CN108872608A - It is a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody - Google Patents
It is a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody, belong to vitro diagnostic techniques field, kit includes the elisa plate for being coated with canine distemper virus recombinant antigen, the canine distemper virus recombinant antigen solution of europium label, analysis buffer, cleaning solution and enhancement solution, canine distemper virus recombinant antigen can be specifically bound with canine distemper virus antibody.Kit of the present invention uses dual-antigen sandwich method, and the sensitivity of detection and accuracy are higher.
Description
Technical field
The invention belongs to vitro diagnostic techniques field more particularly to a kind of times for detecting canine distemper virus antibody point
Distinguish fluorescence immunoassay kit.
Background technique
Canine distemper (CD) is the acute, highly contagious disease as caused by canine distemper virus (CDV) infection dog.Hundstaupe
Fever virus infectiousness is extremely strong, and canine and some other carnivore illness can be caused even dead.Currently, canine distemper virus is anti-
The detection of body is related to the detection of immune effect after canine distemper vaccine inoculation more, and there are many methods, such as colloidal gold immunochromatographimethod
Method, ELISA method etc..It is always one of emphasis and problem of detection for the detection of canine antiviral antibody.
Time-resolved fluoroimmunoassay (Time-resolved Fluorescence Immunoassay,
TRFIA) be a kind of novel external ultramicro-analysis quantitative technique, it using the lanthanide series with unique fluorescent characteristic and its
Chelate is tracer, the fluorescence intensity in reaction product is measured with time-resolved fluoroimmunoassay detector, according to product
The ratio of fluorescence intensity and relative intensity of fluorescence, judges the concentration of analyte in reaction system, to reach quantitative analysis.It gram
Unstable, the chemiluminescence for having taken enzyme marker are only capable of once shining and vulnerable to environmental disturbances, the indirect mark of electrochemical luminescence
The disadvantages of note, makes non-specific signals be reduced to negligible degree, has reached high signal-to-noise ratio, thus considerably more than
The attainable sensitivity of radioactive isotope institute, and also have that marker preparation is easy, storage time is long, "dead" dirt
Contaminate, detect reproducible, operating process is short, standard curve range is wide, not by sample natural fluorescence interference and application range very
The advantages that extensive, becomes a new milestone of on-radiation immunoassay development after the nineties.
The reaction pattern of dual-antigen sandwich method is similar with double antibody sandwich method, is coated with and is prepared with specific antigen and is anti-
Former marker, to detect corresponding antibody.It is not required to dilute by inspection sample in this method, can directly measured, therefore its susceptibility is opposite
Higher than indirect method antibody test.
Therefore, it is necessary to design a kind of novel double antigens sandwich detection kit, which will the time point
It distinguishes in the detection applied to canine distemper virus antibody, keeps the sensitivity of detection and accuracy higher.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and provide a kind of for detecting canine distemper virus antibody
Time-resolved fluoroimmunoassay kit, detection sensitivity and accuracy it is higher.
To achieve the above object, the technical scheme adopted by the invention is as follows：It is a kind of for detect canine distemper virus antibody when
Between resolved fluorometric immune reagent kit comprising be coated with canine distemper virus recombinant antigen elisa plate, europium label hundstaupe pyreticosis
Malicious recombinant antigen solution, analysis buffer, cleaning solution and enhancement solution；The canine distemper virus recombinant antigen and canine distemper virus are anti-
Physical efficiency specific binding；
The elisa plate for being coated with canine distemper virus recombinant antigen is through the following steps that prepared：
Canine distemper virus recombinant antigen is to pass through one section of flexible peptide linker by H protein and the F protein column of canine distemper virus
(GGGGS)nThe recombinant protein of eukaryotic expression is connected and carries out, G is expressed as glycine, and S is expressed as serine, n >=1；First use
The canine distemper virus recombinant antigen is diluted to 1~5 μ g/ml by the carbonate buffer solution of 50mmol/L, pH 9.6, is then added
Coating buffer, 4 DEG C overnight after discard coating buffer；Confining liquid is added, confining liquid is discarded after closing overnight, is patted dry after washing, vacuum-
20 DEG C of freezen protectives.
Kit of the present invention includes the canine distemper virus weight of the elisa plate for being coated with canine distemper virus recombinant antigen, europium label
Group antigenic solution, analysis buffer, cleaning solution and enhancement solution, it is anti-in the solid phase carrier combination sample of antigen by being coated with
Body forms the compound of Ag-Ab；Antigen of the antigen-antibody complex again with europium label is combined, and it is anti-to form antigen-
Body-antigen-europium labeled complex；Finally by fluorescence detection, the content of calculating antibody；The present invention adjusts the canine distemper of europium label
The content and pH value of various components in virus recombinant antigen solution, the canine distemper virus recombinant antigen and antibody for marking europium
It sufficiently combines, is conducive to the sensitivity for improving detection.
As an improvement of the above technical solution, europium label canine distemper virus recombinant antigen through the following steps that into
Row preparation：
S1) canine distemper virus recombinant antigen is placed in the centrifuge tube with filter membrane, then label is added into centrifuge tube and delays
Fliud flushing is collected by centrifugation；
S2 europium labelled reagent) is added in the solution collected to step S1), room temperature concussion is overnight to get europium label after mixing
Canine distemper virus recombinant antigen solution crude product；
S3) the canine distemper virus recombinant antigen solution crude product upper prop for marking the europium, is eluted with eluent, purifying
Afterwards up to the canine distemper virus recombinant antigen solution of europium label.
As a further improvement of the above technical scheme, the label buffer is 20mM NaH2PO4Solution, the label
The pH value of buffer is 8.4.
As a further improvement of the above technical scheme, the eluent is slow for the 50mM Tris-HCl containing 1.2%KCl
Fliud flushing.
The present invention is defined the canine distemper virus recombinant antigen solution that europium marks：Mark the component and pH in buffer
It is worth, the component and pH value in eluent；So that antigen is preferably marked europium, reaches fluorescence volume maximum value.
As an improvement of the above technical solution, the coating buffer is containing 0.6%BSA and 1.3%NaN350mM carbonate
Buffer, the pH value of the coating buffer are 9.6.
As an improvement of the above technical solution, the confining liquid is containing 2%~4%BSA, 1.1%Tween-40 and 0.5%
NaN350mM phosphate buffer, the pH value of the confining liquid is 7.4.
As an improvement of the above technical solution, the analysis buffer is the Tris- that containing 50mmol/L and pH value is 7.8
HCl, 9g/L NaCl, 0.02%BSA (w/v), 0.05%Tween-20 (v/v) and 0.05%NaN3(w/v), surplus is water, point
The pH value for analysing buffer is 7.8~8.3；Cleaning solution is the Tris-HCl buffer of the 50mmol/L containing 0.9%NaCl, cleaning solution
PH value be 7.5 (according to 1：25 times of dilutions use)；Enhancement solution is the buffer system that glacial acetic acid and Potassium Hydrogen Phthalate form,
And contain β-NTA (betanaphthyl formyl trichloroacetone), TOPO (positive trioctylphosphine oxide (TOPO)) and Triton x-100.
The beneficial effects of the present invention are：It is glimmering that the present invention provides a kind of time resolution for detecting canine distemper virus antibody
Light immune reagent kit, kit of the present invention include the canine distemper of the elisa plate for being coated with canine distemper virus recombinant antigen, europium label
Virus recombinant antigen solution, analysis buffer, cleaning solution and enhancement solution, the canine distemper virus antigen that the present invention adjusts europium label are molten
The content and pH value of various components in liquid and analysis buffer, make test antibodies preferably combine be coated with antigen elisa plate and
The antigen of europium label is conducive to the sensitivity and accuracy that improve detection.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
The present embodiment provides a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody, the reagent
Box includes the elisa plate for being coated with canine distemper virus recombinant antigen, the canine distemper virus recombinant antigen solution of europium label, analyzes and delay
Fliud flushing, cleaning solution and enhancement solution；Canine distemper virus recombinant antigen can be specifically bound with canine distemper virus antibody；
The elisa plate of canine distemper virus recombinant antigen is coated with through the following steps that being prepared：
Canine distemper virus recombinant antigen is to pass through one section of flexible peptide linker by H protein and the F protein column of canine distemper virus
(GGGGS)nThe recombinant protein of eukaryotic expression is connected and carries out, G is expressed as glycine, and S is expressed as, serine, n >=1；First use
Above-mentioned canine distemper virus recombinant antigen is diluted to 1 μ g/ml by the carbonate buffer solution of 50mmol/L, pH 9.6, then with 150 μ
The hole l/ is added coating buffer into elisa plate, 4 DEG C overnight after discard coating buffer；Closing is added into elisa plate with 200 holes μ l/ again
Liquid discards confining liquid liquid after closing overnight, pats dry after washing, -20 DEG C of freezen protectives of vacuum；
The antigenic solution of the label of europium described in the present embodiment is through the following steps that prepared：
S1) canine distemper virus recombinant antigen is placed in the centrifuge tube with filter membrane, then label is added into centrifuge tube and delays
Fliud flushing is collected by centrifugation；
S2 europium labelled reagent) is added in the solution collected to step S1), room temperature concussion is overnight to get europium label after mixing
Canine distemper virus recombinant antigen solution crude product；
S3) the canine distemper virus recombinant antigen solution crude product upper prop for marking the europium, is eluted with eluent, purifying
Afterwards up to the canine distemper virus recombinant antigen solution of europium label；
It is 20mM NaH that label buffer, which is label buffer, in the present embodiment2PO4Solution, marks the pH value of buffer to be
8.4；Eluent is the 50mM Tris-HCl buffer containing 1.2%KCl；Coating buffer is containing 0.6%BSA and 1.3%NaN3's
50mM carbonate buffer solution, the pH value of coating buffer are 9.6；Confining liquid is containing 2%BSA, 1.1%Tween-40 and 0.5%NaN3
50mM phosphate buffer, the pH value of confining liquid is 7.4；Analysis buffer containing 50mol/L and pH value be 7.8Tris-HCl,
9g/L NaCl, 0.02%BSA (w/v), 0.05%Tween-20 (v/v) and 0.05%NaN3(w/v), surplus is water, and analysis is slow
The pH value of fliud flushing is 7.8；Cleaning solution is the Tris-HCl buffer of the 50mmol/L containing 0.9%NaCl, and the pH value of cleaning solution is
7.5 (according to 1：25 times of dilutions use)；Enhancement solution is the buffer system that glacial acetic acid and Potassium Hydrogen Phthalate are constituted, and contains β-
NTA, TOPO and Triton x-100.
Embodiment 2
The present embodiment provides a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody, the reagent
Box includes the elisa plate for being coated with canine distemper virus recombinant antigen, the canine distemper virus recombinant antigen solution of europium label, analyzes and delay
Fliud flushing, cleaning solution and enhancement solution；Canine distemper virus recombinant antigen can be specifically bound with canine distemper virus antibody；
The elisa plate of canine distemper virus recombinant antigen is coated with through the following steps that being prepared：
Canine distemper virus recombinant antigen is to pass through one section of flexible peptide linker by H protein and the F protein column of canine distemper virus
(GGGGS)nThe recombinant protein of eukaryotic expression is connected and carries out, G is expressed as glycine, and S is expressed as, serine, n >=1；First use
Above-mentioned canine distemper virus recombinant antigen is diluted to 3 μ g/ml by the carbonate buffer solution of 50mmol/L, pH 9.6, then with 150 μ
The hole l/ is added coating buffer into elisa plate, 4 DEG C overnight after discard coating buffer；Closing is added into elisa plate with 200 holes μ l/ again
Liquid discards confining liquid liquid after closing overnight, pats dry after washing, -20 DEG C of freezen protectives of vacuum；
The antigenic solution of the label of europium described in the present embodiment is through the following steps that prepared：
S1) canine distemper virus recombinant antigen is placed in the centrifuge tube with filter membrane, then label is added into centrifuge tube and delays
Fliud flushing is collected by centrifugation；
S2 europium labelled reagent) is added in the solution collected to step S1), room temperature concussion is overnight to get europium label after mixing
Canine distemper virus recombinant antigen solution crude product；
S3) the canine distemper virus recombinant antigen solution crude product upper prop for marking the europium, is eluted with eluent, purifying
Afterwards up to the canine distemper virus recombinant antigen solution of europium label；
It is 20mM NaH that label buffer, which is label buffer, in the present embodiment2PO4Solution, marks the pH value of buffer to be
8.4；Eluent is the 50mM Tris-HCl buffer containing 1.2%KCl；Coating buffer is containing 0.6%BSA and 1.3%NaN3's
50mM carbonate buffer solution, the pH value of coating buffer are 9.6；Confining liquid is containing 3%BSA, 1.1%Tween-40 and 0.5%NaN3
50mM phosphate buffer, the pH value of confining liquid is 7.4；Analysis buffer containing 50mol/L and pH value be 7.8Tris-HCl,
9g/L NaCl, 0.02%BSA (w/v), 0.05%Tween-20 (v/v) and 0.05%NaN3(w/v), surplus is water, and analysis is slow
The pH value of fliud flushing is 8.1；Cleaning solution is the Tris-HCl buffer of the 50mmol/L containing 0.9%NaCl, and the pH value of cleaning solution is
7.5 (according to 1：25 times of dilutions use)；Enhancement solution is the buffer system that glacial acetic acid and Potassium Hydrogen Phthalate are constituted, and contains β-
NTA, TOPO and Triton x-100.
Embodiment 3
The present embodiment provides a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody, the reagent
Box includes the elisa plate for being coated with canine distemper virus recombinant antigen, the canine distemper virus recombinant antigen solution of europium label, analyzes and delay
Fliud flushing, cleaning solution and enhancement solution；Canine distemper virus recombinant antigen can be specifically bound with canine distemper virus antibody；
The elisa plate of canine distemper virus recombinant antigen is coated with through the following steps that being prepared：
Canine distemper virus recombinant antigen is to pass through one section of flexible peptide linker by H protein and the F protein column of canine distemper virus
(GGGGS)nThe recombinant protein of eukaryotic expression is connected and carries out, G is expressed as glycine, and S is expressed as, serine, n >=1；First use
Above-mentioned canine distemper virus recombinant antigen is diluted to 5 μ g/ml by the carbonate buffer solution of 50mmol/L, pH 9.6, then with 150 μ
The hole l/ is added coating buffer into elisa plate, 4 DEG C overnight after discard coating buffer；Closing is added into elisa plate with 200 holes μ l/ again
Liquid discards confining liquid liquid after closing overnight, pats dry after washing, -20 DEG C of freezen protectives of vacuum；
The antigenic solution of the label of europium described in the present embodiment is through the following steps that prepared：
S1) canine distemper virus recombinant antigen is placed in the centrifuge tube with filter membrane, then label is added into centrifuge tube and delays
Fliud flushing is collected by centrifugation；
S2 europium labelled reagent) is added in the solution collected to step S1), room temperature concussion is overnight to get europium label after mixing
Canine distemper virus recombinant antigen solution crude product；
S3) the canine distemper virus recombinant antigen solution crude product upper prop for marking the europium, is eluted with eluent, purifying
Afterwards up to the canine distemper virus recombinant antigen solution of europium label；
It is 20mM NaH that label buffer, which is label buffer, in the present embodiment2PO4Solution, marks the pH value of buffer to be
8.4；Eluent is the 50mM Tris-HCl buffer containing 1.2%KCl；Coating buffer is containing 0.6%BSA and 1.3%NaN3's
50mM carbonate buffer solution, the pH value of coating buffer are 9.6；Confining liquid is containing 4%BSA, 1.1%Tween-40 and 0.5%NaN3
50mM phosphate buffer, the pH value of confining liquid is 7.4；Analysis buffer containing 50mol/L and pH value be 7.8Tris-HCl,
9g/L NaCl, 0.02%BSA (w/v), 0.05%Tween-20 (v/v) and 0.05%NaN3(w/v), surplus is water, and analysis is slow
The pH value of fliud flushing is 8.3；Cleaning solution is the Tris-HCl buffer of the 50mmol/L containing 0.9%NaCl, and the pH value of cleaning solution is
7.5 (according to 1：25 times of dilutions use)；Enhancement solution is the buffer system that glacial acetic acid and Potassium Hydrogen Phthalate are constituted, and contains β-
NTA, TOPO and Triton x-100
Embodiment 4
The present embodiment provides a kind of for detecting the method and step of canine distemper virus antibody, and the kit is embodiment 1
Kit described in~3 comprising following steps：
1) 25 μ L samples to be tested or standard items are sequentially added into the elisa plate being coated with, and add 200 μ L analysis buffering
Liquid carries out incubation at room temperature after mixing, form compound；
2) supernatant is removed, washing removes unbonded substance；
3) it is slow to add 100 μ L analysis for the canine distemper virus antigenic solution that 150 μ L europiums label is continuously added into ELISA Plate
Fliud flushing is incubated after mixing；
4) supernatant is removed, washing removes unbonded substance；
5) 200 μ L enhancement solutions are added, measures fluorescence intensity on time resolution instrument, antibody content is calculated.
Embodiment 5Linear relationship is investigated
Canine distemper virus antibody standard substance is diluted to 0,1,10,50,100,500,1000ng/ml system with sample diluting liquid
Column reference standard solution calculates linear relationship；As a result, it has been found that the related coefficient of standard curve is R2=0.9995, it is linearly related
Property is good；The detection sensitivity of antibody is 1.2ng/ml.
Embodiment 6Precision is investigated
It is measured using high, normal, basic three standard concentration of the kit of the present invention to accurate quantification, each setting 10 is multiple
Hole；≤ 10%, measurement result is shown in Table 1 to the variation within batch coefficient and interassay coefficient of variation of this kit.
Table 1
As described in Table 1, the precision of kit of the present invention is good.
Embodiment 7The rate of recovery is investigated
The measurement of the rate of recovery, measurement are carried out to high, normal, basic three standard concentrations of accurate quantification using kit of the present invention
It the results are shown in Table 2.
Table 2
As shown in table 2, for the rate of recovery of high, normal, basic three concentration between 96.6%~101.06%, rate of recovery effect is good
It is good.
Embodiment 8
Antibody samples to be measured are measured.Serum sample 200 by canine distemper vaccine inoculation of selection, without connecing
Dog negative serum 10 for planting and being uninfected by canine distemper.Canine distemper in serum is detected with experiment using in the serum of gradient dilution
Antibody titer, while being detected using kit of the present invention.Testing result discovery：200 positive samples are illustrated as the positive,
10 are negative and are illustrated as feminine gender.And the yin and yang attribute critical point of this kit is determined according to this:Sample to be tested antibody concentration >=
3.5ng/ml, for the positive；Sample to be tested antibody concentration≤3.5ng/ml, for feminine gender.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
Claims (7)
1. a kind of for detecting the time-resolved fluoroimmunoassay kit of canine distemper virus antibody, which is characterized in that including coating
There are the elisa plate of canine distemper virus recombinant antigen, the canine distemper virus recombinant antigen solution of europium label, analysis buffer, cleaning solution
And enhancement solution；The canine distemper virus recombinant antigen can be specifically bound with canine distemper virus antibody；
The elisa plate for being coated with canine distemper virus recombinant antigen is through the following steps that prepared：
Canine distemper virus recombinant antigen is to pass through one section of flexible peptide linker (GGGGS) by H protein and the F protein column of canine distemper virusn
The recombinant protein of eukaryotic expression is connected and carries out, G is expressed as glycine, and S is expressed as serine, n >=1；First use 50mmol/L, pH
The canine distemper virus recombinant antigen is diluted to 1~5 μ g/ml by 9.6 carbonate buffer solution, and coating buffer, 4 DEG C of mistakes are then added
Coating buffer is discarded after night；Confining liquid is added, confining liquid is discarded after closing overnight, is patted dry after washing, -20 DEG C of freezings of vacuum are protected
It deposits.
2. time-resolved fluoroimmunoassay kit as described in claim 1, which is characterized in that the hundstaupe pyreticosis of the europium label
Malicious recombinant antigen is through the following steps that prepared：
S1) canine distemper virus recombinant antigen is placed in the centrifuge tube with filter membrane, then label buffer is added into centrifuge tube,
It is collected by centrifugation；
S2 europium labelled reagent) is added in the solution collected to step S1), the dog that room temperature concussion marks overnight to get europium after mixing
Distemper virus recombinant antigen solution crude product；
S3) the canine distemper virus recombinant antigen solution crude product upper prop for marking the europium, is eluted with eluent, is after purification
Obtain the canine distemper virus recombinant antigen solution of europium label.
3. time-resolved fluoroimmunoassay kit as claimed in claim 2, which is characterized in that the label buffer is 20mM
NaH2PO4The pH value of solution, the label buffer is 8.4.
4. time-resolved fluoroimmunoassay kit as claimed in claim 2, which is characterized in that the eluent is containing 1.2%
The 50mM Tris-HCl buffer of KCl.
5. time-resolved fluoroimmunoassay kit as described in claim 1, which is characterized in that the coating buffer is containing 0.6%
BSA and 1.3%NaN350mM carbonate buffer solution, the pH value of the coating buffer is 9.6.
6. time-resolved fluoroimmunoassay kit as described in claim 1, which is characterized in that the confining liquid be containing 2%~
4%BSA, 1.1%Tween-40 and 0.5%NaN350mM phosphate buffer, the pH value of the confining liquid is 7.4.
7. time-resolved fluoroimmunoassay kit as described in claim 1, which is characterized in that the analysis buffer be containing
Tris-HCl, 9g/L NaCl, 0.02%BSA, 0.05%Tween-20 and 0.05%NaN of 50mmol/L and pH value for 7.83,
Surplus is water, and the pH value of analysis buffer is 7.8~8.3；The Tris-HCl that cleaning solution is the 50mmol/L containing 0.9%NaCl is slow
Fliud flushing, the pH value of cleaning solution are 7.5；The enhancement solution is the buffer system that glacial acetic acid and Potassium Hydrogen Phthalate are constituted, and is contained
There are β-NTA, TOPO and Triton x-100.
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Cited By (3)
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| CN109342725A (en) * | 2018-11-29 | 2019-02-15 | 广州优迪生物科技股份有限公司 | A kind of canine distemper, canine parvovirus detection kit |
| CN110568204A (en) * | 2019-06-10 | 2019-12-13 | 青岛海润检测股份有限公司 | Chemiluminescence kit for one-step quantitative detection of canine distemper virus-canine parvovirus antibody |
| CN113009133A (en) * | 2021-03-05 | 2021-06-22 | 上海市农业科学院 | Detection method of canine parvovirus |
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