CN102520196A - Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method - Google Patents
Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method Download PDFInfo
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- CN102520196A CN102520196A CN2011103941963A CN201110394196A CN102520196A CN 102520196 A CN102520196 A CN 102520196A CN 2011103941963 A CN2011103941963 A CN 2011103941963A CN 201110394196 A CN201110394196 A CN 201110394196A CN 102520196 A CN102520196 A CN 102520196A
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Abstract
The invention discloses a neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and a corresponding detection method. The corresponding detection method can not only improve relevance ratio, but also can save time, labor, reagents and samples, has the advantages of being economic and efficient and can be used in screening of a large amount of samples nationwide.
Description
Technical field
The present invention relates to the detection kit field, be specifically related to a kind of neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit and corresponding detecting method.
Background technology
CH disease (CH is called for short the low or first of congenital first and subtracts) is because various innate factors reduce thyroid hormone dyssynthesis, secretion, causes the infant growth disorder, the clinical syndrome that intelligence falls behind.It is one of modal endocrine system disease of paediatrics, once comprises multinomial classification of diseases title: cretinism, EndemicCretinism, goitre property cretinism and spontaneous cretinism.The hypothyroidism that takes place when at present CH is defined as any birth.
Examination of newborn infant diseases result all over the world shows, the low incidence of disease of congenital first is at country variant, and difference is less between the different nationalities, is generally 1:3 000 ~ 1:4 000.The CH infant often lacks the specific manifestations of disease after birth, and generally to arrive for 6 monthly ages just to engender intrinsic clinical symptoms, and increase the weight of day by day, yet, in case the clinical symptoms of disease occurred, show that disease has got into late period.Even treatment, treatment lowly also are difficult to recover; On the contrary, if can find disease soon in birth, make a definite diagnosis treatment, the body and mind of thumping majority infant will be grown normally so, and its intelligence also can reach normal person's level.
The examination of newborn infant diseases; Use a kind of simple as far as possible method exactly, whole ewborn infants is generally investigated, in the hope of finding infant early; Give efficacious therapy after making a definite diagnosis; Thereby guaranteed growing up healthy and sound of infant, how are the curative effect of disease and prognosis, depend on the morning and evening of making a definite diagnosis and treating fully.It is reported that the CH infant if can be made a definite diagnosis and treat in 3 months in birth, the infant intelligence development 80% or more is normal or near normally.
The examination of CH; Dussault at first detected the thyroxine (T4) in neonate's tip blood of 4-7 days in the North America in 1993 with filter paper blood cake radioimmunology; Usefulness filter paper blood cakes such as Lrie in 1975 and Naruse are measured thyroid-stimulating hormone (TSH) and are carried out the examination of CH; After this, the examination of CH in the u s. state, country such as Europe, Australia carries out rapidly.In the Asian-Pacific area, the research that Japan began relevant newborn child's illness in 1966, and, take the lead in having carried out on-radiation examination method---the enzymoimmunoassay of TSH of CH from 1977, to nineteen eighty-three, all Japan examination goes out CH case 400 examples.
The examination of newborn infant diseases work of China is started late.From 1989, provinces and cities such as Beijing, Shanghai, Guangzhou, Tianjin progressively carried out the conventional examination of CH.Clearly propose the clause of " progressively carrying out the examination of newborn infant diseases " in " mother and baby's Protection Code " of country's promulgation in 1994, make the examination of newborn infant diseases go on legal track.At present, China has 27 provinces and cities, the examination of diseases such as neonate CH has been carried out in 81 laboratories, and diagnosis and treatment level also are greatly improved.But as a whole; The examination of newborn infant diseases work of China is also relatively backward; The annual birth in the whole nation 2,100 ten thousand live-born infants only have 540,000 to accept this examination, and coverage rate has only 2.5%; Still have a lot of infants to leave serious intelligence disability, had a strong impact on the raising of the health of newborns because of failing diagnosis in time and treatment.
(Time Resolved Immunofluorometric assay TRIFMA) is the novel ultramicron immuno analytical method that early eighties comes out to time resolution immunofluorescence assay technology.The TIME RESOLVED TECHNIQUE of fluorescence is to utilize the fluorescence lifetime difference of fluorescent emission body, and the fluorescence spectrum that its fluorescence separates is technological.Is that tracer agent replaces radioactive isotope with the lanthanide series, comes labelled antigen or antibody with the lanthanide series with bifunctional group, when lanthanide series that disintegrates down and special amplification liquid formed new chelate, its fluorescence obviously strengthened and lastingly.Can eliminate the background fluorescence of sample self with the way of delay measurements, thereby improve detection sensitivity greatly.
Many countries such as America and Europe, Japan subtract neonate's first and carry out zonal general examination at present; Method is updated, and result's judgement is also more accurate. and the screening method that uses now has following 3 kinds: look into thyrotropic hormone when
thyroxine is low again;
thyroxine and thyrotropic hormone detect simultaneously;
singly looks into thyrotropic hormone.The detection method that neonate TSH is commonly used has EUSA (ELISA), enzyme immunofluorescence assay (EFIA), time resolution immunofluorescence assay (Tr-FIA) and CLIA.
At present domestic all measuring the screening method of TSH as CH, but this method can not detect thyroglobulin lack with hypothalamus hypophysis property first low, subclinical CH that also can omission TSH delay rising.Desirable screening method is to detect TSH and FT4 simultaneously, but cost improves greatly.
The time resolution Double Labelling Technique is to utilize the wavelength of fluorescence between different lanthanide ions different with fluorescence decay time; With two kinds of different antibody of lanthanide series difference mark; Detect different antigens, this method can save time, saving of labor, province's reagent and sample, has outstanding advantages such as economic and efficient.Being used in particular for the detection of multinomial purpose associating examination experiment and some precious samples, is the developing direction that the labelled immune credit is analysed.Eu in the lanthanide series
3+And Sm
3+Be to be usually used in a pair of lanthanide ion that double labeling is analyzed, excitation wavelength is 340nm, all can utilize β-NTA sequestrant to dissociate and strengthens fluorescence intensity.Eu
3+And Sm
3+Maximum emission wavelength be respectively 615nm and 643nm because the emission of narrow peak can fully be differentiated.Other fluorescence lifetime differs significantly, is respectively 820 μ s and 88 μ s, and each other interference ratio is less during detection.
Summary of the invention
The objective of the invention is to the deficiency that exists according in the existing detection kit; Provide a kind of and can not only improve recall rate; And can also save time, the kit of saving of labor, province's reagent and sample, have economical and advantage efficiently, be well suited in a large amount of sample examinations in the whole nation, using.
Another purpose of the present invention is to provide the preparation method of mentioned reagent box.
A further object of the invention is to provide the detection method of mentioned reagent box.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
Kit provided by the present invention is used for the examination of neonate's thyroid hypofunction; A kind of two different monoclonal antibodies can discerning different antigenic determinants on the thyrotropic hormone molecule are provided, with the thyroid-stimulating hormone in the double-antibody sandwich quantitative analysis filter paper dried blood spot.Can also discern the monoclonal antibody of antigenic determinant on the free thyroxine molecule, and thyroxine-BSA connector, with the free thyroxine in the competition law quantitative test filter paper dried blood spot.Method with manufacturer's standard blood speckles and Quality Control blood speckles is provided simultaneously; And with the free thyroxine monoclonal antibody of europium mark thyroid-stimulating hormone monoclonal antibody and samarium mark; The time-resolved fluoroimmunoassay technology that strengthens through dissociating; With sandwich method/competition law, the method for production standard concentration curve.
That the above-mentioned detection kit of the present invention can provide is distinguishing with the detection method of using now, from neonate's heel filter paper dried blood spot; Time-resolved fluoroimmunoassay technology through double-tagging; The method of while quantitative test thyroid-stimulating hormone and free thyroxine; Thereby in the examination process, needn't gather a large amount of blood, just can distinguish out CH and high TSH mass formed by blood stasis.
A kind of neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay method) is characterized in that said kit comprises following component:
(1) experiment damping fluid;
(2) concentrate washing lotion;
(3) strengthen liquid;
(4) encapsulate the micro reaction plate of thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector simultaneously;
(5) contain the filter paper dried blood spot standard items and the quality-control product of thyroid-stimulating hormone and free thyroxine simultaneously;
(6) Eu
3+-N
2The thyroid-stimulating hormone monoclonal antibody and the Sm of-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark
3+-N
2The free thyroxine monoclonal antibody of-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark.
The above-mentioned preparation method who detects the kit (time-resolved fluoroimmunoassay method) of neonate's thyroid-stimulating hormone and free thyroxine simultaneously comprises the steps:
(1) preparation experiment damping fluid, concentrate, enhancing liquid; Said experiment damping fluid is processed with bovine serum albumin(BSA) and the damping fluid of 1 ~ 20g/L; Said concentrate is processed with Tween-20 and the damping fluid of 1.0 ~ 10.0ml/L; Said enhancing liquid is processed with three n-octyl phosphorous oxide and the damping fluid of 1 ~ 5mg/L β-naphthoyltrifluoroacetone, 15 ~ 30mg/L;
(2) prepare the micro reaction plate that encapsulates simultaneously with thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector; Said reaction plate is diluted to TSH monoclonal antibody, FT4-BSA connector with the damping fluid that contains 1 ~ 10ppm antiseptic the coating buffer of 1 ~ 10 μ g/ml; Encapsulate blank micro reaction plate, and process with the protectant confining liquid sealing back drying that contains 1.0 ~ 10.0g/L;
(3) preparation is with filter paper dried blood spot standard items, the quality-control product of the pure article of thyroid-stimulating hormone, the pure article preparation of free thyroxine;
(4) preparation is with Eu
3+-N
2-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark thyroid-stimulating hormone monoclonal antibody, Sm
3+-N
2The plain monoclonal antibody of-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium marked free thyroid;
(5) the above-mentioned experiment damping fluid of packing, concentrated washing lotion, enhancing liquid, standard items and label;
(6) label;
(7) be assembled into finished product;
Wherein, the preparation method of filter paper dried blood spot standard items, quality-control product is following in the step (3): the matrix that standard items and quality-control product are processed the filter paper dried blood spot is human red cell and removes hormone people negative serum; Matrix includes but not limited to human red cell and removes hormone people negative serum; The hematocrit of its mesostroma is 45 ~ 90%; Wherein hematocrit is 50 ~ 55%; Scraps of paper standard items are the point of 3 ~ 10 variable concentrations; Wherein the standard point with 6 variable concentrations is main, but is not limited to 6; Quality-control product is the point of 1 ~ 5 variable concentrations; Wherein with 2 variable concentrations be main, but be not limited to 2; External packing is an aluminium foil bag; The making of thyroid-stimulating hormone, free thyroxine standard items, quality-control product; Be to be reference with TSH national standard article and FT4 national standard article; The human red cell of the hematocrit with 50 ~ 55% and the mixed-matrix that removes the hormone negative serum are prepared into standard items and the C1 of A, B, C, D, E, six debita spissitudos of F, the quality-control product of two concentration of C2 (low, height) respectively with thyroid-stimulating hormone or the pure article of free thyroxine; On A, B, C, D, E, F position and the C1 that drips respectively at filter paper with proper volume, the C2 position; The dried blood filter paper standard items, the quality-control product that behind natural drying at room temperature, obtain are used aluminium foil bag vacuum seal.
The principle of work of neonate's thyroid-stimulating hormone according to the invention/free thyroxine double-tagging detection kit is the time-resolved fluoroimmunoassay method, and concrete method of operating comprises the steps:
(1) reagent is prepared: with the micro reaction plate bar balance of reagent requirement to room temperature; Above-mentioned concentrated washing lotion of 40ml and 960ml purified water are mixed into the work cleansing solution; Before the use, with each personal experiment damping fluid of europium and samarium label by volume 1:100 be diluted to the label working fluid;
(2) standard items, quality-control product or sample are squeezed in the micropore that encapsulates reaction plate with diameter 3.0mm card punch, in every hole, added the label working fluid of above-mentioned europium of 100 ~ 200 μ l and samarium respectively; Reaction plate at room temperature slowly vibrates and hatches 1 ~ 4h; Wash plate 6 times with above-mentioned work cleansing solution, clap and do; In every hole, add 100 ~ 200 μ l and strengthen liquid, the 5min that slowly vibrates carries out the fluorescence counting in the time resolved fluoro-immunoassay appearance; Carry out Fitting Analysis by location parameter, obtain quantitative result.
Kit of the present invention is applicable to the examination of neonate CH, TSH in detection by quantitative neonate's filter paper dried blood spot and FT4 content.
The TSH detection sensitivity can reach 2.0 μ U/mL; The specificity aspect detects 250 U/L national standard article hLH, 250 U/L national standard article hFSH, and 10000 U/L national standard article hCG, the TSH apparent value all is not higher than 0.25 μ U/mL; Linearly dependent coefficient: in the measurement range 1-260 μ U/mL of kit, the dose-response curve linearly dependent coefficient should be not less than 0.9900; The HOOK effect: the HOOK effect of detection is up to 1000 μ U/mL.
FT4 detection sensitivity: be not higher than 5pmol/L; The specificity aspect is not higher than 20% with the cross reacting rate of trilute (T3), is not higher than 20% with the cross reacting rate of coelonychia shape gland original ammonia acid (rT3); Linearly dependent coefficient: in the measurement range of kit, the dose-response curve linearly dependent coefficient should be not less than 0.9900.
Accuracy of measurement: be contrast with TSH national standard article and FT4 national standard article respectively, the actual measurement of kit calibration object is tired and indicated potency ratio mean value should be in 0.900～1.100 scope; Measure accuracy: be no more than 15.0% in the analysis; Be no more than 20.0% between analysis; The quality-control product measured value: measure with two variable concentrations quality-control products, measured value is in allowed band; Stability: after 37 ℃ of bakings in 3 days, testing result meets above technical performance index.
Compared with prior art, the present invention has following beneficial effect:
Kit of the present invention adopts the double-tagging time-resolved fluoroimmunoassay, and one-shot measurement can obtain TSH, FT4 value simultaneously, can improve clinical examination speed and amount of inspection, reduces personal error significantly, alleviates clinical position intensity; And sample consumption to be checked is few, in same reaction system, has also reduced the influence that system interference detects two indexs; And the specificity of raising antidiastole; Establish the good technical basis for double-tagging TRFIA detects, filled up the blank of domestic such reagent simultaneously, reduced cost.
Description of drawings
Fig. 1 is TSH scraps of paper standard items curve map in the kit of the present invention;
Fig. 2 is TF4 scraps of paper standard items curve map in the kit of the present invention;
Fig. 3 is the comparison of neonate's thyroid-stimulating hormone detection kit detection neonate filter paper dried blood spot sample value of PE company on kit of the present invention and the market, and coefficient of relationship is r=0.995;
Fig. 4 detects the comparison of the blood serum sample value of corresponding filter paper dried blood spot for the thyroxine detection kit of Guangzhou Feng Hua bio-engineering corporation on kit of the present invention and the market, and coefficient of relationship is r=0.995.
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
Thyroid-stimulating hormone among the embodiment, the pure article of free thyroxine, thyroid-stimulating hormone, free thyroxine monoclonal antibody, the free thyroxine connector is purchased the company in Sigma; 96 hole micropore blank plates (8 * 12 hole) are the Therom product; Sephadex G-50 is the Pharmacia Company products; The national standard article are available from Nat'l Pharmaceutical & Biological Products Control Institute; β-naphthoyltrifluoroacetone, three n-octyl phosphorous oxide, TritonX-100 purchase the company in Sigma; Other reagent are that homemade analysis is pure; Time resolved fluoro-immunoassay appearance (PE company 1420 types), constant-temperature shaking appearance, full-automatic Enzyme-linked washing-board machine are all purchased in the Guangzhou the biological company limited of Feng Hua.
Embodiment 1 preparation neonate's thyroid-stimulating hormone of the present invention/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay method)
Component comprises:
The experiment damping fluid: bovine serum albumin(BSA) and damping fluid with 1 g/L～20g/L are processed;
Concentrate washing lotion: Tween-20 and damping fluid with 1.0ml/L～10.0ml/L are processed;
Strengthen liquid: three n-octyl phosphorous oxide and damping fluid with 1～5mg/L β-naphthoyltrifluoroacetone, 15～30mg/L are processed.
Reaction plate: reaction plate is diluted to TSH monoclonal antibody, FT4-connector with the damping fluid that contains 1～10ppm antiseptic the coating buffer of 1～10 μ g/ml; Encapsulate blank micro reaction plate, and process with the protectant confining liquid sealing back drying that contains 1.0g/L～10.0g/L.
Scraps of paper standard items, quality-control product: the hematocrit by 50%～55% is by human red cell and go hormone negative serum mixed-matrix that TSH, the pure article of FT4 are prepared into the standard items of 6 debita spissitudos and the quality-control product of low, high two debita spissitudos; Drip respectively on filter paper with 50 μ l volumes, behind natural drying at room temperature, obtain.
Label: TSH, FT4 label make mother liquor with the lanthanide ion chelate by 1:1～1:6 (m/m) mark respectively with TSH monoclonal antibody, FT4 monoclonal antibody, and mother liquor doubly dilutes by 1:10～1:25 with the label dilution and forms.
The main technique that detects TSH, FT4 kit (time resolved fluoro-immunoassay method) simultaneously of the present invention prepares as follows:
1) preparation of common reagent: the experiment damping fluid is for adding the bovine serum albumin(BSA) of 1.0g/L～20.0g/L in the Tris-Hcl solution of the 50mmol/L of the disodium ethylene diamine tetraacetate that contains 0.01g/L, pH7.8; Concentrated washing lotion is to contain the 0.15mol/L of 1.0ml/L～10.0ml/L Tween-20, the Tris-Hcl damping fluid of pH7.8; Strengthen liquid in containing 0.1mol/L Potassium Hydrogen Phthalate-glacial acetic acid solution of 1ml/L TritonX-100, adding the three n-octyl phosphorous oxide of 1mg/L～5mg/L β-naphthoyltrifluoroacetone, 15mg/L～30mg/L.The bovine serum albumin(BSA) of wherein testing damping fluid is 10.0g/L; The Tween-20 that concentrates in the washing lotion is 4.0ml/L.β-naphthoyltrifluoroacetone the 4.0mg/L, three n-octyl phosphorous oxide 20mg/L, the TritonX-100 that strengthen in the liquid are 1ml/L;
2) encapsulate the preparation of plate: use buffering to be diluted to the coating buffer that concentration is 1～10 μ g/ml respectively TSH monoclonal antibody, FT4-BSA connector; In the micropore blank plate in 96 holes, add coating buffer 100～200 μ l/ holes, incubated overnight under 35 ℃ of conditions; The plate that encapsulates of hatching is washed plate 1 time with the work cleansing solution, claps and does; The washed plate that encapsulates is added confining liquid, incubated overnight under 25 ± 2 ℃ of conditions by 250～280 μ l/ holes; Dry, spend the night and dry; Use aluminium foil bag vacuum seal; Put under 2～8 ℃ of conditions and preserve.
Wherein TSH monoclonal antibody, FT4-BSA connector adopt the chessboard titrimetry to confirm to encapsulate concentration, and using damping fluid to be diluted to concentration is that the coating buffer of 10 μ g/ml encapsulates effect and is the best.The coated antibody dilution is for containing 5ppm Proclin300
0.05mol/L, sodium carbonate-sodium bicarbonate buffer liquid of pH9.6; Confining liquid is for containing 5.0g/L trehalose, 0.5%BSA, the sodium carbonate of 0.05mol/L, pH9.6-sodium bicarbonate buffer liquid; Encapsulate the good thermal stability of plate.
4) preparation of standard items, quality-control product: with TSH national standard article and FT4 national standard article is reference; Hematocrit by 50%～55% by human red cell with go hormone negative serum mixed-matrix that TSH, the pure article of FT4 are mixed with into the standard items of 6 debita spissitudos and the quality-control product of low, high two debita spissitudos; On A, B, C, D, E, F position and the C1 that drips respectively at filter paper with 50 μ l volumes, the C2 position; The dried filter paper standard items and the quality-control product that behind natural drying at room temperature, obtain are used aluminium foil bag vacuum seal.
5) preparation of label: the Eu that 1mg TSH monoclonal labelled antibody is added 1mg
3+-N
2Mixing in-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium, 28 ℃ of reaction 44h; (1 * 40cm) the Tris-Hcl buffer solution elution with 50mmol/L, pH7.8 is separated Eu to reactant liquor through Sephadex G-50 post
3+-monoclonal antibody bond and free Eu
3+, adopt full-automatic fraction collector 2ml/ pipe to carry out liquid and collect; On the time resolved fluoro-immunoassay appearance, carry out the photofluorometer number, fluorescence is counted the eluent greater than 10000000 in the collection first peak, adds equal-volume glycerine ,-20 ℃ of preservations behind the mixing; Adopt the chessboard titrimetry to confirm europium markers work liquid concentration, press the 1:100 dilution again with the experiment damping fluid and detect best results;
The Sm that 1mg FT4 monoclonal antibody is added 1mg
3+-N
2Mixing in-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium, 28 ℃ of reaction 44h; (1 * 40cm) the Tris-Hcl buffer solution elution with 50mmol/L, pH7.8 is separated Sm to reactant liquor through Sephadex G-50 post
3+-monoclonal antibody bond and free Sm
3+, adopt full-automatic fraction collector 2ml/ pipe to carry out liquid and collect; On the time resolved fluoro-immunoassay appearance, carry out the photofluorometer number, fluorescence is counted the eluent greater than 1000000 in the collection first peak, adds equal-volume glycerine ,-20 ℃ of preservations behind the mixing; Adopt the chessboard titrimetry to confirm markers work liquid concentration, press the 1:100 dilution again with the experiment damping fluid and detect best results;
6) experiment damping fluid, concentrated washing lotion, enhancing liquid, standard items and the packing of europium label;
7) label;
8) finished product assembling.
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract 3 parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time resolved fluoro-immunoassay method).After accomplishing, assembling also need inspect just can dispatch from the factory after qualified (Fig. 1 ~ 2) by random samples.
The method of application of the kit that embodiment 2 embodiment 1 make
The concrete operations of the neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time resolved fluoro-immunoassay method) of above embodiment 1 preparation are following:
With the micro reaction plate bar balance of reagent requirement to room temperature; Above-mentioned concentrated washing lotion of 40ml and 960ml purified water are mixed into the work cleansing solution; Before the use, with the europium label, the samarium label with the experiment damping fluid by volume 1:100 be diluted to the label working fluid.
Standard items, quality-control product or sample are squeezed in the solid-phase coating reaction plate with diameter 3.0mm card punch, in every hole, added the above-mentioned label working fluid of 100～200 μ l, slowly 1～4h is hatched in vibration; Wash plate 6 times with above-mentioned work cleansing solution, clap and do; In every hole, add 100～200 μ l and strengthen liquid; Slowly vibration is 5 minutes, in the time resolved fluoro-immunoassay appearance, carries out the fluorescence counting; Carry out Fitting Analysis as a result by location parameter, obtain quantitative result.
The analytical performance evaluation index of embodiment 3 kits of the present invention
The performance evaluation index of the neonate's thyroid-stimulating hormone/free thyroxine detection kit (time resolved fluoro-immunoassay method) of above embodiment 1 preparation is following:
The dose-effect curve and the range of linearity: adopt LOG-LOGB principal axis transformation pattern and SPLINE fitting algorithm, standard items content logarithm value is a horizontal ordinate, and respective reaction hole fluorescent value logarithm value is an ordinate, the drawing standard curve.In the measurement range of kit, the dose-response curve linearly dependent coefficient should be not less than 0.9900.
Sensitivity: detect 20 times with zero standard, calculate its concentration average (
x) and standard deviation (
s), with
x + 2
sFor detecting lower bound, then the TSH detection sensitivity can reach 2.0 μ U/mL; FT4 detection sensitivity: be not higher than 5pmol/L.
Specificity: detect 250 U/L national standard article hLH, 250 U/L national standard article hFSH, 10000 U/L national standard article hCG, the TSH apparent value all is not higher than 0.25 μ U/mL; The cross reacting rate of FT4 and trilute (T3) is not higher than 2%, is not higher than 2% with the cross reacting rate of coelonychia shape gland original ammonia acid (rT3);
Accuracy: with national standard article and company standard article is contrast, and the actual measurement of kit standard items is tired and indicated potency ratio mean value should be in 0.900～1.100 scope.
Accuracy: the measurement accuracy of kit is no more than 15.0% in batch; Be no more than 20.0% between batch.
Indoor Quality Control: three variable concentrations Quality Controls, TSH, FT4 measured value are all in allowed band.
The HOOK effect of HOOK effect: TSH is up to 1000 μ U/mL.
Thermal stability (test accelerates the failure): kit is after 37 ℃ of bakings in 3 days, and testing result meets above performance index.
Above performance index show that the range of linearity of " neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time resolved fluoro-immunoassay method) " is wide, highly sensitive, high specificity, accuracy good, accuracy is good and Heat stability is good, meet the requirements fully.
Embodiment 4
The neonate's thyroid-stimulating hormone detection kit (time-resolved fluoroimmunoassay method) of PE company and the thyroxine detection kit (time-resolved fluoroimmunoassay method) of Guangzhou City Fenghua Biological Engineering Co.,Ltd compare experiment on neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay method) that use embodiment 1 makes and the market.
Neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay method) that the filter paper dried blood spot sample that collects is made with this patent and neonate's thyroid-stimulating hormone detection kit (time-resolved fluoroimmunoassay method) of PE company detect; Detect collecting the thyroxine detection kit (time-resolved fluoroimmunoassay method) of the corresponding serum of filter paper dried blood spot with the Guangzhou City Fenghua Biological Engineering Co.,Ltd; To detect the gained result and make correlation analysis; The result sees Fig. 3, Fig. 4.
The result shows that neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay method) and existing kit testing result correlativity that this patent makes are good, can be applied to neonatal screening fully.
Claims (3)
1. neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit is characterized in that said kit comprises following component:
(1) experiment damping fluid;
(2) concentrate washing lotion;
(3) strengthen liquid;
(4) encapsulate the micro reaction plate of thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector simultaneously;
(5) contain the filter paper dried blood spot standard items and the quality-control product of thyroid-stimulating hormone and free thyroxine simultaneously;
(6) Eu
3+-N
2The thyroid-stimulating hormone monoclonal antibody and the Sm of-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark
3+-N
2The free thyroxine monoclonal antibody of-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark.
2. the preparation method of the said neonate's thyroid-stimulating hormone of claim 1/free thyroxine double-tagging detection kit is characterized in that comprising the steps:
(1) preparation experiment damping fluid, concentrate, enhancing liquid; Said experiment damping fluid is processed with bovine serum albumin(BSA) and the damping fluid of 1 ~ 20g/L; Said concentrate is processed with Tween-20 and the damping fluid of 1.0 ~ 10.0ml/L; Said enhancing liquid is processed with three n-octyl phosphorous oxide and the damping fluid of 1 ~ 5mg/L β-naphthoyltrifluoroacetone, 15 ~ 30mg/L;
(2) prepare the micro reaction plate that encapsulates simultaneously with thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector; Said reaction plate is diluted to the coating buffer of 1 ~ 10 μ g/ml with the damping fluid that contains 1 ~ 10ppm antiseptic with TSH monoclonal antibody, FT4-connector, encapsulates blank micro reaction plate, and with the protectant confining liquid sealing that contains 1.0 ~ 10.0g/L afterwards drying process;
(3) preparation is with filter paper dried blood spot standard items, the quality-control product of the pure article of thyroid-stimulating hormone, the pure article preparation of free thyroxine;
(4) preparation is with Eu
3+-N
2-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark thyroid-stimulating hormone monoclonal antibody, Sm
3+-N
2The plain monoclonal antibody of-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium marked free thyroid;
(5) the above-mentioned experiment damping fluid of packing, concentrated washing lotion, enhancing liquid, standard items and label;
(6) label;
(7) be assembled into finished product;
Wherein, the preparation method of filter paper dried blood spot standard items, quality-control product is following in the step (3): the matrix that standard items and quality-control product are processed the filter paper dried blood spot is human red cell and removes hormone people negative serum; Matrix includes but not limited to human red cell and removes hormone people negative serum; The hematocrit of its mesostroma is 45 ~ 90%; Wherein hematocrit is 50 ~ 55%; Scraps of paper standard items are the point of 3 ~ 10 variable concentrations; Wherein the standard point with 6 variable concentrations is main, but is not limited to 6; Quality-control product is the point of 1 ~ 5 variable concentrations; Wherein with 2 variable concentrations be main, but be not limited to 2; External packing is an aluminium foil bag; The making of thyroid-stimulating hormone, free thyroxine standard items, quality-control product; Be to be reference with TSH national standard article and FT4 national standard article; The human red cell of the hematocrit with 50 ~ 55% and the mixed-matrix that removes the hormone negative serum are prepared into standard items and the C1 of A, B, C, D, E, six debita spissitudos of F, the quality-control product of two concentration of C2 (low, height) respectively with thyroid-stimulating hormone or the pure article of free thyroxine; On A, B, C, D, E, F position and the C1 that drips respectively at filter paper with proper volume, the C2 position; The dried blood filter paper standard items, the quality-control product that behind natural drying at room temperature, obtain are used aluminium foil bag vacuum seal.
3. the method for operating of the said neonate's thyroid-stimulating hormone of claim 1/free thyroxine double-tagging detection kit is characterized in that comprising the steps:
(1) reagent is prepared: with the micro reaction plate bar balance of reagent requirement to room temperature; Above-mentioned concentrated washing lotion of 40ml and 960ml purified water are mixed into the work cleansing solution; Before the use, with each personal experiment damping fluid of europium and samarium label by volume 1:100 be diluted to the label working fluid;
(2) standard items, quality-control product or sample are squeezed in the micropore that encapsulates reaction plate with diameter 3.0mm card punch, in every hole, added the label working fluid of above-mentioned europium of 100 ~ 200 μ l and samarium respectively; Reaction plate at room temperature slowly vibrates and hatches 1 ~ 4h; Wash plate 6 times with above-mentioned work cleansing solution, clap and do; In every hole, add 100 ~ 200 μ l and strengthen liquid, the 5min that slowly vibrates carries out the fluorescence counting in the time resolved fluoro-immunoassay appearance; Carry out Fitting Analysis by location parameter, obtain quantitative result.
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Denomination of invention: The invention relates to a neonatal thyrotropin / free thyroxine double label detection kit and a corresponding detection method Effective date of registration: 20210918 Granted publication date: 20130626 Pledgee: Guangzhou Baiyun Branch of China Construction Bank Co.,Ltd. Pledgor: GUANGZHOU FENGHUA BIOENGINEERING Co.,Ltd. Registration number: Y2021980009623 |
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