claims (10)
1. thyroid-stimulating hormone detection by quantitative kit " time-resolved fluoroimmunoassay method " is characterized in that comprising the steps:
(1) through chromatographic column separation antibody bond and free rare earth ion;
(2) adopt bifunctional chelating agent during mark, one of which end chelating rare earth ion, another section can with protein-NH2 is connected, in neutrality or under near the neutral PH condition, bifunctional chelating agent has sufficiently high complex stability to rare earth ion;
(3) in strengthening liquid, add β-NTA, make the fluorescence intensity of final reagent be improved through adjusting its concentration;
(4) Sodium azide that routine is used in the replacement traditional mode of production in analyzing buffer system.
2. method according to claim 1; It is characterized in that the described difunctional chelate that is marked with rare earth ion has chlorosulfonation ortho-terphenyl (BTBCT); Diethylenetriamine pentaacetic acid (DTPA); Isothiocyanic acid diethylamino phenyl amine tetraacethyl (EDTA), preferred isothiocyanic acid benzyl diethylene triamine tetraacethyl (DTTA).
3. method according to claim 1, wherein said rare earth ion has Eu
3+, Tb
3+, Dy
3+, Sm
3+, preferred Eu
3+
4. method according to claim 1 adds β-NTA in the wherein said enhancing liquid, makes the fluorescence intensity of final reagent be greatly improved through adjusting its concentration, has also improved the reaction sensitivity of reagent simultaneously.
5. method according to claim 1, the separation method of wherein said step (1) are to separate preferably Sephadex G-50 chromatographic column through chromatographic column.
6. method according to claim 1; The Sodium azide substitute of wherein said step (4) is Proclin 300; In the analysis buffer system, added Proclin300, prevented the harm that Sodium azide causes environment when producing configuration and test waste discharge.
7. Sephadex G-50 chromatographic column according to claim 6 can be used to produce thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be used to prepare the antibody of other rare earth ion mark.
8. enhancing liquid β-NTA according to claim 4 can be used to produce the enhancing liquid in the thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be used to prepare the enhancing liquid of differentiating At All Other Times in the fluorescent immune method kit.
9. the substitute Proclin300 of Sodium azide according to claim 6; Can be used to produce the buffer solution system in the thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be used to prepare the buffer solution system of differentiating At All Other Times in the fluorescent immune method kit.
10. according to the said method for preparing labelled antibody of claim 1; Can be used to produce the Eu-TSH antibody in the thyroid-stimulating hormone detection by quantitative kit (time-resolved fluoroimmunoassay method), also can be used to research and develop the Eu-antibody in the new time-resolved fluoroimmunoassay standard measure detection kit.
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| CN103575891A (en) * | 2012-08-03 | 2014-02-12 | 河南生生医疗器械有限公司 | Kit for comprehensively detecting HE4 and CA125 and application of kit |
| CN109828107A (en) * | 2019-01-16 | 2019-05-31 | 清华大学 | A kind of polyatom rubidium marking probe and the preparation method and application thereof |
| CN111721931A (en) * | 2020-06-23 | 2020-09-29 | 广州万孚生物技术股份有限公司 | Solid-phase time-resolved fluorescence immune marker and preparation method and application thereof |
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| CN109828107A (en) * | 2019-01-16 | 2019-05-31 | 清华大学 | A kind of polyatom rubidium marking probe and the preparation method and application thereof |
| CN111721931A (en) * | 2020-06-23 | 2020-09-29 | 广州万孚生物技术股份有限公司 | Solid-phase time-resolved fluorescence immune marker and preparation method and application thereof |
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Application publication date: 20120704 |