CN110221067A - A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof - Google Patents
A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof Download PDFInfo
- Publication number
- CN110221067A CN110221067A CN201910644896.XA CN201910644896A CN110221067A CN 110221067 A CN110221067 A CN 110221067A CN 201910644896 A CN201910644896 A CN 201910644896A CN 110221067 A CN110221067 A CN 110221067A
- Authority
- CN
- China
- Prior art keywords
- antigen
- trichina
- test strips
- preparation
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Peptides Or Proteins (AREA)
Abstract
A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof, belong to fluorescence immunoassay detection technique field.In order to more rapidly, stablize, accurately detect trichinzation, the present invention provides a kind of pigs trichina disease antibody test test strips, including sample pad, bonding pad, chromatographic film, water absorption pad and bottom plate, the anti-pig IgG of having time resolved fluorometric microballoon conjugated goat is marked on the bonding pad；The chromatographic film is equipped with detection line and nature controlling line, and the trichina cocktail antigen being made of the Ts-WM5 antigen of recombination muscle larvae phase of prokaryotic expression, the Ts-WN10 antigen of enteric infection larva, the Ts-ZH68 antigen in adult stage and the Ts-T668-C antigen of newborn larvae phase is wherein sprayed in detection line；It is coated with rabbit-anti goat IgG on the nature controlling line, test strips are prepared into after above-mentioned each group subassembly, the early stage that the present invention can be used for Trichinella sui infection is quickly detected.
Description
Technical field
The invention belongs to fluorescence immunoassay detection technique fields, and in particular to a kind of pigs trichina disease antibody test test strips and
Preparation method and application.
Background technique
Trichinosis is a kind of harm Amphixenosis very serious, can not only be caused to Animal husbandry production huge
Economic loss, and grave danger is also constituted to human health, human or animal, which mainly passes through, to be eaten raw or partly eats raw containing rotation
The meat (predominantly pork) of caterpillar and fall ill.
Inspection trichinous for slaughtered animals, frequently with the regulation method of inspection be Microscopical Method For Detection and collection sample digestion method.
However there are certain drawbacks in above two method, Microscopical Method For Detection is time-consuming and laborious and sensibility is poor, and sensibility is polypide in meat
Density can detect when reaching every gram of 3 polypide.Though collection sample digestion method be greatly improved recall rate, by polypide recall rate improve to
1 polypide of every gram of meat, but this method is still very complicated, still needed to when finding positive sample to positive group using digestion method carry out by
Head detection.From the perspective of sensibility, there is security risk in the meat of the missing inspection as caused by Microscopical Method For Detection sum aggregate sample digestion method
And the infection (infection that 75 polypides of intake can cause people) of the mankind can be caused.Using indirect fluorescent antibody, immuno-enzymatic
Dye test, western blot test, the technologies such as immunosorbent adsorption test (ELISA) detect trichina antibody, operate it is more complex, often
Need 2-3 hour to detect as a result, and need expensive instrument and efficient staff to complete in laboratory, cannot apply
The detection of pigs trichina disease is carried out with grass-roots unit on site.
Currently, cultivation of larvae of Trichinella spiralis from muscle excretion secretion ES antigen is unique as defined in OIE and International Trichinella Committee
Standard antigen for serology antibody test.However ES antigenic component is complicated, prepares that cumbersome, the production cycle is long, batch quality
Unevenness, and the problems such as there are serious diagnosis blind area (can not detect before 19d after infection) and cross reactions, thus hinder it
Practical application.
Summary of the invention
For how quickly, stablize, accurate detection Trichinella sui infection conditions, the present invention provides a kind of pigs trichina diseases
Antibody test test strips, including sample pad, bonding pad, chromatographic film, water absorption pad and bottom plate, label having time point on the bonding pad
Distinguish the anti-pig IgG of fluorescent microsphere conjugated goat；Well is provided in the sample pad；The chromatographic film is equipped with detection line and matter
Control line, be wherein coated with trichina cocktail antigen in detection line, the trichina cocktail antigen by the muscle larvae phase Ts-
WM5 recombinant antigen, the Ts-WN10 recombinant antigen of enteric infection larva, the Ts-ZH68 recombinant antigen and newborn larvae in adult stage
The Ts-T668-C recombinant antigen of phase forms；Rabbit-anti goat IgG is coated on the nature controlling line.
It further limits, the sample pad material is glass fibre element film XQ-Y8；The bonding pad material is glass fibers
Tie up plain film Ahistrom8964；The chromatography membrane material is nitrocellulose filter Millipore135.
It further limits, the Ts-WM5 of the recombination muscle larvae phase of prokaryotic expression is anti-in the trichina cocktail antigen
Original, the Ts-WN10 antigen of enteric infection larva, the Ts-ZH68 antigen in adult stage and newborn larvae phase Ts-T668-C antigen
Concentration ratio be (0.5-1): (0.5-1): (0.5-0.75): (0.5-0.75) mg/mL.
The present invention also provides the preparation methods of above-mentioned trichinosis fluorescence immune chromatography test strip, including walk as follows
It is rapid:
1) preparation of trichina cocktail antigen: Ts-WM5 recombinant protein, the enteric infection of muscle larvae phase will be expressed respectively
The weight of C-terminal in the Ts-WN10 recombinant protein of property larva, the Ts-ZH68 recombinant protein and the Ts-T668 of newborn larvae phase in adult stage
Each recombinant plasmid transformed host strain of histone, after inducing expression, precipitating, urea dissolution is collected by centrifugation in ultrasonication thallus
Then inclusion body obtains the Ts-WM5 recombinant antigen of muscle larvae phase, the Ts-WN10 of enteric infection larva respectively through affinity purification
Recombinant antigen, the Ts-zh68 recombinant antigen in adult stage, the Ts-T668-C recombinant antigen of newborn larvae phase, each recombinant antigen is pressed
According to concentration than obtaining trichina cocktail antigen after mixing；
2) preparation of bonding pad: the anti-pig IgG of time-resolved fluorescence microballoon conjugated goat is sprayed on bonding pad, and vacuum is taken out
Kept dry is spare after dry；
3) preparation of detection line and nature controlling line: by the trichina cocktail antigen obtained in step 1) and 0.5-1mg/mL rabbit
Anti goat igg is sprayed in chromatographic film respectively, forms detection line and nature controlling line, and 37 DEG C of dry 2-3h are impregnated dry using sealer
Chromatographic film 1-2h after dry, subsequent 37 DEG C dry 1-2h are sealed spare；The muscle larvae phase in the trichina cocktail antigen
Ts-WM5 antigen, the Ts-WN10 antigen of enteric infection larva, the Ts-ZH68 antigen in adult stage and the Ts- of newborn larvae phase
T668-C antigen, final concentration are respectively 0.5-1mg/mL, 0.5-1mg/mL, 0.5-0.75mg/mL and 0.5-0.75mg/mL；
4) assembling of test strips: chromatography direction when on bottom plate along sample detection is successively by sample pad, bonding pad, chromatography
Pad, water absorption pad are pasted on bottom plate, and test strips are made.
It further limits, the Ts-WM5 recombinant antigen of step 1) muscle larvae phase primer such as SEQ used when preparing
Shown in ID NO.1-SEQ ID NO.2；Primer such as SEQ used when the Ts-WN10 recombinant antigen preparation of enteric infection larva
Shown in ID NO.3-SEQ ID NO.4；Primer such as SEQ ID NO.5- used when the Ts-ZH68 recombinant antigen preparation in adult stage
Shown in SEQ ID NO.6；Primer such as SEQ ID NO.7- used when the Ts-T668-C recombinant antigen preparation of newborn larvae phase
Shown in SEQ ID NO.8.
It further limits, the step 2) spraying, point sample speed is 50 μ L/cm.
It further limits, the step 3) spraying, point sample speed is 1 μ L/cm.
The present invention also provides application of the above-mentioned pigs trichina disease test strip in trichinosis detection.
It further limits, refers to after diluting test serum with sample diluting liquid, be added in test strips well, room
Temperature stands 5~10min, if with the observation of ultraviolet light irradiation test strips as a result, sample to be tested nature controlling line and detection line develop the color,
It is determined as the positive；If only nature controlling line develops the color, and detection line does not develop the color, then is determined as feminine gender.
Vocabulary of terms illustrates:
The Ts-WM5 antigen of muscle larvae phase: Immunological Screening is carried out by the cDNA library to cultivation of larvae of Trichinella spiralis from muscle, is obtained
The antigen protein of one strong reactionogenicity of high abundance, is named as WM5, and bioinformatics sequence analysis WM5 albumen belongs to serine
Protein inhibitor.
The Ts-WN10 antigen of enteric infection larva: it is carried out by the cDNA library to trichina enteric infection larva
Immunological Screening obtains the antigen protein of a strong reactionogenicity of high abundance, is named as WN10, the analysis of bioinformatics sequence
WN10 albumen has 3 structural domains for being similar to cystatin, it is believed that the albumen belongs to cystatin sample egg
It is white.
The Ts-ZH68 antigen in adult stage: Immunological Screening is carried out by the cDNA library to 3 age in days adult of trichina, is obtained
The antigen protein for obtaining a strong reactionogenicity of high abundance, is named as ZH68, and bioinformatics sequence analysis ZH68 albumen belongs to silk
Serine protease.
The Ts-T668-C antigen of newborn larvae phase: immunology sieve is carried out by the cDNA library to File of Newborn Larvae of Trichinella spiralis
Choosing, obtains the antigen protein of a strong reactionogenicity of high abundance, is named as T668, and bioinformatics sequence analyzes T668 albumen category
In serine protease protein.For pepscan analysis shows its C-terminal is immunodominant regions, i.e., main antigenic domains are of the invention
The immunodominant regions for using its C-terminal, are denoted as Ts-T668-C.
Beneficial effect
The present invention utilizes the prokaryotic expression recombinant protein of the diagnostic antigen gene of trichina different development stage: muscle larvae phase
Ts-WM5 recombinant protein, the Ts-WN10 recombinant protein of enteric infection larva, the Ts-ZH68 recombinant protein in adult stage and new
Antibody test antigen of the Ts-T668 recombinant protein of raw larval phase as pigs trichina disease, prepares cocktail antigen, is established
Trichina antibody capture fluorescence immune chromatography test strip detection method has high sensitivity and spy compared with ES ELISA
The opposite sex significantly shortens check frequency.
Test strips prepared by the present invention have antigen preparation simple, the ES such as easy to operate, quick and easy interpretation of result
The advantages of ELISA does not have, while cross reaction does not occur with other helminth positive serums when detecting, it is specific and sensitive
Degree is high；And having the characteristics that steady in a long-term, practicability is relatively strong, is easy to save, has market Development volue.With being widely popularized sky
Between, great market prospects.
Detailed description of the invention
Fig. 1 test strips structure schematic diagram of the present invention；Wherein 1 is sample pad；2 be bottom plate；3 be bonding pad；4 be cellulose nitrate
Plain film；5 be detection line；6 be nature controlling line；7 be water absorption pad；
Fig. 2 test strips testing result of the present invention determines schematic diagram；Wherein 1 is well；2 be detection line；3 be nature controlling line；
It a) is positive findings schematic diagram；It b) is negative findings schematic diagram；It c) is invalid schematic diagram.
Specific embodiment
In trichina context of detection not yet using trichina cocktail antigen prepared by the present invention as diagnostic antigen
The fluorescence immune chromatography test paper bar of pigs trichina disease antibody is detected, the present invention is using time resolution immunofluorescence microballoon as label
Object is established a kind of based on trichina chicken by the optimization of selection and reaction system and reaction condition to various solid phase materials
Pigs trichina disease antibody test fluorescence immune chromatography test paper bar of the tail wine antigen as diagnostic antigen.It, will when making test strips
The trichina cocktail antigen of preparation is sprayed in NC film detection line, and rabbit-anti goat IgG is sprayed on its nature controlling line, together with spraying
Goat-anti pig bonding pad, sample pad, blotting paper and the bottom plate of having time resolved fluorometric microballoon carry out pigs trichina disease antibody test examination
The assembling of paper slip.Measuring samples can be mobile to the top of test strips using the capillarity of NC film, if measuring samples are positive, examination
The detection line and nature controlling line of paper slip develop the color；If measuring samples are negative, test strips i.e. only nature controlling line colour developing；If test strips
The i.e. test strips that nature controlling line do not develop the color have failed.The test strips have the characteristics that special, sensitive, quick, simple, do not need higher
Professional skill and other reagents, new technology platform is provided for trichina antibody test, can be widely applied to pigs trichina disease
Base scene quick and precisely primary dcreening operation.Trichinosis test strip of the present invention and preparation method thereof is detailed below
With application.
Reagent or instrument and equipment of the present invention can be bought by commercialization approach and be obtained.
1. pigs trichina disease antibody test test strips of embodiment.
Pigs trichina disease antibody test test strips described in the present embodiment, including sample pad, bonding pad, chromatographic film, water suction
Pad and bottom plate mark the anti-pig IgG of having time resolved fluorometric microballoon conjugated goat on the bonding pad；It is provided in the sample pad
Well；The chromatographic film is equipped with detection line and nature controlling line, and trichina cocktail antigen is wherein coated in detection line, described
Trichina cocktail antigen is by the Ts-WM5 antigen of muscle larvae phase, the Ts-WN10 antigen of enteric infection larva, adult stage
Four kinds of recombinant antigen compositions of Ts-ZH68 antigen and the Ts-T668-C antigen of newborn larvae phase (end T688C immunodominant regions).
The recombination Ts-WM5 recombinant antigen of muscle larvae phase of prokaryotic expression in the trichina cocktail antigen, enteric infection larva
The concentration ratio of the Ts-T668-C antigen of Ts-WN10 recombinant antigen, the Ts-ZH68 recombinant antigen in adult stage and newborn larvae phase is
(0.5-1): (0.5-1): (0.5-0.75): (0.5-0.75) mg/mL.Rabbit-anti goat IgG is coated on the nature controlling line, it is described
Sample pad material is glass fibre element film XQ-Y8；The bonding pad material is glass fibre element film Ahistrom8964；The layer
Analysis membrane material is nitrocellulose filter Millipore135.
The preparation method of 2. pigs trichina disease antibody test test strips of embodiment.
1. the preparation of trichina cocktail antigen: Ts-WM5 recombinant protein, the enteric infection of muscle larvae phase will be expressed respectively
Property the Ts-WN10 recombinant protein of larva, the Ts-ZH68 recombinant protein in adult stage and the Ts-T668-C of newborn larvae phase recombinate egg
White each recombinant plasmid transformed host strain, after inducing expression, precipitating is collected by centrifugation in ultrasonication thallus, and urea dissolution is forgiven
Then body obtains the Ts-WM5 recombinant antigen of muscle larvae phase, the Ts-WN10 recombination of enteric infection larva respectively through affinity purification
Antigen, the Ts-ZH68 recombinant antigen in adult stage, the Ts-T668-C recombinant antigen of newborn larvae phase, by each recombinant antigen according to dense
Degree is than obtaining trichina cocktail antigen after mixing.
The specific method is as follows:
1) four kinds of recombinant antigens the preparation method is as follows:
A. the extraction of polypide total serum IgE:
Extract the total serum IgE of muscle larvae (ML), 3 age in days adults (Ad3) and newborn larvae (NBL) respectively with TRIzoL reagent.
Extracting method is as follows:
(1) 1mL TRIzoL reagent is added in the sample of every 50-100mg.
(2) glass homogenizer is homogenized 20min, and tissue is smashed in grinding to pieces.It is stored at room temperature 5min.
(3) 0.2mL chloroform/mL is added, acutely vibrates 15s, is stored at room temperature 3min.
(4) 12000g, 4 DEG C of centrifugation 15min, mixed liquor is divided into upper, middle and lower-ranking after centrifugation.Upper layer colourless aqueous phase is main
For RNA, middle layer milky is mainly the ingredients such as genomic DNA, albumen and polysaccharide, and lower layer's red organic phase is mainly phenol, chloroform.
(5) upper layer colourless aqueous phase is drawn in sterile centrifugation tube, and 0.5mL isopropanol/mL is added, is mixed by inversion, room temperature is quiet
Set 10min.
(6) 12000g, 4 DEG C of centrifugation 10min abandon supernatant.
(7) 75% ethyl alcohol of 1mL (preparation of DEPC water) is added and washs RNA precipitate.
(8) 7500g, 4 DEG C of centrifugation 5min abandon supernatant.
(9) liquid is exhausted, air-dries RNA, dissolve RNA, 55 DEG C of water-bath 10min without the DEPC water of RNase with 30 μ L.
The integrality of total serum IgE is extracted in the identification of (10) 1% agarose gel electrophoresis, and nucleic acid-protein analyzer detects its purity.
Each group sample total rna concentration is diluted to 60ng/ μ L, and -70 DEG C save backup.
B. reverse transcription reaction:
Each 0.3 μ g of different development stage polypide total serum IgE is taken, following reaction system is added:
RNAase-free water to 25 μ L,
In 42 DEG C of reaction 1h after mixing.- 20 DEG C of preservations.
C. design of primers
Respectively according to Ts-WM5 antigen gene in GenBank database, accession number: DQ864973, trichina Ts-WN10 are anti-
Protogene, accession number: 68 gene of EU263325, Ts-ZH, accession number: EU263332 and Ts-T668 gene, accession number:
AY491941 (present invention expands Ts-T668 gene C section region, and primer is as follows) sequence design target gene PCR special primer, send
It is synthesized by the raw work in Shanghai.
Template used Ts-WM5 antigen gene amplification is muscle larvae cDNA, and amplimer is as follows:
TsWM5-F:5’-TAACGAATTCATGGA AACAGAAATT-3'；
TsWM5-R:5’-GACGCTCGAGTTAACATTCAACAGTTG-3'；
Template used Ts-WN10 antigen gene amplification is 3 age in days adult cDNA, and amplimer is as follows:
TsWN10-F:5’-TAACGAATTCCAGATACTTGGTGA-3'；
TsWN10-R:5’-GACGCTCGAGTTAACATTCAACA-3'；
Template used Ts-ZH68 gene magnification is 3 age in days adult cDNA, and amplimer is as follows:
Tszh68-F:5’-TAACGAATTCATTATGAATGTGGCACCTTA-3'；
Tszh68-R:5’-GACGCTCGAGTTAACGGAAAAAAGTG-3'；
The template used gene C section region Ts-T668 amplification is newborn larvae cDNA, and amplimer is as follows:
TsT668-F:5’-TAACGAATTCGAAAATTCTCCTGAAG-3'；
TsT668-R:5’-GACGCTCGAG TTACTTAGAAAAGTG-3’。
Underscore part is EcoRI, XhoI restriction enzyme site introduced.
D. the building of expression vector:
The cDNA (muscle larvae, adult, newborn larvae) obtained with reverse transcription is that template expands Ts-WM5, Ts-WN10 respectively,
Ts-ZH68 and Ts-T668-C.
PCR reaction system (50uL) is as follows:
Reaction condition is 95 DEG C of initial denaturations 5min, 95 DEG C of 45s, 53 DEG C of 45s, 72 DEG C of 45s, recycles 30, and 72 DEG C extend eventually
10min.PCR product glue is recycled.Target gene Ts-WM5, Ts-WN10, Ts-ZH68 and Ts-T668-C that glue is recycled
And prokaryotic expression carrier pET28a carries out double digestion respectively, digestion system is as follows:
Meanwhile double digestion is carried out to prokaryotic expression carrier pET28a, digestion system is as follows:
Endonuclease reaction system is set into 37 DEG C of water-baths and stands 2h, carries out glue recycling later.By the target gene after double digestion point
It is not attached with pET28a carrier, system are as follows: 10 × T4DNA Ligase Buffer 1uL, target gene 4uL after digestion,
PET28a 1.5uL after digestion, T4DNA ligase 1uL, ddH2O 2.5uL.16 DEG C of connections are overnight.Connection product is totally converted
Ecoli DH5a competent cell, picking single colonie carry out PCR identification and sequencing.Positive recombinant plasmid converts BL21 (DE3) sense
By state cell.
E. recombinant protein inducing expression and purifying:
Correctly it is transferred to 4 expression bacterium point of plasmid Ts-WM5, Ts-WN10, Ts-ZH68 and Ts-T668-C respectively to verifying
It does not expand culture, 10mL bacterium solution is taken to be inoculated in the fresh LB liquid medium of 1L containing 50 μ g/mL Kan, in concussion shaking table
In 37 DEG C of 180rpm/min cultivate to bacterium solution OD600Reach 0.4~0.6, be added the IPTG of final concentration of 0.5mM, and by shaking table temperature
Degree is set as 37 DEG C, continues to pour into bacterium solution in 50mL centrifuge tube after cultivating 5h, after trim, 4 DEG C, 3000rpm, is centrifuged 30min.
It discards supernatant, precipitating PBS washing thalline 1 time of 5mL, after being then resuspended with 40mL bacterial lysate, after multigelation 3-4 times
Ultrasonication.Ultrasound condition is power 400W, and ultrasonic time 4 seconds, the intermittent time 5 seconds, all times were about 30 minutes.Ultrasound is broken
Broken thallus is sub-packed in 1.5mL centrifuge tube, and 4 DEG C of 10000g are centrifuged 10min, discard supernatant, stay precipitating, add 2moL/L urea 1mL/
Pipe, 4 DEG C of resuspensions 2min, 4 DEG C of 10000g centrifugation 10min discard supernatant, stay precipitating.It is purged 2-3 times repeatedly with 2moL/L urea, 4
DEG C 10000g centrifugation discards supernatant and retains precipitating, and weighing, adding 8moL/L urea dissolution inclusion body precipitating, (every 0.01g inclusion body adds
50 μ L 8moL/L urea).
Use the expression egg of 100 system of the AKTA Purifier purifying His label of GE HeaLthcare company, the U.S.
It is white.His-Trap HP column is connect with system, flow velocity is that the Bingding Buffer of 5-10 column volume of 1mL/min is balanced
His-Trap HP column is steady to ultraviolet absorption curve.Albumen loading is carried out with loading ring after ultraviolet absorption peak zeroing, when
Elution Buffer is used instead after Bingding Buffer eluting peak balance to be eluted, and postpones 500 μ L use after there is eluting peak
Sterile test tube collects eluent, by the recombinant antigen of elution, successively obtains trichina after dialysis and the concentration of 3KDa super filter tube
Recombinant antigen Ts-WM5, recombinant antigen Ts-WN10, recombinant antigen Ts-ZH68 and recombinant antigen Ts-T668-C, through SDS-PAGE
Examine purification result.
Bacterial lysate: Tris alkali 6.055g, NaCL 5.844, Na2EDTA·2H2O 0.37g, lysozyme 100mg,
PMSF 0.17g adjusts pH to 7.4, is settled to 1000mL.
Elution Buffer:NaCl 14.61g, imidazoles 17g, NaH2PO4·2H2O 0.2964g, Na2HPO4·12H2O
2.009g, urea 240g are dissolved in 450mL ddH2O adjusts pH to 7.4, is settled to 500mL.
Bingding Buffer:NaCl 14.61g, imidazoles 1g, NaH2PO4·2H2O 0.2964g, Na2HPO4·12H2O
2.009g, urea 240g are dissolved in 450mL ddH2O adjusts pH to 7.4, is settled to 500mL.
Detect the preparation of antigen:
Respectively by the Ts-WM5 of muscle larvae phase of purifying, the Ts-WN10 of enteric infection larva, the Ts-ZH68 in adult stage
It is prepared by mixing into " cocktail " antigen in proportion with the Ts-T668-C (end T688C immunodominant regions) of newborn larvae phase, it is dense eventually
Degree is respectively 1mg/mL, 1mg/mL, 0.5mg/mL and 0.5mg/mL.
2. the preparation of bonding pad: the anti-pig IgG of time-resolved fluorescence microballoon conjugated goat being sprayed on bonding pad, 4 DEG C true
Empty pump is dry, spare in 4 DEG C of kept dries；Method particularly includes:
Using the carboxyl on EDC and NHS activation microballoon, the carboxyl after activation promotes Eu-CM in conjunction with the amino on albumen
Albumen in coupling.
A) into 100 μ L (1mg, 1% (w/v)) time-resolved fluorescence microballoon, 400 μ L 0.05moL/L 2- morpholines are added
Ethanesulfonic acid MES solution, and dispersing by ultrasonic device, be then added 50 μ L n-hydroxysuccinimide NHS (16mg/mL) and
50 μ L 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide EDC (8mg/mL) are mixed, and are incubated at room temperature 1-2h；Reaction terminates
Afterwards, supernatant, 0.02M phosphate buffer (PB, 1.9mL NaH are abandoned after centrifugation2PO4.H2O(2.76g/L),8.1mL 3.56g/L
(Na2HPO4.2H2O)), after rinsing 2 times, 500 μ LPB are resuspended.
B) the anti-pig IgG of 5mg/mL goat (dilution of 0.01moL/L PBS buffer solution) of 100 μ L is added, is incubated at room temperature 1h.
C) the 0.1moL/L glycine solution of 100 μ L is added, is incubated at room temperature 30min.
D) 4 DEG C, 8000 × g is centrifuged 15min, abandons supernatant.
E) 8000 μ L coupling storing liquid is added to be resuspended.(ProcLin300,0.05mL, BSA 1g, polyethylene glycol 400,2g,
Add 0.05moL/L PBS solution to 100mL.
F) 4 DEG C of ultrasonic disperse 1min are kept in dark place under the conditions of 4 DEG C.
The above-mentioned fluorescent microsphere containing the anti-pig IgG of coupled antibody goat prepared is sprayed film instrument with test paper to be sprayed into
On 300 × 5mm glass fibre element film Ahistrom8964, speed is 50 μ L/cm, and 4 DEG C of vacuum are drained, standby in 4 DEG C of kept dries
With.
3. the preparation of detection line and nature controlling line: trichina cocktail antigen, wherein Ts-WM5 antigen, the enteron aisle of muscle larvae phase
The Ts-T668-C antigen of the Ts-WN10 antigen of infective larvae, the Ts-zh68 antigen in adult stage and newborn larvae phase, final concentration
Respectively 1mg/mL, 1mg/mL, 0.5mg/mL and 0.5mg/mL, as detection line reagent, the rabbit-anti goat IgG of 0.5mg/mL,
As nature controlling line reagent, two kinds of reagents are put on nitrocellulose filter Millipore135 respectively with test paper spray film instrument, point sample
Speed is 1 μ L/cm, 37 DEG C of dry 2h after spraying, uses sealer (BSA that phosphate buffer PBS is 2% containing mass fraction) leaching
Nitrocellulose filter MiLLipore135 1-2h, 37 DEG C of dry 1-2h after bubble is dry, 4 DEG C be sealed it is spare.
4. the assembling of test strips: on bottom plate along detection when chromatography direction successively by sample pad, bonding pad, chromatography pad,
Water absorption pad is pasted on bottom plate, and test card is made, method particularly includes: by shown in Fig. 1 schematic diagram, by the dedicated black floor of fluorescence,
Sample pad XQ-Y8, bonding pad Ahistrom8964, water absorption pad H5072, nitrocellulose filter Millipore135 stick together,
And the test strips of 3.5mm wide are cut into test strips cutting machine, test strips are put into test card, by test card be put into containing
In the hermetic bag of desiccant, saved backup after sealing in 4 DEG C.
Investigate pigs trichina disease antibody test test strips sensitivity, specificity and stability prepared by the present invention.
1. sensitivity is tested:
Anti-trichinella antibody in the Swine serum under different infectious conditions is detected with trichina test strip, will test result
It is compared with the OIE enzyme-linked immunosorbent antibodies detection method of standard provided.The test strips applied in ensuring to detect are from same
One batch.The step of standard Enzyme-linked Immunosorbent Assay method that OIE is provided, is as follows:
(1) coating buffer dilutes cross reaction to 5 μ g/mL, and every 0.5 μ g of hole is coated with, in 4 DEG C of standing 12h.
(2) coating buffer in ELISA Plate is discarded, cleaning solution washs 3 times.
(3) 100uL is added by 1:50 dilution treated Swine serum, is incubated at room temperature 30min.
(4) step (2) are repeated.
(5) the rabbit-anti pig IgG of 100uL HRP label is added, 1:1000 times dilutes, and is incubated for 30min.
(6) after repeating step (2), the tmb substrate developing solution of 100uL is added.
(7) after 10min, absorbance detection is carried out under 450nm wavelength condition, if it find that acquired results are negative 4
It is determined as the positive again.
Test strips of the present invention and ELISA detect 60 parts of pig infection trichina serum respectively, and ELISA detects 58 parts as the result is shown
Serum is the positive, and 2 parts of serum are feminine gender, and the detection of " cocktail " recombinant antigen test strips may also detect that 58 parts of positive serums,
Wherein 1 part of ES ELISA is feminine gender, and " cocktail " recombinant antigen test strips are the positive.90 parts of negative serums, ELISA 86 are
Feminine gender, 89 parts of " cocktail " antigen are feminine gender.Illustrate two methods concordance rate and goodness of fit in terms of detecting animal trichinosis
It is relatively strong；The no significant difference of test strips and ELISA in sensibility, " cocktail " recombinant antigen can reduce vacation simultaneously
Positive rate.Illustrate pigs trichina disease antibody detection method (ELISA) that sensibility of the invention and OIE are recommended quite, and it is special
Property be higher than ES ELISA.The advantages of not having but also with ELISA such as easy to operate, the quick and easy interpretations of result simultaneously has wide
General popularization space, great market prospects.
2. the specific detection of trichinosis test strip:
Other helminth positive serums are detected, mainly there is ascaris suum, toxoplasma, pork measles, Schistosoma japonicum and Hua Zhi testis
Fluke positive serum and negative serum, and carry out specific analysis.In addition to Trichinella sui positive serum, other serum are yin
Property, i.e. trichinosis test strip does not occur cross reaction with other helminths, meets its specific requirements.
3. pig infects various dose and the detection of time point trichina:
Anti-trichinella antibody detection to different time points Swine serum after infection trichina, in severe infection (20000
Item/only) digestion method is detected as every gram of Muscle Larva number 636.7-1257.6Lpg, ES ELISA 20-25 days and can detecte rotation
Caterpillar infection, and cocktail antigen paper item can detect trichinzation at 16-20 days.In grade and moderate infection (1000/
Only) digestion method is detected as every gram of Muscle Larva number 30.7-98.5Lpg, ES ELISA 30-40 days and can detecte trichina sense
Dye, and cocktail antigen paper item can detect trichinzation at 20-30 days.In minuent infection (100/only) digestion
Method, which is detected as every gram of Muscle Larva number 1.7-7.2Lpg, ES ELISA 35-50 days, can detecte trichinzation, and this hair
The bright test strips can detect trichinzation at 25-35 days, compared with ES ELISA, the test paper based on cocktail antigen
Detection sensitivity significantly improves, and can early detect Anti-trichinella antibody in trichinzation.Further to trichina sense
The serum in dye later period is that 90,120,150 days serum is detected after infecting, and ES ELSIA and test strips are in the trichina not same feeling
Stain amount may detect that trichina antibody.It can be seen that the Immunofluorescence test paper strip pair of antigen preparation of the present invention
Trichinosis early diagnosis has certain values.
4. field sample analysis:
Using ELISA and test strip prepared by the present invention to the 13200 bull stable breeding of Yunnan trichinosis hotspot
Pig carries out antibody test, and ES ELISA detects pig totally 12 of trichinzation seropositivity, while test strips can also be
It is positive.And 3 ELISA ELISA are detected as feminine gender, test strips test positive.15 positive serum pigs are examined through digestion method
12 detectable trichina polypides are surveyed, show the test strips of the present invention using cocktail antigen compared to existing
ELISA and conventional digestion method testing result are sensitiveer, are suitble to the large area generaI investigation and on-site test of the infection of base's Trichinella sui.
5. the repeatability of trichinosis test strip:
The test strips of different batches are selected to carry out repeated analysis.It is detected respectively with the test strips of 4 different batches, warp
30 parts of Trichinella suis infection positive serum and 20 parts of normal pig negative serums after ELISA detection；The test strips of 4 different batches
It is the positive to 30 parts of positive serum testing results, is feminine gender to 20 parts of negative serum testing results, shows that the present invention is good
Good sensitivity and specificity, and stability and repeatability are good.
6. the Detection of Stability of trichinosis test strip:
Test strips are placed in 4 DEG C and room temperature preservation, the pig positive and each 10 parts of negative serum is detected respectively every two weeks, examines altogether
It surveys 12 times, sensibility and specificity is 100%, shows that test strips at least can be reserved for 6 months at 4 DEG C, i.e., of the invention is effective
Phase is at least 6 months.The 2nd week to the 16th week result sensibility and specificity of test strips of room temperature preservation is 100%, and is examined
Apparent difference is not present in the color of survey line and control line.From the 18th week, the color of T line and C line shoaled, therefore room temperature ring
It can be reserved for 16 weeks under border.It is steady in a long-term to prove that the present invention has the characteristics that, practicability is relatively strong, is easy to save, has market exploitation
Value.
7. the stability verification result of trichinosis test strip:
Test strips are stored in room temperature and 4 DEG C respectively after detection, under room temperature testing result 4 months when it is distinguishable clear, 4
It is still high-visible at testing result 6 months under the conditions of DEG C, illustrate that the testing result holding time of the invention is room temperature at least four
The moon and 4 DEG C of at least six moons, present invention feature steady in a long-term was shown again.
The preparation method of 3. pigs trichina disease test strip of embodiment.
Embodiment 2 is repeated, is with the difference of embodiment 2, the trichina in this implementation in step 3 as detection line reagent
Cocktail antigen, wherein the Ts-WM5 antigen of muscle larvae phase, the Ts-WN10 antigen of enteric infection larva, the Ts- in adult stage
The Ts-T668-C antigen of ZH68 antigen and newborn larvae phase, final concentration are respectively 0.5mg/mL, 0.5mg/mL, 0.75mg/mL and
0.75mg/mL；As the rabbit-anti goat IgG of nature controlling line reagent, concentration 0.75mg/mL.Using inspection described in embodiment 2
Survey method investigates sensitivity, specificity and the stability of pigs trichina disease antibody test test strips manufactured in the present embodiment, knot
Fruit shows: being detected detection sensitivity equally can be improved compared with ES ELSIA at this concentration, reached and early detect
Trichinella sui antiserum, but fluorescence intensity is weaker than embodiment 2 in T line and C line, in severe and grade and moderate infection, detection spirit
Quick property is identical, and (100 /) the present embodiment can detect trichinzation at 30-35 days in minuent infection.To field
Identical as ES ELSIA testing result when pattern detection, both ES ELISA and digestion method were detected as the pig of trichinzation, implemented
Example is the positive, and the stability of test strips and repeated result are the same as embodiment 2.
Application of the pigs trichina disease test strip of the present invention of embodiment 4. in trichinosis detection.
Sample dilution: respectively with sample diluting liquid by trichina Positive Sera (such as pig infect trichina serum), yin
Property serum (such as normal swine serum) and serum to be checked (such as Swine serum) sample dilute 10 times, i.e., 10 μ L are added into 90 μ L dilutions
Swine serum is mixed with pipettor.Pigs trichina disease antibody test test strips prepared by the present invention are lain against into operating table surface, are loaded
Upward, the blood serum sample after 100 μ L dilution is added into well is stored at room temperature 5~10min in hole.The sample diluting liquid: chlorine
Change sodium (NaCL) 9g, bovine serum albumin(BSA) (BSA) 2g, distilled water is added to be formulated to 1000mL.It is shone with ultraviolet lamp 360-370nm
Penetrate test strips observation result.
Result judgement should carry out in 20min after sample-adding.If positive serum samples nature controlling line and detection line develop the color；Yin
Property serum only nature controlling line develops the color, and detection line does not develop the color；The colour developing of sample to be tested nature controlling line, then test establishment, as a result effectively.
If sample to be tested nature controlling line and detection line develop the color, it is determined as the positive；If only nature controlling line develops the color, and detects
Line does not develop the color, then is determined as feminine gender；If nature controlling line does not develop the color, then detection is invalid, and Ying Genghuan test strips detect again, fluorescence immunoassay
Chromatograph test strip testing result determines schematic diagram as shown in Figure 2.
Nucleotides sequence list
<110>Jilin University
<120>a kind of pigs trichina disease antibody test test strips and the preparation method and application thereof
<130>
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> TsWN10-F
<400> 1
taacgaattc cagatacttg gtga 24
<210> 2
<211> 23
<212> DNA
<213> TsWN10-R
<400> 2
gacgctcgag ttaacattca aca 23
<210> 3
<211> 25
<212> DNA
<213> TsWM5-F
<400> 3
taacgaattc atggaaacag aaatt 25
<210> 4
<211> 27
<212> DNA
<213> TsWM5-R
<400> 4
gacgctcgag ttaacattca acagttg 27
<210> 5
<211> 30
<212> DNA
<213> TsZH68-F
<400> 5
taacgaattc attatgaatg tggcacctta 30
<210> 6
<211> 26
<212> DNA
<213> TsZH68-R
<400> 6
gacgctcgag ttaacggaaa aaagtg 26
<210> 7
<211> 26
<212> DNA
<213> TsT668-F
<400> 7
taacgaattc gaaaattctc ctgaag 26
<210> 8
<211> 25
<212> DNA
<213> TsT668-R
<400> 8
gacgctcgag ttacttagaa aagtg 25
Claims (9)
1. a kind of pigs trichina disease antibody test test strips, including sample pad, bonding pad, chromatographic film, water absorption pad and bottom plate, special
Sign is, the anti-pig IgG of having time resolved fluorometric microballoon conjugated goat is marked on the bonding pad；It is provided with and adds in the sample pad
Sample hole；The chromatographic film is equipped with detection line and nature controlling line, and trichina cocktail antigen, the rotation are wherein coated in detection line
Ts-WM5 recombinant antigen, the Ts-WN10 recombinant antigen of enteric infection larva, adult of the caterpillar cocktail antigen by the muscle larvae phase
The Ts-ZH68 recombinant antigen and the Ts-T668-C recombinant antigen of newborn larvae phase of phase forms；Rabbit-anti is coated on the nature controlling line
Goat IgG.
2. pigs trichina disease antibody test test strips according to claim 1, which is characterized in that the sample pad material is
Glass fibre element film XQ-Y8；The bonding pad material is glass fibre element film Ahistrom8964；The chromatography membrane material is nitre
Acid cellulose film Millipore135.
3. pigs trichina disease antibody test test strips according to claim 1, which is characterized in that the trichina cocktail
The recombination Ts-WM5 antigen of muscle larvae phase, the Ts-WN10 antigen of enteric infection larva, adult stage of prokaryotic expression in antigen
The concentration ratio of Ts-ZH68 antigen and the Ts-T668-C antigen of newborn larvae phase is (0.5-1): (0.5-1): (0.5-0.75):
(0.5-0.75)mg/mL。
4. the preparation method of pigs trichina disease antibody test test strips described in claim 1, which is characterized in that including walking as follows
It is rapid:
1) preparation of trichina cocktail antigen: Ts-WM5 recombinant protein, the enteric infection children of muscle larvae phase will be expressed respectively
The recombination egg of C-terminal in the Ts-WN10 recombinant protein of worm, the Ts-ZH68 recombinant protein in adult stage and the Ts-T668 of newborn larvae phase
White each recombinant plasmid transformed host strain, after inducing expression, precipitating is collected by centrifugation in ultrasonication thallus, and urea dissolution is forgiven
Then body obtains the Ts-WM5 recombinant antigen of muscle larvae phase, the Ts-WN10 recombination of enteric infection larva respectively through affinity purification
Antigen, the Ts-ZH68 recombinant antigen in adult stage, the Ts-T668-C recombinant antigen of newborn larvae phase, by each recombinant antigen according to dense
Degree is than obtaining trichina cocktail antigen after mixing；
2) preparation of bonding pad: the anti-pig IgG of time-resolved fluorescence microballoon conjugated goat is sprayed on bonding pad, after vacuum is drained
Kept dry is spare；
3) preparation of detection line and nature controlling line: by the trichina cocktail antigen obtained in step 1) and 0.5-1mg/mL rabbit-anti mountain
Sheep IgG is sprayed in chromatographic film respectively, forms detection line and nature controlling line, 37 DEG C of dry 2-3h, after impregnating drying using sealer
Chromatographic film 1-2h, subsequent 37 DEG C dry 1-2h are sealed spare；The muscle larvae phase in the trichina cocktail antigen
Ts-WM5 antigen, the Ts-WN10 antigen of enteric infection larva, the Ts-ZH68 antigen and the Ts- of newborn larvae phase in adult stage
T668-C antigen, final concentration are respectively 0.5-1mg/mL, 0.5-1mg/mL, 0.5-0.75mg/mL and 0.5-0.75mg/mL；
4) assembling of test strips: chromatography direction when on bottom plate along sample detection successively by sample pad, bonding pad, chromatography pad,
Water absorption pad is pasted on bottom plate, and test strips are made.
5. the preparation method according to claim 4, which is characterized in that the Ts-WM5 recombination of step 1) the muscle larvae phase is anti-
Primer used is as shown in SEQ ID NO.1-SEQ ID NO.2 when original preparation；The Ts-WN10 recombination of enteric infection larva is anti-
Primer used is as shown in SEQ ID NO.3-SEQ ID NO.4 when original preparation；When the Ts-ZH68 recombinant antigen preparation in adult stage
Primer used is as shown in SEQ ID NO.5-SEQ ID NO.6；The Ts-T668-C recombinant antigen of newborn larvae phase prepares when institute
Primer is as shown in SEQ ID NO.7-SEQ ID NO.8.
6. the preparation method according to claim 4, which is characterized in that the step 2) spraying, point sample speed are 50 μ L/
cm。
7. the preparation method according to claim 4, which is characterized in that the step 3) spraying, point sample speed are 1 μ L/cm.
8. application of the pigs trichina disease antibody test test strips described in claim 1 in trichinosis detection.
9. application according to claim 8, which is characterized in that after diluting test serum with sample diluting liquid, be added to
In test strips well, it is stored at room temperature 5~20min, if observed with ultraviolet light irradiation test strips as a result, sample to be tested nature controlling line
It develops the color with detection line, is then determined as the positive；If only nature controlling line develops the color, and detection line does not develop the color, then is determined as feminine gender.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910644896.XA CN110221067A (en) | 2019-07-16 | 2019-07-16 | A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910644896.XA CN110221067A (en) | 2019-07-16 | 2019-07-16 | A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110221067A true CN110221067A (en) | 2019-09-10 |
Family
ID=67813516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910644896.XA Pending CN110221067A (en) | 2019-07-16 | 2019-07-16 | A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110221067A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734495A (en) * | 2019-09-30 | 2020-01-31 | 吉林大学 | hybridoma cell strains, monoclonal antibody of trichina-resistant serine protease in new larva stage and application |
| CN110954704A (en) * | 2019-12-30 | 2020-04-03 | 北京维德维康生物技术有限公司 | Trichina antibody detection test strip based on Ts31 gene and application thereof |
| CN111187754A (en) * | 2020-01-21 | 2020-05-22 | 吉林大学 | Hybridoma cell strain, anti-trichina intestinal serine protease monoclonal antibody produced by hybridoma cell strain and application of anti-trichina intestinal serine protease monoclonal antibody |
| CN111303276A (en) * | 2019-12-20 | 2020-06-19 | 吉林大学 | B cell epitope polypeptide of trichina intestinal cysteine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
| CN111333709A (en) * | 2020-03-17 | 2020-06-26 | 吉林大学 | B cell epitope polypeptide of trichina muscle larva serine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
| CN111505295A (en) * | 2020-04-23 | 2020-08-07 | 吉林大学 | Trichinosis antibody detection kit based on competitive monoclonal antibody and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007159489A (en) * | 2005-12-14 | 2007-06-28 | Gifu Univ | Species-specific antigen of trichinella and method for testing infection of trichinella utilizing the antigen |
| CN103305523A (en) * | 2013-06-24 | 2013-09-18 | 吉林大学 | Trichina Tsp-05904 recombinant protein antigen and preparation method thereof |
| CN107098955A (en) * | 2017-06-28 | 2017-08-29 | 潍坊汉唐生物工程有限公司 | It is a kind of to be used to diagnose recombinant antigen composition of toxoplasma antibody and preparation method thereof |
-
2019
- 2019-07-16 CN CN201910644896.XA patent/CN110221067A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007159489A (en) * | 2005-12-14 | 2007-06-28 | Gifu Univ | Species-specific antigen of trichinella and method for testing infection of trichinella utilizing the antigen |
| CN103305523A (en) * | 2013-06-24 | 2013-09-18 | 吉林大学 | Trichina Tsp-05904 recombinant protein antigen and preparation method thereof |
| CN107098955A (en) * | 2017-06-28 | 2017-08-29 | 潍坊汉唐生物工程有限公司 | It is a kind of to be used to diagnose recombinant antigen composition of toxoplasma antibody and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| 刘丽等: "《胶体金免疫层析技术》", 30 September 2017 * |
| 吴英松等: "《时间分辨荧光免疫技术》", 30 January 2009 * |
| 唐斌: "WN10蛋白生物学功能的研究及旋毛虫病免疫学检测方法的建立", 《中国博士学位论文全文数据库农业科技辑》 * |
| 张小波: "旋毛虫病荧光免疫层析抗体检测试纸条的建立", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 王春: "上转发光免疫层析技术快速检测猪抗旋毛虫lgG抗体方法的建立", 《中国兽医学报》 * |
| 翟铖铖: "旋毛虫两个发育时期丝氨酸蛋白酶的结构及功能的比较与分析", 《中国生物制品学杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734495A (en) * | 2019-09-30 | 2020-01-31 | 吉林大学 | hybridoma cell strains, monoclonal antibody of trichina-resistant serine protease in new larva stage and application |
| CN111303276A (en) * | 2019-12-20 | 2020-06-19 | 吉林大学 | B cell epitope polypeptide of trichina intestinal cysteine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
| US11634481B2 (en) | 2019-12-20 | 2023-04-25 | Jilin University | B-cell epitope of Trichinella spiralis cysteine protease inhibitor, hybridoma cell line, monoclonal antibody and uses thereof |
| CN110954704A (en) * | 2019-12-30 | 2020-04-03 | 北京维德维康生物技术有限公司 | Trichina antibody detection test strip based on Ts31 gene and application thereof |
| CN111187754A (en) * | 2020-01-21 | 2020-05-22 | 吉林大学 | Hybridoma cell strain, anti-trichina intestinal serine protease monoclonal antibody produced by hybridoma cell strain and application of anti-trichina intestinal serine protease monoclonal antibody |
| CN111333709A (en) * | 2020-03-17 | 2020-06-26 | 吉林大学 | B cell epitope polypeptide of trichina muscle larva serine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
| CN111333709B (en) * | 2020-03-17 | 2023-09-08 | 吉林大学 | B cell epitope polypeptide of serine protease inhibitor in trichina larval stage, hybridoma cell strain, monoclonal antibody and application |
| CN111505295A (en) * | 2020-04-23 | 2020-08-07 | 吉林大学 | Trichinosis antibody detection kit based on competitive monoclonal antibody and preparation method thereof |
| CN111505295B (en) * | 2020-04-23 | 2021-04-16 | 吉林大学 | Trichinosis antibody detection kit based on competitive monoclonal antibody and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110221067A (en) | A kind of pigs trichina disease antibody test test strips and the preparation method and application thereof | |
| CN110642926B (en) | African swine fever virus p72 recombinant protein, monoclonal antibody and test paper | |
| CN110658339B (en) | Test paper and kit for detecting African swine fever virus and preparation method thereof | |
| CN112964884B (en) | Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus past infection detection | |
| CN105606826B (en) | A kind of kit of enzyme linked immunosorbent detection fowl chlamydia psittaci | |
| CN111518176B (en) | Paired antigen for novel coronavirus antibody double-antigen sandwich detection, detection test paper and preparation method thereof | |
| Marcilla et al. | Leucine aminopeptidase is an immunodominant antigen of Fasciola hepatica excretory and secretory products in human infections | |
| CN113215107B (en) | Time-resolved fluoroimmunoassay kit for detecting novel coronavirus and preparation method thereof | |
| CN111007257A (en) | Porcine pseudorabies virus gE protein antibody detection immune blocking chromatography kit and application thereof | |
| CN114736290B (en) | Nanometer antibody capable of recognizing porcine pseudorabies virus with high accuracy and sensitivity, preparation method and application | |
| CN110221066B (en) | Trichinosis fluorescence immunochromatographic assay test strip and preparation method and application thereof | |
| CN109374887A (en) | Bovine viral diarrhea virus antigen colloidal gold detection kit and its application | |
| CN103616509B (en) | Detect E III-indirect ELISA antibody assay kit and the application of Latex agglutination test | |
| CN111537741A (en) | Double-antigen sandwich immunofluorescence chromatography kit for detecting African swine fever virus CD2v protein antibody | |
| CN109810191B (en) | Monoclonal antibody for resisting sheep skeletal muscle troponin I and application thereof | |
| CN102533663B (en) | Foot-and-mouth disease hybridoma cell line, monoclonal antibody, detection reagent and test kit | |
| CN107884578A (en) | For echinococcosis antibody test card in Quantitative detection serum | |
| CN101403746B (en) | Conjugate used for immunity detection | |
| CN102175861A (en) | Indirect enzyme linked immunosorbent assay (ELISA) detection method for European- and American-type porcine reproductive and respiratory syndrome virus (PRRSV) antibodies | |
| CN111505295B (en) | Trichinosis antibody detection kit based on competitive monoclonal antibody and preparation method thereof | |
| CN103820471A (en) | Recombined chlamydia trachomatis protein and application thereof | |
| CN113721035B (en) | Colloidal gold immunochromatographic test paper card for detecting African swine fever virus antibody | |
| CN106188249B (en) | For detecting the antigen and method and kit of PEDV variation strain antibody | |
| CN108761091A (en) | Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof | |
| CN109975541A (en) | A kind of detection card and preparation method thereof of quick detection canine distemper virus antigen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190910 |
|
| RJ01 | Rejection of invention patent application after publication |