CN103499697A - FT3 in-vitro diagnostic kit and application method thereof - Google Patents
FT3 in-vitro diagnostic kit and application method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G01N2800/046—Thyroid disorders
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses an FT3 in-vitro diagnostic kit, which is used for detection on haptens which have strong polarity and small molecular weight and cannot be coated by solid-phase carriers. The kit is convenient to use, and helps to solve the case that haptens cannot coated well, and the kit is convenient for rapid and sensitive FT3 detection.
Description
Technical field
The present invention relates to a kind of FT3 detection kit, be specifically related to a kind of FT3 external diagnosis reagent case and using method thereof.
Background technology
People's hypothalamic-pituitary-thyroid axis controls and regulates organism metabolism by secreting hormone.Wherein by the thyrotropic hormone (TSH) of pituitary, promoted the synthetic and secretion of thyroid hormone, and be subject to the T3 in blood, the feedback regulation of T4 content, stable with the relative concentration of the free T3 in maintenance blood, T4 (FT3, FT4).
FT3, FT4 are the forms of thyroid hormone performance active function.In blood circulation 99.5% of T4, T3 with blood in the combinations such as TBG, albumin, free T3 only accounts for 0.2-0.4%.Serum FT 3 ranges of normal value are different different because of assay method.The clinical meaning that FT3 measures: the diagnosis that hyperthyroidism, first subtract, it is diagnosis of hyperthyroidism and the sensitiveest index of T3 type hyperthyroidism that FT3 raises, and is not subject to the impact that in blood, TBG changes.Be one of leading indicator of estimating thyroid function and research hypothalamus-hypophysis-hypothalamic pituitary thyroidal axis, can be used for auxiliary diagnosis and the therapeutic evaluation of primary and Secondary cases thyroid disease.
FT3 used radiommunoassay (RIA) in the past always, it had both had immunoreactive high specific radiommunoassay, there is again radiometric high sensitivity, so various materials with immunocompetent denier of energy Accurate Measurement, but there are the problems such as radiation and pollution in radiommunoassay, fast and convenient by the ELISA method, the advantage of existing radiommunoassay, also reduced pollution simultaneously.
Competition law can be used for measuring antigen, also can be used for measuring antibody.The mensuration antigen of take is example, is examined antigen and solid phase antigen competition desmoenzyme labeling antibody, and the enzyme labelled antibody amount that therefore is incorporated into solid phase is inversely proportional to the amount of being examined antigen.Operation steps is as follows: (1) is connected specific antigen with solid phase carrier, forms solid phase antigen.Washing.(2) treat in test tube to add the mixed solution of being examined sample and a certain amount of enzyme labelled antibody, make it to react with solid phase antigen.As examined in sample without antigen, enzyme labelled antibody can successfully be combined with solid phase antigen.Contained antigen as examined in sample, be combined with solid phase antigen with same chance with enzyme labelled antibody, the chance that enzyme labelled antibody is combined with solid phase carrier that accounted for competitively, reduce the binding capacity of enzyme labelled antibody and solid phase carrier.Only add enzyme labelled antibody in reference tube, after insulation, the combination of enzyme labelled antibody and solid phase antigen can reach amount the most fully.Washing.(3) add the luminescent solution colour developing: the enzyme labelled antibody due to combination in reference tube is maximum, and signal value is the highest.Reference tube signal value and the size for the treatment of the test tube signal value, the amount of sample antigen is examined in representative.Treat that the test tube signal value is lower, mean that in sample, antigenic content is more.
Because haptens is all little molecule, the antigenicity that itself is difficult to have, can only induced animal produce very weak immune response, thereby with carrier protein, to be coupled be very important.
Usually at many on the market FT3 kits, radioimmunological kit and ELISA kit are arranged, there are many places in radioimmunological kit, comprises following aspect:
One, radioactivity
Some radioactive isotope is unfavorable for because the half life period is short storing and transportation, simultaneously because the pollution of radiomaterial has also caused certain harm to staff's health and environment, need the equipment such as special protection and refuse processing, increased greatly cost.
Two, sensitivity.Usually the sensitivity for analysis of ELISA kit can reach 10
-13mol/L, the sensitivity for analysis of solid phase radioimmunological kit is carved and is reached 10
-16mol/L.
Three, the term of validity
In general, put and exempt from can only preserve 3 months, the ELISA kit is preserved 6 months.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of FT3 external diagnosis reagent case and using method thereof for the defect of above-mentioned existence.The present invention is strong for those polarity, and the haptens that molecular weight is little can't be coated in solid phase carrier and be detected, and a kind of kit easy to use is provided, and has solved the coated not good situation of haptens, is convenient to quick, the sensitive detection of carrying out FT3.
A kind of FT3 external diagnosis reagent case of the present invention and using method technical scheme thereof are that this FT3 external diagnosis reagent case, comprise microwell plate, calibration object, quality-control product, enzyme conjugates, luminous substrate liquid and concentrated cleaning solution; Wherein in microwell plate, contain little molecule haptens FT3 and be coupled the little molecule coupled protein that carrier protein obtains; The FT3 antigen that calibration object and quality-control product are concentration known; Contain the FT3 enzyme labelled antibody in enzyme conjugates.
Luminous substrate liquid comprises luminous substrate liquid A and luminous substrate liquid B, and luminous substrate liquid A contains luminol, and luminous substrate liquid B contains horseradish peroxidase stabilizing agent, hydrogen peroxide; Contain sodium chloride and Tween-20 in concentrated cleaning solution.
The little molecule coupled protein contained in microwell plate is that little molecule haptens FT3 is coupled to the little molecule coupled protein that carrier protein BSA obtains.
Described little molecule coupled protein, be coupled by little molecule haptens FT3 and carrier protein BSA the little molecule coupled protein obtained for using EDC.
Little molecule haptens FT3 and carrier protein BSA are coupled to concrete grammar is:
1. balance EDC, NHS is to room temperature;
2. EDC, NHS, BSA are dissolved with the activation damping fluid respectively;
3. by the EDC solution of 2-4 parts by volume with after 5-10 parts by volume BSA solution mixes, add the NHS solution with EDC equivalent, room temperature reaction 10-20 minute, after add again the EDC of 1-2 parts by volume; Room temperature reaction 10-20 minute again;
4. add mercaptoethanol to stop the EDC reaction, obtain activated protein EDC-BSA;
5. add and the little molecule haptens of the equimolar purpose of activated protein FT3, room temperature reaction 1.5-3 hour;
6. add the Tris cessation reaction, obtain coupled protein BSA-FT3;
7. add desalting column, the storage liquid of the inside discharged, after then adding PBS with centrifugal 3-5 time of same rotating speed, add the coupled protein of reaction, centrifugal after, be and be coupled product.
Step 2. middle activation damping fluid comprises: the MES of 0.1M, and the NACl of 0.5M, regulating pH is 6.0.
Step 2. middle EDC is dissolved with the activation damping fluid, and final concentration is 45-55mg/mL, and NHS dissolves with activating damping fluid equally, and final concentration is 45-55mg/mL, and BSA dissolves with the activation damping fluid, and final concentration is 4.5-5.5mg/mL.
Step 6. middle Tris is 20-50 mmol/L.
The coated mode of the albumen be coupled prepares kit, and step is:
The coating buffer that 1. will contain quantitative little molecule coupled protein joins in microwell plate, and 37 degree are placed 2 hours;
2. dry lath, add confining liquid, 37 degree are placed 2 hours;
3. after washing once, put into vacuum drying chamber 4-6 hour;
4. after vacuum sealing bag, put into 4 degree refrigerators.
Wherein, the coating buffer of step in 1. is that the mixed aqueous solution of sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, wherein can also contain antiseptic.
The using method of above-mentioned FT3 external diagnosis reagent case, is characterized in that,
1. add calibration object, quality-control product and sample to be tested respectively in microwell plate, then add enzyme conjugates in microwell plate, fully mix, 37 ℃ of reaction 50-80 minute;
2. add cleansing solution washing 3-6 time, add amount of liquor is 300-400uL at every turn;
3. then in microwell plate, add luminous substrate liquid, put into the chemiluminescent analyzer reading, computational data, draw the amount of sample to be tested small molecular haptens FT3.
Concentrated cleaning solution adds the pure water dilution for cleansing solution (example: the pure water of 20 * concentrated cleaning solution 50ml+950ml is 1 * cleansing solution).
Beneficial effect of the present invention is: kit of the present invention is easy to use, and the present invention uses the chemiluminescence standard measure to detect, and can make its sensing range broader, increases its sensitivity, and Precision Experiment also is better than general ELISA method.And contrast chemical method immue quantitative detection reagent box on the market, what kit of the present invention was used is indirect method, by coated little molecule haptens, adopts competition law to detect FT3.
Little molecular antigen, be called again haptens, and these little molecules itself do not have immunogenicity, when the large molecule of formation after carrier is combined, and can adaptive immune originality.The immunogene that haptens and carrier form, when the hapten molecule amount minimum, when or polarity is larger, little molecular antigen often is not easy direct solid phase adsorption on lath, uses the little molecule of BSA coupling, can form larger molecular weight, by hydrophobic effect, just easily be adsorbed on solid phase carrier, such method is highly sensitive, and specificity and the sensitivity of height can make analytic process simplify.
EDC is coupled FT3 and carrier.EDC can form a kind of active ester structure with carrier protein BSA, on the group of haptens FT3, carboxylic group is arranged, and can make it to react with BSA-EDC, can form the BSA-FT3 conjugate.
Adopt competition law to detect little molecule haptens FT3, the benefit of the method is simple to operate, one step completes, be coated with and generally do not there will be excessive being coated with to cause IC50 value (concentration of a suppressed half inhibitor) higher, and free antigen is easier and antibody mark is reacted, and sensitivity and degree of accuracy are higher.And the method that adopts coated antibody is easy to cause coated excessive affect IC50 value, and micromolecular mark can not adopt classical sodium periodate method usually, other chemical method of needs employing, and EDC, NHS/EDC etc., have a certain impact to the activity of enzyme.
Kit of the present invention also has following characteristics:
1. the on-radiation chemical luminescence reagent kit has been abandoned radioactive isotope fully, has therefore not only eliminated the harm to environment and operator, and the stability of reagent and the term of validity are extended greatly.Because luminous signal is died away in the short time again, therefore easily realize continuously simultaneously, dynamically, replication.
2. the sensitivity for analysis of high sensitivity chemistry luminescence reagent box can reach 10-18mol/L.If take the application of sample amount as 50ul at every turn, by mole constant, be 6.02 * 1023 calculating, tens testing molecules are now only arranged in sample, this sensitivity has reached the limit of immunoassay technology.
3. grow the validity chemiluminescence and abandoned radioactive isotope fully, therefore thoroughly broken away from the restriction of half life period, the stability of reagent and the term of validity have been extended greatly.Chemiluminescent kit can reach more than 1 year.
the accompanying drawing explanation:
Figure 1 shows that the competition law schematic diagram;
Figure 2 shows that the calibration object trend map.
embodiment:
In order to understand better the present invention, below with instantiation, describe technical scheme of the present invention in detail, but the present invention is not limited thereto.
Embodiment 1
Material:
Activation damping fluid: 0.1M MES, 0.5M NACl, pH6.0;
Carrier protein: BSA;
Destination protein: little molecule haptens FT3;
NHS; Mercaptoethanol; Desalting column
Little molecule haptens FT3 and carrier protein are coupled,
1. balance EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), NHS(N-maloyl imines) to room temperature.
2. EDC is dissolved with the activation damping fluid, final concentration is 50mg/mL, and NHS dissolves with the activation damping fluid equally, and final concentration is 50mg/mL, the BSA(bovine serum albumin(BSA)) with the activation damping fluid, dissolve, final concentration is 5mg/mL.
3. by 200uL EDC solution with after 500uL BSA solution mixes, add 200uL NHS solution, room temperature reaction 15 minutes, after add 100uL EDC.React again 15 minutes.
4. add the 1.4uL mercaptoethanol to stop the EDC reaction, obtain activated protein (EDC-BSA).
5. add and the equimolar destination protein of activated protein (little molecule haptens FT3), room temperature reaction 2 hours.
6. add 30 mmol/L Tris cessation reactions, obtain coupled protein (BSA-FT3).
7. add desalting column, desalting column is first with 1000g, and 2min discharges the storage liquid of the inside, after then adding 0.01M PBS with centrifugal 4 times of same rotating speed, adds the coupled protein of reaction, centrifugal after, be and be coupled product.
The preparation of kit microwell plate:
The coating buffer that 1. will contain quantitative little molecule coupled protein joins in microwell plate, and 37 degree are placed 2 hours;
2. dry lath, add confining liquid 200uL.37 degree are placed 2 hours.
3. after washing once, put into vacuum drying chamber 5 hours.
4. after vacuum sealing bag, put into 4 degree refrigerators.
Wherein, the coating buffer of step in 1. is to contain sodium chloride 8.5g in the 1L pure water, sodium dihydrogen phosphate 0.58g, sodium hydrogen phosphate 5.8g.
The preparation of calibration object, quality-control product: with FT3 antigen, sample diluting liquid (1%BSA is dissolved in the PBS solution of PH7.4) respectively compound concentration be 0.5,2,10,30,50,6 calibration objects, 10 of 100 pmol/ml, 2 quality-control products of 50pmol/ml.
The enzyme conjugates preparation:
Be mixed with certain density enzyme conjugates with FT3 enzyme labelled antibody, enzyme dilution (20% calf serum is mixed in the PBS solution of pH7.4).
The use of kit:
1. add 50uL calibration object, quality-control product and sample to be tested respectively in microwell plate, then add the 100uL enzyme labelled antibody in microwell plate, fully mix with calibration object, quality-control product and sample to be tested respectively, 37 ℃ are reacted 60 minutes.
2. to add pure water dilution be 1 * cleansing solution (example: the pure water of 20 * concentrated cleaning solution 50ml+950ml is 1 * cleansing solution) to concentrated cleaning solution, adds cleansing solution washing four times, and add amount of liquor is 350uL at every turn.
3. then in microwell plate, add luminous substrate liquid, put into the chemiluminescent analyzer reading, computational data draws the amount of FT3 antigen in sample to be tested.
Luminous substrate liquid comprises luminous substrate liquid A and luminous substrate liquid B, and luminous substrate liquid A contains luminol, and luminous substrate liquid B contains the agent of horseradish peroxidase temperature, hydrogen peroxide; Contain sodium chloride and Tween-20 in concentrated cleaning solution.
Experimental result:
Table 1: calibration object result
Table 2: sample to be tested result
Wherein sample value is that Roche 411 instruments record, cutoff=6.8, sample value and kit detected value correlativity of the present invention: 98.32 %.
Embodiment 2
Material:
Activation damping fluid: 0.1M MES, 0.5M NACl, pH6.0
Carrier protein: BSA;
Destination protein: little molecule haptens FT3
NHS; Mercaptoethanol; Desalting column
Little molecule haptens FT3 and carrier protein are coupled,
1. balance EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), NHS(N-maloyl imines) to room temperature.
2. EDC is dissolved with the activation damping fluid, final concentration is 45mg/mL, and NHS dissolves with the activation damping fluid equally, and final concentration is 55mg/mL, the BSA(bovine serum albumin(BSA)) with the activation damping fluid, dissolve, final concentration is 4.5mg/mL.
3. by 200uL EDC solution with after 500uL BSA solution mixes, add 200uL NHS solution, room temperature reaction 15 minutes, after add 100uL EDC.React again 15 minutes.
4. add the 1.4uL mercaptoethanol to stop the EDC reaction, obtain activated protein (EDC-BSA).
5. add and the equimolar destination protein of activated protein (little molecule haptens FT3), room temperature reaction 2 hours.
6. add 20 mmol/L Tris cessation reactions, obtain coupled protein (BSA-FT3).
7. add desalting column, desalting column is first with 1000g, and 2min discharges the storage liquid of the inside, after then adding 0.01M PBS with centrifugal 3 times of same rotating speed, adds the coupled protein of reaction, centrifugal after, be and be coupled product.
The preparation of kit microwell plate:
The coating buffer that 1. will contain quantitative little molecule coupled protein joins in microwell plate, and 37 degree are placed 2 hours;
2. dry lath, add confining liquid 200uL.37 degree are placed 2 hours.
3. after washing once, put into vacuum drying chamber 5 hours.
4. after vacuum sealing bag, put into 4 degree refrigerators.
Wherein, the coating buffer of step in 1. is to contain sodium chloride 8.5g in the 1L pure water, sodium dihydrogen phosphate 0.58g, sodium hydrogen phosphate 5.8g.
Calibration object, quality-control product preparation and enzyme conjugates preparation are with embodiment 1.
The use of kit:
1. add 50uL calibration object (or sample) successively in microwell plate, 100uL enzyme mark (antigen), fully mix, and 37 ℃ are reacted 60 minutes.
2. wash five times, each application of sample amount is 350uL.
3. then add luminescent solution, put into the chemiluminescent analyzer reading, computational data draws the amount of FT3 antigen in sample to be tested.
Luminous substrate liquid comprises luminous substrate liquid A and luminous substrate liquid B, and luminous substrate liquid A contains luminol, and luminous substrate liquid B contains the agent of horseradish peroxidase temperature, hydrogen peroxide; Contain sodium chloride and Tween-20 in concentrated cleaning solution.
Experimental result:
Table 3: calibration object result
Table 4: sample to be tested result
Wherein sample value is that Roche 411 instruments record, cutoff=6.8, sample value and detected value correlativity: 98.08 %.
Embodiment 3
Material:
Activation damping fluid: 0.1M MES, 0.5M NACl, pH6.0
Carrier protein: BSA;
Destination protein: little molecule haptens FT3
NHS; Mercaptoethanol; Desalting column
Little molecule haptens FT3 and carrier protein are coupled to method is,
1. balance EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), NHS(N-maloyl imines) to room temperature.
2. EDC is dissolved with the activation damping fluid, final concentration is 55mg/mL, and NHS dissolves with the activation damping fluid equally, and final concentration is 45mg/mL, the BSA(bovine serum albumin(BSA)) with the activation damping fluid, dissolve, final concentration is 5.0mg/mL.
3. by 200uL EDC solution with after 500uL BSA solution mixes, add 200uL NHS solution, room temperature reaction 15 minutes, after add 100uL EDC.React again 15 minutes.
4. add the 1.4uL mercaptoethanol to stop the EDC reaction, obtain activated protein (EDC-BSA).
5. add and the equimolar destination protein of activated protein (little molecule haptens FT3), room temperature reaction 2 hours.
6. add 50 mmol/L Tris cessation reactions, obtain coupled protein (BSA-FT3).
7. add desalting column, desalting column is first with 1000g, and 2min discharges the storage liquid of the inside, after then adding 0.01M PBS with centrifugal 2 times of same rotating speed, adds the coupled protein of reaction, centrifugal after, be and be coupled product.
The preparation of kit microwell plate:
The coating buffer that 1. will contain quantitative little molecule coupled protein joins in microwell plate, and 37 degree are placed 2 hours;
2. dry lath, add confining liquid 200uL.37 degree are placed 2 hours.
3. after washing once, put into vacuum drying chamber 5 hours.
4. after vacuum sealing bag, put into 4 degree refrigerators.
Wherein, the coating buffer of step in 1. is to contain sodium chloride 8.5g in the 1L pure water, sodium dihydrogen phosphate 0.58g, sodium hydrogen phosphate 5.8g.
Calibration object, quality-control product preparation and enzyme conjugates preparation are with embodiment 1.
The use of kit:
1. add 50uL calibration object (or sample) successively in microwell plate, 100uL enzyme mark (antigen), fully mix, and 37 ℃ are reacted 60 minutes.
2. wash four times, each application of sample amount is 350uL.
3. then add luminescent solution, put into the chemiluminescent analyzer reading, computational data draws the amount of FT3 antigen in calibration object or sample.
Luminous substrate liquid comprises luminous substrate liquid A and luminous substrate liquid B, and luminous substrate liquid A contains luminol, and luminous substrate liquid B contains the agent of horseradish peroxidase temperature, hydrogen peroxide; Contain sodium chloride and Tween-20 in concentrated cleaning solution.
Experimental result:
Table 5: calibration object result
Table 6: sample to be tested result
Wherein sample value is that Roche 411 instruments record, cutoff=6.8, sample value and detected value correlativity: 98.17%.
From above embodiment, kit of the present invention is simple to operate, one step completes, be coated with and generally do not there will be excessive being coated with to cause IC50 value (concentration of a suppressed half inhibitor) higher, and free antigen is easier and antibody mark is reacted, sensing range is broader, and detection sensitivity and degree of accuracy are higher.
Claims (10)
1. a FT3 external diagnosis reagent case, is characterized in that, comprises microwell plate, calibration object, quality-control product, enzyme conjugates, luminous substrate liquid and concentrated cleaning solution; Wherein in microwell plate, contain little molecule haptens FT3 and be coupled the little molecule coupled protein that carrier protein obtains; The FT3 antigen that calibration object and quality-control product are concentration known; Contain the FT3 enzyme labelled antibody in enzyme conjugates.
2. FT3 external diagnosis reagent case according to claim 1, is characterized in that, luminous substrate liquid comprises luminous substrate liquid A and luminous substrate liquid B, and luminous substrate liquid A contains luminol, and luminous substrate liquid B contains horseradish peroxidase stabilizing agent, hydrogen peroxide; Contain sodium chloride and Tween-20 in concentrated cleaning solution.
3. FT3 external diagnosis reagent case according to claim 1, is characterized in that, the little molecule coupled protein contained in microwell plate is that little molecule haptens FT3 is coupled to the little molecule coupled protein that carrier protein BSA obtains.
4. a kind of FT3 external diagnosis reagent case according to claim 3, is characterized in that, described little molecule coupled protein is coupled by little molecule haptens FT3 and carrier protein BSA the little molecule coupled protein obtained for using EDC.
5. FT3 external diagnosis reagent case according to claim 4, is characterized in that, little molecule haptens FT3 and carrier protein BSA is coupled to concrete grammar be:
1. balance EDC, NHS is to room temperature;
2. EDC, NHS, BSA are dissolved with the activation damping fluid respectively;
3. by the EDC solution of 2-4 parts by volume with after 5-10 parts by volume BSA solution mixes, add the NHS solution with EDC equivalent, room temperature reaction 10-20 minute, after add again the EDC of 1-2 parts by volume; Room temperature reaction 10-20 minute again;
4. add mercaptoethanol to stop the EDC reaction, obtain activated protein EDC-BSA;
5. add and the little molecule haptens of the equimolar purpose of activated protein FT3, room temperature reaction 1.5-3 hour;
6. add the Tris cessation reaction, obtain coupled protein BSA-FT3;
7. add desalting column, the storage liquid of the inside discharged, after then adding PBS with centrifugal 3-5 time of same rotating speed, add the coupled protein of reaction, centrifugal after, be and be coupled product.
6. FT3 external diagnosis reagent case according to claim 5, is characterized in that, step 2. middle activation damping fluid comprises: the MES of 0.1M, and the NACl of 0.5M, regulating pH is 6.0.
7. FT3 external diagnosis reagent case according to claim 5, it is characterized in that, step 2. middle EDC is dissolved with the activation damping fluid, final concentration is 45-55mg/mL, NHS dissolves with the activation damping fluid equally, final concentration is 45-55mg/mL, and BSA dissolves with the activation damping fluid, and final concentration is 4.5-5.5mg/mL.
8. FT3 external diagnosis reagent case according to claim 5, is characterized in that, step 6. middle Tris is 20-50 mmol/L.
9. FT3 external diagnosis reagent case according to claim 5, is characterized in that, the coated mode of the albumen be coupled prepares kit, and step is:
The coating buffer that 1. will contain quantitative little molecule coupled protein joins in microwell plate, and 37 degree are placed 2 hours;
2. dry lath, add confining liquid, 37 degree are placed 2 hours;
3. after washing once, put into vacuum drying chamber 4-6 hour;
4. after vacuum sealing bag, put into 4 degree refrigerators.
10. the using method of FT3 external diagnosis reagent case as claimed in claim 1, is characterized in that,
1. add calibration object, quality-control product and sample to be tested respectively in microwell plate, then add enzyme conjugates in microwell plate, fully mix, 37 ℃ of reaction 50-80 minute;
2. add cleansing solution washing 3-6 time, add amount of liquor is 300-400uL at every turn;
3. then in microwell plate, add luminous substrate liquid, put into the chemiluminescent analyzer reading, computational data, draw the amount of sample to be tested small molecular haptens FT3.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0265411A2 (en) * | 1986-10-22 | 1988-04-27 | Monsanto Company | In vitro method to detect allergic reactions to low molecular weight chemicals |
| CN101054410A (en) * | 2005-03-29 | 2007-10-17 | 河南科技学院 | Antigen epitope III of neutrality B cell of chicken IBDV VP2 protein and application thereof |
| CN102095882A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for free triiodothyronine |
| CN102095878A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for follicle-stimulating hormone |
| WO2012019107A1 (en) * | 2010-08-05 | 2012-02-09 | Abbott Point Of Care Inc. | Magnetic immunosensor and method of use |
| CN102520155A (en) * | 2011-12-13 | 2012-06-27 | 潍坊市康华生物技术有限公司 | Clenbuterol hydrochloride assay kit and its preparation method and use method |
| CN102692408A (en) * | 2012-04-26 | 2012-09-26 | 北京北方生物技术研究所 | One-step chemiluminiscence quantitative detection kit for hyaluronic acid |
| CN102980999A (en) * | 2008-11-27 | 2013-03-20 | 上海交通大学 | Chemiluminescence immunodetection kit for detecting ractopamine |
-
2013
- 2013-09-06 CN CN201310403123.5A patent/CN103499697B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0265411A2 (en) * | 1986-10-22 | 1988-04-27 | Monsanto Company | In vitro method to detect allergic reactions to low molecular weight chemicals |
| CN101054410A (en) * | 2005-03-29 | 2007-10-17 | 河南科技学院 | Antigen epitope III of neutrality B cell of chicken IBDV VP2 protein and application thereof |
| CN102980999A (en) * | 2008-11-27 | 2013-03-20 | 上海交通大学 | Chemiluminescence immunodetection kit for detecting ractopamine |
| CN102095882A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for free triiodothyronine |
| CN102095878A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for follicle-stimulating hormone |
| WO2012019107A1 (en) * | 2010-08-05 | 2012-02-09 | Abbott Point Of Care Inc. | Magnetic immunosensor and method of use |
| CN102520155A (en) * | 2011-12-13 | 2012-06-27 | 潍坊市康华生物技术有限公司 | Clenbuterol hydrochloride assay kit and its preparation method and use method |
| CN102692408A (en) * | 2012-04-26 | 2012-09-26 | 北京北方生物技术研究所 | One-step chemiluminiscence quantitative detection kit for hyaluronic acid |
Non-Patent Citations (1)
| Title |
|---|
| 宋娟等: "半抗原的设计、修饰及人工抗原的制备", 《分析化学》, vol. 38, no. 8, 31 August 2010 (2010-08-31) * |
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