CN110261615A - Cystic echinococcosis diagnostic kit and its application - Google Patents
Cystic echinococcosis diagnostic kit and its application Download PDFInfo
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- G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
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Abstract
The invention discloses cystic echinococcosis diagnostic kit and its applications.The kit includes envelope antigen, and the envelope antigen is made of eB2-3 conjugate and/or eB3-3 conjugate；The eB2-3 conjugate is the comlete antigen obtained by eB2-3 and carrier protein couplet；The eB3-3 conjugate is the comlete antigen obtained by eB3-3 and carrier protein couplet；Polypeptide shown in the eB2-3 sequence 1, the eVP1-2 are polypeptides shown in sequence 2.Kit specificity is high, sensibility is high, and accuracy is high, easy to operate, quick, suitable for base's Veterinary office at different levels and Entry-Exit Inspection and Quarantine Bureau to the rapid, high volume selective mechanisms of Echinococcus granulosus serum antibody.
Description
Technical field
The present invention relates to cystic echinococcosis diagnostic kit and its applications.
Background technique
Echinococcosis is the abbreviation of hydatidosis, is the parasitized larvae by echinococcus in people beast caused in people or cattle and sheep body
Suffer from parasitic disease altogether.Echinococcosis is mainly propagated by canids such as dog and foxes, and echinococcosis granulosa, Echinococcus moltilocularis are divided into
Disease saves hydatidosis and Fu Shi hydatidosis less.China is primarily present the bladder type Echinococcus hydatid cyst as caused by the larva of Echinococcus granulosus
The larva of sick (echinococcosis granulosa) and Echinococcus multilocularis causes alveolar echinococcosis (echinococcosis multilocularis), echinococcosis multilocularis
Harm is serious, and lethality is high, and be otherwise known as " worm cancer ".
Echinococcus granulosus (Echinococcus granulosus) also known as wraps raw tapeworm.Adult parasitizes Canidae food meat
Animal, and larva (echinococcus) main parasitic causes a kind of serious Amphixenosis in the plant-eating animals such as people and Niu, sheep,
Referred to as echinococcosis granulosa.
Domestic and foreign scholars have done a large amount of research to the advantage diagnostic antigen of screening Echinococcus Granulosus Cysts, and main research is burnt at present
Point is antigen B (AgB).Antigen B is the heat-resisting lipoprotein that one of echinococcosis cyst fluid molecular weight is 120~160kDa, is had
High degree of immunogenicity, and be considered as currently used in Immunodiagnostic Assays have compared with hypersensitivity and specificity antigen it
One.The polymer that AgB is made of the subunit of several about 8kDa, immunoblotting show that its minimum subunit (8kDa) is most to examine
The target antigen of disconnected value.Encoding AgB gene is a gene family, contains AgB1, AgB2, AgB3, AgB4 gene.
River jasmine etc. utilizes pET32a carrier, pET28a carrier and pGEX4T-1 carrier, expression China's Echinococcus granulosus point
From two subunit genes of AgB1 and AgB2 cloned in strain (Xinjiang source and Gan Suyuan), the result shows that AgB1 is in pET28a carrier
In cannot express, and can be expressed in pGEX4T-1 and pET32a, but it is insoluble for expressing albumen.AgB2 is in 3 kinds of carriers
It can express, express the dissolubility of albumen successively are as follows: insoluble-part is solvable-solvable.
It, need to be by laboratory since echinococcosis granulosa is with having similar clinical symptoms with echinococcosis multilocularis
Detection technique makes a definite diagnosis the disease.
Summary of the invention
A technical problem to be solved by this invention be how high specific and high sensitivity detection Echinococcus Granulosus Cysts sense
Antibody is contaminated more accurately to diagnose echinococcosis granulosa.
In order to solve the above technical problems, the present invention provides echinococcosis granulosa diagnostic kit or detection Echinococcus Granulosus Cysts
The kit of antibody (Echinococcus granulosus antibody or echinococcosis granulosa antibody).
Echinococcosis granulosa diagnostic kit provided by the present invention or detection Echinococcus Granulosus Cysts antibody (Echinococcus Granulosus Cysts sense
Contaminate antibody or echinococcosis granulosa antibody) kit, include envelope antigen, the envelope antigen by eB2-3 conjugate and/
Or eB3-3 conjugate composition；The eB2-3 conjugate is the comlete antigen obtained by eB2-3 and carrier protein couplet；It is described
EB3-3 conjugate is the comlete antigen obtained by eB3-3 and carrier protein couplet；The eB2-3 is the more of P11, P12 or P13
Peptide:
The eB2-3 is the polypeptide of P11, P12 or P13:
P11, the polypeptide that amino acid sequence is sequence 1 in sequence table,
P12, the 2-17 polypeptides that amino acid sequence is sequence 1 in sequence table,
P13, P12 polypeptide amino terminal or carboxyl terminal connection amino acid residue to be obtained with carrier protein couplet
Polypeptide；
The eB3-3 is the polypeptide of P21, P22 or P23:
P21, the polypeptide that amino acid sequence is sequence 2 in sequence table,
P22, the 2-14 polypeptides that amino acid sequence is sequence 2 in sequence table,
P23, P22 polypeptide amino terminal or carboxyl terminal connection amino acid residue to be obtained with carrier protein couplet
Polypeptide.
In mentioned reagent box, sequence 1 is made of 17 amino acid residues in sequence table, and the 1st cysteine residues are
In order to be connect with carrier protein, in the linking arm of the amino terminal addition of the antigen epitope polypeptide of the AgB2 of Echinococcus Granulosus Cysts, the
The 2-17 AgB2 from Echinococcus Granulosus Cysts；Sequence 2 is made of 14 amino acid residues in sequence table, the 1st cysteine
Residue is in order to connect with carrier protein, in the connection of the amino terminal addition of the antigen epitope polypeptide of the AgB3 of Echinococcus Granulosus Cysts
Arm, the 2-14 AgB3 from Echinococcus Granulosus Cysts.
In mentioned reagent box, in the envelope antigen, the mass ratio of the eB2-3 conjugate and the eB3-3 conjugate
Those skilled in the art can determine according to Echinococcus Granulosus Cysts antibody test effect, such as can be 4:6.
In mentioned reagent box, the kit can be the time-resolved fluoroimmunoassay reagent of diagnosis echinococcosis granulosa
Box or the time-resolved fluorescence for detecting Echinococcus Granulosus Cysts antibody (Echinococcus granulosus antibody or echinococcosis granulosa antibody) are exempted from
Epidemic disease assay kit.
In mentioned reagent box, the kit further includes other reagents needed for carrying out time-resolved fluoroimmunoassay,
The secondary antibody marked such as rare earth element.
In mentioned reagent box, the rare earth element can be europium (Eu3+), dysprosium (De3+), terbium (Te3+), samarium (Sm3+).
In mentioned reagent box, the kit can also be thin for the enzyme linked immunological kit of diagnosis echinococcosis granulosa or detection
The enzyme linked immunological kit of grain echinococcus antibody.
In mentioned reagent box, the kit can further include other reagents needed for carrying out ELISA, such as enzyme mark
Secondary antibody.
In order to solve the above technical problems, the present invention also provides echinococcosis granulosa diagnosis test paper or detection Echinococcus Granulosus Cysts
The test paper of antibody.
Echinococcosis granulosa diagnosis test paper or detection Echinococcus Granulosus Cysts antibody (Echinococcus granulosus provided by the present invention
Antibody or echinococcosis granulosa antibody) test paper include envelope antigen, the envelope antigen by above-mentioned eB2-3 conjugate and/
Or above-mentioned eB3-3 conjugate composition.
In above-mentioned test paper, in the envelope antigen, the mass ratio sheet of the eB2-3 conjugate and the eB3-3 conjugate
Field technical staff can determine according to Echinococcus Granulosus Cysts antibody test effect, such as can be 4:6.
The application of above-mentioned envelope antigen, complete polypeptide or independent polypeptide in reagent preparation box also belongs to protection of the invention
Range.The kit is that (Echinococcus granulosus is anti-for echinococcosis granulosa diagnostic kit or detection Echinococcus Granulosus Cysts antibody
Body or echinococcosis granulosa antibody) kit；The complete polypeptide is made of above-mentioned eB2-3 and above-mentioned eB3-3.It is described independent
Polypeptide is above-mentioned eB2-3 or above-mentioned eB3-3.
Above, the quality of eB2-3 described in the complete polypeptide and eB3-3 can basis than those skilled in the art
Echinococcus Granulosus Cysts antibody test effect is determined, such as can be 4:6.
In above-mentioned application, the kit can be the time-resolved fluoroimmunoassay kit of diagnosis echinococcosis granulosa
Or the time-resolved fluoroimmunoassay kit of detection Echinococcus Granulosus Cysts antibody, the kit includes envelope antigen, described
Envelope antigen is made of the eB2-3 conjugate and/or the eB3-3 conjugate.
In above-mentioned application, the kit can be enzyme linked immunological kit or the detection particulate spine of diagnosis echinococcosis granulosa
The enzyme linked immunological kit of ball larva of a tapeworm or the cercaria of a schistosome antibody (Echinococcus granulosus antibody or echinococcosis granulosa antibody), the kit include
Above-mentioned envelope antigen.
Above-mentioned envelope antigen, above-mentioned complete polypeptide or above-mentioned independent polypeptide also belong to the present invention in the application prepared in test paper
Protection scope.
In above-mentioned application, the test paper can diagnose test paper or detection Echinococcus Granulosus Cysts antibody (particulate for echinococcosis granulosa
Echinococcus infects antibody or echinococcosis granulosa antibody) test paper.
In above-mentioned application, the test paper includes envelope antigen, and the envelope antigen is by the eB2-3 conjugate and/or institute
State eB3-3 conjugate composition.
Mentioned reagent box or above-mentioned test paper also belong to the present invention in the application prepared in echinococcosis granulosa monitoring reagent box
Protection scope.
It is demonstrated experimentally that in the detection serum for using BSA-eB2-3 and/or BSA-eB3-3 to prepare as envelope antigen respectively
The kit of Echinococcus Granulosus Cysts antibody is to sheep echinococcosis granulosa negative serum, sheep echinococcosis multilocularis positive serum and sheep capsule tail
The equal no cross reaction of larva of a tapeworm or the cercaria of a schistosome disease positive serum, the TRFIA method 1 of the present invention using BSA-eB2-3 and BSA-eB3-3 as envelope antigen
Total coincidence rate with clinical diagnosis is 91.67% (positive coincidence rate 91.11%, negative match-rate 92.22%), with BSA-
EB2-3 is as total coincidence rate of the TRFIA method 2 of the present invention and sheep Echinococcus Granulosus Cysts serum neutralization test method of envelope antigen
89.44% (positive coincidence rate 84.44%, negative match-rate 94.44%), using BSA-eB3-3 as the sheet of envelope antigen
(positive coincidence rate is total coincidence rate of invention TRFIA method 3 and sheep Echinococcus Granulosus Cysts serum neutralization test method for 87.22%
81.11%, negative match-rate 93.33%).
The antibody assay kit specificity that the present invention is prepared using BSA-eB2-3 and/or BSA-eB3-3 as coating antigen
By force, sensibility is high, and accuracy is high, easy to operate, quick, is suitable for base's Veterinary office at different levels and Entry-Exit Inspection and Quarantine Bureau pair
The rapid, high volume selective mechanisms of Echinococcus granulosus serum antibody.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Examples provided below can be used as the art ordinary skill
The guide that personnel are further improved, is not construed as limiting the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Material as used in the following examples
Material, reagent etc., are commercially available unless otherwise specified.
Europium labelled element (Eu3+) in following embodiments is Guangzhou Da Rui Biotechnology Ltd. product, rabbit-anti
Goat secondary antibody is Sigma Products, and 1235 time-resolved fluorescence detector of Auto-DELFIA is purchased from PerkinElmer share
Co., Ltd, ELISA Plate are purchased from U.S. Costar company.
In following embodiments, clinical diagnosis echinococcosis granulosa positive serum, echinococcosis multilocularis positive serum, particulate spine
The method of ball larva of a tapeworm or the cercaria of a schistosome disease negative serum and echinococcosis multilocularis negative serum is as follows: slaughterhouse dissection is daily to butcher sheep only, finds trunk
Body organ has the sheep of echinococcosis granulosa packing lesion (bladder type echinococcus lesion) to be only determined as infecting the sheep of echinococcosis granulosa
Only, the serum of the sheep is diagnosed as echinococcosis granulosa positive serum, is on the contrary negative serum；It was found that trunk organ has more room spine balls
The sheep of larva of a tapeworm or the cercaria of a schistosome disease packing lesion (alveolitoid echinococcus lesion) is only determined as infecting the sheep of echinococcosis multilocularis, and the serum of the sheep is made a definite diagnosis
It is on the contrary negative serum for echinococcosis multilocularis positive serum；It was found that trunk organ has the sheep of cysticercus lesion to be only determined as feeling
The sheep of cysticercosis is contaminated, the serum of the sheep is diagnosed as cysticercus positive serum, is on the contrary negative serum.
Embodiment 1, time-resolved fluoroimmunoassay (TRFIA) detect Echinococcus granulosus antibody
The present inventor is in R&D process, it is intended to according to following document method using pET32a (+), pET28a (+) and
AgB1, AgB2, AgB3, AgB4 of pGEX4T-1 expression Echinococcus Granulosus Cysts in e. coli bl21 (DE3), do not give expression to
Destination protein.Inventor has respectively selected 4 dominant antigen epitope polypeptides from AgB1, AgB2, AgB3, AgB4 of Echinococcus Granulosus Cysts
(eB1-1, eB1-2, eB1-3, eB2-1, eB2-2, eB2-3, eB3-1, eB3-2, eB3-3, eB4-1, eB4-2 and eB4-3), point
Time-resolved fluorescence immunoassay method is not established as envelope antigen with after BSA coupling, specificity is significantly improved,
Echinococcus Granulosus Cysts positive serum and Echinococcus moltilocularis positive serum, cysticercus positive serum can effectively be distinguished.By eB2-3 with
The conjugate (BSA-eB2-3) of BSA and the conjugate (BSA-eB3-3) of eB3-3 and BSA are mixed to get according to the mass ratio of 4:6
The time-resolved fluorescence immunoassay method established as envelope antigen of mixing comlete antigen, specificity obtained significantly mentioning
Height can effectively distinguish Echinococcus Granulosus Cysts positive serum and Echinococcus moltilocularis positive serum, cysticercus positive serum, sensibility
It significantly improves.Specific experimental method is as follows:
1 includes the preparation of antigen
The present embodiment is prepared for following 13 kinds of envelope antigens: 1) BSA-eB2-3+BSA-eB3-3, and 2) BSA-eB2-3 (eB2-
3 conjugates), 3) BSA-eB3-3 (eB3-3 conjugate), 4) BSA-eB1-1 (eB1-1 conjugate), 5) BSA-eB1-2 (eB1-2
Conjugate), 6) BSA-eB1-3 (eB1-3 conjugate), 7) BSA-eB2-1 (eB2-1 conjugate), 8) (eB2-2 is even by BSA-eB2-2
Join object), 9) BSA-eB3-1 (eB3-1 conjugate), 10) BSA-eB3-2 (eB3-2 conjugate), 11) (eB4-1 is even by BSA-eB4-1
Join object), 12) BSA-eB4-2 (eB4-2 conjugate), 13) BSA-eB4-3 (eB4-3 conjugate).
1.1 dominant antigen epitope polypeptides
The selective advantage antigen epitope polypeptide from AgB1, AgB2, AgB3, AgB4 albumen of Echinococcus Granulosus Cysts, by Beijing six
Close Hua Da Gene Tech. Company Limited synthesis C-terminal or N-terminal be connected with polypeptide eB1-1, eB1-2 of cysteine, eB1-3,
EB2-1, eB2-2, eB2-3, eB3-1, eB3-2, eB3-3, eB4-1, eB4-2 and eB4-3 (table 1).Purity is greater than 95%, freeze-drying
It saves.
1. polypeptide of table
| Title | Sequence |
| eB1-1 | DDGLTSTSRSVMKMFG*C |
| eB1-2 | C*KYFFERDPLGQKVV |
| eB1-3 | C*EEVFQLLRKKLRMA |
| eB2-1 | KDEPKAHMGQV*C |
| eB2-2 | C*ELRDFFRNDPLRQR |
| eB2-3 | C*KKYVKNLVEEKDDDSK |
| eB3-1 | DDDEVTKTKKGVMKAIS*C |
| eB3-2 | C*KHFFQSDPLGKKLV |
| eB3-3 | C*LKEYVRKLVKEDE |
| eB4-1 | C*KAEPERCKCLIMRKLG |
| eB4-2 | C*IRDFFRSDPLGQKL |
| eB4-3 | C*KYVKDLLEEEDEDDLK |
Note: * C or C* in sequence are cysteine residues, are in order to connect with carrier protein, in Echinococcus Granulosus Cysts
The linking arm of amino terminal or the carboxyl terminal addition of the antigen epitope polypeptide of AgB；Other amino acid residues derive from particulate spine
The antigen of the AgB of the ball larva of a tapeworm or the cercaria of a schistosome.
The preparation of 1.2 13 kinds of envelope antigens
By eB1-1, eB1-2, eB1-3, eB2-1, eB2-2, eB2-3, eB3-1, eB3-2, eB3-3, eB4-1, eB4-2 and
This 12 kinds of polypeptides of eB4-3 are coupled to obtain 12 kinds of envelope antigens respectively with BSA: 2) BSA-eB2-3 (conjugate of BSA and eB2-3),
3) BSA-eB3-3 (conjugate of BSA and eB3-3), 4) BSA-eB1-1 (conjugate of BSA and eB1-1), 5) BSA-eB1-2
(conjugate of BSA and eB1-2), 6) BSA-eB1-3 (conjugate of BSA and eB1-3), 7) BSA-eB2-1 (BSA and eB2-1's
Conjugate), 8) BSA-eB2-2 (conjugate of BSA and eB2-2), 9) BSA-eB3-1 (conjugate of BSA and eB3-1), 10)
BSA-eB3-2 (conjugate of BSA and eB3-2), 11) BSA-eB4-1 (conjugate of BSA and eB4-1), 12) BSA-eB4-2
(conjugate of BSA and eB4-2), 13) BSA-eB4-3 (conjugate of BSA and eB4-3).Specifically the preparation method is as follows: using beauty
BSA label coupling reagent kit (ReadilinkTM BSA Conjugation Kit) article No. of KPL company, state production: 5501, batch
Number: 148045, it requires to be coupled the polypeptide fragment of synthesis to specifications, is prepared into above-mentioned 12 kinds of envelope antigens.
BSA-eB2-3 and BSA-eB3-3 is mixed to get envelope antigen BSA-eB2-3+BSA- according to the mass ratio of 4:6
eB3-3。
2 utilize the time-resolved fluoroimmunoassay kit or detection Echinococcus Granulosus Cysts antibody for diagnosing echinococcosis granulosa
Time-resolved fluoroimmunoassay kit carry out time-resolved fluoroimmunoassay
Present embodiments provide the time-resolved fluoroimmunoassay kit or detection of 13 kinds of diagnosis echinococcosis granulosas
The time-resolved fluoroimmunoassay kit of Echinococcus Granulosus Cysts antibody.This 13 kinds of kits include envelope antigen, europium label
Secondary antibody, coating buffer, cleaning solution, secondary antibody diluent.The difference of this 13 kinds of kits is only that envelope antigen is different, other groups
Divide identical.
This 13 kinds of kits are respectively the diagnosis particulate spine ball for the BSA-eB2-3+BSA-eB3-3 that envelope antigen is step 1
The time-resolved fluoroimmunoassay kit of larva of a tapeworm or the cercaria of a schistosome disease or the time-resolved fluoroimmunoassay examination for detecting Echinococcus Granulosus Cysts antibody
Agent box, kit 1 hereinafter referred to as of the invention；Envelope antigen be step 1 BSA-eB2-3 diagnosis echinococcosis granulosa when
Between resolved fluorometric immunoassay kits or detect Echinococcus Granulosus Cysts antibody time-resolved fluoroimmunoassay kit, below
Kit 2 referred to as of the invention；The time resolution for the diagnosis echinococcosis granulosa that envelope antigen is the BSA-eB3-3 of step 1 is glimmering
Light immunoassay kits or the time-resolved fluoroimmunoassay kit for detecting Echinococcus Granulosus Cysts antibody, hereinafter referred to as this hair
Bright kit 3；The time-resolved fluoroimmunoassay point for the diagnosis echinococcosis granulosa that envelope antigen is the BSA-eB1-1 of step 1
It analyses kit or detects the time-resolved fluoroimmunoassay kit of Echinococcus Granulosus Cysts antibody, hereinafter referred to as contrast agents box 1；
The time-resolved fluoroimmunoassay kit for the diagnosis echinococcosis granulosa that envelope antigen is the BSA-eB1-2 of step 1 or inspection
Survey the time-resolved fluoroimmunoassay kit of Echinococcus Granulosus Cysts antibody, hereinafter referred to as contrast agents box 2；Envelope antigen is step
The time-resolved fluoroimmunoassay kit or detection Echinococcus Granulosus Cysts of the diagnosis echinococcosis granulosa of rapid 1 BSA-eB1-3
The time-resolved fluoroimmunoassay kit of antibody, hereinafter referred to as contrast agents box 3；Envelope antigen is the BSA- of step 1
The time-resolved fluoroimmunoassay kit of the diagnosis echinococcosis granulosa of eB2-1 or the time for detecting Echinococcus Granulosus Cysts antibody
Resolved fluorometric immunoassay kits, hereinafter referred to as contrast agents box 4；Envelope antigen is that the diagnosis of the BSA-eB2-2 of step 1 is thin
The time-resolved fluoroimmunoassay kit of grain hydatidosis or the time-resolved fluoroimmunoassay for detecting Echinococcus Granulosus Cysts antibody
Assay kit, hereinafter referred to as contrast agents box 5；The diagnosis echinococcosis granulosa that envelope antigen is the BSA-eB3-1 of step 1
Time-resolved fluoroimmunoassay kit or the time-resolved fluoroimmunoassay kit for detecting Echinococcus Granulosus Cysts antibody, with
Lower abbreviation contrast agents box 6；The time-resolved fluorescence for the diagnosis echinococcosis granulosa that envelope antigen is the BSA-eB3-2 of step 1
Immunoassay kits or the time-resolved fluoroimmunoassay kit for detecting Echinococcus Granulosus Cysts antibody, hereinafter referred to as control examination
Agent box 7；The time-resolved fluoroimmunoassay reagent for the diagnosis echinococcosis granulosa that envelope antigen is the BSA-eB4-1 of step 1
Box or the time-resolved fluoroimmunoassay kit for detecting Echinococcus Granulosus Cysts antibody, hereinafter referred to as contrast agents box 8；Coating is anti-
It originally was the time-resolved fluoroimmunoassay kit or detection particulate of the diagnosis echinococcosis granulosa of the BSA-eB4-2 of step 1
The time-resolved fluoroimmunoassay kit of echinococcus antibody, hereinafter referred to as contrast agents box 9；Envelope antigen is step 1
The time-resolved fluoroimmunoassay kit of the diagnosis echinococcosis granulosa of BSA-eB4-3 detects Echinococcus Granulosus Cysts antibody
Time-resolved fluoroimmunoassay kit, hereinafter referred to as contrast agents box 10.
The rabbit-anti goat secondary antibody (abbreviation europium marks secondary antibody) of europium label: Epstein-Barr virus core is prepared according to the method in following document
The south the development medical courses in general of antigen (NA1) IgA antibody and Zta protein I gA antibody time-resolved fluoroimmunoassay detection reagent are big
Learn 2012 grades of master thesis.
Be coated with buffer: the sodium carbonate-bicarbonate buffer (pH9.6) of 0.05mol/L, solvent is water, solute and its
Concentration is as follows: Na2CO31.59g/L and NaHCO3 2.93g/L。
Cleaning solution is PBST cleaning solution.PBST cleaning solution is prepared as follows: being 0.01M, pH value 7.4 in concentration
PBS buffer solution in be added polysorbas20 to polysorbas20 content be 5mL/L, obtain PBST cleaning solution.
Confining liquid is 1%BSA confining liquid.1%BSA confining liquid is prepared as follows: being 0.01M, pH value in concentration
1%BSA confining liquid is obtained until the volumn concentration of BSA is 1% for the BSA solution for adding 10% in 7.4 PBS buffer solution.
Secondary antibody diluent: being 0.01M in concentration, the content that BSA to BSA is added in the PBS buffer solution that pH value is 7.4 is
1% (volumn concentration), obtains secondary antibody diluent.
Wherein, concentration 0.01M, the preparation for the PBS buffer solution that pH value is 7.4: 8.5g NaCl, 0.2g KCl, 2.9g
Na2HPO4·12H2O、0.59g NaH2PO4·2H2O, 1L deionized water.
2.1 utilize kit 1 of the invention, establish by optimization experiment with BSA-eB2-3+BSA-eB3-3 as coating
The time-resolved fluorescence immunoassay method (TRFIA method 1 hereinafter referred to as of the present invention) of antigen, specific as follows:
2.1.1 it is coated with: with the BSA-eB2-3+BSA-eB3-3 to BSA-eB2-3+ in coating buffer dilution step 1
The concentration (total mass concentration of BSA-eB2-3+BSA-eB3-3) of BSA-eB3-3 is 1.0 μ g/ml, obtains envelope antigen solution,
It is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.1.2 wash: incline hole endoperidium original solution, is washed 5 times with PBST cleaning solution, each 3min；It pats dry.
2.1.3 it closes: 1%BSA confining liquid, 250 holes μ L/, 37 DEG C of incubation 2h is added.
2.1.4 it is loaded:
2.1.4.1 sample well
Sheep echinococcosis granulosa positive serum is diluted 50 times with coating buffer, obtains test serum.100 μ L are to be measured
Serum is added on ELISA Plate, 37 DEG C of reaction 1h, then liquid in hole of inclining is washed 5 times with cleaning solution.
Sheep echinococcosis granulosa positive serum is to use above-mentioned clinical diagnosis for the sheep blood serum with sheep echinococcosis granulosa.
2.1.4.2 blank control wells
Difference with 2.1.4.1, which is only that, replaces with test serum isometric high purity water, and other steps are constant.
Plus europium element mark secondary antibody 2.1.5: addition carries out the diluted europium of 1:50000 with secondary antibody diluent and marks rabbit-anti goat
IgG, 100 holes μ L/, 37 DEG C of 30min.
2.1.6 it develops the color: TMB is added, 10min is reacted in 100 holes μ L/.
2.1.7 it terminates: 0.2mol/L H is added2SO4Solution terminates reaction, 100 holes μ L/.
2.1.8 it measures: reading the fluorescent measurement in each hole with time-resolved fluorescence immunoassay instrument.
2.1.9 the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.1.1-2.1.8 (by the sheep particulate in 2.1.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 portions of sheep are thin
Grain hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value X of 400 parts of sheep echinococcosis granulosa negative serums is that 2217, SD is
114, therefore the critical fluorescent measurement of yin and yang attributeIt is 2559.
2.2 utilize kit 2 of the invention, establish the time using BSA-eB2-3 as envelope antigen by optimization experiment
Resolved fluorometric immunoassay method (TRFIA method 2 hereinafter referred to as of the present invention) is as follows:
2.2.1 it is coated with: being 1.0 μ g/ml with the BSA-eB2-3 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.2.2 wash: incline hole endoperidium original solution, is washed 5 times with PBST cleaning solution, each 3min；It pats dry.
2.2.3 it closes: 1%BSA confining liquid, 250 holes μ L/, 37 DEG C of incubation 2h is added.
2.2.4 it is loaded:
2.2.4.1 sample well
Sheep echinococcosis granulosa positive serum is diluted 50 times with coating buffer, obtains test serum.100 μ L are to be measured
Serum is added on ELISA Plate, 37 DEG C of reaction 1h, then liquid in hole of inclining is washed 5 times with cleaning solution.
Sheep echinococcosis granulosa positive serum is to use above-mentioned clinical diagnosis for the sheep blood serum with sheep echinococcosis granulosa.
2.2.4.2 blank control wells
Difference with 2.2.4.1, which is only that, replaces with test serum isometric high purity water, and other steps are constant.
Plus europium element mark secondary antibody 2.2.5: addition carries out the diluted europium of 1:50000 with secondary antibody diluent and marks rabbit-anti goat
IgG, 100 holes μ L/, 37 DEG C of 30min.
2.2.6 it develops the color: TMB is added, 10min is reacted in 100 holes μ L/.
2.2.7 it terminates: 0.2mol/L H is added2SO4Solution terminates reaction, 100 holes μ L/.
2.2.8 it measures: reading the fluorescent measurement in each hole with time-resolved fluorescence immunoassay instrument.
2.2.9, the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.2.1-2.2.8 (by the sheep particulate in 2.2.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 portions of sheep are thin
Grain hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 1879, SD
106, therefore the critical fluorescent measurement of yin and yang attributeIt is 2197.
2.3 utilize kit 3 of the invention, establish the time using BSA-eB3-3 as envelope antigen by optimization experiment
Resolved fluorometric immunoassay method (TRFIA method 3 hereinafter referred to as of the present invention) is as follows:
2.3.2 it is coated with: being 1.0 μ g/ml with the BSA-eB3-3 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.3.2 wash: incline hole endoperidium original solution, is washed 5 times with PBST cleaning solution, each 3min；It pats dry.
2.3.3 it closes: 1%BSA confining liquid, 250 holes μ L/, 37 DEG C of incubation 2h is added.
2.3.4 it is loaded:
2.3.4.1 sample well
Sheep echinococcosis granulosa positive serum is diluted 50 times with coating buffer, obtains test serum.100 μ L are to be measured
Serum is added on ELISA Plate, 37 DEG C of reaction 1h, then liquid in hole of inclining is washed 5 times with cleaning solution.
Sheep echinococcosis granulosa positive serum is to use above-mentioned clinical diagnosis for the sheep blood serum with sheep echinococcosis granulosa.
2.3.4.2 blank control wells
Difference with 2.3.4.1, which is only that, replaces with test serum isometric high purity water, and other steps are constant.
Plus europium element mark secondary antibody 2.3.5: addition carries out the diluted europium of 1:50000 with secondary antibody diluent and marks rabbit-anti goat
IgG, 100 holes μ L/, 37 DEG C of 30min.
2.3.6 it develops the color: TMB is added, 10min is reacted in 100 holes μ L/.
2.3.7 it terminates: 0.2mol/L H is added2SO4Solution terminates reaction, 100 holes μ L/.
2.3.8 it measures: reading each hole fluorescent measurement with time-resolved fluorescence immunoassay instrument.
2.3.9 the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.3.2-2.3.8 (by the sheep particulate in 2.3.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 portions of sheep are thin
Grain hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 1796, SD
98, therefore the critical fluorescent measurement of yin and yang attributeIt is 2090.
2.4 utilize contrast agents box 1, establish the time resolution using BSA-eB1-1 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 1) is as follows:
2.4.1 it is coated with: being 1.0 μ g/ml with the BSA-eB1-1 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.4.2 it washs: same to 2.2.2.
2.4.3 it closes: same to 2.2.3.
2.4.4 it is loaded: same to 2.2.4.
2.4.5 plus europium element mark secondary antibody: same to 2.2.5.
2.4.6 it develops the color: same to 2.2.6.
2.4.7 it terminates: same to 2.2.7.
2.4.8 it measures: same to 2.2.8.
2.4.9 the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.4.1-2.4.8 (by the sheep particulate in 2.4.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 parts of sheep particulates
Hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 2987, SD
232, therefore the critical fluorescent measurement of yin and yang attributeIt is 3683.
2.5 utilize contrast agents box 2, establish the time resolution using BSA-eB1-2 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 2) is as follows:
2.5.1 it is coated with: being 1.0 μ g/ml with the BSA-eB1-2 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.5.2 it washs: same to 2.2.2.
2.5.3 it closes: same to 2.2.3.
2.5.4 it is loaded: same to 2.2.4.
2.5.5 plus europium element mark secondary antibody: same to 2.2.5.
2.5.6 it develops the color: same to 2.2.6.
2.5.7 it terminates: same to 2.2.7.
2.5.8 it measures: same to 2.2.8.
2.5.9, the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.5.1-2.5.8 (by the sheep particulate in 2.5.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 portions of sheep are thin
Grain hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 3354, SD
287, therefore the critical fluorescent measurement of yin and yang attributeIt is 4215.
2.6 utilize contrast agents box 3, establish the time resolution using BSA-eB1-3 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 3) is as follows:
2.6.1 it is coated with: being 1.0 μ g/ml with the BSA-eB1-3 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.6.2 it washs: same to 2.2.2.
2.6.3 it closes: same to 2.2.3.
2.6.4 it is loaded: same to 2.2.4.
2.6.5 plus europium element mark secondary antibody: same to 2.2.5.
2.6.6 it develops the color: same to 2.2.6.
2.6.7 it terminates: same to 2.2.7.
2.6.8 it measures: same to 2.2.8.
2.6.9 the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.6.1-2.6.8 (by the sheep particulate in 2.6.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 portions of sheep are thin
Grain hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 4150, SD
352, therefore the critical fluorescent measurement of yin and yang attributeIt is 5206.
2.7 utilize contrast agents box 4, establish the time resolution using BSA-eB2-1 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 4) is as follows:
2.7.1 it is coated with: being 1.0 μ g/ml with the BSA-eB2-1 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.7.2 it washs: same to 2.2.2.
2.7.3 it closes: same to 2.2.3.
2.7.4 it is loaded: same to 2.2.4.
2.7.5 plus europium element mark secondary antibody: same to 2.2.5.
2.7.6 it develops the color: same to 2.2.6.
2.7.7 it terminates: same to 2.2.7.
2.7.8 it measures: same to 2.2.8.
2.7.9 the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.7.1-2.7.8 (by the sheep particulate in 2.7.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation of the fluorescent measurement of 400 parts of sheep echinococcosis granulosa negative serums
Poor (SD).It is judged to the positive；It is judged to feminine gender.400 portions of sheep are thin
Grain hydatidosis negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 3694, SD
325, therefore the critical fluorescent measurement of yin and yang attributeIt is 4669.
2.8 utilize contrast agents box 5, establish the time resolution using BSA-eB2-2 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 5) is as follows:
2.8.1 it is coated with: being 1.0 μ g/ml with the BSA-eB2-2 in coating buffer dilution step 1 to its concentration, wrapped
By antigenic solution, it is coated with experimental port with the envelope antigen solution, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.8.2 it washs: same to 2.2.2.
2.8.3 it closes: same to 2.2.3.
2.8.4 it is loaded: same to 2.2.4.
2.8.5 plus europium element mark secondary antibody: same to 2.2.5.
2.8.6 it develops the color: same to 2.2.6.
2.8.7 it terminates: same to 2.2.7.
2.8.8 it measures: same to 2.2.8.
2.8.9, the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.8.1-2.8.8 (by the sheep particulate in 2.8.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation (SD) of 400 parts of sheep echinococcosis granulosa negative serums.It is judged to the positive；It is judged to feminine gender.400 parts of sheep Echinococcus Granulosus Cysts
Sick negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 4154, SD
379, therefore the critical fluorescent measurement of yin and yang attributeIt is 5291.
2.9 utilize contrast agents box 6, establish the time resolution using BSA-eB3-1 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 6) is as follows:
2.9.1 be coated with: the concentration with the BSA-eB3-1 in coating buffer dilution step 1 is 1.0 μ g/ml, is coated with
Antigenic solution is coated with experimental port with the envelope antigen solution, and 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.9.2 it washs: same to 2.2.2.
2.9.3 it closes: same to 2.2.3.
2.9.4 it is loaded: same to 2.2.4.
2.9.5 plus europium element mark secondary antibody: same to 2.2.5.
2.9.6 it develops the color: same to 2.2.6.
2.9.7 it terminates: same to 2.2.7.
2.9.8 it measures: same to 2.2.8.
2.9.9 the determination of yin and yang attribute critical value
By 400 parts of sheep echinococcosis granulosa negative serums using step 2.9.1-2.9.8 (by the sheep particulate in 2.9.4.1
Hydatidosis positive serum replaces with 400 parts of sheep echinococcosis granulosa negative serums respectively, and other steps are identical) method into
Row TRFIA detection, calculates the average value (X) and standard deviation (SD) of 400 parts of sheep echinococcosis granulosa negative serums.It is judged to the positive；It is judged to feminine gender.400 parts of sheep Echinococcus Granulosus Cysts
Sick negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 3107, SD
221, therefore the critical fluorescent measurement of yin and yang attributeIt is 3770.
2.10 utilize contrast agents box 7, establish the time resolution using BSA-eB3-2 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 7) is as follows:
2.10.1 it is coated with: being 1.0 μ g/ml with the BSA-eB3-2 in coating buffer dilution step 1 to its concentration, obtain
Envelope antigen solution is coated with experimental port with the envelope antigen solution, and 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.10.2 washing: same to 2.2.2.
2.10.3 closing: same to 2.2.3.
2.10.4 sample-adding: same to 2.2.4.
2.10.5 plus europium element mark secondary antibody: same to 2.2.5.
2.10.6 colour developing: same to 2.2.6.
2.10.7 terminating: same to 2.2.7.
2.10.8 measurement: same to 2.2.8.
2.10.9 the determination of yin and yang attribute critical value
400 parts of sheep echinococcosis granulosa negative serums are (thin by the sheep in 2.10.4.1 using step 2.10.1-2.10.8
Grain hydatidosis positive serum replace with 400 parts of sheep echinococcosis granulosa negative serums respectively, other steps are identical) method
TRFIA detection is carried out, the average value (X) and standard deviation (SD) of 400 parts of sheep echinococcosis granulosa negative serums are calculated.It is judged to the positive；It is judged to feminine gender.400 parts of sheep Echinococcus Granulosus Cysts
Sick negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 2895, SD
243, therefore the critical fluorescent measurement of yin and yang attributeIt is 3624.
2.11 utilize contrast agents box 8, establish the time resolution using BSA-eB4-1 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 8) is as follows:
2.11.1 it is coated with: being 1.0 μ g/ml with the BSA-eB4-1 in coating buffer dilution step 1 to its concentration, obtain
Envelope antigen solution is coated with experimental port with the envelope antigen solution, and 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.11.2 washing: same to 2.2.2.
2.11.3 closing: same to 2.2.3.
2.11.4 sample-adding: same to 2.2.4.
2.11.5 plus europium element mark secondary antibody: same to 2.2.5.
2.11.6 colour developing: same to 2.2.6.
2.11.7 terminating: same to 2.2.7.
2.11.8 measurement: same to 2.2.8.
2.11.9, the determination of yin and yang attribute critical value
400 parts of sheep echinococcosis granulosa negative serums are (thin by the sheep in 2.11.4.1 using step 2.11.1-2.11.8
Grain hydatidosis positive serum replace with 400 parts of sheep echinococcosis granulosa negative serums respectively, other steps are identical) method
TRFIA detection is carried out, the average value (X) and standard deviation (SD) of 400 parts of sheep echinococcosis granulosa positive serums are calculated.It is judged to the positive；It is judged to feminine gender.400 parts of sheep Echinococcus Granulosus Cysts
Sick negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa positive serumsIt is for 4859, SD
376, therefore the critical fluorescent measurement of yin and yang attributeIt is 5987.
2.12 utilize contrast agents box 9, establish the time resolution using BSA-eB4-2 as envelope antigen by optimization experiment
Fluorescence immune analysis method (hereinafter referred to as control TRFIA method 9) is as follows:
2.12.1 it is coated with: being 1.0 μ g/ml with the BSA-eB4-2 in coating buffer dilution step 1 to its concentration, obtain
Envelope antigen solution is coated with experimental port with the envelope antigen solution, and 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.12.2 washing: same to 2.2.2.
2.12.3 closing: same to 2.2.3.
2.12.4 sample-adding: same to 2.2.4.
2.12.5 plus europium element mark secondary antibody: same to 2.2.5.
2.12.6 colour developing: same to 2.2.6.
2.12.7 terminating: same to 2.2.7.
2.12.8 measurement: same to 2.2.8.
2.12.9 the determination of yin and yang attribute critical value
400 parts of sheep echinococcosis granulosa negative serums are (thin by the sheep in 2.12.4.1 using step 2.12.1-2.12.8
Grain hydatidosis positive serum replace with 400 parts of sheep echinococcosis granulosa negative serums respectively, other steps are identical) method
TRFIA detection is carried out, the average value (X) and standard deviation (SD) of 400 parts of sheep echinococcosis granulosa negative serums are calculated.It is judged to the positive；It is judged to feminine gender.400 parts of sheep Echinococcus Granulosus Cysts
Sick negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 4213, SD
334, therefore the critical fluorescent measurement of yin and yang attributeIt is 5215.
2.13 utilize contrast agents box 10, establish by optimization experiment using BSA-eB4-3 as the time of envelope antigen point
Distinguish that fluorescence immune analysis method (hereinafter referred to as control TRFIA method 10) is as follows:
2.13.1 it is coated with: being 1.0 μ g/ml with the BSA-eB4-3 in coating buffer dilution step 1 to its concentration, obtain
Envelope antigen solution is coated with experimental port with the envelope antigen solution, and 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.
2.13.2 washing: same to 2.2.2.
2.13.3 closing: same to 2.2.3.
2.13.4 sample-adding: same to 2.2.4.
2.13.5 plus europium element mark secondary antibody: same to 2.2.5.
2.13.6 colour developing: same to 2.2.6.
2.13.7 terminating: same to 2.2.7.
2.13.8 measurement: same to 2.2.8.
2.13.9 the determination of yin and yang attribute critical value
400 parts of sheep echinococcosis granulosa negative serums are (thin by the sheep in 2.13.4.1 using step 2.13.1-2.13.8
Grain hydatidosis positive serum replace with 400 parts of sheep echinococcosis granulosa negative serums respectively, other steps are identical) method
TRFIA detection is carried out, the average value (X) and standard deviation (SD) of 400 parts of sheep echinococcosis granulosa negative serums are calculated.It is judged to the positive；It is judged to feminine gender.400 parts of sheep Echinococcus Granulosus Cysts
Sick negative serum is the sheep blood serum for using above-mentioned clinical diagnosis to be not suffering from sheep echinococcosis granulosa.
The result shows that the mean fluorecence detected value of 400 parts of sheep echinococcosis granulosa negative serumsIt is for 3280, SD
254, therefore the critical fluorescent measurement of yin and yang attributeIt is 4042.
3, specific test
TRFIA method 1-3 (abbreviation the method for the present invention 1-3) of the present invention and control TRFIA method 1-10 using step 2
(abbreviation contrast method 1-10) is to each 10 parts of sheep echinococcosis granulosa negative serum, sheep echinococcosis multilocularis positive serum and sheep
Cysticercosis positive serum is detected, and observation has no cross reaction with Other diseases.The result shows that the result shows that the present invention
TRFIA method 1-3 is to sheep echinococcosis granulosa negative serum, sheep echinococcosis multilocularis positive serum and cysticercus ovis disease positive blood
Clear equal no cross reaction, illustrates that kit 1-3 of the invention can be to sheep echinococcosis granulosa negative serum, sheep Echinococcus moltilocularis
Sick positive serum and cysticercus ovis disease positive serum are accurately distinguished.(table 2-4).
The testing result of 2. the method for the present invention 1-3 of table, contrast method 1-10 to 10 parts of sheep echinococcosis granulosa negative serums
Testing result of the table 3. the method for the present invention 1-3 and contrast method 1-10 to 10 parts of sheep echinococcosis multilocularis positive serums
Testing result of the table 4. the method for the present invention 1-3 and contrast method 1-10 to 10 parts of cysticercus ovis disease positive serums
4, sheep echinococcosis granulosa positive serum is carried out doubling dilution by sensitivity test, and TRFIA of the present invention is respectively adopted
Method 1-3 (abbreviation the method for the present invention 1-3) and control TRFIA method 1-10 (abbreviation contrast method 1-10) are detected, and are obtained
Greatest dilution when positive critical value.
The result shows that the highest extension rate of TRFIA method 1-3 detection positive serum of the present invention is respectively 1:1024 times, 1:
512 times, 1:512 times；The highest extension rate for compareing TRFIA method 1-10 detection positive serum is respectively 1:256 times, 1:512
Again, 1:1024 times, 1:256 times, 1:128 times 1:256 times, 1:512 times, 1:1024 times, 1:512 times, 1:512 times.
5, repetitive test
Using TRFIA method 1-3 of the present invention respectively to 6 parts of sheep echinococcosis granulosa positive serums in same batch plate and difference
It is detected, is measured in parallel 5 times respectively on batch plate, calculate batch interior, interassay coefficient of variation (CV).The results show that of the invention
The coefficient of variation is repeated in TRFIA method 1-3 batches, and the coefficient of variation is repeated less than 4%, between batch less than 6% (table 5-7).The result shows that
Kit 1-3 of the invention has good repeatability to sheep echinococcosis granulosa positive serum.
1 repetitive test of TRFIA method of the present invention of table 5.
2 repetitive test of TRFIA method of the present invention of table 6.
3 repetitive test of TRFIA method of the present invention of table 7.
6, accordance test (accuracy experiment)
Using above-mentioned methods for clinical diagnosis from China Animal Disease Control And Prevention Center (in agriculture rural area portion Disease Diagnosis of Veterinary
The heart) sheep blood serum (come from Xinjiang, China) that saves picked out 90 parts of sheep echinococcosis granulosa positive serums and 90 parts of sheep particulate spine
Ball larva of a tapeworm or the cercaria of a schistosome disease negative serum.To this 180 parts of sheep blood serums be respectively adopted TRFIA method 1-3 (abbreviation the method for the present invention 1-3) of the present invention and
Control TRFIA method 1-10 (abbreviation contrast method 1-10) is detected, and the coincidence rate with clinical diagnosis is calculated.
Sensibility (True Positive Rate, TPR, true positive rate, positive coincidence rate): the practical positive (suffers from particulate spine
Ball larva of a tapeworm or the cercaria of a schistosome disease) and correctly it is judged as positive percentage by testing standard, sensibility is the bigger the better, and ideal sensibility is 100%.
Specific (True Negative Rate, TNR, true negative rate, negative match-rate): practical feminine gender (is not suffering from particulate
Hydatidosis) and correctly it is judged as negative percentage by testing standard, specificity is the bigger the better, and desired specificity is
100%.
TPR=TP/ (TP+FN).TNR=TN/ (TN+FP).ACC=(TP+TN)/(TP+FP+FN+TN).TP: true positives.
TN: true negative.FP: false positive.FN: false negative.ACC: accuracy.
The result shows that total coincidence rate of 180 parts of sheep blood serums, TRFIA method 1 of the present invention and clinical diagnosis is 91.67%
(positive coincidence rate 91.11%, negative match-rate 92.22%), TRFIA method 2 of the present invention and sheep Echinococcus Granulosus Cysts serum
Total coincidence rate of neutralization test method is 89.44% (positive coincidence rate 84.44%, negative match-rate 94.44%), this hair
Total coincidence rate of bright TRFIA method 3 and sheep Echinococcus Granulosus Cysts serum neutralization test method is 87.22%, and (positive coincidence rate is
81.11%, negative match-rate 93.33%).Compare 1 testing result of TRFIA method and the serum neutralization test of sheep Echinococcus Granulosus Cysts
Total coincidence rate of method is 83.33% (positive coincidence rate 86.67%, negative match-rate 80%), compares TRFIA method 2
Total coincidence rate of testing result and sheep Echinococcus Granulosus Cysts serum neutralization test method is 80.56%, and (positive coincidence rate is
75.56%, negative match-rate 85.56%), control 3 testing result of TRFIA method and the serum neutralization test of sheep Echinococcus Granulosus Cysts
Total coincidence rate of method is 83.33% (positive coincidence rate 76.67%, negative match-rate 90%), compares TRFIA method 4
Total coincidence rate of testing result and sheep Echinococcus Granulosus Cysts serum neutralization test method is 80.56% (positive coincidence rate 80%, yin
Property coincidence rate be 81.11%), compare 5 testing result of TRFIA method and sheep Echinococcus Granulosus Cysts serum neutralization test method total symbol
Conjunction rate is 78.89% (positive coincidence rate 92.22%, negative match-rate 65.56%), compares 6 testing result of TRFIA method
Total coincidence rate with sheep Echinococcus Granulosus Cysts serum neutralization test method is 80.56% (positive coincidence rate 82.22%, feminine gender symbol
Conjunction rate is 78.89%), to compare total coincidence rate of 7 testing result of TRFIA method and sheep Echinococcus Granulosus Cysts serum neutralization test method
For 81.67% (positive coincidence rate 76.67%, negative match-rate 86.67%), 8 testing result of TRFIA method and sheep are compareed
Total coincidence rate of Echinococcus Granulosus Cysts serum neutralization test method is that 77.78% (positive coincidence rate 80%, negative match-rate are
75.56%) it, compares 9 testing result of TRFIA method and total coincidence rate of sheep Echinococcus Granulosus Cysts serum neutralization test method is
82.78% (positive coincidence rate 74.44%, negative match-rate 91.11%) compares 10 testing result of TRFIA method and sheep
Total coincidence rate of Echinococcus Granulosus Cysts serum neutralization test method is 77.78% (positive coincidence rate 91.11%, negative match-rate
For 64.44%) (table 8-20).
Illustrate respectively to prepare BSA-eB2-3+BSA-eB3-3, BSA-eB2-3 and BSA-eB3-3 as envelope antigen
The time resolution for diagnosing the time-resolved fluoroimmunoassay kit or detection Echinococcus Granulosus Cysts antibody of echinococcosis granulosa is glimmering
Light immunoassay kits and total coincidence rate of clinical diagnosis are significantly higher than respectively with BSA-eB1-1, BSA-eB1-2, BSA-
EB1-3, BSA-eB2-1, BSA-eB2-2, BSA-eB3-1, BSA-eB3-2, BSA-eB4-1, BSA-eB4-2 and BSA-eB4-3
The time-resolved fluoroimmunoassay kit or detection particulate spine ball of diagnosis echinococcosis granulosa as envelope antigen preparation
The time-resolved fluoroimmunoassay kit of larva of a tapeworm or the cercaria of a schistosome antibody.
The TRFIA method 1 of the present invention of table 8. is to sheep blood serum sample detection result
The TRFIA method 2 of the present invention of table 9. is to sheep blood serum sample detection result
The TRFIA method 3 of the present invention of table 10. is to sheep blood serum sample detection result
Table 11. compares TRFIA method 1 to sheep blood serum sample detection result
Table 12. compares TRFIA method 2 to sheep blood serum sample detection result
Table 13. compares TRFIA method 3 to sheep blood serum sample detection result
Table 14. compares TRFIA method 4 to sheep blood serum sample detection result
Table 15. compares TRFIA method 5 to sheep blood serum sample detection result
Table 16. compares TRFIA method 6 to sheep blood serum sample detection result
Table 17. compares TRFIA method 7 to sheep blood serum sample detection result
Table 18. compares TRFIA method 8 to sheep blood serum sample detection result
Table 19. compares TRFIA method 9 to sheep blood serum sample detection result
Table 20. compares TRFIA method 10 to sheep blood serum sample detection result
The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and
Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range
Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further.
In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen
Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims,
It can carry out the application of some essential characteristics.
<110>China Animal Disease Control And Prevention Center's (butchering technique center in agriculture rural area portion)
<120>cystic echinococcosis diagnostic kit and its application
<130> GNCFH190885
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Cys Lys Lys Tyr Val Lys Asn Leu Val Glu Glu Lys Asp Asp Asp Ser
1 5 10 15
Lys
<210> 2
<211> 14
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Cys Leu Lys Glu Tyr Val Arg Lys Leu Val Lys Glu Asp Glu
1 5 10
Claims (10)
1. echinococcosis granulosa diagnostic kit or the kit for detecting Echinococcus Granulosus Cysts antibody, it is characterised in that: the reagent
Box includes envelope antigen, and the envelope antigen is made of eB2-3 conjugate and/or eB3-3 conjugate；The eB2-3 conjugate
It is the comlete antigen obtained by eB2-3 and carrier protein couplet；The eB3-3 conjugate is by eB3-3 and carrier protein couplet
Obtained comlete antigen；
The eB2-3 is the polypeptide of P11, P12 or P13:
P11, the polypeptide that amino acid sequence is sequence 1 in sequence table,
P12, the 2-17 polypeptides that amino acid sequence is sequence 1 in sequence table,
P13, P12 polypeptide amino terminal or carboxyl terminal connection amino acid residue it is more to be obtained with carrier protein couplet
Peptide；
The eB3-3 is the polypeptide of P21, P22 or P23:
P21, the polypeptide that amino acid sequence is sequence 2 in sequence table,
P22, the 2-14 polypeptides that amino acid sequence is sequence 2 in sequence table,
P23, P22 polypeptide amino terminal or carboxyl terminal connection amino acid residue it is more to be obtained with carrier protein couplet
Peptide.
2. kit according to claim 1, it is characterised in that: the kit is the time for diagnosing echinococcosis granulosa
Resolved fluorometric immunoassay kits or the time-resolved fluoroimmunoassay kit for detecting Echinococcus Granulosus Cysts antibody.
3. kit according to claim 1, it is characterised in that: the kit is the enzyme-linked of diagnosis echinococcosis granulosa
Immune reagent kit or the enzyme linked immunological kit for detecting Echinococcus Granulosus Cysts antibody.
4. echinococcosis granulosa diagnoses test paper or detects the test paper of Echinococcus Granulosus Cysts antibody, it is characterised in that: the test paper includes
Envelope antigen, eB3- described in envelope antigen eB2-3 conjugate as described in claim 1 and/or claim 1
3 conjugates composition.
5. the application of envelope antigen described in claim 1, complete polypeptide or independent polypeptide in reagent preparation box, feature
Be: the kit is echinococcosis granulosa diagnostic kit or the kit for detecting Echinococcus Granulosus Cysts antibody；It is described complete
Polypeptide is made of the eB2-3 described in claim 1 and the eB3-3 described in claim 1；
The independent polypeptide is eB3-3 described in eB2-3 described in claim 1 or claim 1.
6. application according to claim 5, it is characterised in that: the kit is the time point for diagnosing echinococcosis granulosa
It distinguishes fluorescence immunoassay kit or detects the time-resolved fluoroimmunoassay kit of Echinococcus Granulosus Cysts antibody, the reagent
Box includes envelope antigen, described in envelope antigen eB2-3 conjugate as described in claim 1 and/or claim 1
EB3-3 conjugate composition.
7. application according to claim 5, it is characterised in that: the kit is that diagnosis the enzyme-linked of echinococcosis granulosa is exempted from
Epidemic disease kit or the enzyme linked immunological kit for detecting Echinococcus Granulosus Cysts antibody, the kit includes envelope antigen, the coating
Antigen by described in claim 1 eB2-3 conjugate and/or claim 1 described in eB3-3 conjugate form.
8. envelope antigen described in claim 1, complete polypeptide or independent polypeptide exist preparing the application in test paper, feature
In: the test paper is the test paper that echinococcosis granulosa diagnoses test paper or detection Echinococcus Granulosus Cysts antibody, and the complete polypeptide is by weighing
Benefit requires eB3-3 described in eB2-3 described in 1 and claim 1 to form, and the independent polypeptide is institute in claim 1
EB3-3 described in the eB2-3 or claim 1 stated.
9. application according to claim 8, it is characterised in that: the test paper includes envelope antigen, the envelope antigen by
The composition of eB3-3 conjugate described in eB2-3 conjugate described in claim 1 and/or claim 1.
10. kit as claimed in any one of claims 1-3 or test paper as claimed in claim 4 are preparing echinococcosis granulosa prison
Application in test agent box.
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| CN113341160A (en) * | 2021-06-09 | 2021-09-03 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | ELISA kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep |
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