CN102520196B - 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
- Publication number
- CN102520196B CN102520196B CN 201110394196 CN201110394196A CN102520196B CN 102520196 B CN102520196 B CN 102520196B CN 201110394196 CN201110394196 CN 201110394196 CN 201110394196 A CN201110394196 A CN 201110394196A CN 102520196 B CN102520196 B CN 102520196B
- Authority
- CN
- China
- Prior art keywords
- stimulating hormone
- thyroid
- liquid
- damping fluid
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
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 detection method.
Background technology
CH disease (CH is called for short the low or first of congenital first and subtracts), be to reduce because various innate factors make 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 occurred when CH is defined as any birth at present.
Examination of newborn infant diseases result all over the world shows, the low incidence of disease of congenital first is at country variant, and between different nationalities, difference is less, 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 increasingly increase the weight of, yet, once the clinical symptoms of disease occur, show that disease has entered late period.Even treatment, treatment lowly also is difficult to recover; On the contrary, if can find soon disease 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 exactly a kind of simple as far as possible method, whole ewborn infants is generally investigated, to finding early infant, give effective treatment 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, the CH infant is if made a definite diagnosis and treated in 3 months in birth, and the infant intelligent development 80% or more is normal or approach normally.
The examination of CH, Dussault is in the thyroxine (T4) at first detected with filter paper blood cake radioimmunology in North America in 1993 in neonate's peripheral blood of 4-7 days, the use 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 u s. state, the country such as Europe, Australia carries out rapidly.In the Asian-Pacific area, the research that Japan started relevant newborn child's illness in 1966, and, from 1977, take the lead in having carried out on-radiation examination method---the enzymoimmunoassay of TSH of CH, to nineteen eighty-three, all Japan examination goes out CH case 400 examples.
The Screening Disease in Neonates of China is started late.From 1989, the provinces and cities such as Beijing, Shanghai, Guangzhou, Tianjin progressively carried out the CH routine screening.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 the 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 Screening Disease in Neonates 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, coverage rate only has 2.5%, still have a lot of infants to leave serious intelligent disability because failing in time diagnosis and treatment, had a strong impact on the raising of the health of newborns.
Time resolution immunofluorescence assay technology (Time Resolved Immunofluorometric assay, TRIFMA) is the novel ultramicron immuno analytical method that early eighties comes out.The TIME RESOLVED TECHNIQUE of fluorescence is to utilize the fluorescence lifetime difference of fluorescent emission body, the fluorescence spectroscopy technique that its fluorescence is separated.Take lanthanide series as tracer agent replaces radioactive isotope, with the lanthanide series with bifunctional group, come labelled antigen or antibody, when the lanthanide series disintegrated down and special amplification liquid form new chelate, its fluorescence obviously strengthens and lastingly.Can eliminate the background fluorescence of sample self by the way of delay measurements, thereby greatly improve detection sensitivity.
At present many countries such as American-European, Japanese subtract neonate's first and carry out zonal general examination, and method is updated, and the judgement of result is also more accurate. and the screening method of use has following 3 kinds now:
look into again thyrotropic hormone when thyroxine is low;
thyroxine and thyrotropic hormone detect simultaneously;
singly look into thyrotropic hormone.The detection method that Neonatal TSH is commonly used has enzyme linked immunosorbent assay (ELISA), enzyme immunofluorescence assay (EFIA), time resolution immunofluorescence assay (Tr-FIA) and chemiluminescent immunoassay(CLIA).
At present domestic all using measure the screening method of TSH as CH, but this method can not detect that thyroglobulin lacks and hypothalamus hypophysis first is low, subclinical CH that also can undetected 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 antigen, the method can save time, saving of labor, province's reagent and sample, has the economic and efficient outstanding advantages that waits.Being used in particular for the United screening experiment of entry and the detection of some precious samples, is the developing direction of labelled immune Epidemiological Analysis.Eu in 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 dissociated and strengthen fluorescence intensity.Eu
3+and Sm
3+maximum emission wavelength be respectively 615nm and 643nm, due to narrowed emission, can fully differentiate.Other fluorescence lifetime differs significantly, is respectively 820 μ s and 88 μ s, and during detection, interference each other is smaller.
Summary of the invention
The object of the invention is to the deficiency existed according in 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, there is economic and advantage efficiently, be well suited for applying in a large amount of sample examinations in the whole nation.
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 by the following technical programs:
Kit provided by the present invention is for the Neonatal Congenital Hypothyroidism examination; A kind of two different monoclonal antibodies can identifying different antigenic determinants on the thyrotropic hormone molecule are provided, with the thyroid-stimulating hormone in double-antibody sandwich quantitative analysis Filter Paper Dry Blood sheet.Can also identify the monoclonal antibody of antigenic determinant on the free thyroxine molecule, and thyroxine-BSA connector, with the free thyroxine in competition law quantitative test Filter Paper Dry Blood sheet.Method with manufacturer's standard blood speckles and Quality Control blood speckles is provided simultaneously, and by the free thyroxine monoclonal antibody of europium mark thyroid-stimulating hormone monoclonal antibody and samarium mark, by the time-resolved fluoroimmunoassay technology of dissociating and strengthening, 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 Dry Blood sheet, time-resolved fluoroimmunoassay technology by double-tagging, the method of Simultaneous Quantitative Analysis thyroid-stimulating hormone and free thyroxine, thereby needn't gather a large amount of blood in the examination process, just can distinguish 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) is characterized in that described kit comprises following component:
(1) experiment damping fluid;
(2) concentrated washing lotion;
(3) strengthen liquid;
(4) be coated with the micro reaction plate of thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector simultaneously;
(5) contain Filter Paper Dry Blood sheet standard items and the quality-control product of thyroid-stimulating hormone and free thyroxine simultaneously;
(6) Eu
3+-N
2thyroid-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 simultaneously detects the kit (time-resolved fluoroimmunoassay) of neonate's thyroid-stimulating hormone and free thyroxine comprises the steps:
(1) preparation experiment damping fluid, concentrate, enhancing liquid; Described experiment damping fluid is made with bovine serum albumin(BSA) and the damping fluid of 1 ~ 20g/L; Described concentrate is made with Tween-20 and the damping fluid of 1.0 ~ 10.0ml/L; Described for enhancing liquid three n-octyl phosphorous oxide and the damping fluid of 1 ~ 5mg/L β-naphthoyltrifluoroacetone, 15 ~ 30mg/L make;
(2) preparation is with thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector coated micro reaction plate simultaneously; Described reaction plate is diluted to TSH monoclonal antibody, FT4-BSA connector the coating buffer of 1 ~ 10 μ g/ml with the damping fluid that contains 1 ~ 10ppm antiseptic, coated blank micro reaction plate, and make by drying after the protectant confining liquid sealing that contains 1.0 ~ 10.0g/L;
(3) preparation is with Filter Paper Dry Blood sheet standard items, the quality-control product of thyroid-stimulating hormone sterling, the preparation of free thyroxine sterling;
(4) preparation is with Eu
3+-N
2-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium mark thyroid-stimulating hormone monoclonal antibody, Sm
3+-N
2-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium marked free thyroid element monoclonal antibody;
(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, in step (3), the preparation method of Filter Paper Dry Blood sheet standard items, quality-control product is as follows: the matrix that standard items and quality-control product are made the Filter Paper Dry Blood sheet 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%, the point that scraps of paper standard items are 3 ~ 10 variable concentrations, wherein take the standard point of 6 variable concentrations as main, but be not limited to 6, the point that quality-control product is 1 ~ 5 variable concentrations, wherein take 2 variable concentrations as main, but be not limited to 2, external packing is aluminium foil bag, thyroid-stimulating hormone, the free thyroxine standard items, the making of quality-control product, that to take TSH national standard and FT4 national standard be reference, the human red cell of the hematocrit with 50 ~ 55% and go the mixed-matrix of hormone negative serum that thyroid-stimulating hormone or free thyroxine sterling are prepared into respectively to A, B, C, D, E, standard items and the C1 of six debita spissitudos of F, C2(is low, height) quality-control product of two concentration, the A dripped respectively at filter paper with proper volume, B, C, D, E, F position and C1, on the C2 position, the dry blood filter paper standard items that obtain after natural drying at room temperature, quality-control product, use aluminium foil bag vacuum seal.
The principle of work of neonate's thyroid-stimulating hormone of the present invention/free thyroxine double-tagging detection kit is time-resolved fluoroimmunoassay, and concrete method of operating comprises the steps:
(1) reagent is prepared: by the micro reaction plate bar balance of reagent requirement to room temperature; The above-mentioned concentrated washing lotion of 40ml and 960ml purified water are mixed into to the work cleansing solution; Before use, by 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 into diameter 3.0mm card punch in the micropore of coated reaction plate, to the label working fluid that adds respectively the above-mentioned europium of 100 ~ 200 μ l and samarium in every hole; Reaction plate at room temperature, slow oscillation incubation 1 ~ 4h; Wash plate 6 times with above-mentioned work cleansing solution, pat dry; In every hole, add 100 ~ 200 μ l to strengthen liquid, the 5min that slowly vibrates carries out the fluorescence counting in the time resolved fluoro-immunoassay instrument; Carry out Fitting Analysis by location parameter, obtain quantitative result.
Kit of the present invention is applicable to the examination of neonate CH, quantitatively detects TSH and FT4 content in neonate's Filter Paper Dry Blood sheet.
The TSH detection sensitivity can reach 2.0 μ U/mL; The specificity aspect, detect 250 U/L national standard hLH, 250 U/L national standard hFSH, and 10000 U/L national standard hCG, the TSH apparent value is not all 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: not higher than 5pmol/L; The specificity aspect, with the cross reacting rate of trilute (T3) not higher than 20%, with the cross reacting rate of coelonychia shape gland original ammonia acid (rT3) not higher than 20%; 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: take respectively TSH national standard and FT4 national standard is contrast, the actual measurement of kit calibration object tire with indicate potency ratio mean value should be in 0.900～1.100 scope; Precise engineering surveying: be no more than 15.0% in analysis; Be no more than 20.0% between analysis; The quality-control product measured value: with two variable concentrations quality-control products, measured, 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, and the decrease personal error alleviates clinical position intensity; And sample consumption to be checked is few, in same reaction system, also reduced the impact that system interference detects two indexs, and the specificity of raising antidiastole, establish good technical foundation for double-tagging TRFIA detects, filled up the blank of domestic such reagent simultaneously, reduced cost.
The accompanying drawing explanation
Fig. 1 is TSH scraps of paper standard items curve map in kit of the present invention;
Fig. 2 is TF4 scraps of paper standard items curve map in kit of the present invention;
Neonate's thyroid-stimulating hormone detection kit that Fig. 3 is PE company on kit of the present invention and market detects the comparison of neonate's Filter Paper Dry Blood sheet sample value, and coefficient of relationship is r=0.995;
Fig. 4 is the comparison that kit of the present invention and the thyroxine detection kit of Fenghua bio-engineering corporation in Guangzhou on market detect the serum sample performance number of corresponding Filter Paper Dry Blood sheet, and coefficient of relationship is r=0.995.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not limit in any form to the present invention.
Thyroid-stimulating hormone in embodiment, free thyroxine sterling, thyroid-stimulating hormone, free thyroxine monoclonal antibody, the free thyroxine connector is purchased from Sigma company; 96 hole micropore blank plates (8 * 12 hole) are the Therom product; Sephadex G-50 is Pharmacia company product; National standard is purchased from Nat'l Pharmaceutical & Biological Products Control Institute; β-naphthoyltrifluoroacetone, three n-octyl phosphorous oxide, TritonX-100 are purchased from Sigma company; Other reagent are that domestic analysis is pure; Time resolved fluoro-immunoassay instrument (PE company 1420 types), constant temperature oscillation instrument, full-automatic ELIAS microplate washer all are purchased from the biological company limited of Guangzhou Fenghua.
embodiment 1 preparation neonate's thyroid-stimulating hormone of the present invention/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay)
Component comprises:
Experiment damping fluid: make with bovine serum albumin(BSA) and the damping fluid of 1 g/L～20g/L;
Concentrated washing lotion: make with Tween-20 and the damping fluid of 1.0ml/L～10.0ml/L;
Strengthen liquid: make with three n-octyl phosphorous oxide and the damping fluid of 1～5mg/L β-naphthoyltrifluoroacetone, 15～30mg/L.
Reaction plate: reaction plate is diluted to TSH monoclonal antibody, FT4-connector the coating buffer of 1～10 μ g/ml with the damping fluid that contains 1～10ppm antiseptic; coated blank micro reaction plate, and make by drying after the protectant confining liquid sealing 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, FT4 sterling are prepared into to 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, obtain after natural drying at room temperature.
Label: TSH, for the FT4 label TSH monoclonal antibody, FT4 monoclonal antibody respectively with the lanthanide ion chelate by 1:1～1:6(m/m) mark makes mother liquor, mother liquor doubly dilutes and forms by 1:10～1:25 with the label dilution again.
The main technique that simultaneously detects TSH, FT4 kit (time resolved fluoro-immunoassay method) of the present invention is prepared as follows:
1) preparation of common reagent: the bovine serum albumin(BSA) that adds 1.0g/L～20.0g/L in the Tris-Hcl solution of experiment damping fluid for 50mmol/L, the pH7.8 of the disodium ethylene diamine tetraacetate containing 0.01g/L; The Tris-Hcl damping fluid that concentrated washing lotion is 0.15mol/L, pH7.8 containing 1.0ml/L～10.0ml/L Tween-20; Strengthen liquid for add the three n-octyl phosphorous oxide of 1mg/L～5mg/L β-naphthoyltrifluoroacetone, 15mg/L～30mg/L in the 0.1mol/L Potassium Hydrogen Phthalate-glacial acetic acid solution that contains 1ml/L TritonX-100.The bovine serum albumin(BSA) of wherein testing damping fluid is 10.0g/L; Tween-20 in concentrated washing lotion is 4.0ml/L.β in enhancing liquid-naphthoyltrifluoroacetone 4.0mg/L, three n-octyl phosphorous oxide 20mg/L, TritonX-100 are 1ml/L;
2) preparation of coated plate: TSH monoclonal antibody, FT4-BSA connector are diluted to the coating buffer that concentration is 1～10 μ g/ml by buffering respectively; Add coating buffer 100～200 μ l/ holes in the micropore blank plate in 96 holes, overnight incubation under 35 ℃ of conditions; The coated plate of hatching is washed plate 1 time with the work cleansing solution, pats dry; Washed coated plate is added to confining liquid by 250～280 μ l/ holes, overnight incubation under 25 ± 2 ℃ of conditions; 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 determine coated concentration, and it is best being diluted to damping fluid the coated effect of coating buffer that concentration is 10 μ g/ml.The coated antibody dilution is for containing 5ppm Proclin300
?the sodium carbonate-sodium bicarbonate buffer liquid of 0.05mol/L, 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; The good thermal stability of coated plate.
4) preparation of standard items, quality-control product: take TSH national standard and FT4 national standard is reference, hematocrit by 50%～55% by human red cell with go hormone negative serum mixed-matrix that TSH, FT4 sterling are mixed with into to 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 dripped respectively at filter paper with 50 μ l volumes, C2 position, the dry filter paper standard items and the quality-control product that after natural drying at room temperature, obtain, use aluminium foil bag vacuum seal.
5) preparation of label: the Eu that 1mg TSH monoclonal labelled antibody is added to 1mg
3+-N
2mix 28 ℃ of reaction 44h in-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium; Reactant liquor is the Tris-Hcl buffer solution elution with 50mmol/L, pH7.8 through Sephadex G-50 post (1 * 40cm), separates Eu
3+-monoclonal antibody bond and free Eu
3+, adopt full-automatic fraction collector 2ml/ pipe to carry out liquid collection; Carry out the photofluorometer number on the time resolved fluoro-immunoassay instrument, collect the eluent that in first peak, the fluorescence counting is greater than 10000000, add equal-volume glycerine ,-20 ℃ of preservations after mixing; Adopt the chessboard titrimetry to determine europium markers work liquid concentration, press again the 1:100 dilution with the experiment damping fluid and detect best results;
The Sm that 1mg FT4 monoclonal antibody is added to 1mg
3+-N
2mix 28 ℃ of reaction 44h in-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium; Reactant liquor is the Tris-Hcl buffer solution elution with 50mmol/L, pH7.8 through Sephadex G-50 post (1 * 40cm), separates Sm
3+-monoclonal antibody bond and free Sm
3+, adopt full-automatic fraction collector 2ml/ pipe to carry out liquid collection; Carry out the photofluorometer number on the time resolved fluoro-immunoassay instrument, collect the eluent that in first peak, the fluorescence counting is greater than 1000000, add equal-volume glycerine ,-20 ℃ of preservations after mixing; Adopt the chessboard titrimetry to determine markers work liquid concentration, press again the 1:100 dilution with the experiment damping fluid and detect best results;
6) experiment damping fluid, concentrated washing lotion, enhancing liquid, standard items and Europium label packing;
7) label;
8) finished product assembling.
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract 3 parts out and just can be assembled into neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit (time resolved fluoro-immunoassay method) through specificity, accuracy, sensitivity and stability assay approval.Also need to inspect by random samples qualified rear just can dispatch from the factory (Fig. 1 ~ 2) after having assembled.
the using method of the kit that embodiment 2 embodiment 1 make
The concrete operations of neonate's thyroid-stimulating hormone of above embodiment 1 preparation/free thyroxine double-tagging detection kit (time resolved fluoro-immunoassay method) are as follows:
By the micro reaction plate bar balance of reagent requirement to room temperature; The above-mentioned concentrated washing lotion of 40ml and 960ml purified water are mixed into to the work cleansing solution; Before use, by 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 oscillation incubation 1～4h; Wash plate 6 times with above-mentioned work cleansing solution, pat dry; In every hole, add 100～200 μ l to strengthen liquid; Slowly vibration is 5 minutes, carries out the fluorescence counting in the time resolved fluoro-immunoassay instrument; 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 Evaluating Indexes of neonate's thyroid-stimulating hormone of above embodiment 1 preparation/free thyroxine detection kit (time resolved fluoro-immunoassay method) is as follows:
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 horizontal ordinate, and respective reaction hole fluorescent value logarithm value is 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: with zero standard, detect 20 times, calculate its concentration average (
x) and standard deviation (
s), with
x + 2
sfor detecting lower bound, the TSH detection sensitivity can reach 2.0 μ U/mL; FT4 detection sensitivity: not higher than 5pmol/L.
Specificity: detect 250 U/L national standard hLH, 250 U/L national standard hFSH, 10000 U/L national standard hCG, the TSH apparent value is not all higher than 0.25 μ U/mL; The cross reacting rate of FT4 and trilute (T3) is higher than 2%, with the cross reacting rate of coelonychia shape gland original ammonia acid (rT3) not higher than 2%;
Accuracy: take national standard and company standard product is contrast, the actual measurement of kit standard items tire with indicate potency ratio mean value should be in 0.900～1.100 scope.
Accuracy: the precise engineering surveying of kit is no more than 15.0% in batch; Be no more than 20.0% between batch.
Internal 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 is good, accuracy is good and Heat stability is good, meet the requirements fully.
embodiment 4
On the neonate's thyroid-stimulating hormone that uses embodiment 1 to make/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay) and market, neonate's thyroid-stimulating hormone detection kit (time-resolved fluoroimmunoassay) of PE company and the thyroxine detection kit (time-resolved fluoroimmunoassay) of Guangzhou City Fenghua Biological Engineering Co.,Ltd are carried out the contrast experiment.
Neonate's thyroid-stimulating hormone detection kit (time-resolved fluoroimmunoassay) of neonate's thyroid-stimulating hormone that the Filter Paper Dry Blood sheet sample collected is made with this patent/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay) and PE company is detected, by collecting the corresponding serum of Filter Paper Dry Blood sheet, by the thyroxine detection kit (time-resolved fluoroimmunoassay) of Guangzhou City Fenghua Biological Engineering Co.,Ltd, detected, to detect acquired results and make correlation analysis, the results are shown in Figure 3, Fig. 4.
Result shows that neonate's thyroid-stimulating hormone that this patent makes/free thyroxine double-tagging detection kit (time-resolved fluoroimmunoassay) and existing kit testing result correlativity are good, can be applied to neonatal screening fully.
Claims (1)
1. the preparation method of neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit is characterized in that consisting of of described kit:
(1) experiment damping fluid;
(2) concentrated washing lotion;
(3) strengthen liquid;
(4) be coated with the micro reaction plate of thyroid-stimulating hormone monoclonal antibody and free thyroxine BSA connector simultaneously;
(5) contain Filter Paper Dry Blood sheet standard items and the quality-control product of thyroid-stimulating hormone and free thyroxine simultaneously;
(6) Eu
3+-N
2thyroid-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 preparation method of described neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit comprises the steps:
(a) preparation experiment damping fluid, concentrate, enhancing liquid; Described experiment damping fluid is made with bovine serum albumin(BSA) and the damping fluid of l ~ 20g/L; Described concentrate is made with Tween-20 and the damping fluid of 1.0 ~ 10.0ml/L; Described for enhancing liquid three n-octyl phosphorous oxide and the damping fluid of 1 ~ 5mg/L β-naphthoyltrifluoroacetone, 15 ~ 30mg/L make;
(b) the coated plate of preparation: TSH monoclonal antibody, FT4-BSA connector are diluted to the coating buffer that concentration is 1～10 μ g/ml by buffering respectively; Add coating buffer 100～200 μ l/ holes in the micropore blank plate in 96 holes, overnight incubation under 35 ℃ of conditions; The coated plate of hatching is washed plate 1 time with the work cleansing solution, pats dry; Washed coated plate is added to confining liquid by 250～280 μ l/ holes, overnight incubation under 25 ± 2 ℃ of conditions; 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 determine coated concentration, the sodium carbonate that the coated antibody dilution is 0.05mol/L, pH9.6 containing 5ppm Proclin300-sodium bicarbonate buffer liquid; Confining liquid is for containing 5.0g/L trehalose, 0.5%BSA, the sodium carbonate of 0.05mol/L, pH 9.6-sodium bicarbonate buffer liquid;
(c) preparation is with Filter Paper Dry Blood sheet standard items, the quality-control product of thyroid-stimulating hormone sterling, the preparation of free thyroxine sterling;
(d) preparation of label: the Eu that 1mg TSH monoclonal labelled antibody is added to 1mg
3+-N
2mix 28 ℃ of reaction 44h in-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium; Reactant liquor, through the Tris-Hcl buffer solution elution of Sephadex G-50 50mmol/L, pH7.8 for post, separates Eu
3+-monoclonal antibody bond and free Eu
3+, adopt full-automatic fraction collector 2ml/ pipe to carry out liquid collection; Carry out the photofluorometer number on the time resolved fluoro-immunoassay instrument, collect the eluent that in first peak, the fluorescence counting is greater than 10000000, add equal-volume glycerine ,-20 ℃ of preservations after mixing; Adopt the chessboard titrimetry to determine europium markers work liquid concentration;
The Sm that 1mg FT4 monoclonal antibody is added to 1mg
3+-N
2mix 28 ℃ of reaction 44h in-[P-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium; Reactant liquor, through the Tris-Hcl buffer solution elution of Sephadex G-50 50mmol/L, pH7.8 for post, separates Sm
3+-monoclonal antibody bond and free Sm
3+, adopt full-automatic fraction collector 2ml/ pipe to carry out liquid collection; Carry out the photofluorometer number on the time resolved fluoro-immunoassay instrument, collect the eluent that in first peak, the fluorescence counting is greater than 1000000, add equal-volume glycerine ,-20 ℃ of preservations after mixing; Adopt the chessboard titrimetry to determine markers work liquid concentration;
(e) the above-mentioned experiment damping fluid of packing, concentrated washing lotion, enhancing liquid, standard items and label;
(f) label:
(g) be assembled into finished product;
The method of operating of described neonate's thyroid-stimulating hormone/free thyroxine double-tagging detection kit comprises the steps:
(i) reagent is prepared: by the micro reaction plate bar balance of reagent requirement to room temperature; The above-mentioned concentrated washing lotion of 40ml and 960ml purified water are mixed into to the work cleansing solution; Before use, by each personal experiment damping fluid of europium and samarium label by volume l:100 be diluted to the label working fluid;
(ii) standard items, quality-control product or sample are squeezed into diameter 3.0mm card punch in the micropore of coated reaction plate, to the label working fluid that adds respectively the above-mentioned europium of 100 ~ 200 μ l and samarium in every hole; Reaction plate at room temperature, slow oscillation incubation l ~ 4h; Wash plate 6 times with above-mentioned work cleansing solution, pat dry; In every hole, add 100 ~ 200 μ l to strengthen liquid, the 5min that slowly vibrates carries out the fluorescence counting in the time resolved fluoro-immunoassay instrument; Carry out Fitting Analysis by location parameter, obtain quantitative result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110394196 CN102520196B (en) | 2011-12-02 | 2011-12-02 | Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110394196 CN102520196B (en) | 2011-12-02 | 2011-12-02 | Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102520196A CN102520196A (en) | 2012-06-27 |
| CN102520196B true CN102520196B (en) | 2013-06-26 |
Family
ID=46291184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110394196 Active CN102520196B (en) | 2011-12-02 | 2011-12-02 | Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102520196B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103105496A (en) * | 2012-09-20 | 2013-05-15 | 河南生生医疗器械有限公司 | Time-resolved fluorescence comprehensive detection esophagus cancer kit and application thereof |
| CN103869073A (en) * | 2014-04-01 | 2014-06-18 | 广州市丰华生物工程有限公司 | Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen |
| CN104730247A (en) * | 2015-03-12 | 2015-06-24 | 广州市丰华生物工程有限公司 | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit |
| CN105445451B (en) * | 2016-01-08 | 2017-04-12 | 广州市丰华生物工程有限公司 | Sample adding method suitable for time resolution fluorescence immunoassay method |
| CN105807070B (en) * | 2016-06-08 | 2018-03-09 | 广西中医药大学附属瑞康医院 | A kind of kit for detecting hepatitis B |
| CN108982836A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of total thyroxin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| CN109307663A (en) * | 2018-09-13 | 2019-02-05 | 广州俊通生物科技有限公司 | A kind of micro reaction plate preparation method, the detection method of kit and kit |
| CN109342719A (en) * | 2018-12-10 | 2019-02-15 | 广州俊通生物科技有限公司 | A kind of preparation method of thyroid function multi-link detection reagent kit |
| CN109443876A (en) * | 2018-12-14 | 2019-03-08 | 郑州安图生物工程股份有限公司 | Hepatic fibrosis markers quality-control product and preparation method thereof |
| CN111965352A (en) * | 2020-06-28 | 2020-11-20 | 广州市丰华生物工程有限公司 | Kit and method for screening progressive muscular dystrophy of newborn |
| CN117169519B (en) * | 2023-10-26 | 2024-01-30 | 艾康生物技术(杭州)有限公司 | Dissociation agent and kit for detecting TT3 and/or TT4 in sample |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993020054A1 (en) * | 1992-04-06 | 1993-10-14 | Nordion International Inc. | Europium and terbium chelators for time-resolved fluorometric assays |
-
2011
- 2011-12-02 CN CN 201110394196 patent/CN102520196B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993020054A1 (en) * | 1992-04-06 | 1993-10-14 | Nordion International Inc. | Europium and terbium chelators for time-resolved fluorometric assays |
Non-Patent Citations (15)
| Title |
|---|
| Europium as a label in time-resolved immunofluorometric assays.;Ilkka Hemmla et al.;《Analytical Biochemistry》;19841231;第137卷;第336页 * |
| Feng-Bo Wu et al..Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels..《Analytical Biochemistry》.2003,第314卷 |
| Ilkka Hemmla et al..Europium as a label in time-resolved immunofluorometric assays..《Analytical Biochemistry》.1984,第137卷 |
| Katsutoshi Ito et al..Simultaneous determination of alpha-fetoprotein,human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay..《Journal of Pharmaceutical and Biomedical Analysis》.1999,第20卷 |
| Kauko Matsumoto et al..Simultaneous determination of α-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay..《Analytical Biochemistry》.1999,第276卷 |
| Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels.;Feng-Bo Wu et al.;《Analytical Biochemistry》;20031231;第314卷;第88页右侧栏－第91页右侧栏"材料和方法"部分 * |
| Simultaneous determination of alpha-fetoprotein,human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay.;Katsutoshi Ito et al.;《Journal of Pharmaceutical and Biomedical Analysis》;19991231;第20卷;第169－178页 * |
| Simultaneous determination of α-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay.;Kauko Matsumoto et al.;《Analytical Biochemistry》;19991231;第276卷;第81－87页 * |
| 付志锋等.多组分免疫传感及其应用.《化学传感器》.2006,(第04期), |
| 多组分免疫传感及其应用;付志锋等;《化学传感器》;20061230(第04期);第1－11页 * |
| 尿清蛋白、β_2微球蛋白的双标记TrFIA法的建立;马智鸿等;《临床检验杂志》;20090915(第05期);第360－362页 * |
| 张丽民等.甲状腺素的时间分辨荧光免疫测定.《第二军医大学学报》.1991,(第06期), |
| 田国力，朱伟明，朱蕾，仇效坤.国产筛查滤纸与进口滤纸制备促甲状腺素（TSH）标准干血片的方法及比较.《光电子技术与信息》.1994,(第05期), * |
| 甲状腺素的时间分辨荧光免疫测定;张丽民等;《第二军医大学学报》;19911230(第06期);第567页右侧栏－第568页左侧栏"材料和方法"部分，以及第568页左侧栏最后一段 * |
| 马智鸿等.尿清蛋白、β_2微球蛋白的双标记TrFIA法的建立.《临床检验杂志》.2009,(第05期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102520196A (en) | 2012-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102520196B (en) | Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method | |
| CN110862881B (en) | Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN101819206A (en) | AFP (Alpha-Fetoprotein) testing kit (time-resolved fluoroimmunoassay) for prenatal screening and preparation method thereof | |
| CN103076455B (en) | Quantitative detection serum amyloid A protein kit and Synthesis and applications thereof | |
| CN105785016A (en) | Double-tagging time resolution fluoroimmunoassay reagent kit based on PG magnetic particle | |
| CN107543932A (en) | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of calcitonin | |
| CN102798725A (en) | Diagnostic kit for determination of serum total IgE, preparation method and application method | |
| CN101363860A (en) | Syphilis helicoid antibody chemiluminescence immune assay determination kit and method for preparing same | |
| CN107462726B (en) | Dual quantitative ELISA detection method is immunized in a kind of magnetic enzyme sandwich based on double monoclonal antibodies | |
| CN104090105A (en) | Method and kit for detecting hepatitis E virus (HEV) antibody and method for preparing kit | |
| CN103278651A (en) | Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit | |
| CN101639478A (en) | Kit for detecting estradiol by utilizing magnetic particle chemiluminescence immunoassay | |
| CN101377504A (en) | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody | |
| CN101551389A (en) | Magnetic particle chemiluminescence detection kit of free thyroxine and application thereof | |
| CN103293299A (en) | Method and kit for broadening double-antibody sandwich immunodetection concentration range | |
| CN103033619A (en) | Protein chip reagent kit and method for comprehensively detecting lung cancer marker | |
| CN102520195A (en) | Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof | |
| CN103792356A (en) | Preparation and detection method for ELISA kit detecting Fumonisins | |
| CN102998442A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for aldosterone, and preparation method of kit | |
| CN109187971A (en) | Neuronspecific enolase chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN101750502A (en) | TRFIA for synchronously detecting AFP and AFP-IgM and reagent kit thereof | |
| CN105929157A (en) | Diagnostic kit for jointly detecting HIV antigen and HIV antibody and preparation method of diagnostic kit | |
| CN101592660A (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit | |
| CN101819210B (en) | Beta-human chorionic gonadotrophin test kit (time-resolved fluoroimmunoassay) for prenatal screening and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
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 |
|
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 510000 No. 6, Yinyi street, economic and Technological Development Zone, Guangzhou, Guangdong Patentee after: Guangzhou Fenghua Biological Co.,Ltd. Address before: 510730, No. 6, silver friendship Street, Guangzhou economic and Technological Development Zone, Guangzhou, Guangdong Patentee before: GUANGZHOU FENGHUA BIOENGINEERING Co.,Ltd. |