CN103983782A - ELISA kit for detecting hog cholera virus Erns IgM antibody - Google Patents
ELISA kit for detecting hog cholera virus Erns IgM antibody Download PDFInfo
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Abstract
The invention discloses an ELISA kit for detecting a hog cholera virus Erns IgM antibody, and belongs to the fields of a biotechnology and diagnosis research of an animal-borne disease. The ELISA kit comprises an elisa plate of enveloping an anti-swine IgM monoclonal antibody, a sample diluent, a positive control and a negative control, a detection antigen (hog cholera virus Erns protein), washing concentrate, an enzyme conjugate, an enzyme chromogenic substrate and a stopping solution. The IgM antibody is detected by adopting a capture method, the specific hog cholera virus Erns IgM antibody is detected by adopting a sandwich method, the specificity of the prepared kit can be up to 100%, the sensitivity is 1:800, and the ELISA kit can be applied to diagnosis of swine fever virus infection and assessment of immune efficacy of a hog cholera vaccine.
Description
Technical field
The invention belongs to biotechnology and zoonosis diagnosis research field, be specifically related to a kind of ELISA kit that detects CSFV Erns IgM antibody.
Background technology
Swine fever is a kind of height contagious disease being caused by CSFV (Classical swine fever virus, CSFV), serious to pig industry harm, is one of Infectious Diseases of the World Food Programme and national governments' monitor closely and quarantine.
CSFV belongs to flaviviridae pestivirus member, is the single strand plus RNA virus that has cyst membrane, and the about 12.3kb of Genome Size, only contains a large Open reading frame (ORF).This ORF translates into containing 3898 amino acid residues, the polyprotein of the about 438kDa of molecular weight, and further under the effect of virus and host cell proteins enzyme, being processed into structural proteins and non-structural protein, its structural proteins and the non-structural protein coded sequence on viral RNA is Npro, C, Erns (E0), E1, E2, P7, NS2-3, NS4A, NS4B, NS5A, NS5B.Wherein, except C, Erns, E1 and E2 are structural proteins, all the other are non-structural protein.
Pig mainly produces the antibody for structural glycoprotein E0, E2 and non-structural protein NS3 after infecting CSFV.Glycoprotein E 0 and E2 are two main protection antigen that virus induction body produces neutralizing antibody, are also the indispensable proteins that viruses adsorption enters sensitive cells simultaneously.
Erns is viral a kind of membrane glycoprotein, also claims gp44/48, is once called as E0.Have 9 possible glycosylation sites, the about 26KDa of albumen molecule of the skeleton amount of desaccharification base, is made up of Glu268-Ala494 in virus O RF.In infection cell, Erns accumulates in endoplasmic reticulum, and can be present in surface of cell membrane or be secreted into outside born of the same parents.In virion, Erns is present in cyst membrane surface with the homodimer form of 97KDa.Owing to lacking hydrophobic film anchorage zone in its molecule, a little less than virus envelope adhesion, easily dissociate to get off from cyst membrane.Erns can induce the neutralizing antibody that produces swine fever, and immune swine can be induced the protective immunity producing lethal quantity CSFV.Because the nucleotide sequence of coding Erns is high in the conservative degree of sequence that belongs to internal ratio coding E2, therefore Erns can be used as a kind of target protein of control swine fever.
The fast detecting of CSFV has the meaning of particular importance for the diagnosis of swine fever.CSFV detection method mainly contains application Virus Isolation, immunohistochemical method, reverse transcription-polymerase chain reaction (RT-PCR) and Elisa detection etc. at present.Wherein Elisa detection method, with its advantage such as quick, convenient, responsive, is shown one's talent in numerous methods.But the Elisa kit in market mostly is the kit that detects anti-E2 antibody, and the research of the aspect of nothing to Erns IgM antibody.
Summary of the invention
Primary and foremost purpose of the present invention is to provide a kind of ELISA kit that detects CSFV (CSFV) Erns IgM antibody.
The present invention also aims to the using method of the ELISA kit that above-mentioned detection CSFV Erns IgM antibody is provided.
To achieve these goals, the following technical scheme that the present invention adopts:
An ELISA kit that detects CSFV Erns IgM antibody, includes: ELISA Plate, sample diluting liquid, positive control and the negative control of coated anti-pig IgM monoclonal antibody, antigen, concentrated cleaning solution, enzyme conjugates, enzyme chromogenic substrate and stop buffer for detection.
Described detection antigen is restructuring CSFV Erns (CSFV-Erns) albumen, with sample diluting liquid dissolved dilution to 5-10 μ g/mL; Preferably, described Recombinant Swine pestivirus Erns albumen prepares by the method that comprises following steps: extract the RNA of CSFV and be template by the reverse transcription PCR Erns albumen genes of interest that increase, by genes of interest and be connected to the Recombinant Swine pestivirus Erns albumen that proceeds to the BL21 (DE3) that contains PGrO7 expression of recombinant plasmid chaperone GroEL on pET30a carrier again and carry out prokaryotic expression, purifying and obtain purifying; Wherein amplimer is:
CSFV-Erns upstream region of gene primer: 5 '-CG
gGATCCgAAAATATAACTCAGTGGAACCTGA-3 ',
CSFV-Erns gene downstream primer: 5 '-CG
cTCGAGgGCATAGGCACCAAACCAG-3 '.
The ELISA Plate of described coated anti-pig IgM monoclonal antibody, the amount of the anti-pig IgM monoclonal antibody being coated with is preferably 0.1-0.5 μ g.Described anti-pig IgM monoclonal antibody is commercially available, or prepare according to the method that comprises following steps: with the pig IgM immunity Balb/c mouse of purifying, getting the successful mouse boosting cell of immunity and murine myeloma cell SP2/0 merges, through clone and ELISA screening, the final hybridoma cell strain that can identify pig IgM that filters out respectively, is injected to mouse peritoneal production ascites and purifies ascites with sad ammonium sulfate precipitation method with this hybridoma cell strain and obtain anti-pig IgM monoclonal antibody.The ELISA Plate of coated anti-pig IgM monoclonal antibody preferably obtains by the method that comprises following steps: by resisting the coated damping fluid (carbonate buffer solution of 0.05mol/L, pH9.6) of pig IgM monoclonal antibody to be diluted to 1-5 μ g/mL, add 100 μ L toward every hole in ELISA Plate, 4 DEG C of coated spending the night; Every hole adds 150 μ L confining liquids (5% BSA, with 0.01mol/L, the phosphate buffer of pH7.2-7.4 (PBS) is prepared), 37 DEG C of incubation 1h again.
Described sample diluting liquid is preferably containing 1%BSA, 0.1% deep-sea fish gelatin, 0.02% sodium azide (NaN
3) PBS, wherein said PBS is preferably 0.01mol/L, the PBS that pH value is 7.2-7.4.
Described positive control be with sample diluting liquid 100 times of dilutions containing CSFV Erns IgM antibody pig positive serum, this serum be with hog cholera lapinised virus seedling immunity acquisition; Described negative control is the health pig negative serum that did not infect CSFV and do not cross with hog cholera vaccine immunity with 100 times of dilutions of sample diluting liquid.
Described concentrated cleaning solution (10 ×) is preferably containing the 0.1mol/L of 0.5%Tween20, the PBS of pH7.2-7.4, before using, does 10 times of dilutions with deionized water.
Described enzyme conjugates is preferably the swine fever virus resistant Erns monoclonal antibody of horseradish peroxidase-labeled, and its working concentration is 10-20 μ g/mL, preferably by the method preparation that comprises following steps:
1) prepare swine fever virus resistant Erns monoclonal antibody: with the Recombinant Swine pestivirus Erns protein immunization Balb/c mouse of purifying, three exempt from after, getting Elisa mouse boosting cell and the murine myeloma cell SP2/0 that reaches 1:10000 that tire merges, after cultivating with HAT nutrient culture media selectivity, carry out indirect Elisa detection, pick out positive hole and carry out subclone; Get again subclone hole and carry out indirect Elisa detection, pick out positive hole and proceed to 24 orifice plates and carry out subclone for the second time; Two subcellular fraction strains are carried out to the monoclonal hybridoma strain that indirect Elisa detection and immunofluorescence screening go out swine fever virus resistant Erns albumen; This hybridoma cell strain is carried out frozen, make a call to 3 mouse simultaneously and prepare ascites, the mouse ascites making, with sad ammonium sulfate precipitation method purifying, dialysis, is made to swine fever virus resistant Erns protein monoclonal antibody.
2) swine fever virus resistant Erns monoclonal antibody is carried out to horseradish peroxidase-labeled, the steps include: to take 5mg HRP and be dissolved in 0.5mL distilled water, add freshly prepared 0.06mol/L NaIO
4aqueous solution 0.5mL, mix and put refrigerator 4 degree reaction 30 minutes, taking-up adds 0.16mol/L glycol water 0.5mL, after 30 minutes, add the aqueous solution 1mL containing 5mg swine fever virus resistant Erns monoclonal antibody in the reaction of room temperature lucifuge, mix and fill bag filter and 0.05mol/L, pH9.5 carbonate buffer solution and slowly stir dialysis 6h or spend the night, make it combination; Then sucking-off adds NaBH4 solution (5mg/mL) 0.2mL, puts refrigerator 4 and spends 2h, takes out and adds equal-volume saturated ammonium sulfate; Put refrigerator 4 and spend after 30min centrifugally, gained sediment is dissolved in a small amount of 0.02mol/L, pH7.4PBS, and dialysed overnight; Next day is the centrifugal insolubles of removing again, obtains the swine fever virus resistant Erns monoclonal antibody of HRP mark; Add to 5mL with 0.02mol/L, pH7.4PBS, then measure concentration with Bradford method, be then diluted to 10-20 μ g/mL with enzyme labelled antibody dilution (commercially available) and make enzyme conjugates working fluid.
Described enzyme chromogenic substrate is preferably TMB nitrite ion.
Described stop buffer is preferably the H of 1mol/L
2sO
4solution.
The using method of the ELISA kit of above-mentioned detection CSFV (CSFV) Erns IgM antibody, comprises the steps:
(1) test serum or other sample are pressed to 1:100 dilution with sample diluting liquid, every hole 100 μ L add in ELISA Plate, and 2～6 holes of parallel application of sample arrange positive controls, negative control group and blank group simultaneously; Wherein positive controls adds 100 μ L positive control solutions, negative control group to add 100 μ L negative controls, blank group to add 100 μ L sample diluting liquids, and every group contrasts 2～6 holes of parallel application of sample.
(2) after application of sample completes, the ELISA Plate of gently shaking mixes sample in hole, hatches 1 hour for 37 DEG C.
(3) discard solution in hole, cleansing solution cleans 3～5 times, pats dry for the last time on thieving paper.
(4) every hole adds 100 μ L to detect with antigen (Recombinant Swine pestivirus Erns albumen, sample diluting liquid dissolves, concentration 5-10 μ g/mL), hatches 1 hour for 37 DEG C.
(5) repeating step (3), every hole adds enzyme conjugates 100 μ L, hatches 1 hour for 37 DEG C.
(6) repeating step (3), every hole adds enzyme chromogenic substrate 100 μ L, room temperature lucifuge reaction 10-15 minute.
(7) every hole adds 100 μ L stop buffers, ELISA Plate is placed in to microplate reader and measures 450nm absorbance (OD
450nm).
(8) result is calculated and is judged: in detection, the difference of the OD value of positive control serum and the OD value of negative control sera must be greater than at 0.4 o'clock, detects and is considered to effective; Cut off (CO value)=negative control absorbance value OD
450nm× 2.1 times, sample value=sample absorbance value OD
450nm/ CO value, wherein, sample value > 1 is positive; Sample value≤1 is negative.
Compared with prior art, kit of the present invention has the following advantages and effect:
(1) the ELISA kit of detection CSFV (CSFV) Erns IgM antibody of the present invention adopts pig IgM monoclonal antibody to prepare ELISA Plate, and then the kit of composition double antibodies sandwich method Elisa detection CSFV Erns specific IgM antibodies, significantly improve the sensitivity and the specificity that detect.
(2) the present invention, by adopting chaperone expression system prokaryotic expression CSFV (CSFV) Erns albumen, successfully makes Erns albumen express in supernatant, and has improved expression.
(3) the present invention adopts prize law to detect IgM antibody, sandwich method detects special (CSFV) Erns IgM antibody, made kit specificity reaches 100%, highly sensitive in 1:800, can be used for diagnosis to swine fever virus infection and the Efficacy evaluation of hog cholera vaccine.
Brief description of the drawings
Recombinant Swine pestivirus (CSFV) Erns albumen prepared by Fig. 1 the present invention is expressed SDS-PAGE figure in a small amount.
SDS-PAGE figure after Recombinant Swine pestivirus (CSFV) Erns protein purification prepared by Fig. 2 the present invention.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.
The preparation of embodiment 1 Recombinant Swine pestivirus Erns albumen
(1) according to Classical Swine Fever Virus Shimen Strain whole genome sequence (accession number AF333000.1) in NCBI GeneBank, taking the gene order in Classical Swine Fever Virus Shimen Strain envelope protein Erns complete sequence and HeD/A district of B/C district of E2 major antigen district as stencil design synthetic primer
CSFV-Erns upstream region of gene primer is: 5 '-CG
gGATCCgAAAATATAACTCAGTGGAACCTGA-3 ',
CSFV-Erns gene downstream primer is: 5 '-CG
cTCGAGgGCATAGGCACCAAACCAG-3 ';
Wherein, underscore part is the restriction enzyme site of introducing, and the restriction enzyme site that upstream primer is introduced is BamHI, and the restriction enzyme site that downstream primer is introduced is XhoI.Carry out reverse transcription PCR taking the RNA extracting as template from Classical Swine Fever Virus Shimen Strain, obtain Erns albumen genes of interest.
(2) build recombinant expression carrier: expression plasmid pET30a is carried out to double digestion with Erns albumen genes of interest BamHI and the XhoI of restriction enzyme and above-mentioned amplification, enzyme is cut product after agarose electrophoresis, reclaim kit (purchased from Invitrogen company) with gel and reclaim DNA fragmentation, connect the DNA fragmentation reclaiming with T4DNA ligase.
(3) by step 2) middle gained connection product conversion bacillus coli DH 5 alpha, screens picking list spot, bacterium colony PCR Preliminary Identification according to the mark of recombinant vector (anti-Kan or blue hickie).
(4) qualification of recombinant expression carrier: extract plasmid, carry out double digestion qualification, obtained the DNA fragmentation that is about 680bp, in the same size with the CSFV-Erns (681bp) of expection.CSFV-Erns positive colony in line endonuclease bamhi is delivered to the order-checking of Hua Da genome company, and position, direction, size, reading frame and nucleotide sequence that result is inserted are all correct, by this recombinant expression carrier called after pET30a-Erns.
(5) making of chaperone competence expressive host bacterium: the probability of expressing at supernatant in order to improve CSFV-Erns, is transformed into the correct recombinant plasmid pET30a-Erns of order-checking in BL21 (DE3) competent cell that contains PGrO7 expression of recombinant plasmid chaperone GroEL.
(6) abduction delivering in a small amount: picking transforms single colony inoculation on flat board in 3mL LB nutrient solution (need add kanamycins and two kinds of microbiotic of chloromycetin of final concentration 0.05-0.1g/L), 37 DEG C, 180rpm by microbe growth to OD
600=0.2; Add derivant tetracycline or the Arabinose of 0.1-0.5mmol/L chaperone, 37 DEG C, 180rpm continue microbe growth to OD
600=1.0; Adding final concentration is the IPTG of 0.5mM, and 30 DEG C, 180rpm induction spend the night.
(7) qualification of expressing protein: the Host Strains that induction is spent the night is collected, fragmentation, carries out SDS-PAGE electrophoresis; After electrophoresis finishes, dyed and decolouring is rear to be found, the pET30a-Erns recombinant bacterium of abduction delivering has obvious chaperone GroEL to express at 60KD place, have obvious recombinant protein Erns to express at 26KD place, and instruction card is reached merit (Fig. 1).
(8) a large amount of abduction deliverings of recombinant C SFV-Erns albumen: the Escherichia coli that contain correction carrier are inoculated in a large amount of nutrient solutions to induce in a small amount the same terms abduction delivering, cultivation finishes rear centrifugal collection bacterium liquid, the clarification of ultrasonic treatment bacterium liquid bacterium liquid or evacuation state; 11000rpm, 4 DEG C of centrifugal 30min, by supernatant and precipitate and separate, be stored in 4 DEG C.Get supernatant and deposit sample and carry out SDS-PAGE, find that recombinant C SFV-Erns expresses at supernatant.
(9) purifying of recombinant C SFV-Erns albumen: according to Ni-NTA agarose column (Qiagen, article No. 30210) instructions purifying, the SDS-PAGE electrophoresis recombinant C SFV-Erns protein SDS-PAGE of purifying the results are shown in Figure 2.
The preparation of embodiment 2 swine fever virus resistant Erns albumen (CSFV-Erns) IgG monoclonal antibodies
(1) by the recombinant C SFV-Erns protein immunization Balb/c mouse of embodiment 1 purifying, three exempt from after, get Elisa mouse boosting cell and the murine myeloma cell SP2/0 that reaches 1:10000 that tire and merge, with 37 DEG C of HAT nutrient culture media, saturated humidity, 5%CO
2cultivate fused cell.
(2) after fusion, within the 4th day, start to observe colony, at the bottom of colony grows to hole, approximately 1/6 o'clock Ban amount is changed HT nutrient culture media, and carry out indirect Elisa detection with recombinant C SFV-Erns albumen wrapper sheet next day respectively, picks out positive hole and carry out subclone.
(3) subclone: the positive porocyte of Microscopic observation, select the good colony of cellular morphology to carry out subclone with limiting dilution assay.Subclone plate carries out indirect Elisa detection with CSFV positive serum 1:1000 wrapper sheet on the 5th day, picks out positive hole and proceeds to 24 orifice plates and carry out subclone for the second time.
(4) secondary subclone the 5th day, get culture supernatant and synchronously carry out immunofluorescence (IF) and following three kinds of Elisa detection:
A. detect with the indirect Elisa of recombinant C SFV-Erns albumen wrapper sheet.
B. detect with the indirect Elisa of CSFV positive serum 1:1000 wrapper sheet.
C. first with in CSFV-Erns and the positive hole supernatant of anti-CSFV-Erns, the supernatant after neutralization is carried out to B detection.
Finishing screen is selected and is met immunofluorescence (IF) positive and A, the B detection positive and the negative anti-CSFV-Erns hybridoma cell strain of C detection simultaneously, and this hybridoma cell strain is the cell line that can identify CSFV Erns albumen.
(5) the frozen and ascites of cell line preparation: the above-mentioned hybridoma cell strain of successfully building strain is carried out frozen, make a call to 3 mouse simultaneously and prepare ascites, every mouse peritoneal injection cell concentration is 5 × 10
5～1 × 10
6individual/0.5mL.
(6) by the mouse ascites making with sad ammonium sulfate precipitation method purifying, dialysis, make swine fever virus resistant Erns albumen (CSFV-Erns) IgG monoclonal antibody.
Embodiment 3 detects the preparation of the ELISA kit of CSFV (CSFV) Erns IgM antibody
(1) ELISA Plate of coated anti-pig IgM monoclonal antibody: will resist pig IgM monoclonal antibody to be diluted to 1 μ g/mL to be coated with damping fluid, every hole adds 100 μ L, 4 DEG C of coated spending the night; With cleansing solution washing 2～3 times, pat dry; Every hole adds 150 μ L confining liquids, 37 DEG C of incubation 1h, and cleansing solution washing 2～3 times, pats dry.Wherein, coated damping fluid is 0.05mol/L pH9.6 carbonate buffer solution, and it is formulated as: accurately take 1.59g sodium carbonate, and 2.93g sodium bicarbonate, adding distil water is to 1000mL; Confining liquid is the concentration of dissolving taking the phosphate buffer of 0.01mol/L, pH7.2-7.4 (PBS) BSA as 5%; Cleansing solution is the working fluid of making after concentrated cleaning solution dilutes 10 times.
(2) sample diluting liquid: add 1% (m/v) BSA, 0.1% (m/v) deep-sea fish gelatin, 0.02% (m/v) sodium azide (NaN3) formulated in the phosphate buffer (PBS) of 0.01mol/L, pH7.2-7.4.
(3) positive control solution: gather the pig positive serum containing CSFV Erns IgM antibody obtaining with the immunity of hog cholera lapinised virus seedling, with 100 times of dilutions of sample diluting liquid.Negative controls: gather the health pig serum that did not infect CSFV and do not cross with hog cholera vaccine immunity, with 100 times of dilutions of sample diluting liquid.
(4) detect with antigen be the recombinant C SFV-Erns albumen of embodiment 1 purifying, with sample diluting liquid dissolved dilution to 5-10 μ g/mL.
(5) concentrated cleaning solution (10 ×): adding volume ratio in the PBS of 0.1mol/L, pH7.2-7.4 is 0.5% Tween20, does 10 times of dilutions with deionized water before using.
(6) enzyme conjugates is the swine fever virus resistant Erns monoclonal antibody of horseradish peroxidase (HRP) mark, and the swine fever virus resistant Erns protein I gG monoclonal antibody that embodiment 2 is made is carried out horseradish peroxidase-labeled, the steps include:
1) take 5mg HRP and be dissolved in 0.5mL distilled water, add freshly prepared 0.06mol/L NaIO
4aqueous solution 0.5mL, mixes and puts refrigerator 4 degree reaction 30 minutes;
2) take out and add 0.16mol/L glycol water 0.5mL, in room temperature lucifuge reaction 30 minutes;
3) add the aqueous solution 1mL containing 5mg swine fever virus resistant Erns protein I gG monoclonal antibody, mix and fill bag filter and 0.05mol/L, pH9.5 carbonate buffer solution and slowly stir dialysis 6h or spend the night, make it combination;
4) sucking-off adds NaBH
4solution (5mg/ml) 0.2mL, puts refrigerator 4 and spends 2h, takes out and adds equal-volume saturated ammonium sulfate; Put refrigerator 4 and spend after 30min centrifugally, gained sediment is dissolved in a small amount of 0.02mol/L, pH7.4PBS, and dialysed overnight;
5) next day the centrifugal insolubles of removing again, obtain the swine fever virus resistant Erns monoclonal antibody of HRP mark, add to 5mL with 0.02mol/L, pH7.4PBS, then measure concentration with Bradford method, be then diluted to 10-20 μ g/mL with enzyme labelled antibody dilution (commercially available).
(7) enzyme chromogenic substrate: be the single solution TMB of the commercial instant nitrite ion (article No.: T8665) purchased from Sigma
(8) stop buffer: get 54.3mL concentration and be 95% the concentrated sulphuric acid, adding distil water, to 1000mL, is made the H that concentration is 1mol/L
2sO
4solution.
Embodiment 4 detects the using method of the ELISA kit of CSFV Erns IgM antibody
Utilize the ELISA kit of detection CSFV Erns IgM antibody of the present invention in the application detecting in CSFV, the steps include:
(1) from kit, take out ELISA Plate, test serum or other sample are pressed to 1:100 dilution with sample diluting liquid, every hole 100 μ L add in ELISA Plate, 2～6 holes of parallel application of sample, positive controls, negative control group and blank group are set simultaneously, wherein positive controls adds 100 μ L positive control solutions, negative control group to add 100 μ L negative controls, blank group to add 100 μ L sample diluting liquids, and every group contrasts 2～6 holes of parallel application of sample.
(2) after application of sample completes, the ELISA Plate of gently shaking mixes sample in hole, hatches 1 hour for 37 DEG C.
(3) discard solution in hole, cleansing solution cleans 3～5 times, pats dry for the last time on thieving paper.
(4) every hole adds 100 μ L detection antigens, hatches 1 hour for 37 DEG C.
(5) repeating step (3), every hole adds enzyme conjugates 100 μ L, hatches 1 hour for 37 DEG C.
(6) repeating step (3), every hole adds enzyme chromogenic substrate 100 μ L, room temperature lucifuge reaction 10-15 minute.
(7) every hole adds 100 μ L stop buffers, ELISA Plate is placed in to microplate reader and measures 450nm absorbance (OD
450nm).
(8) result is calculated and is judged: in detection survey, the difference of the OD value of positive control serum and the OD value of negative control sera must be greater than at 0.4 o'clock, detects and is considered to effective; Cut off (CO value)=negative control absorbance value OD
450nm× 2.1 times, sample value=sample absorbance value OD
450nm/ CO value, wherein, sample value > 1 is positive; Sample value≤1 is negative.
Embodiment 5 detects specificity, the sensitivity test of the ELISA kit of CSFV Erns IgM antibody
(1) specific detection
Press the detection method described in embodiment 4, utilize the kit that embodiment 3 makes to detect respectively hog cholera lapinised virus vaccine (HCLV) Initial stage of immunization (7-14 days) serum, CSFV (CSFV) infects the serum of early stage (7-14 days), the CSFV-Erns IgG antibody (embodiment 2 makes) of purifying, porcine reproductive and respiratory syndrome virus (PRRSV, claim again pig blue-ear disease poison) infect serum, the serum that porcine circovirus 2 type (PCV2) infects, the serum that PRV (PRV) infects, serum and CSFV negative serum that Latex agglutination test (JEV) infects.
Three holes of every kind of parallel application of sample of serum, measure OD
450nmvalue, calculates its OD
450nmmean value.Result is as shown in the table:
Note: PC: positive control, NC: negative control.
As seen from the above table, OD
450nm(PC)-OD
450nm(NC)=1.164 > 0.4, represent that result is effective; CO value=OD
450nm(NC) × 2.1=0.164, the Positive Sera of PRRSV, PCV2, PRV, JEV, the CSFV-Erns IgG antibody of purifying and CSFV negative serum OD
450mean value is all less than CO value, negative; Hog cholera antibody positive serum (serum of HCLV Initial stage of immunization serum, CSFV early infection) OD
450mean value is greater than CO value, positive.Show the generation of kit of the present invention and CSFV-Erns IgG antibody no cross reaction, can specific detection CSFV-Erns IgM antibody, it is 100% to swine fever poison recall rate simultaneously.
(2) sensitivity detects
Press the detection method described in embodiment 4, utilize the kit that embodiment 3 makes to detect respectively different dilution positive reference serums (gathering the pig positive serum containing CSFV Erns IgM antibody obtaining with the immunity of hog cholera lapinised virus seedling).
Positive reference serum is detected with following gradient dilution 1:100,1:200,1:400,1:800,1:1600,1:3200 with sample diluting liquid.3 holes of the parallel application of sample of each gradient, measure OD
450nmvalue, calculates its OD
450nmmean value.Result is as shown in the table:
Note: PC: positive control, NC: negative control.
As seen from the above table, OD
450nm(PC)-OD
450nm(NC)=1.044 > 0.4, represent that result is effective; CO value=OD
450nm(NC) × 2.1=0.158, as seen from the above table, when positive reference serum dilutes 800 times, kit of the present invention still can effectively detect the positive, and the OD value recording is still higher, estimate that the detectability of the positive reference material of kit of the present invention should be greater than 800 times.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Chopper Biology Co., Ltd.
Mono-kind of <120> detects the ELISA kit of CSFV Erns IgM antibody
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> CSFV-Erns upstream region of gene primer
<400> 1
cgggatccga aaatataact cagtggaacc tga 33
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> CSFV-Erns gene downstream primer
<400> 2
cgctcgaggg cataggcacc aaaccag 27
Claims (10)
1. an ELISA kit that detects CSFV Erns IgM antibody, is characterized in that including: ELISA Plate, sample diluting liquid, positive control and the negative control of coated anti-pig IgM monoclonal antibody, antigen, concentrated cleaning solution, enzyme conjugates, enzyme chromogenic substrate and stop buffer for detection; Described detection antigen is CSFV Erns albumen.
2. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: the concentration of described detection antigen is 5-10 μ g/mL; Described CSFV Erns albumen prepares by the method that comprises following steps: extract the RNA of CSFV and be template by the reverse transcription PCR Erns albumen genes of interest that increase, by genes of interest and be connected to the Recombinant Swine pestivirus Erns albumen that proceeds to the BL21 that contains PGrO7 expression of recombinant plasmid chaperone GroEL on pET30a carrier again and carry out prokaryotic expression, purifying and obtain purifying; Wherein amplimer is:
CSFV-Erns upstream region of gene primer: 5 '-CGGGATCCGAAAATATAACTCAGTGGAACCTGA-3 ',
CSFV-Erns gene downstream primer: 5 '-CGCTCGAGGGCATAGGCACCAAACCAG-3 '.
3. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: the amount of the anti-pig IgM monoclonal antibody that ELISA Plate was coated with of described coated anti-pig IgM monoclonal antibody is 0.1-0.5 μ g.
4. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: described sample diluting liquid is the PBS containing 1% BSA, 0.1% deep-sea fish gelatin, 0.02% sodium azide.
5. the ELISA kit of detection according to claim 1 CSFV Erns IgM antibody, is characterized in that: described positive control be with 100 times of dilutions of sample diluting liquid containing CSFV Erns IgM antibody pig positive serum; Described negative control is the health pig negative serum that did not infect CSFV and do not cross with hog cholera vaccine immunity with 100 times of dilutions of sample diluting liquid.
6. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: described concentrated cleaning solution is 0.1mol/L, the PBS of pH7.2-7.4 containing 0.5% Tween20, before using, does 10 times of dilutions with deionized water.
7. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: the swine fever virus resistant Erns monoclonal antibody that described enzyme conjugates is horseradish peroxidase-labeled, its concentration is 10-20 μ g/mL.
8. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: described enzyme chromogenic substrate is TMB nitrite ion.
9. the ELISA kit of detection CSFV Erns IgM antibody according to claim 1, is characterized in that: the H that described stop buffer is 1mol/L
2sO
4solution.
10. the using method of the ELISA kit of the detection CSFV Erns IgM antibody described in claim 1-9 any one, is characterized in that comprising the steps:
(1) test serum or other sample are pressed to 1:100 dilution with sample diluting liquid, every hole 100 μ L add in ELISA Plate, and 2～6 holes of parallel application of sample arrange positive controls, negative control group and blank group simultaneously; Wherein positive controls adds 100 μ L positive control solutions, negative control group to add 100 μ L negative controls, blank group to add 100 μ L sample diluting liquids, and every group contrasts 2～6 holes of parallel application of sample;
(2) after application of sample completes, the ELISA Plate of gently shaking mixes sample in hole, hatches 1 hour for 37 DEG C;
(3) discard solution in hole, cleansing solution cleans 3～5 times, pats dry for the last time on thieving paper;
(4) every hole adds 100 μ L detection antigens, hatches 1 hour for 37 DEG C;
(5) repeating step (3), every hole adds enzyme conjugates 100 μ L, hatches 1 hour for 37 DEG C;
(6) repeating step (3), every hole adds enzyme chromogenic substrate 100 μ L, room temperature lucifuge reaction 10-15 minute;
(7) every hole adds 100 μ L stop buffers, ELISA Plate is placed in to microplate reader and measures 450nm absorbance;
(8) result is calculated and is judged: in detection, the difference of the OD value of positive control serum and the OD value of negative control sera must be greater than at 0.4 o'clock, detects and is considered to effective; CO value=negative control absorbance value OD
450nm× 2.1 times, sample value=sample absorbance value OD
450nm/ CO value, wherein, sample value > 1 is positive; Sample value≤1 is negative.
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| CN115975052A (en) * | 2022-12-01 | 2023-04-18 | 北京标驰泽惠生物科技有限公司 | Fusion protein of classical swine fever virus and application thereof |
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