CN109283348A

CN109283348A - Detect the double-antibody sandwich elisa kit and preparation method and application of arch insect circulating antigen in many animals serum

Info

Publication number: CN109283348A
Application number: CN201811375417.0A
Authority: CN
Inventors: 刘晶; 李祎; 刘群
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-11-19
Filing date: 2018-11-19
Publication date: 2019-01-29

Abstract

The invention discloses the double-antibody sandwich elisa kits and preparation method and application of arch insect circulating antigen in a kind of detection many animals serum, the double-antibody sandwich elisa kit includes: capture antibody, the capture antibody is toxoplasma GRA1 monoclonal antibody, and the potency of the capture antibody is 5 × 10⁶, antibody subtype IgG1；And the detection antibody of HRP label, the detection antibody are source of mouse toxoplasma GRA1 polyclonal antibody, the potency of the detection antibody is 1.28 × 10⁵；The double-antibody sandwich elisa kit is easy to operate, small by such environmental effects, no species specificity, has higher sensibility and specificity compared to the diagnostic method that polyclonal antibody is established.

Description

Detect the double-antibody sandwich elisa examination of arch insect circulating antigen in many animals serum Agent box and preparation method and application

Technical field

The present invention relates to Serologic detection fields, recycle especially with regard to toxoplasma in a kind of detection many animals serum The double-antibody sandwich elisa kit and preparation method and application of antigen.

Background technique

Toxoplasma gondii (Toxoplasma gondii) is a kind of special sexual cell endoparasitism protozoon, in the world extensively General propagation, it is estimated that there are about the crowds of one third to show the toxoplasma serum antibody positive in the whole world.It is same popular wide in domestic animal It is general, wherein the infection rate with pig is higher.Arch insect infection can according to host fall ill the order of importance and emergency be divided into acute toxoplasmosis and Chronic toxoplasmosis, acute toxoplasmosis are in contrast bigger to the harm of domestic animal.The acute toxoplasmosis of pig often causes sow to be flowed The breeding difficultys such as production, stillborn foetus, piglet fever, expiratory dyspnea even death etc., cause huge economic loss to aquaculture；And The pig meat products source important as the mankind, has played important function in the propagation of people's toxoplasmosis.Therefore, it researches and develops a kind of high Effect, easily acute In Diagnostic Technology of Toxoplasmosis is very necessary.

The common diagnostic method of acute toxoplasmosis includes aetology, serology, molecular biology and immunopathology etc.. Aetology and molecular biological testing (such as PCR) result are although intuitive and reliable, but etiological diagnosis is complicated for operation, recall rate It is low, the period is long, false negative easily occurs, molecular biological testing is needed using special equipment, detection time and costly. The common method of serodiagnosis has affinity of antibody test, indirect immunofluorescence assay (IFA) and Enzyme-linked Immunosorbent Assay examination (ELISA) etc. is tested, but also has different degrees of defect.The variation of affinity of antibody has not been with arch insect infection process It is consistent entirely, such as IgG affinity may will not rise in some time after polypide infects during gestation, it is possible to cause false sun Property testing result.IFA detection toxoplasma early infection is to be diagnosed by internal IgM level, but the sensibility of its detection is far not It such as ELISA method, and tests and needs special instrument and equipment, there is subjectivity to the judgement of result, false positive rate is high, at present It is mainly used for laboratory testing.ELISA method has a sensitive advantages such as special, easy to operate, reproducible, and can be with Detection for base's high-volume clinical sample.

The diagnosis of toxoplasmosis is often to detect in serum IgG antibody as target, but the presence of high-affinity IgG antibody is only It can indicate that arch insect infection at least occurs 3-4 months before detection.Acute toxoplasmosis is clinically commonly indicated at present It is IgM antibody, but is only that the IgM antibody positive cannot determine whether recent arch insect infection, because IgM can exists in body Several months even several years, cause false positive diagnostic result.And arch insect circulating antigen (CAg) refers to that toxoplasma enters in host Invade, move and breeding in the metabolism that generates and pyrolysis product, usually in the serum of arch insect infection early stage or active stage Occur, and time of occurrence is generally earlier than IgM, it may be said that it is the positive evidence for making a definite diagnosis acute toxoplasmosis, compares antibody test, CAg can more accurately diagnose acute toxoplasmosis.

Toxoplasma dense granule protein 1 (GRA1) is the important component of CAg, has good immunogenicity, is one The good vaccine candidate molecule of kind and toxoplasmosis diagnose common candidate antigens.Toxoplasma when invading host cell and GRA1 can be secreted after invasion to receiving in worm vacuole (PV), adjusts calcium ion concentration in PV, play adjusting, worm vacuole membrane is received in modification The attack of host's lysosome is resisted in the effect of (Parasitophorous vacuole membrane, PVM), is that toxoplasma is raw It is long to develop essential critical function albumen；And equal can stablize of the toxoplasma of different development stage and different genotype is divided It secretes, expression quantity is higher, this is very beneficial to the detection of toxoplasma.

Currently, domestic and foreign scholars had been established it is a variety of about change of serum C Ag detection method, such as based on the dual anti-of SAG3 monoclonal antibody Body sandwich ELISA (bandit Jinhua), but the offline standard positive sample to dilute 640 times is detected, not to following in positive sample Ring antigen carries out quantitative detection；Rui Chen etc. establishes one-step method sandwich ELISA method, detects offline in serum 31.2ng/mL circulating antigen, but this method uses detection antibody and capture antibody is that the full worm of toxoplasma is mostly anti-.

In conclusion inventing, a kind of high specificity, sensibility are high, and sandwich ELISA method easy to operate is to many animals The diagnosis of acute toxoplasmosis has great importance.

The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.

Summary of the invention

The purpose of the present invention is to provide a kind of double-antibody sandwiches of arch insect circulating antigen in detection many animals serum ELISA kit and preparation method and application, the double-antibody sandwich elisa kit is easy to operate, small by such environmental effects, Without species specificity, there is higher sensibility and specificity compared to the diagnostic method that polyclonal antibody is established.

To achieve the above object, the present invention provides it is a kind of detection many animals serum in arch insect circulating antigen it is dual anti- Body sandwich ELISA lcits, the double-antibody sandwich elisa kit include: capture antibody, and the capture antibody is toxoplasma GRA1 (that is, dense granule protein 1) monoclonal antibody, the potency of the capture antibody are 5 × 10⁶, antibody subtype IgG1；And The detection antibody of HRP label, the detection antibody are source of mouse toxoplasma GRA1 polyclonal antibody, and the potency of the detection antibody is 1.28×10⁵。

The present invention is detection target with GRA1 antigen component in CAg, wherein GRA1 is a kind of with good immunogenicity Soluble antigen, toxoplasma development each period can great expression, it participate in receiving worm vacuole membrane formation and related function The performance of energy.

In a preferred embodiment, the double-antibody sandwich elisa kit further include: 96 hole enzyme reaction plates； Coating buffer, the coating buffer are the 50mM carbonate buffer solutions that pH value is 9.6；Confining liquid and sample diluting liquid, the confining liquid And sample diluting liquid is 5% defatted milk；Cleaning solution, the cleaning solution are the phosphate buffers containing 0.5%Tween-20； Tmb substrate developing solution；Terminate liquid, the terminate liquid are the H of 2mol/L₂SO₄Solution；Standard positive serum；And standard female blood Clearly.

The present invention also provides the double-antibody sandwich elisa examinations of arch insect circulating antigen in above-mentioned detection many animals serum The preparation method of agent box, comprising: (1) it is described capture antibody the preparation method comprises the following steps: being exempted from using toxoplasma GRA1-His recombinant protein Epidemic disease mouse, filters out positive hybridoma cell, which is injected to mouse peritoneal, continuously collects ascites, pure Change ascites in toxoplasma GRA1 monoclonal antibody to get；(2) the detection antibody of HRP label the preparation method comprises the following steps: will bow After shape worm GRA1-His recombinant protein adds Freund's adjuvant to emulsify, mouse is immunized, acquires blood, separate serum, in purified blood serum Toxoplasma GRA1 polyclonal antibody, by the toxoplasma GRA1 polyclonal antibody after purifying using HRP mark to get；(3) described Coating buffer the preparation method comprises the following steps: weighing Na₂CO₃1.43g and NaHCO₃3.066g is dissolved in 1000mL distilled water, and 4 DEG C of preservations are standby With；(4) confining liquid and sample diluting liquid the preparation method comprises the following steps: weighing 5g skimmed milk power are dissolved in 100mL PBS (phosphate are slow Fliud flushing) in, it is ready-to-use；(5) cleaning solution the preparation method comprises the following steps: in 1LPBS be added 0.5mL Tween-20；(6) institute State tmb substrate developing solution the preparation method comprises the following steps: weighs TMB 200mg, and dehydrated alcohol 100mL is added, adjusts pH value 5.0, adds double steamings Water is settled to 1000mL, and substrate A liquid is made；In addition citric acid 9.33g, Na are weighed₂HPO₄6.4mL H is added in 14.60g₂O₂, adjust PH value 5.0, adds distilled water to be settled to 1000mL, and substrate B liquid is made, by the substrate A liquid and substrate B liquid with the volume ratio of 1:1 Colour developing is added；(7) terminate liquid is made the preparation method comprises the following steps: concentrated sulfuric acid 22.2mL is poured into distilled water 178.8mL；(8) institute Stating standard positive serum is the FBS (fetal calf serum) that joined 100ng/mL toxoplasma GRA1-His recombinant protein；And (9) institute Stating standard female serum is the FBS for not adding toxoplasma GRA1-His recombinant protein.

In a preferred embodiment, it is described capture antibody the preparation method is as follows: with toxoplasma GRA1-His recombinate Protein immunization mouse, three exempt from after take mouse splenocyte and SP2/0 cell fusion, be packet with toxoplasma GRA1-GST recombinant protein Indirect ELISA method screening positive hybridoma cell is established by object, which is injected to mouse peritoneal, continuously Ascites is collected, and then using the monoclonal antibody in Protein G gravity prepackage column purification ascites, is identified by SDS-PAGE pure Change effect, obtains the capture antibody.

In a preferred embodiment, the detection antibody of HRP label the preparation method is as follows: using toxoplasma After GRA1-His recombinant protein adds same volume Freund's complete adjuvant to emulsify, according to 100 μ g/ first immunisation BALB/c mouses, so It is emulsified afterwards with not formula Freund's incomplete adjuvant, BALB/c mouse is only immunized according to 50 μ g/, be immunized twice, each immunization interval 2 weeks, two Exempt from ten days eye socket acquisition blood after exempting from three, separate serum and detect antibody titer, titre reaches 1 × 10⁶By mouse after above Eyeball blood sampling separation serum is plucked, and then using the polyclonal antibody in Protein G gravity prepacked column purified blood serum, by SDS- PAGE purification Identification effect, further by the toxoplasma GRA1 polyclonal antibody after purifying using HRP mark to get.

In a preferred embodiment, the toxoplasma GRA1 polyclonal antibody after purifying is using improvement sodium periodate Method carries out HRP label to it；Preferably, the step of the toxoplasma GRA1 polyclonal antibody after the purifying is marked using HRP It is rapid as follows: to carry out the label of horseradish peroxidase to the polyclonal antibody after purifying using improvement Over-voltage protection: by 5mg HRP is dissolved in 0.5mL distilled water, and 2min is stirred at room temperature；The 0.06mol/LNaIO of 0.5mL Fresh is added₄Aqueous solution mixes Even to be placed on 4 DEG C of refrigerators and be protected from light 30min, solution is slowly changed into green；It is water-soluble to add 0.5mL 0.16mol/L ethylene glycol Liquid is stored at room temperature 30min, terminates reaction；The 1mL aqueous solution of toxoplasma GRA1 polyclonal antibody after being purified containing 5mg It is added in aforesaid liquid, is fitted into after mixing in preactivated bag filter, be put into the carbonate buffer of 0.05mol/LpH9.5 Liquid is slowly stirred, and 4 DEG C are dialyzed overnight；Liquid in bag filter is transferred out, the NaBH of 5mg/mL is added₄Solution 0.5mL, sets 6h is restored in 4 DEG C of refrigerators, enzyme labelled antibody is made to be reduced to stable conjugate；Then isometric saturated ammonium sulfate is added, in 4 DEG C Refrigerator overnight, 4 DEG C of 3000r/min are centrifuged 10min, and precipitating is dissolved in 0.02mol/LpH7.4PBS, and is dialysed with PBS；Dialysis 4 DEG C of 3000r/min of liquid afterwards are centrifuged 10min, and suitable PBS is added according to precipitation capacity and dissolves；Added according to final liquid volume Enter the mixing of equivalent glycerol, in -20 DEG C of long-term preservations.

In a preferred embodiment, the toxoplasma GRA1-His recombinant protein and toxoplasma GRA1-GST recombinate egg White to obtain in the following way: (1) according to the coding gene sequence of toxoplasma GRA1, (GRA1 is in ToxoDB database Accession number is TGGT1_270250) polypide RNA, reverse transcription cDNA are extracted, target fragment is expanded, TgGRA1 is named as；(2) will The target fragment is recombinated with carrier pET-28a and pGEX-6P-1 respectively, is assembled into pET-28a-TgGRA1 and pGEX- PET-28a-TgGRA1 the and pGEX-6P-1-TgGRA1 recombinant plasmid is further transferred to by 6P-1-TgGRA1 recombinant plasmid In Transetta (DE3) competent cell；And (3) have been transferred to recombinant plasmid pET-28a-TgGRA1 and pGEX- for identified The competent cell of 6P-1-TgGRA1 obtains toxoplasma GRA1-His recombinant protein (that is, TgGRA1- through inducing expression, purifying His recombinant protein) and toxoplasma GRA1-GST recombinant protein (that is, TgGRA1-GST recombinant protein).

In a preferred embodiment, the size of the toxoplasma GRA1-His recombinant protein is 30KDa, the arch The size of worm GRA1-GST recombinant protein is 50kDa.

The present invention also provides the double-antibody sandwich elisa reagents of arch insect circulating antigen in above-mentioned survey many animals serum The application method of box, comprising the following steps: (1) antibody will be captured with coating buffer and carry out 200 times of dilutions, in 96 hole enzyme reaction plates In every hole in 100 μ L are added, in 4 DEG C of coating 14h then at 37 DEG C of coating 1h, knockout plate, and washed 4 times with cleaning solution, every time between Every 3min；(2) it is added confining liquid, 100 μ L of every hole closes 1h at 37 DEG C, washed with cleaning solution；(3) measuring samples are added, in 37 DEG C effect 1h, washed with cleaning solution；(4) the detection antibody of 400 times of diluted HRP labels is added, every 100 μ L of hole makees in 37 DEG C With 1h, washed with cleaning solution；(5) 100 μ LTMB substrate developing solutions are added in every hole, and develop the color 10min；(6) it is terminated with terminate liquid anti- It answers, every 50 μ L of hole；And (7) read OD value at wavelength 450nm: when OD value>0.157, determine that sample is the positive, 0.131< When OD < 0.157, sample is suspicious specimen, when OD < 0.131, determines that sample is feminine gender.

The present invention also provides the double-antibody sandwich elisa examinations of arch insect circulating antigen in above-mentioned detection many animals serum Application of the agent box in Serologic detection field.

Compared with prior art, the invention has the following beneficial effects:

(1) present invention is detection target with GRA1 antigen component in CAg, double-antibodies sandwich ELISA is established, to acute Toxoplasmosis makes diagnosis, has the characteristics that the diagnostic method tool for single epitope, established compared to polyclonal antibody There is higher sensibility and specificity；

(2) present invention will test the direct marker enzyme of antibody, eliminate the limitation to test sample kind, can be used for a variety of dynamic The detection of object blood serum sample；

(3) toxoplasma GRA1 has good immunogenicity, and existing a large amount of research confirms toxoplasma GRA1 in ESA Presence in (excreted/secreted antigen, excretory-secretory antigen), and compared to phases such as common diagnostic antigen SAG1 Specific proteins, the energy great expression of each period that GRA1 is developed in toxoplasma, have more the diagnosis of toxoplasmosis pervasive Property.

Detailed description of the invention

Fig. 1 is the SDS-PAGE qualification figure after antibody purification according to an embodiment of the present invention；

Fig. 2 is TgGRA1-His recombinant protein according to an embodiment of the present invention through SDS-PAGE coomassie brilliant blue R250 Effect picture after dyeing and decolourizing；

Fig. 3 is TgGRA1-GST recombinant protein according to an embodiment of the present invention through SDS-PAGE coomassie brilliant blue R250 Effect picture after dyeing and decolourizing.

Main appended drawing reference explanation:

The purifying of 1- monoclonal antibody IgG；The purifying of 2- source of mouse GRA1 polyclonal antibody；1', 1 "-do not induce bacterium solution；2', Bacterium solution after 2 "-inductions；3, supernatant after 3'- bacterium solution ultrasound is cracked and is centrifuged；4, inclusion body after 4'- bacterium solution ultrasound is cracked and is centrifuged； 5, the albumen after 5'- is purified and is concentrated；M- albumen Marker.

Specific embodiment

With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention Shield range is not limited by the specific implementation.

Unless otherwise explicitly stated, otherwise in entire disclosure and claims, term " includes " or its change Changing such as "comprising" or " including " etc. will be understood to comprise stated element or component, and not exclude other members Part or other component parts.

The experimental method involved in the present invention arrived is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

E.coli Transetta (DE3) bacterial strain: it is purchased from Beijing Quanshijin Biotechnology Co., Ltd；

Nickel affinity chromatography filler and glutathione S epharose 4B filler: it is purchased from U.S. GE company；

Protein G gravity prepacked column suit: it is purchased from Beijing Bo Aolong Immune Technology Corp.；

Mouse monoclonal antibody subtype identification kit: it is purchased from U.S. SigmaAldrich company；

The detachable enzyme reaction plate in 96 holes: it is purchased from Guangzhou Jie Te biofiltration limited liability company；

Toxoplasma RH tachyzoite is saved by laboratory；

6~8 week old female BAl BIcs/c mouse ties up tonneau China experimental animal technology Co., Ltd purchased from Beijing；

Experiment pig is purchased from Beijing along prosperous agriculture limited liability company.

The preparation of embodiment 1:TgGRA1

Toxoplasma antigen is TgGRA1 in the present invention (its accession number in ToxoDB database is TGGT1_270250) (http://toxodb.org/toxo/app/record/gene/TGGT1_270250), the genetic fragment for encoding the albumen are 495bp。

The clone of 1.TgGRA1 gene and the building of recombinant plasmid

Design primer expands TgGRA1 gene, and primer is as shown in the table.Primer both ends contain restriction enzyme site, and 5 end ' of primer increases Base is protected, 3 ends ' are GRA1 gene order.

1 TgGRA1PCR amplimer of table

Using GT1 plants of toxoplasma of cDNA as template, TgGRA1 is expanded, reaction system is as follows:

2 TgGRA1 PCR amplification system of table

PCR reaction condition: 94 DEG C of initial denaturation 10min carry out 31 wheel circulations, and circulation includes 94 DEG C of denaturation 30sec, and 56 DEG C are moved back Fiery 30sec, 72 DEG C of extension 1min.Last 72 DEG C of extensions 10min.

PCR product sampling carries out agarose gel electrophoresis detection, identifies whether it expands success.It will be with target fragment size The PCR product being consistent is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit.

With BamH I and III double digestion pGEX-6P-1 skeleton of Hind and BamH I and I double digestion pET-28a skeleton of Xho, And carry out nucleic acid electrophoresis and purification and recovery.Specific digestion system is as follows:

3 endonuclease reaction system of table

TgGRA1 sequence after purification is subjected to single slice connection with double digestion skeleton, specific reaction system is as follows:

4 single slice linked system of table

Remarks: DNA concentration, most suitable segment usage amount are estimated by the method for more each segment brightness of nucleic acid electrophoresis before connection =[0.4 × segment base logarithm] ng (0.06pmol), most suitable carrier usage amount=[0.2 × segment base logarithm] ng (0.03pmol)。

The expression of 2.TgGRA1 recombinant protein

Two kinds of recombinant plasmids are transferred in expression competence Transetta (DE3), ice bath 30min, 42 DEG C of heat shock 1min, Ice bath 3-5min, be added 500 μ l non-resistants LB liquid medium, 37 DEG C are shaken bacterium 1h, take 100-200 μ l bacterium solution be coated on containing On the LB solid medium of kalamycin resistance, 37 DEG C are incubated overnight.Next day picking single bacterium colony, is added containing kanamycin 37 DEG C of LB liquid medium are cultivated to logarithmic growth phase (OD_600nmValue is about 0.6-0.8), IPTG is added, keeps its final concentration of 0.8mM.Continue after cultivating 4-6h, by 4 DEG C of centrifugation 20min of bacterium solution 8000rpm, collects bacterial sediment.40ml PBS is added to be resuspended Bacterial sediment carries out ultrasonic cracking, and work 2s, interval 4s, cracks 10min.Then 4 DEG C of centrifugation 20min of 12000rpm, are received respectively Collect supernatant precipitating, sampling carries out SDS-PAGE electrophoresis.Identified, TgGRA1-His and TgGRA1-GST are primarily present in In clear, the albumen in supernatant is carried out according to the explanation of nickel affinity chromatography filler and glutathione S epharose 4B filler respectively Purifying.As a result see Fig. 2.It is saved backup for -80 DEG C after the albumen packing of purifying.

Embodiment 2: the preparation of toxoplasma GRA1 antibody

1. the preparation of polyclonal antibody

After adding same volume Freund's complete adjuvant to emulsify with toxoplasma GRA1-His recombinant protein, only exempt from for the first time according to 100 μ g/ Then epidemic disease BALB/c mouse is emulsified with not formula Freund's incomplete adjuvant, BALB/c mouse is only immunized according to 50 μ g/, is immunized twice, every time Immunization interval 2 weeks, two exempted from ten days eye socket acquisition blood after exempting from three, separate serum and detect antibody titer, were detected with ELISA anti- Body titre, titre reach 1 × 10⁶After above, mouse is plucked into eyeball blood sampling separation serum, utilizes Protein G gravity prepacked column It is mostly anti-in purified blood serum, and SDS-PAGE purification Identification effect is used, which is 1.28 × 10⁵, the result is shown in Figure 1.

2. the preparation of monoclonal antibody

With recombination GRA1-His protein immunization mouse, three exempt from after take mouse splenocyte and SP2/0 cell fusion, use GRA1-GST establishes indirect ELISA method screening positive monoclonal hybridoma；By the positive hybridoma cell filtered out to Mouse peritoneal injection, continuously collects ascites, pre-installs column purification Monoclonal Antibodies in Mice Ascites using Protein G gravity, is used in combination SDS-PAGE purification Identification effect.The result is shown in Figure 1.Monoclonal antibody after purification is measured it with the coated ELISA method of GRA1-GST Potency carries out the identification of antibody subtype with commercialization mouse monoclonal antibody subtype identification kit.The results show that antibody titer It is 5 × 10⁶, antibody subtype IgG1 is specifically shown in Table 5 and table 6.

The measurement of 5 titer of ascites of table

The identification of 6 antibody subtype of table

Embodiment 3: the preparation of toxoplasma acute infection animal sample

1. the preparation of toxoplasma acute infection mouse blood serum sample

The RH strain of Toxoplasma gondii tachyzoite of the purifying of fresh release is collected, totally 20 cleaning grade 6-8 week old are female for experimental group BALB/c mouse, every is injected intraperitoneally 100, separately sets up 2 mouse of control group, sterile PBS is injected intraperitoneally.It is seen daily after inoculation Clinical symptoms are examined, 2 mouse is taken to pluck eyeball blood sampling at random.

2. the preparation of toxoplasma acute infection pig anteserum sample

The RH strain of Toxoplasma gondii tachyzoite 2 × 10 of experiment pig intraperitoneal injection after purification⁷It is a, daily vena cava anterior blood sampling 10mL, 3mL separates serum, and remaining addition sodium citrate anticoagulant utilizes commercialization whole blood, histocyte genome extraction kit Extract whole blood DNA.

Embodiment 4: the foundation of arch insect circulating antigen sandwich ELISA detection method

1. the preparation and evaluation of the how grand antibody of horseradish peroxidase-labeled

The label of horseradish peroxidase is carried out to polyclonal antibody using improvement Over-voltage protection.5mg HRP is dissolved in In 0.5mL distilled water, 2min is stirred at room temperature；The 0.06mol/L NaIO of 0.5mL Fresh is added₄Aqueous solution mixes postposition It is protected from light 30min in 4 DEG C of refrigerators, solution is slowly changed into green；Add 0.5mL 0.16mol/L glycol water, room temperature 30min is stood, reaction is terminated；1mL aqueous solution containing 5mg antibody purification is added in aforesaid liquid, is packed into after mixing pre- In the bag filter first activated, the carbonate buffer solution for being put into 0.05mol/LpH9.5 is slowly stirred, and 4 DEG C are dialyzed overnight；It will dialysis Liquid in bag is transferred out, and the NaBH of 5mg/mL is added₄Solution 0.5mL is placed in 4 DEG C of refrigerator reduction 6h, makes enzyme labelled antibody also It originally was stable conjugate；Then isometric saturated ammonium sulfate is added, in 4 DEG C of refrigerator overnights, 4 DEG C of 3000r/min centrifugations 10min, precipitating is dissolved in 0.02mol/LpH7.4PBS, and is dialysed with PBS；4 DEG C of 3000r/min centrifugations of liquid after dialysis 10min is added suitable PBS according to precipitation capacity and dissolves；Equivalent glycerol is added according to final liquid volume to mix, it is long in -20 DEG C Phase saves.

2. the optimization of monoclonal antibody peridium concentration and enzyme labelled antibody dilution

Monoclonal antibody peridium concentration and enzyme labelled antibody optimum dilution degree are determined using chessboard method.Monoclonal antibody and enzyme labelled antibody are subjected to ladder Degree dilution, coating condition are 4 DEG C and stay overnight；Next day knockout plate, and washed 4 times with 0.5%PBST, every minor tick 3min；Closing is added Liquid (diluted 5% skimmed milk of PBS), 100 μ L of every hole close 1h at 37 DEG C；It washs again, method is same as above；Addition standard yin-yang 1h is acted in property sample and 37 DEG C, standard positive sample is the FBS serum that joined 100ng/mLTgGRA1-His recombinant protein, Standard female serum is the FBS for not adding albumen.Washing is same as above.Final choice P value is 1.0 or so, and the maximum condition of P/N value is Optimum antibody uses concentration.The results show that 200 times of monoclonal antibody dilutions, when 400 times of dilutions of enzyme labelled antibody, P value is 1.0 or so, and P/ N value is maximum, is shown in Table 7.

The determination of table 7 monoclonal antibody peridium concentration and enzyme labelled antibody optimum dilution degree

3. the optimization of coating buffer type

It is single with tri- kinds of buffer coatings of carbonate buffer solution, Tris-HCl pH7.2 and the PBS pH7.4 of pH9.6 respectively Anti-, concentration is best peridium concentration, sets up yin and yang attribute control, each 4 repetitions, while carrying out using best enzyme labelled antibody dilution Sandwich ELISA experiment, other operating procedures are the same as 2.Final choice P value is 1.0 or so, and the maximum condition of P/N value is best packet By liquid.The results show that P value is 1.0 or so, and P/N value is maximum, is shown in Table 8 when being coated with using carbonate buffer solution.

The optimization of 8 coating buffer type of table

4. the optimization of the condition of coating

Sandwich ELISA experiment is carried out using the coating buffer of optimization, peridium concentration and enzyme labelled antibody dilution, sets up 3 kinds not 4 repetitions are done in same coating condition, respectively 4 DEG C of 14h, 37 DEG C of 2h, 4 DEG C of 14h+37 DEG C of 1h, every group of yin and yang attribute control, other Operating procedure is the same as 2.Final choice P value is 1.0 or so, and the maximum condition of P/N value is best coating condition.The results show that packet When by condition being 4 DEG C of 14h+37 DEG C of 1h, P value is 1.0 or so, and P/N value is maximum, is shown in Table 9.

The optimization of the coating condition of table 9

5. the determination of confining liquid

Carry out sandwich ELISA using the experiment condition that has optimized, set up 6 kinds of different confining liquids, be respectively 1% gelatin, 4 repetitions are done in DMEM, 5% defatted milk, 1%BSA, 2% horse serum, 1%BSA+2% horse serum, every group of yin and yang attribute control, His operating procedure is the same as 2.Final choice P value is 1.0 or so, and the maximum condition of P/N value is best confining liquid.The results show that envelope When to close liquid be 5% defatted milk, P value is 1.0 or so, and P/N value is maximum, is shown in Table 10.

The optimization of 10 confining liquid of table

6. the determination of developing time

Sandwich ELISA is carried out using the experiment condition optimized, chromogenic substrate is added after completed washings, is developing the color respectively 5min, 10min, 15min, 20min terminate reaction, and 4 repetitions are done in every group of yin and yang attribute control, other operating procedures are the same as 2.Finally Select P value 1.0 or so, and the maximum condition of P/N value is best developing time.The results show that P value exists when colour developing 10min 1.0 or so, and P/N value is maximum, is shown in Table 11.

The optimization of 11 developing time of table

7. the determination of yin and yang attribute critical value

Sandwich ELISA experiment is carried out using all conditions that the above optimization is completed, uses the negative serum of 6 kinds of separate sources It is incubated for, 10 times of dilutions use, and calculate yin and yang attribute critical value according to formula.Calculation formula: cut off value=negative control OD_450nmAverage value+3 × standard deviation (SD).It is computed, cut off value is 0.157.It is shown in Table 12；Meanwhile we be greater than 0.131 It is suspicious specimen range less than 0.157.

The determination of 12 yin and yang attribute critical value of table

Embodiment 5: arch insect circulating antigen sandwich ELISA detection method effect assessment

1. sensitivity experiments

It is tested, 10 times of doubling dilutions of standard positive sample with cut using the sandwich ELISA that early period has optimized Off value determines antigen concentration limit.The results show that the sandwich ELISA method after optimization can detecte in FBS 1.563ng/mL GRA1 antigen below.It is shown in Table 13.

13 sensitivity Detection of table

2. specificity experiments

Specific detection is carried out using the FBS for being separately added into neospora and toxoplasma tachyzoite, while setting up feminine gender Control, is tested according to established sandwich ELISA method early period.The results show that joined the FBS detection knot of neospora Fruit is feminine gender, it was demonstrated that this method specificity is good.

3. repeated experiment

Select between 10 parts of serum progress double-antibodies sandwich ELISAs batch and criticize interior repetitive test.The results show that in criticizing Repetitive test coefficient of variation maximum value is 9.231%, and it is 9.295% that coefficient of variation maximum value is repeated between batch, illustrates this method It is repeated preferable.It is shown in Table 14.

14 repetitive test of table

4. detecting the sandwich ELISA lcits of arch insect circulating antigen and the comparison of coherence of nPCR detection method

Using the reports Testis formula PCR method such as royal power, the gene of toxoplasma in experimental animal whole blood is identified.nPCR The specific operation method is as follows: the interior outer primer of synthesis nPCR is designed by document report, particular sequence is as follows:

ITS outer primer F1:5 ' ACCTTTGAATCCCAAGCA 3 ' (SEQ ID NO.:5)

ITS inner primer R1:5 ' TAAATCGGACAAACGCCC 3 ' (SEQ ID NO.:6)

ITS outer primer F2:5 ' TTTGCATTCAAGAAGCGTG 3 ' (SEQ ID NO.:7)

ITS inner primer R2:5 ' AAGGTGCCATTTGCGTTC 3 ' (SEQ ID NO.:8)

PCR amplification is carried out to toxoplasma ITS gene.Reaction system is as follows:

15 nPCR amplification system of table

First round PCR amplification condition: the Taq archaeal dna polymerase amplified reaction of 25 μ L systems, 94 DEG C of initial denaturation 5min, 94 DEG C It is denaturalized 50s, 60 DEG C of annealing 40s, 72 DEG C of extension 45s, totally 30 circulations, 72 DEG C extend 10min eventually.Second wheel PCR: by the first round PCR product takes 2 μ L for template after carrying out 500 times of dilutions, and other conditions are constant.Two-wheeled pcr amplification product is coagulated in 2% agarose Electrophoresis is carried out in glue, observes result under gel imager.Meanwhile with above-mentioned sandwich ELISA operating procedure to the arch in serum Insect circulating antigen is detected.The experimental results showed that mouse can detect toxoplasma cdna from 4d in whole blood, and pig exists 1d can be detected after infection, such as table 16 and table 17.Since the presence of toxoplasma tachyzoite in the circulatory system is existing for CAg Premise, only when toxoplasma reaches certain quantity, circulating antigen can be just successfully detected, and nPCR detection target is Toxoplasma cdna group is not CAg, and therefore, detection of the testing result nPCR positive earlier than ELISA circulating antigen is normal and must A kind of right phenomenon.

16 mouse toxoplasma acute infection testing result of table

17 pig toxoplasma acute infection testing result of table

5. compared with domestic commercial kit detects mice serum circulating antigen effect

To compare using GRA1 as the effect of the double-antibodies sandwich ELISA of test-target and commercial goods kit, The mouse blood serum sample of above-mentioned acquisition is detected with two kinds of domestic reagent boxes.The results show that with domestic commercial kit phase Than testing result of the invention shows apparent regularity and higher sensibility, and credibility is higher, is shown in Table 18.

Table 18 is compared with domestic commercial kit detects mice serum circulating antigen effect

Embodiment 6: the assembling and detection method of the sandwich ELISA lcits of arch insect circulating antigen are detected

ELISA detection kit includes following components:

1. capturing antibody is GRA1 monoclonal antibody (4H10), detection antibody is source of mouse GRA1 mostly anti-；

2. coating buffer (pH value 9.650mM carbonate buffer solution): Na₂CO₃1.43g NaHCO₃3.066g being dissolved in 1000mL In distilled water, 4 DEG C are saved backup；

3. confining liquid and sample diluting liquid (diluted 5% skimmed milk of PBS): weighing 5g skimmed milk power and be dissolved in 100mLPBS In, it is ready-to-use.

4. cleaning solution (0.5%PBST): 0.5mLTween-20 being added in 1LPBS；

5.TMB substrate developing solution: by substrate A liquid (weigh TMB 200mg, dehydrated alcohol 100mL be added, adjust pH value 5.0, Distilled water is added to be settled to 1000mL), (citric acid 9.33g, Na are weighed with substrate B liquid₂HPO₄6.4mL H is added in 14.60g₂O₂, PH value 5.0 is adjusted, distilled water is added to be settled to 1000mL) 1:1 addition colour developing；

6. terminate liquid (the 2M concentrated sulfuric acid): distilled water 178.8mL is added in concentrated sulfuric acid 22.2mL；

7. standard positive serum；

8. standard female serum.

Above-mentioned material is assembled up to the present invention.

The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.

Sequence table

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Claims

1. the double-antibody sandwich elisa kit of arch insect circulating antigen in a kind of detection many animals serum, which is characterized in that The double-antibody sandwich elisa kit includes:

Antibody is captured, the capture antibody is toxoplasma GRA1 monoclonal antibody, and the potency of the capture antibody is 5 × 10⁶, antibody Hypotype is IgG1；And

The detection antibody of HRP label, the detection antibody are source of mouse toxoplasma GRA1 polyclonal antibody, the potency of the detection antibody It is 1.28 × 10⁵。

2. detecting the double-antibody sandwich elisa reagent of arch insect circulating antigen in many animals serum as described in claim 1 Box, which is characterized in that the double-antibody sandwich elisa kit further include:

96 hole enzyme reaction plates；

Coating buffer, the coating buffer are the 50mM carbonate buffer solutions that pH value is 9.6；

Confining liquid and sample diluting liquid, the confining liquid and sample diluting liquid are 5% defatted milks；

Cleaning solution, the cleaning solution are the phosphate buffers containing 0.5%Tween-20；

Tmb substrate developing solution；

Terminate liquid, the terminate liquid are the H of 2mol/L₂SO₄Solution；

Standard positive serum；And

Standard female serum.

3. the double-antibody sandwich elisa of arch insect circulating antigen tries in detection many animals serum as claimed in claim 1 or 2 The preparation method of agent box characterized by comprising

(1) it is described capture antibody the preparation method comprises the following steps: using toxoplasma GRA1-His recombinant protein be immunized mouse, filter out the positive The positive hybridoma cell is injected to mouse peritoneal, continuously collects ascites, purify the toxoplasma in ascites by hybridoma GRA1 monoclonal antibody to get；

(2) the detection antibody of HRP label the preparation method comprises the following steps: toxoplasma GRA1-His recombinant protein is added Freund's adjuvant cream After change, mouse is immunized, acquisition blood separates serum, the toxoplasma GRA1 polyclonal antibody in purified blood serum, after purifying Toxoplasma GRA1 polyclonal antibody using HRP mark to get；

(3) coating buffer the preparation method comprises the following steps: weighing Na₂CO₃1.43g and NaHCO₃3.066g is dissolved in 1000mL distilled water In, 4 DEG C save backup；

(4) confining liquid and sample diluting liquid are dissolved in 100mLPBS the preparation method comprises the following steps: weighing 5g skimmed milk power, now with existing With；

(5) cleaning solution the preparation method comprises the following steps: in 1LPBS be added 0.5mL Tween-20；

(6) the tmb substrate developing solution the preparation method comprises the following steps: weigh TMB 200mg, dehydrated alcohol 100mL is added, adjusts pH value 5.0, add distilled water to be settled to 1000mL, substrate A liquid is made；In addition citric acid 9.33g, Na are weighed₂HPO₄14.60g being added 6.4mL H₂O₂, adjust pH value 5.0, add distilled water to be settled to 1000mL, substrate B liquid is made, by the substrate A liquid and substrate B liquid with Colour developing is added in the volume ratio of 1:1；

(7) terminate liquid is made the preparation method comprises the following steps: concentrated sulfuric acid 22.2mL is poured into distilled water 178.8mL；

(8) standard positive serum is the FBS that joined 100ng/mL toxoplasma GRA1-His recombinant protein；And

(9) the standard female serum is the FBS for not adding toxoplasma GRA1-His recombinant protein.

4. detecting the double-antibody sandwich elisa reagent of arch insect circulating antigen in many animals serum as claimed in claim 3 The preparation method of box, which is characterized in that it is described capture antibody the preparation method is as follows:

With toxoplasma GRA1-His recombinant protein be immunized mouse, three exempt from after take mouse splenocyte and SP2/0 cell fusion, with bow Shape worm GRA1-GST recombinant protein is that coating object establishes indirect ELISA method screening positive hybridoma cell, which is hybridized Oncocyte is injected to mouse peritoneal, is continuously collected ascites, and then purify the monoclonal antibody in ascites, is identified by SDS-PAGE Purification effect obtains the capture antibody.

5. detecting the double-antibody sandwich elisa reagent of arch insect circulating antigen in many animals serum as claimed in claim 3 The preparation method of box, which is characterized in that the detection antibody of HRP label the preparation method is as follows:

After adding same volume Freund's complete adjuvant to emulsify with toxoplasma GRA1-His recombinant protein, according to 100 μ g/ first immunisations Then BALB/c mouse is emulsified with not formula Freund's incomplete adjuvant, BALB/c mouse is only immunized according to 50 μ g/, is immunized twice, exempts from every time Epidemic disease interval 2 weeks, two exempt from ten days eye socket acquisition blood after exempting from three, separate serum and detect antibody titer, and titre reaches 1 × 10⁶ Mouse is plucked into eyeball blood sampling separation serum, and then the polyclonal antibody in purified blood serum after above, is identified by SDS-PAGE pure Change effect, further by the toxoplasma GRA1 polyclonal antibody after purifying using HRP mark to get.

6. detecting the double-antibody sandwich elisa reagent of arch insect circulating antigen in many animals serum as claimed in claim 5 The preparation method of box, which is characterized in that the toxoplasma GRA1 polyclonal antibody after purifying is using improvement Over-voltage protection pair It carries out HRP label.

7. detecting the double-antibody sandwich elisa reagent of arch insect circulating antigen in many animals serum as claimed in claim 4 The preparation method of box, which is characterized in that the toxoplasma GRA1-His recombinant protein and toxoplasma GRA1-GST recombinant protein are It obtains in the following way:

(1) polypide RNA, reverse transcription cDNA are extracted according to the coding gene sequence of toxoplasma GRA1, expands target fragment, name For TgGRA1；

(2) target fragment is recombinated with carrier pET-28a and pGEX-6P-1 respectively, is assembled into pET-28a- TgGRA1 and pGEX-6P-1-TgGRA1 recombinant plasmid, further by the pET-28a-TgGRA1 and pGEX-6P-1-TgGRA1 Recombinant plasmid is transferred in Transetta (DE3) competent cell；And

(3) by the identified competent cell for being transferred to recombinant plasmid pET-28a-TgGRA1 and pGEX-6P-1-TgGRA1 through luring Expression, purifying are led, toxoplasma GRA1-His recombinant protein and toxoplasma GRA1-GST recombinant protein are obtained.

8. such as the double-antibody sandwich of arch insect circulating antigen in the described in any item detection many animals serum of claim 4-7 The preparation method of ELISA kit, which is characterized in that the size of the toxoplasma GRA1-His recombinant protein is 30KDa, described The size of toxoplasma GRA1-GST recombinant protein is 50kDa.

9. the double-antibody sandwich elisa of arch insect circulating antigen tries in detection many animals serum as claimed in claim 1 or 2 The application method of agent box, which comprises the following steps:

(1) it is diluted, is added in every hole in 96 hole enzyme reaction plates, after 4 DEG C of coatings again by antibody is captured with coating buffer It is coated in 37 DEG C, knockout plate, and is washed with cleaning solution；

(2) confining liquid is added in every hole, and closing is washed with cleaning solution；

(3) measuring samples are added, acts in 37 DEG C, is washed with cleaning solution；

(4) the detection antibody of the HRP label after dilution is added, acts in 37 DEG C, is washed with cleaning solution；

(5) tmb substrate developing solution, colour developing is added in every hole；

(6) it is terminated and is reacted with terminate liquid；And

(7) OD value is read at wavelength 450nm: when OD value>0.157, determining that sample is the positive, when 0.131<OD<0.157, Sample is suspicious specimen, when OD < 0.131, determines that sample is feminine gender.

10. the double-antibody sandwich elisa examination of arch insect circulating antigen in detection many animals serum of any of claims 1 or 2 Application of the agent box in Serologic detection field.