CN108181464A - A kind of preparation method of TTR time resolutions immunofluorescent reagent box - Google Patents
A kind of preparation method of TTR time resolutions immunofluorescent reagent box Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
Present application relates generally to a kind of preparation methods of TTR time resolutions immunofluorescent reagent box, the kit is mainly used in the detection of molecular marked compound and diagnostic application in the clinical infant serum of Kawasaki disease (KD), and the KD infants detection to promote intravenous gamma globulin (IVIG) responsive type and absorbing wave maker provides technical foundation as the clinical generaI investigation project of a routine.Including step：(1) preparation of anti-human transthyretin TTR monoclonal antibodies；(2) screening of anti-TTR pairings antibody and the optimization of testing conditions；(3) TRFIA ELISA methods are established and are assessed；(4) the KD infant positive samples for preserving hospital examine verification test for the extensive sample of kit.
Description
Technical field
Present application relates generally to a kind of preparation method of TTR time resolutions immunofluorescent reagent box, which mainly should
Detection and diagnostic application for molecular marked compound in the clinical infant serum of Kawasaki disease (KD), to promote intravenous gamma globulin
(IVIG) detection of the KD infants of responsive type and absorbing wave maker becomes the clinical generaI investigation project offer technical foundation of a routine.
Background technology
In our previous work, gamma globulin is controlled by proteomics 2D electrophoresis and Western Blot technologies
The screening (Fig. 1-2 is the experimental result of part KD infants) of monitoring index, screens before and after treatment Patients with Kawasaki Disease (serum sample)
There were significant differences for the albumen such as RBP4, APCS and TTR, is found by statistical analysis, and IVIG responsive type KD infants are treated in IVIG
TTR contents in preceding serum are substantially less than normal child, and albumen TTR substantially returns to normal level in serum after medication；
For IVIG absorbing wave maker KD infants before and after medication, the TTR contents in serum do not change substantially.It can be seen that serum
In albumen TTR can be used as a species specific molecular marked compound, exploitation is used for the quick IVIG responsive types and reactionless distinguished
The early diagnosis kit of the KD infants of type will have good application prospect, and correlated results is published in《Biochem Biophys Res Commun》。
Invention content
The purpose of the present invention is establish the KD infants of a kind of easy, quick diagnosis IVIG responsive types and IVIG absorbing wave makers
Kit, by analyzing TTR contents in serum, the quick KD infants for distinguishing IVIG responsive types and absorbing wave maker, so as to quick accurate
Really evaluation IVIG adjusts the therapeutic scheme of IVIG absorbing wave maker KD infants, avoids coronal dynamic in time to the therapeutic effect of KD infants
The appearance of arteries and veins lesion, the life quality to improve infant lay the foundation.
The preparation method of the TTR time resolution immunofluorescent reagent boxes of the application includes：
First, the preparation of anti-human transthyretin TTR monoclonal antibodies
1st, BALB/C mice is immunized：Using TTR albumen as immunogene, carry out BALB/C mice and be immunized.First immunisation back
Multiple spot is subcutaneously injected, comlete antigen and equivalent Freund's complete adjuvant mixing and emulsifying, and later each time immune, and comlete antigen and equivalent are not
Family name's Freund's incomplete adjuvant mixing and emulsifying is divided into 14d between immunization time.
2nd, the preparation of anti-TTR monoclonal antibodies：Under aseptic condition, spleen is taken to fusion mouse, shreds spleen, crosses 200 mesh sieve
It nets spare；SP2/0 cell of the selection in exponential phase, centrifuges for several times after being mixed with splenocyte, is added in 1min in advance
The PEG of heat, side edged lightly stir rapidly, then terminate the reaction of PEG, stand several minutes；HAT cultures are resuspended in after centrifugation
It in base, is added in the culture plate for being covered with feeder cells, 37 DEG C, cultivate in 5%CO2 incubators.The screening of EL I SA methods is positive
Hole carries out it limiting dilution subclone for several times, to obtain the single plant cell of antibody needed for energy stably excreting；Selection 8~10 weeks
Age health BALB/C mice, as immunosuppressor, the injection of 2 points of abdominal cavity is in 2 points of 7~10d abdominal cavities injection paraffin oil in advance
Logarithmic phase single plant cell, ascites pass through affinity chromatography column purification, identification potency, purity etc..
2nd, the screening of anti-TTR pairings antibody and the optimization of testing conditions
From the anti-TTR monoclonal antibodies of above-mentioned preparation, using double-antibody sandwich EL I SA methods as principle, using TTR as target egg
In vain, it takes turns and screens by 2-3, tested by cross match, obtain the pairing antibody number pair of energy specific bond TTR target proteins.Pass through
Antibody conjugates are tested：The pairing antibody that thick pairing and perfect match experiment screening can be applied in double antibodies sandwich EL I SA.Optimization
Double antibodies sandwich EL I SA testing conditions：It determines coated antibody and detection antibody concentration, determines cleaning solution, washing intensity and reaction
Condition etc..So as to obtain and TTR high-affinities, high specific pairing antibody.
3rd, the foundation and assessment of TRF IA-EL I SA methods
1st, the preparation of solid-phase coating antibody：Coated antibody is diluted to debita spissitudo by carbonate buffer solution, is added to 96 holes
In plate, 4 DEG C overnight, coating buffer of inclining, and adds in confining liquid closing, -20 DEG C of freezen protectives of vacuum.
2nd, the preparation of Eu3+ labelled antibodies：Labelled antibody is added in the centrifuge tube with filter membrane of Millipore companies,
Centrifugation, then with label buffer solution repeated washing for several times.By labelled antibody and the abundant mixing of europium labelled reagent, room temperature concussion is overnight.
3rd, the purifying of Eu3+ labelled antibodies：Column separating purification is chromatographed with Sephadex G-50 after the completion of label, eluent is washed
It is de-, efflux is collected simultaneously, absorbance is measured by pipe, merges peak pipe, protein content is measured, according to Eu3+ labelling kit explanations
The formula that book is provided measures mark rate and protein recovery.
4th, the foundation and assessment of TRF IA-EL I SA methods：Optimize the reaction condition of TRF IA detections TTR, determine label
Antibody concentration, reaction buffer pH value, confining liquid composition, storage formula of liquid etc. establish optimal TRF IA-EL I SA reactions
Pattern.According to optimal TRF IA-EL I SA reaction patterns, detection performance index is determined.The kit measurement of different batches is same
The result of a sample carries out the comparison of precision；After kit is placed different time at different temperatures, its detection property is measured
Can, so as to obtain its stability；TTR standard items are carried out to the dilution of various concentration, measure the detection numerical value of various concentration TTR,
So as to obtain the range of linearity.The detection albumen similar with TTR or in the sample other simultaneous albumen, determine kit
Detection specificity.
4th, the KD infant positive samples for preserving hospital examine verification test for the extensive sample of kit；With
A variety of detection methods such as ELISA detection method, real-time fluorescence quantitative PCR and colloidal gold are compared, the assay kit
To the comprehensive detection effect of the KD infants of IVI G responsive types and IVI G absorbing wave makers.
3. the advantages of invention：
1st, at present for the limitation of the diagnostic method of KD including clinical indication, ultrasonic image and laboratory examination with
And in place of the deficiencies of subjectivity, utilize the serum molecules marker TTR albumen rapid evaluation IVI G couple for possessing independent intellectual property right
The therapeutic effect of KD infants is a kind of important method innovation.
2nd, the antibody library of anti-TTR albumen voluntarily built, not only possesses independent intellectual property right, antibody producing is at low cost
It is honest and clean, greatly reduce the cost of kit.
3rd, detection kit is easy to operate, at low cost, the clinical diagnosis demand for being suitble to China current, before having wide market
Scape.
4th, TRF IA technologies are applied in the EL I SA detection kits of transthyretin TTR, concentrated
TRF IA are sensitive, the advantage of both easy, quick technologies of quantitative and EL I SA, realize quantitative, quick, easy, sensitive
Clinical diagnosis requirement.
Description of the drawings
Fig. 1：To the KD infants of IVIG responsive types after (- IVIG) and IVIG are treated before IVIG treatments in (+IVIG) serum
The variation of the expression quantity of RBP4, APCS and TTR：(A) variation of the expression quantity of RBP4, APCS and TTR albumen；(B) and (C) is right
Western Blot results carry out the statistical analysis after gray scale scanning, and B is pair analysis, and C is non-pair analysis；Pass through analysis
It was found that the expression quantity variation of only TTR is either matched in (+IVIG) serum after (- IVIG) and IVIG are treated before IVI G treatments
Pair and non-matching analysis, be all statistically that difference is extremely significant.
Fig. 2：To the KD infants of IVIG absorbing wave makers (+IVIG) serum after (- IVIG) and IVIG are treated before IVIG treatments
The variation of the expression quantity of middle TTR：(A) variation of the expression quantity of TTR albumen；(B) and (C) carries out ash to Western Blot results
Statistical analysis after degree scanning, B is pair analysis, and C is non-pair analysis；It is found by analysis, before IVIG treatments (-
IVIG) and after IVIG treatments the expression quantity variation either pairing and non-matching of TTR is analyzed in (+IVIG) serum, statistically
All be do not have it is discrepant.
Fig. 3 is kit Technology Roadmap.
Fig. 4 is TTR-TRF IA standard curves.
Specific embodiment
Refer to the attached drawing, the preparation method of the TTR time resolution immunofluorescent reagent boxes of the application include：
First, the preparation of anti-human transthyretin TTR monoclonal antibodies
1st, BALB/C mice is immunized：Using TTR albumen as immunogene, carry out BALB/C mice and be immunized.First immunisation back
Multiple spot is subcutaneously injected, comlete antigen and equivalent Freund's complete adjuvant mixing and emulsifying, and later each time immune, and comlete antigen and equivalent are not
Family name's Freund's incomplete adjuvant mixing and emulsifying is divided into 14d between immunization time.
2nd, the preparation of anti-TTR monoclonal antibodies：Under aseptic condition, spleen is taken to fusion mouse, shreds spleen, crosses 200 mesh sieve
It nets spare；SP2/0 cell of the selection in exponential phase, centrifuges for several times after being mixed with splenocyte, is added in 1min in advance
The PEG of heat, side edged lightly stir rapidly, then terminate the reaction of PEG, stand several minutes；HAT cultures are resuspended in after centrifugation
It in base, is added in the culture plate for being covered with feeder cells, 37 DEG C, cultivate in 5%CO2 incubators.The screening of EL ISA methods is positive
Hole carries out it limiting dilution subclone for several times, to obtain the single plant cell of antibody needed for energy stably excreting；Selection 8~10 weeks
Age health BALB/C mice, as immunosuppressor, the injection of 2 points of abdominal cavity is in 2 points of 7~10d abdominal cavities injection paraffin oil in advance
Logarithmic phase single plant cell, ascites pass through affinity chromatography column purification, identification potency, purity etc..
2nd, the screening of anti-TTR pairings antibody and the optimization of testing conditions
From the anti-TTR monoclonal antibodies of above-mentioned preparation, using double-antibody sandwich EL ISA methods as principle, using TTR as target egg
In vain, it takes turns and screens by 2-3, tested by cross match, obtain the pairing antibody number pair of energy specific bond TTR target proteins.Pass through
Antibody conjugates are tested：The pairing antibody that thick pairing and perfect match experiment screening can be applied in double antibodies sandwich EL I SA.Optimization
Double antibodies sandwich ELI SA testing conditions：It determines coated antibody and detection antibody concentration, determines cleaning solution, washing intensity and reaction item
Part etc..So as to obtain and TTR high-affinities, high specific pairing antibody.
3rd, the foundation and assessment of TRFIA-ELISA methods
1st, the preparation of solid-phase coating antibody：Coated antibody is diluted to debita spissitudo by carbonate buffer solution, is added to 96 holes
In plate, 4 DEG C overnight, coating buffer of inclining, and adds in confining liquid closing, -20 DEG C of freezen protectives of vacuum.
2nd, the preparation of Eu3+ labelled antibodies：Labelled antibody is added in the centrifuge tube with filter membrane of Millipore companies,
Centrifugation, then with label buffer solution repeated washing for several times.By labelled antibody and the abundant mixing of europium labelled reagent, room temperature concussion is overnight.
3rd, the purifying of Eu3+ labelled antibodies：Column separating purification is chromatographed with Sephadex G-50 after the completion of label, eluent is washed
It is de-, efflux is collected simultaneously, absorbance is measured by pipe, merges peak pipe, protein content is measured, according to Eu3+ labelling kit explanations
The formula that book is provided measures mark rate and protein recovery.
4th, the foundation and assessment of TRFIA-ELISA methods：Optimize the reaction condition of TRFIA detections TTR, determine labelled antibody
Concentration, reaction buffer pH value, confining liquid composition, storage formula of liquid etc., establish optimal TRFIA-ELISA reaction patterns.It presses
According to optimal TRFIA-ELISA reaction patterns, detection performance index is determined.The knot of the kit measurement same sample of different batches
Fruit carries out the comparison of precision；After kit is placed different time at different temperatures, its detection performance is measured, so as to obtain
Its stability；TTR standard items are carried out to the dilution of various concentration, measure the detection numerical value of various concentration TTR, it is linear so as to obtain
Range.The detection albumen similar with TTR or in the sample other simultaneous albumen, determine that the detection of kit is special
Property.
4th, the KD infant positive samples for preserving hospital examine verification test for the extensive sample of kit；With
A variety of detection methods such as ELISA detection method, real-time fluorescence quantitative PCR and colloidal gold are compared, the assay kit
To the comprehensive detection effect of the KD infants of IVIG responsive types and IVIG absorbing wave makers.
1.TTR-TRFIA standard curves prepare the standard solution of TTR albumen, concentration is divided into 0,0.5,5,50,200,
1000ng/ml, draws standard curve, and coefficient R 2=0.9991 shows that linear dependence is good.With zero reference standard product
As sample duplicate measurements 20 times, calculate its fluorescence mean value x and the fluorescent value obtained by standard deviation s, x+2s substitutes into standard curve side
Journey is calculated its sensitivity and reaches 0.25ng/ml.
2. the specificity of method chooses unrelated protein or the albumen that may occur simultaneously as sample, surveyed with this kit
It is fixed, the results are shown in Table 1, this kit measurement thyronine T4 albumen (T4), c reactive protein (CRP), thyroglobulin (Tg),
During retinol-binding proteins (RBP4) albumen, measured concentration far below theoretical concentration, illustrates the specificity of this method preferably.
1 specificity experiments of table
3. Precision Experiment is measured high, normal, basic three standard concentrations of accurate quantification using this kit, respectively set
Put 10 each multiple holes.The variation within batch coefficient and interassay coefficient of variation of this kit≤10% (being shown in Table 2), meet kit regulation
It is required that.
2 Precision Experiment testing result of table
4. accuracy experiment (recovery experiment) conventionally carries out recovery experiment.3 are the results are shown in Table, high, normal, basic three dense
The rate of recovery of degree is between 100.35-100.56%, average recovery rate 100.42%.
3 recovery experiment of table
5. viability is the TTR albumen of high concentration according to 1：With this kit measurement after 2~64 doubling dilutions, after conversion
TTR protein contents are very close.TTR albumen reference standards product is replaced to draw standard with the protein sample of above-mentioned doubling dilution bent
The slope of standard curve of line, slope and standard items is basically identical, shows that TTR-TRFIA has good viability.
KD infants before 5 IVIG are treated, KD infants and the blood of 5 normal children after 5 IVIG treatments are acquired respectively；
After 2000rpm centrifugations 5min, it is spare to take out serum；
Specific detecting step is as follows：The elisa plate being coated with is taken, adds in 25 μ l reference standards product or test serum, then add
Entering 200 μ l analysis buffers, concussion incubates 1h, cleaning solution washs 4 times, adds with analysis buffer 1：50 diluted Eu3+
200 μ l/ holes of labelled antibody, concussion incubate 1h, cleaning solution wash 6 times, be eventually adding 200 μ l/ holes of enhancement solution concussion 5min after
It is detected on time-resolved fluorescence detector.
Compared with healthy children, the TTR levels of children significantly reduce before treatment, after treatment, No. 1, No. 2 and No. 5 youngster
Virgin TTR levels rise to normal level, are IVIG responsive type infants；And the TTR levels of No. 3 and No. 4 children do not become substantially
Change, it is really the KD infants of IVIG absorbing wave makers to make a definite diagnosis No. 3 and No. 4 children with reference to its clinical symptoms etc..Thus illustrate, Ke Yitong
Cross pretherapy and post-treatment TTR concentration variation determine infant whether be IVIG absorbing wave makers KD infants.
The foregoing is merely the preferred embodiment of the application, not to limit the application, it is all in spirit herein and
Within principle, any modification, equivalent replacement, improvement and so on should be included within the protection domain of the application.
Claims (10)
1. a kind of preparation method of TTR time resolutions immunofluorescent reagent box, it is characterised in that：
Including step：(1) preparation of anti-human transthyretin TTR monoclonal antibodies；(2) sieve of anti-TTR pairings antibody
Choosing and the optimization of testing conditions；(3) TRFIA-ELISA methods are established and are assessed；(4) the KD infants for preserving hospital are positive
Sample examines verification test for the extensive sample of kit.
2. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 1, it is characterised in that：
Wherein step (1) is immunized including BALB/C mice and then prepares anti-TTR monoclonal antibodies.
3. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 1, it is characterised in that：
Wherein step (2) includes：From the anti-TTR monoclonal antibodies of preparation, using double antibody sandwich ELISA as principle, using TTR as target
Albumen is taken turns by 2-3 and is screened, tested by cross match, obtains the pairing antibody number pair of energy specific bond TTR target proteins, leads to
Cross antibody conjugates experiment, the pairing antibody that thick pairing and perfect match experiment screening can be applied in double crush syndrome, optimization
Double crush syndrome testing conditions：It determines coated antibody and detection antibody concentration, determines cleaning solution, washing intensity and reaction item
Part, so as to obtain and TTR high-affinities, high specific pairing antibody.
4. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 1, it is characterised in that：
The preparation established TRFIA-ELISA methods and assessed includes the 1, preparation of solid-phase coating antibody, 2, Eu3+ labels it is anti-
The preparation of body, 3, the purifying of Eu3+ labelled antibodies.
5. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 1, it is characterised in that：
Wherein step (4) including being compared with ELISA detection method, real-time fluorescence quantitative PCR and colloidal gold detection method, comment by analysis
The valency kit is to the comprehensive detection effect of the KD infants of IVIG responsive types and IVIG absorbing wave makers.
6. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 2, it is characterised in that：
Wherein BALB/C mice is immunized is immunized using TTR albumen as immunogene, to carry out BALB/C mice, first immunisation back multiple spot skin
Lower injection, comlete antigen and equivalent Freund's complete adjuvant mixing and emulsifying, later each time immune, and comlete antigen and equivalent Freund are endless
Full adjuvant mixing and emulsifying is divided into 14 days between immunization time.
7. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 2, it is characterised in that：
The preparation of anti-TTR monoclonal antibodies is included aseptically, takes spleen to fusion mouse, shreds spleen, it is standby to cross 200 mesh screens
With；SP2/0 cell of the selection in exponential phase, centrifuges for several times after being mixed with splenocyte, adds in what is be preheated in 1min
PEG, side edged stir rapidly, then terminate the reaction of PEG, stand several minutes；It is resuspended in HAT culture mediums, adds in after centrifugation
It to being covered in the culture plate of feeder cells, is cultivated in 37 DEG C, 5%CO2 incubators, positive hole is screened using ELISA method, it is right
It carries out limiting dilution for several times and is subcloned, to obtain the single plant cell of antibody needed for energy stably excreting；Select 8~10 week old health
BALB/C mice, 7~10 days in advance 2 points of abdominal cavity injection paraffin oils are as immunosuppressor, and the injection of 2 points of abdominal cavity is in logarithmic phase
Single plant cell, ascites pass through affinity chromatography column purification, identification potency, purity.
8. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 4, is characterized in that：Its
The preparation of middle solid-phase coating antibody includes coated antibody is diluted to debita spissitudo with carbonate buffer solution, is added to 96 orifice plates
In, 4 DEG C overnight, coating buffers of inclining, add in confining liquid closing, -20 DEG C of freezen protectives of vacuum.
9. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 4, is characterized in that：Eu3
The preparation of+labelled antibody includes adding in labelled antibody in the centrifuge tube with filter membrane of Millipore companies, centrifugation, then uses
Mark buffer solution repeated washing for several times, by labelled antibody and the abundant mixing of europium labelled reagent, room temperature concussion is overnight.
10. a kind of preparation method of TTR time resolutions immunofluorescent reagent box according to claim 4, is characterized in that：
The foundation of TRFIA-ELISA methods and the reaction condition for being evaluated as optimization TRFIA detections TTR, determine labelled antibody concentration, reaction
PH of cushioning fluid, confining liquid composition, storage formula of liquid, establish optimal TRFIA-ELISA reaction patterns, according to optimal
TRFIA-ELISA reaction patterns, determine detection performance index, and the result of the kit measurement same sample of different batches carries out essence
The comparison of density；After kit is placed different time at different temperatures, its detection performance is measured, so as to obtain its stabilization
Property；TTR standard items are carried out to the dilution of various concentration, measure the detection numerical value of various concentration TTR, so as to obtain the range of linearity,
The detection thyronine T4 albumen (T4) similar with TTR, c reactive protein (CRP), thyroglobulin (Tg), retinol combine
(RBP4) albumen of albumen 4 or in the sample other simultaneous albumen determine the detection specificity of kit.
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Application publication date: 20180619 |