CN102608330A - Immunochromatographic strip for detecting alveolar echinococcosis and preparation method thereof - Google Patents
Immunochromatographic strip for detecting alveolar echinococcosis and preparation method thereof Download PDFInfo
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- CN102608330A CN102608330A CN2012100627786A CN201210062778A CN102608330A CN 102608330 A CN102608330 A CN 102608330A CN 2012100627786 A CN2012100627786 A CN 2012100627786A CN 201210062778 A CN201210062778 A CN 201210062778A CN 102608330 A CN102608330 A CN 102608330A
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Abstract
The invention belongs to the field of biological engineering, and discloses an immunochromatographic strip for detecting alveolar echinococcosis, which comprises a sample pad and a water-absorbing pad. A cellulose membrane is disposed between the sample pad and the water-absorbing pad. A gold label pad is disposed between the sample pad and the cellulose membrane. The sample pad, the gold label pad and the cellulose membrane are tightly connected together. A quality control line is disposed at one end, close to the water-absorbing pad, of the cellulose membrane. A detection line is disposed on the cellulose membrane and between the quality control line and the gold label pad. The detection line contains a recombinant antigen Em18 of alveolar echinococcosis. A probe with a colloidal gold label is disposed on the gold label pad. The quality control line contains an antibody or anti-antibody which can be specifically bound to the probe. The immunochromatographic strip for detecting alveolar echinococcosis has the advantages of simplicity, sensitiveness, specificity and rapidness, and is applicable to clinical and field uses.
Description
Technical field
The present invention relates to bioengineering field, relate in particular to a kind of immunity-chromatography test strip and preparation method, is a kind of immunity-chromatography test strip and preparation method who detects alveolar echinococcosis specifically.
Background technology
Hydatidosis (echinococcosis) is claimed echinococcosis (Hydatid disease or Hydatidosis) again; Be that larva by the Echinococcus tapeworm parasitizes a kind of infecting both domestic animals and human parasitic disease that causes in the people, animal body; Can infect human Echinococcus tapeworm and have 4 kinds: Echinococcus granulosus (Echinococcus granulosus; Eg), Echinococcus multilocularis (Echinococcusmultilocularis saves echinococcus (E.oligarthrus) and Fu Shi echinococcus (E.vogeli) Em), less.The adult of these 4 kinds of echinococcus with larval phase form different, can cause dissimilar hydatidosis.China have two kinds of hydatidosis be echinococcosis granulosa (cysticechinococ-cosis CE) claims cystic echinococcosis again, and echinococcosis multilocularis (alveolarechinococcosis AE) claims alveolar echinococcosis again.
Echinococcosis is an important worldwide public health problem, is widely current in Europe, Asia and Africa and mediterranean region coastwise contries, and Australia and South America.In China, echinococcosis mainly is popular in 12 provinces, municipal pastoral area and farming and pastoral areas such as western part, and wherein Xinjiang, Qinghai, Gansu, Ningxia, Tibet, Inner Mongol and western Sichuan are the most serious, is the highest area of echinococcosis morbidity rate in the world.In addition, province some areas such as Jilin, Shaanxi, Yunnan, Guizhou and Henan also have popular among a small circle.The compromised population in the popular district of echinococcosis about 7,000 ten thousand.Ministry of Public Health's " sick on-site investigation of national human body important parasite " report shows that this 12 province (district) echinococcosis morbidity rate is 1.084%, calculates that in view of the above present popular district has patient 380,000 people approximately.Because the B ultrasonic diagnostic result has certain loss, the actual diseased number possibly reach more than 600,000 people.Crowd's infection rate average out to 11.98% is found in Serological testing, and number of the infected is about 7,000,000 people, compromised population about 7,000 ten thousand.
Echinococcosis need be performed the operation or long-term medication treatment, and effect is limited.Wherein the cystic echinococcosis capsules rupture can cause irritated shock and cause death.It is a kind of lethal parasitic disease that alveolar echinococcosis is called as " pernicious echinococcosis ", and the case fatality rate of 10-15 is up to more than 90%.The echinococcosis patient who fails to pinpoint a disease in diagnosis loses treatment opportunity and threat to life.Echinococcosis is not only brought great misery and heavy medical treatment burden to the patient; And the peak of morbidity is the person between twenty and fifty in 20-50 year, and serious patient loses the labour, brings great pressure to society; Be that the western farming and pastoral area masses drive into poverty by medical crises the major reason of backing into poverty by medical crises.In addition, owing to the infection rate quite high (can reach 90%) of echinococcosis cattle and sheep, annual economic loss of also causing for China's livestock products because of echinococcosis reaches billions of.Therefore the echinococcosis serious harm people's life and health not only, and have a strong impact on social and economic development, stability in border areas.
The infection of echinococcosis for a long time (several years or more than ten years) asymptomatic, in case symptom manifests medication effect poor (reaching effective concentration because of there being two-layer abundant capsule parcel to make medicine be difficult to get into), and the treatment of having to undergo surgery, even will undergo surgery repeatedly.Therefore, studying suitable effective immune diagnostic technique and be used for early diagnosis, to improve medication effect, is the fundamental way that effectively reduces M & M most, is problem demanding prompt solution in the echinococcosis control.
Diagnosis to echinococcosis at present greatly relies on the iconography method, like physical diagnostic methods such as X-ray examination, Type B Ultrasonic Diagnosis, CT scan, nuclear magnetic resonance.But these methods can not be carried out early diagnosis (the Echinococcus hydatid cyst patient that imaging diagnosis goes out not too is fit to drug therapy), and can not effectively differentiate and mistaken diagnosis some tumour, abscess or lump.Because image check equipment carries inconvenience, expensive, and higher to operating personnel's technical requirement in addition, the application of (electric power supply all is restricted in the backcountry especially) is restricted in medical diagnosis on disease and epidemiology survey.
The application of immunological method in the echinococcosis diagnosis more and more comes into one's own; The most effectively amynologic diagnostic method is that application of purified native antigen or recombinant antigen detect Echinococcus hydatid cyst patient specific antibody, and detection method commonly used has indirect hemagglutination method (Indirect haemagglutinationassay; IHA), enzyme linked immunosorbent assay (ELISA), enzyme linked immunological electrotransfer imprinting (Enzyme-linked immunoelectrotransfer blot assay; And gold-marking immunity point imprinting (Gold-labelled dotblotting EITBA); GDB; Be commonly called as embrane method or percolation).But these methods or waste time and energy or need the cold chain system to preserve reagent or instrument and equipment and having relatively high expectations of operating personnel are not suitable for on-the-spot the use.
The use of antigen has directly determined the susceptibility and the specificity that detect in above-mentioned immunization method.Crude antigen, purifying antigen and recombinant antigen three phases have been experienced in the research of echinococcosis immunodiagnosis antigen.
Particulate echinococcus packing liquid crude antigen (HCF) is widely used in the diagnosis of cystic echinococcosis, is characterized in that the susceptibility that detects is higher, can reach 75%-95%, but be easy to other diseases such as cysticercosis etc. cross reaction takes place.Adopt diverse ways that capsule liquid crude antigen is separated and purifying to the many scholars of this problem, significantly improved the susceptibility and the specificity that detect.
Em18 antigen is alveolar sphere larva of a tapeworm or the cercaria of a schistosome species-specific antigen component; There is report to show the diagnosis that the natural Em18 antigen of purifying is used for alveolar echinococcosis; Susceptibility and specificity are respectively up to 90.91% and 93.80%; Positive desired value and negative desired value are respectively 83.33% and 96.80%, can also distinguish activity and inactivity focus to a certain extent, thereby are a kind of have diagnosis and immune diagnostic antigens of following up a case by regular visits to meaning; But natural Em18 extracts from artificial challenge's alveolar sphere larva of a tapeworm or the cercaria of a schistosome protoscolex; Extract that difficulty is big, output is few, can not satisfy the diagnosis needs, thereby the reorganization EM18 antigen of synthetic becomes a kind of specific diagnosis antigen that using value is arranged in alveolar echinococcosis antidiastole and the course of disease are followed up a case by regular visits to.
Summary of the invention
The purpose of this invention is to provide a kind of immunity-chromatography test strip and preparation method who detects alveolar echinococcosis; The method that the immunity-chromatography test strip of described this detection alveolar echinococcosis and preparation method will solve detection cystic echinococcosis of the prior art and alveolar echinococcosis is complicated, and technical matters that can not early diagnosis.
The invention provides a kind of immunity-chromatography test strip that detects alveolar echinococcosis; Comprise a sample pad and an adsorptive pads; Between described sample pad and adsorptive pads, be provided with a cellulose membrane; Described adsorptive pads closely is connected with cellulose membrane, between described sample pad and described cellulose membrane, is provided with a gold mark pad, closely connects between described sample pad, gold mark pad and the cellulose membrane; End away from gold mark pad on said cellulose membrane is provided with nature controlling line; On the cellulose membrane between described nature controlling line and the gold mark pad, a detection line is set, described detection line is made up of the antigen Em18 of the alveolar echinococcosis of reorganization, and described gold mark pad is provided with the probe of colloid gold label; The probe of described colloid gold label is to be antibody or staphylococcal protein A or the streptococcal protein G that antigen-immunized animal obtains with the human IgG, and described nature controlling line contains the antibody or the antiantibody that can combine with the colloid gold label probe specificity.
Further, an end of described cellulose membrane is arranged on the downside of described gold mark pad, and an end of described gold mark pad is arranged on the downside of described sample pad.
Further, described sample pad, gold mark pad, cellulose membrane and adsorptive pads are arranged on the backboard.
Further, the colloid gold label probe in the said gold mark pad is the monoclonal antibody that obtains as the antigen immune mouse with human IgG, and said nature controlling line contains the sheep anti mouse antiantibody.
Further, the colloid gold label probe in the said gold mark pad can be staphylococcal protein A or streptococcal protein G, and said nature controlling line contains anti-staphylococcal protein A antibody or streptococcus G protein antibodies.
Further, the protein concentration of the antigen Em18 of the alveolar echinococcosis of reorganization is 0.1-10mg/mL, and quantity for spray is 8-12 μ L/cm; The concentration of colloid gold label probe is counted 1-5mg/15-20mL with protein concentration.
Further, the concentration of sheep anti mouse antiantibody solution is 0.1-5mg/mL, and quantity for spray is 8-12 μ L/cm.
Further, anti-staphylococcal protein A antibody or streptococcus G protein antibodies solution concentration are 0.1-5mg/mL, and quantity for spray is 8-12 μ L/cm.
Further; The plain film of described dimension is selected from nitrocellulose filter or CAM; The gold mark pad of said support colloid gold label probe is selected from glass fibre membrane or polyester film, and said sample pad is selected from hemofiltration film, spun glass or thieving paper, and said adsorptive pads is a thieving paper.
The present invention also provides above-mentioned a kind of preparation method who detects the immunity-chromatography test strip of alveolar echinococcosis; The step of antiantibody of step and preparation and colloid gold label probe specific bond of probe of anti-human IgG of step, a preparation mark of antigen Em18 that comprises the alveolar echinococcosis of a preparation reorganization; After above-mentioned steps is accomplished; The antigen Em18 solution spraying of the alveolar echinococcosis of recombinating is formed detection line to cellulose membrane, can to cellulose membrane, form nature controlling line with the antibody or the antiantibody solution spraying of colloid gold label probe specific bond, the adjacent setting of described nature controlling line and detection line near adsorptive pads; With the tunica fibrosa that is coated with detection line, nature controlling line in the environment of relative humidity below 40% dry 1-2 hour; The step for preparing gold mark pad then in the step of described preparation gold mark pad, immerses glass fibre membrane or polyester film in the probe solution of colloid gold label; After the taking-up with gold mark pad in the environment of relative humidity below 40% dry 1-2 hour; Said cellulose membrane is sticked on the middle part of a backboard, and adsorptive pads sticks on the end near nature controlling line of said cellulose membrane, gold mark pad is sticked on the end near detection line of cellulose membrane; Sample pad is pasted on the end away from cellulose membrane that the gold mark fills up, obtains detecting the immunity-chromatography test strip of alveolar echinococcosis.
Further; The antigen Em18 of the alveolar echinococcosis of described reorganization prepares as follows: many rooms of sheep liver echinococcus protoscolex that will infect alveolar echinococcosis carries out the extracting of the total RNA of protoscolex with kit; Carry out synthetic cDNA first chain of reverse transcription with the reverse transcription kit, the upstream primer sequence is shown in SEQ ID NO:1, and the downstream primer sequence is shown in SEQ ID NO:2; In upstream primer, introduced the BamHI site; In downstream primer, having introduced the EcoRI restriction enzyme site, is template with synthetic cDNA first chain, RT-PCR method amplifying target genes fragment.
The present invention also provides a kind of kit; Constitute by a box body; The immunity-chromatography test strip of above-mentioned detection alveolar echinococcosis is arranged in the described box body; A side of described box body is provided with a well and a viewport, and described well is positioned at the top of sample end adsorptive pads, and described viewport is positioned at the top of described detection line and nature controlling line.
Can infect human Echinococcus tapeworm 4 kinds of echinococcus adult with larval phase form different, can cause dissimilar hydatidosis.Contain the anti-echinococcus antibody of the echinococcus antigen that can specificity combines to be infected in echinococcosis patient's body inner blood, detect in the subject inner blood whether contain anti-echinococcus antibody, can act on the index whether experimenter suffers from echinococcosis.
The immunity-chromatography test strip of detection echinococcosis of the present invention and pathogen kind is applicable to whole blood sample and the blood serum sample that extracts in the alveolar echinococcosis patient body.For whole blood sample, the sample pad in the immunity-chromatography test strip of detection alveolar echinococcosis of the present invention and pathogen kind adopts hemofiltration membrane sample pad; If only detect blood serum sample, the sample pad in the detection alveolar echinococcosis immunity-chromatography test strip of the present invention can adopt spun glass or thieving paper sample pad.
The present invention is fixed on Echinococcus hydatid cyst antigen on the holders such as cellulose membrane as solid phase antigen, in order to catch the anti-many rooms echinococcus antigen-antibody in experimenter person's blood sample.The antigen Em18 of the alveolar echinococcosis that the present invention just recombinates is fixed on and forms detection line on the tunica fibrosa, and this solid phase antigen can be caught corresponding anti-alveolitoid Echinococcus hydatid cyst antigen EM18 antibody in experimenter's blood sample, forms the antigen antibody complex deposition in the detection line position.
The advantage antibody type of the anti-many rooms echinococcus antibody that contains in echinococcosis patient's body inner blood is an IgG antibody.The colloid gold label probe adopts anti-human IgG antibody or staphylococcal protein A or streptococcal protein G preparation; This anti-human IgG antibody or staphylococcal protein A or streptococcal protein G can be the commercialization antibody of buying, or prepare voluntarily according to conventional method.
Adopting the colloid gold label streptococcal protein G in the preferred scheme of the present invention is detector probe, and this colloid gold label antibody is attached on the gold mark pad.In the testing process, the colloid gold label antibody of redissolution can combine with the human IgG antibody in experimenter's blood sample, thereby when having the echinococcosis specific antibody in experimenter's blood sample, forms colour band in the detection line position.
Colloid gold label antibody is not limited to staphylococcal protein A or streptococcal protein G also can adopt the monoclonal antibody of mouse-anti human IgG or how anti-; Perhaps adopt other animals such as rabbit anti-human igg's antibody; Can realize goal of the invention of the present invention equally, this is that one of ordinary skill in the art is all known.
Antibody or antiantibody that the present invention will combine with the colloid gold label probe specificity are fixed on the cellulose membrane as nature controlling line.In preferred scheme of the present invention, the colloid gold label probe is a streptococcal protein G, and is corresponding, and nature controlling line adopts the IgY antibody of the anti-SPG of chicken.No matter whether contain anti-echinococcus antibody in the sample to be checked; Nature controlling line position total energy forms colour band in the immunity-chromatography test strip of detection echinococcosis of the present invention and pathogen kind, and this colour band is to judge the whether normal standard that whether goes bad with immunity-chromatography test strip of testing process.
Nature controlling line is not limited to the IgY antibody of the anti-SPG of chicken, as long as can combine with selected colloid gold label probe specificity, can realize goal of the invention of the present invention equally, and this is that the those skilled in the art in field know.
Detection principle of the present invention is: during mensuration sample serum or whole blood are added on the hemofiltration membrane sample pad; Drip (about 50 μ L) sample dilution after 1 minute; Dilution drives sample and moves by sample pad, the golden direction of marking pad, nitrocellulose filter, adsorptive pads; In flow through when pad gold mark, redissolved the colloid gold label antibody on the gold mark pad, and drive it and move to nitrocellulose filter, adsorptive pads.The colloid gold label probe can combine with antibody or the antibody subclass in the sample, forms immune complex.When this immune complex flow to detection line; If in the sample antibody or the antibody subclass (antibody of anti-alveolar echinococcosis antigen) to antigen EM18 arranged; The specificity solid phase antigen that is seized survey line is caught, and the red lines that detect are showed in the detection line position on nitrocellulose filter; When this immune complex is flowed through nature controlling line, promptly caught by the insolubilized antibody of nature controlling line, red Quality Control lines are showed in the nature controlling line position on nitrocellulose filter.Positive sample had both shown detection line, showed nature controlling line again; Negative sample does not have detection line, only shows nature controlling line.If nature controlling line does not show, represent that then strip lost efficacy.
Immunity-chromatography test strip of the present invention has the following advantages: (1) susceptibility and specificity are high: the laboratory result of appraisal show; The susceptibility that immunity-chromatography test strip of the present invention detects the alveolar echinococcosis patients serum is 94% (the total routine number of the positive routine number/alveolitoid of alveolitoid), and specificity is 100% (the non-echinococcosis patients negative example number/total routine number of non-echinococcosis patient); (2) detection method is simple, quick: sample disposal is simple in the testing process, and serum or whole blood can directly use, and need not to handle; Do not need specialized equipment and staff training, non-specialized-technical personnel can operate to specifications, and observations rapidly; Antibody in the sample is behind the chromatography about 10 minutes; Macroscopic detection line can occur, thereby strive for the time, be well suited for on-the-spot and basic unit's use for Echinococcus hydatid cyst patient's treatment; (3) preparation method is simple, and is with low cost, is easy to carry out suitability for industrialized production.The present invention will play a significant role in the diagnosis of the detection of echinococcosis and relevant disease thereof and treatment, have a extensive future.
Description of drawings
Fig. 1 is the Facad structure synoptic diagram that detects the immunity-chromatography test strip of alveolar echinococcosis.
Fig. 2 is the vertical section structure synoptic diagram that detects the immunity-chromatography test strip of alveolar echinococcosis.
Wherein:
1 is sample pad; 2 is gold mark pad; 3 is cellulose membrane; 4 is adsorptive pads;
5 is detection line; 6 is nature controlling line; 7 is backboard.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Method therefor is conventional method if no special instructions in the following example, and said percentage composition is mass/volume percentage composition or volume percentage composition if no special instructions.
A kind of immunity-chromatography test strip that detects alveolar echinococcosis of the present invention; Comprise a backboard 7; One end of the upside of described backboard 7 is provided with a sample pad 1; An other end of the upside of described backboard 8 is provided with an adsorptive pads 4, between described sample pad 1 and adsorptive pads 4, is provided with a cellulose membrane 3, and described adsorptive pads 4 closely is connected with cellulose membrane 3; Between described sample pad 1 and described cellulose membrane 3, be provided with a gold mark pad 2; Closely connect between described sample pad 1, gold mark pad 2 and the cellulose membrane 3, the end away from gold mark pad 2 on said cellulose membrane 3 is provided with nature controlling line 6, on the cellulose membrane 3 between described nature controlling line 6 and the gold mark pad 2, detection line 5 is set; Described detection line 5 is made up of the antigen Em18 of the alveolar echinococcosis of reorganization; Described gold mark pad 2 is provided with the probe of colloid gold label, and the probe of described colloid gold label is to be antibody or staphylococcal protein A or the streptococcal protein G that antigen-immunized animal obtains with the human IgG, and described nature controlling line 7 contains the antibody or the antiantibody that can combine with the colloid gold label probe specificity.
Further, an end of described cellulose membrane 3 is arranged on the downside of described gold mark pad 2, and an end of described gold mark pad 2 is arranged on the downside of described sample pad 1.
Further, the colloid gold label probe in the said gold mark pad 2 is the monoclonal antibody that obtains as the antigen immune mouse with human IgG, and said nature controlling line 6 contains the sheep anti mouse antiantibody.
Further, the colloid gold label probe in the said gold mark pad 2 can be staphylococcal protein A or streptococcal protein G, and said nature controlling line 7 contains anti-staphylococcal protein A antibody or streptococcus G protein antibodies.
1, the antigen Em18 of the alveolar echinococcosis of reorganization prepares as follows
1.1 the clone of genes of interest, conversion
Gather many rooms of sheep liver echinococcus protoscolex of natural infection in Xinjiang.(E.Z.N.A Kit I) carries out the extracting of the total RNA of protoscolex with kit.Carry out synthetic cDNA first chain of reverse transcription with reverse transcription kit (PROMEGA).According to document design primer, BamHI (upstream primer) and EcoRI (downstream primer) restriction enzyme site (tilted letter is represented) in primer, have been introduced respectively for ease of directed cloning.Primer sequence is following: Em18 F (5 '-GCGGATCCAAGGAGTCTGACTTAGCGGA-3 ') and Em18 R (5 '-GCGAATTCGGCTTCACTTTCATCATCCTG-3 '), primer is given birth to worker company by Shanghai and is synthesized.With synthetic cDNA first chain is template, RT-PCR method amplifying target genes fragment, and the nucleotide sequence of the antigen Em18 of the alveolar echinococcosis of described reorganization is shown in SEQ ID NO:3.Adopt HiFi DNA Amplification Kit (BBST) kit, polymerase is Pfu.Amplification condition: be reflected in the 50ul. system and carry out, loop parameter is 94 ℃ of sex change 3min, 94 ℃ of 30s, and 50 ℃ of 45s, 72 ℃ of 60s, totally 35 circulations, 72 ℃ are extended 10min.The product of amplification is checked with 1% agarose gel electrophoresis.Enzyme is cut product carry out 1.2% agarose gel electrophoresis respectively; Under uviol lamp, the purpose fragment in the gel (380bp) (5000bp) is downcut with the pGEX-3X expression vector; Ago-Gel DNA with Solarbio company reclaims the kit purifying; Genes of interest is connected with the T4 ligase with expression vector, and linked system is following:
Coupled reaction condition: the connection of spending the night under 16 ℃
Get 5 μ L and connect product Transformed E .coli DH5a cell, concrete grammar is: with connection product and the 200 μ L E.coli DH5a competent cell mixings of 5 μ L, ice bath 30 minutes; 42 ℃ of 90 seconds of water-bath heat shock, placed again 1-2 minute on ice afterwards, add the SOC nutrient culture media of 800 μ L preheatings then; 37 ℃; The 200rpm concussion was cultivated 45 minutes, got 200 μ L bacterium liquid and was coated with the LB+Amp flat board, was inverted in 37 ℃ of incubator incubated overnight.
Picking 5-10 single bacterium colony in flat board; Place the 5mLLB+Amp fluid nutrient medium respectively, 37 ℃, 200rpm spends the night to shake and cultivates; Send the order-checking of living worker Bioisystech Co., Ltd to detect to connect whether success; With the recombinant vector of successful connection,, get 1 μ L plasmid according to above method Transformed E .coli BL21 (DE3) cell with the Plasmid MiniKit I extracting plasmid of OMEGA company.Get 1 μ L PGEX-3X empty carrier Transformed E .coli DH5a cell simultaneously.
2 Recombinant Protein Expression and purifying
2.1. a small amount of of recombinant protein is expressed
The single bacterium colony of E.coli BL21 (DE3) of getting the conversion recombinant vector respectively and containing the PGEX-3X empty carrier is in 3mLLB (containing 100 μ g/mL Amp) nutrient culture media, and 37 ℃, the 200rpm shaken cultivation is spent the night; Then 1mL bacterium liquid is joined in the fresh LB of 2mL (the containing 100 μ g/mL Amp) nutrient culture media; 37 ℃, 200rpm shaken cultivation 2h, then add IPTG to final concentration be 1mM; 37 ℃, 200rpm abduction delivering 3h.Whether centrifugal collection thalline washes twice with PBS solution, and both are carried out the 10%SDS-PAGE electrophoresis simultaneously, observe recombinant protein and express.
2.2 the great expression of recombinant protein
Get definite single bacterium colony of E.coli BL21 (DE3) of expressing in 100mLLB (containing 100 μ g/mL Amp) nutrient culture media, 37 ℃, the 200rpm shaken cultivation is spent the night; Then 100mL bacterium liquid is joined in the fresh LB of 1000mL (the containing 100 μ g/mL Amp) nutrient culture media; 37 ℃, 200rpm shaken cultivation 2h, then add IPTG to final concentration be 1mM; 28 ℃, 200rpm abduction delivering 4h.Centrifugal collection thalline washes twice with PBS solution, then multigelation three times in liquid nitrogen and 37 ℃ of water-baths; Add the long-pending PBS solution of 20 multiplication of voltages, ultrasonic 8 times (each 30s, 1min at interval); Then adding 4mL 20%TritonX-100 solution to final concentration is 1%, stir 1h on ice after, the centrifugal 30min of 20000g; Cleer and peaceful deposition in the collection is carried out the 10%SDS-PAGE electrophoresis, with the dissolubility of check albumen.
2.3. the purifying of recombinant protein
Because this albumen is present in the supernatant; So the Glutathione Sepharose 4B purification column purifying supernatant with GE Healthcare company obtains recombinant protein; Concrete operation method carries out according to the instructions of purification column, and the concentration that obtains recombinant protein is 2.6mg/mL.
3, streptococcal protein G can be bought acquisition.
4, preparation immune colloid gold probe and gold mark pad
The immune colloid gold probe and the gold mark pad that prepare the streptococcal protein G mark with following method:
1) adopt the citrate reducing process to prepare colloid gold particle, concrete grammar is: with HAuCl
4(Shanghai examination board is purchased the Chemical Reagent Co., Ltd., Sinopharm Group in Shanghai) is mixed with 0.01% WS; Get 100mL and be heated to boiling; 1% of accurate adding 1.6mL trisodium citrate aqueous solution under stirring, it is red to treat that liquid color is stablized into grape wine, obtains colloidal gold solution.
2) confirm collaurum coupling probe saturation concentration
Use 0.2M K
2CO
3Regulating step 1) the pH value to 6.0 of the colloidal gold solution of preparation is prepared 5 clean tube, adds the 1mL colloidal gold solution respectively.The purified streptococcal protein G dilution of step 1) preparation is 1mg/mL; In 4 test tubes, add 20 μ L, 25 μ L, 30 μ L, 35 μ L respectively; Another is a blank, in room temperature held 5 minutes, adds the 10%NaCl WS behind the mixing; Mixing leaves standstill after 10-20 minute and observes liquid color.The contained minimum optimum concentration of stablizing 1mL colloidal gold solution desirable proteins that is increased by 20% protein content based on this and is the colloidal gold probe saturated solution when colloidal gold solution color was constant.The result: keeping the constant protein content of colloidal gold solution color is 20 μ L, and promptly concentration and probe concentration is 20 μ g/mL.
3) preparation of the immune colloid gold probe of streptococcal protein G (SPG) mark and gold mark pad
Get the 50mL colloidal gold solution, use 0.2M K
2CO
3Regulating the pH value is 6.0, adds the staphylococcal protein A of purifying by 25 μ g/mL, obtains containing the immune colloid gold probe solution 50mL that concentration is 25 μ g/mL streptococcal protein Gs; Stirred 1 hour, adding final concentration again is 0.05% PEG20000, stirs 1 hour; Centrifugal 30 minutes of 10000rpm abandons supernatant, with the washing precipitation of 20mM borate buffer; And it is stored in 10mL contains in the 10mMHEPES damping fluid of 15% sucrose, obtain the colloidal gold probe solution of streptococcal protein G mark.The colloidal gold probe solution of getting 5mL streptococcal protein G mark evenly is added on the glass fibre membrane, and 40% time drying of relative humidity 1 hour obtains gold mark pad.
5, detect the preparation of the immunity-chromatography test strip of alveolar echinococcosis
The preparation method of this strip may further comprise the steps:
1) the NC film encapsulates
Recombinant antigen EM18: the recombinant antigen EM18 that uses 0.01M pH7.4 PBS dilution embodiment 1 preparation is 0.5mg/mL to final concentration, is used to encapsulate detection line 5;
The anti-SPG IgY of Quality Control antibody chicken encapsulates: use 0.01M pH7.4 PBS dilution sheep anti-mouse igg to final concentration to be 0.5mg/mL, be used to encapsulate nature controlling line;
The XZ1000 of BIODOT company Membrane jetter is with being sprayed on respectively on the nitrocellulose filter that 300mm is long, 25mm is wide (available from Sartorius company), and quantity for spray is 10 μ L/cm, forms a detection line and a nature controlling line, relative humidity 40% time, dry 2 hours.
2) preparation of the immunity-chromatography test strip of detection alveolar echinococcosis
Be coated with the PVC backboard 7 of adhesive sticker with a single face, the NC film of handling well on middle stickup the 3; NC film 3 is closely pasted adsorptive pads 4 (purchasing the company in MILLIPORE) near an end of nature controlling line 6; NC film 3 is closely pasted gold mark pad 2 away from an end of nature controlling line 6; Gold mark pad 2 other ends are closely pasted hemofiltration membrane sample pad 1 (available from WHATMAN company).Obtain detecting the immunity-chromatography test strip motherboard of alveolar echinococcosis, can cut, add behind the drying agent sealing and preserve by required size.
6, detect the preparation of the immune chromatography reagent kit of alveolar echinococcosis
Use for ease, the immunity-chromatography test strip of the detection alveolar echinococcosis of above-mentioned preparation packed in the reagent box body, add drying agent after sealing preserve.A side of described box body is provided with a well and a viewport, and described well is positioned at the top of sample end adsorptive pads, and described viewport is positioned at the top of described detection line and nature controlling line.
Goat anti-human igg with buying the industry Lik-Sang thing Science and Technology Ltd. from Shanghai prepares the colloid gold label detector probe, encapsulates nature controlling line with buying from the anti-sheep IgG of the rabbit of Jie Ning biotech firm, and other steps are with embodiment 1.
1, detection method
Hemofiltration membrane sample pad in the immunity-chromatography test strip of embodiment 2 preparation adds test serum, adds sample dilution (PBS) 1 (about 50 μ L) after 1 minute, begins observations after 5 minutes, observes in 15 minutes to stop.The result judges: if detection line 5 red stripes all occurs with nature controlling line 6, promptly be judged to alveolar echinococcosis, if only there is nature controlling line 6 red stripes to occur, promptly be judged to feminine gender, if nature controlling line does not have red stripes, promptly strip lost efficacy.
2, experimental result
As shown in table 1 with the immunity-chromatography test strip of the detection alveolar echinococcosis of the embodiment of the invention 2 preparation chamber result of appraisal that experimentize.Above-mentioned testing result shows that immunity-chromatography test strip of the present invention can be used for the fast detecting of alveolar echinococcosis.
Table 1 the present invention detects the laboratory result of appraisal of the immunity-chromatography test strip of alveolar echinococcosis
With the immunity-chromatography test strip chamber of the experimentizing examination of the detection alveolar echinococcosis of the embodiment of the invention 2 preparation, the result does not have significant difference shown in result and the table 1.
Scope of the present invention does not receive the restriction of said specific embodiments, and said embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating various aspects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to the description and the accompanying drawing of preceding text.Said improvement also falls within the scope of appended claims.
Claims (14)
1. immunity-chromatography test strip that detects alveolar echinococcosis; Comprise a sample pad and an adsorptive pads; Between described sample pad and adsorptive pads, be provided with a cellulose membrane, adsorptive pads closely is connected with described cellulose membrane, between sample pad and cellulose membrane, is provided with a gold mark pad; Closely connect between described sample pad, gold mark pad and the cellulose membrane; End near adsorptive pads on cellulose membrane is provided with nature controlling line, on the cellulose membrane between described nature controlling line and the gold mark pad, a detection line is set, and it is characterized in that: described detection line contains the antigen Em18 of the alveolar echinococcosis of reorganization; Described gold mark pad is provided with the probe of colloid gold label, and described nature controlling line contains the antibody or the antiantibody that can combine with the colloid gold label probe specificity.
2. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 1 is characterized in that: described sample pad, gold mark pad, cellulose membrane and adsorptive pads are arranged on the backboard.
3. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 1 is characterized in that: an end of described cellulose membrane is arranged on the downside of described gold mark pad, and an end of described gold mark pad is arranged on the downside of described sample pad.
4. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 1 is characterized in that: the probe of described colloid gold label is to be antibody or staphylococcal protein A or the streptococcal protein G that antigen-immunized animal obtains with the human IgG.
5. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 4 is characterized in that: described colloid gold label probe is the monoclonal antibody that obtains as the antigen immune mouse with human IgG, and described nature controlling line contains the sheep anti mouse antiantibody.
6. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 4 is characterized in that: described nature controlling line contains anti-staphylococcal protein A antibody or streptococcus G protein antibodies.
7. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 1; It is characterized in that: the protein concentration of the antigen Em18 of the alveolar echinococcosis of reorganization is 0.1-10mg/mL; Quantity for spray is 8-12 μ L/cm, and the concentration of colloid gold label probe is counted 1-5mg/15-20mL with protein concentration.
8. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 5 is characterized in that: the concentration of described sheep anti mouse antiantibody solution is 0.1-5mg/mL, and quantity for spray is 8-12 μ L/cm.
9. the immunity-chromatography test strip of detection alveolar echinococcosis as claimed in claim 6 is characterized in that: anti-staphylococcal protein A antibody or streptococcus G protein antibodies solution concentration are 0.1-5mg/mL, and quantity for spray is 8-12 μ L/cm.
10. method for preparing the immunity-chromatography test strip of the described detection alveolar echinococcosis of claim 1; It is characterized in that: the step of antiantibody of step and preparation and colloid gold label probe specific bond of step, a preparation colloid gold label probe of antigen Em18 that comprises the alveolar echinococcosis of a preparation reorganization; After above-mentioned steps is accomplished; The antigen Em18 solution spraying of the alveolar echinococcosis of recombinating is formed detection line to cellulose membrane; Can to the cellulose membrane near adsorptive pads, form nature controlling line with the antibody or the antiantibody solution spraying of colloid gold label probe specific bond, the adjacent setting of described nature controlling line and detection line was with the tunica fibrosa that is coated with detection line, nature controlling line in the environment of relative humidity below 40% dry 1-2 hour; The step for preparing gold mark pad then; In the step of described preparation gold mark pad, glass fibre membrane or polyester film are immersed in the probe solution of colloid gold label, after the taking-up with gold mark pad in the environment of relative humidity below 40% dry 1-2 hour; Adsorptive pads sticks on the end near nature controlling line of said cellulose membrane; The gold mark is filled up the end near detection line that sticks on cellulose membrane, sample pad is pasted on the end away from cellulose membrane that the gold mark fills up, obtain detecting the immunity-chromatography test strip of alveolar echinococcosis.
11. preparation as claimed in claim 10 detects the method for the immunity-chromatography test strip of alveolar echinococcosis; It is characterized in that: the antigen Em18 of the alveolar echinococcosis of described reorganization prepares as follows: many rooms of sheep liver echinococcus protoscolex that will infect alveolar echinococcosis carries out the extracting of the total RNA of protoscolex with kit; Carry out synthetic cDNA first chain of reverse transcription with the reverse transcription kit, the upstream primer sequence is shown in SEQ ID NO:1, and the downstream primer sequence is shown in SEQ IDNO:2; In upstream primer, introduced the BamHI site; In downstream primer, having introduced the EcoRI restriction enzyme site, is template with synthetic cDNA first chain, RT-PCR method amplifying target genes fragment.
12. kit; Comprise a box body; It is characterized in that: the immunity-chromatography test strip of the described detection alveolar echinococcosis of claim 1 is arranged in the described box body; A side of described box body is provided with a well and a viewport, and described well is positioned at the top of sample end adsorptive pads, and described viewport is positioned at the top of described detection line and nature controlling line.
13. a reagent that detects alveolar echinococcosis is characterized in that: comprise following component:
1) the antigen Em18 of the alveolar echinococcosis of reorganization, the nucleotide sequence of the antigen Em18 of the alveolar echinococcosis of described reorganization is shown in SEQ ID NO:3;
2) colloid gold label probe.
14. the reagent of detection alveolar echinococcosis as claimed in claim 13; It is characterized in that: the antigen Em18 of the alveolar echinococcosis of described reorganization prepares as follows: many rooms of sheep liver echinococcus protoscolex that will infect alveolar echinococcosis carries out the extracting of the total RNA of protoscolex with kit; Carry out synthetic cDNA first chain of reverse transcription with the reverse transcription kit, the upstream primer sequence is shown in SEQ ID NO:1, and the downstream primer sequence is shown in SEQ ID NO:2; In upstream primer, introduced the BamHI site; In downstream primer, having introduced the EcoRI restriction enzyme site, is template with synthetic cDNA first chain, RT-PCR method amplifying target genes fragment.
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| CN2012100627786A CN102608330A (en) | 2012-03-12 | 2012-03-12 | Immunochromatographic strip for detecting alveolar echinococcosis and preparation method thereof |
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| CN2012100627786A CN102608330A (en) | 2012-03-12 | 2012-03-12 | Immunochromatographic strip for detecting alveolar echinococcosis and preparation method thereof |
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| CN111796103A (en) * | 2020-07-17 | 2020-10-20 | 青海省人民医院 | Colloidal gold test strip for detecting echinococcosis and preparation method thereof |
| CN112062858A (en) * | 2020-07-17 | 2020-12-11 | 青海省畜牧兽医科学院 | Tandem protein for diagnosing alveolar echinococcosis and cloning expression method thereof |
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Application publication date: 20120725 |