CN109342711B - The ELISA kit of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination - Google Patents

The ELISA kit of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination Download PDF

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CN109342711B
CN109342711B CN201811336380.0A CN201811336380A CN109342711B CN 109342711 B CN109342711 B CN 109342711B CN 201811336380 A CN201811336380 A CN 201811336380A CN 109342711 B CN109342711 B CN 109342711B
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antibody
dna
detection
beta
rabbit
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CN109342711A (en
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任洪林
水一鸣
胡盼
卢士英
柳增善
柳溪林
李岩松
周玉
常江
王路鹿
张士军
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Jilin University
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Jilin University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Abstract

The present invention relates to the ELISA kits of a kind of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination, belong to field of biological technology detection.The polyclonal detection antibody of the anti-IL-1R in cavy source marked including anti-il-i-beta and IL-1Ra rabbit source polyclonal capture antibody pre-coated elisa plate, IL-1 β and IL-1Ra recombinant protein standard items, dilution, 20 × PBS-T concentrated cleaning solution, the cavy source anti-il-i-beta polyclonal detection antibody of blue-fluorescence microballoon label, green fluorescent microspheres.The present invention has many advantages, such as that high sensitivity, high specificity, detection species range are wide, it can be used for sheep, people, ox, horse, pig, mouse, rabbit anteserum, IL-1 β and IL-1Ra assay in blood plasma and other tissue samples, and its ratio can be calculated, the synchronous detection of two kinds of objects reduces the error of ratio calculation, sensitivity reaches pg grades, this kit measurement IL-1Ra and IL-1 β ratio can be used for brucellosis natural infection and vaccine immunity difference diagnosis and the other and ratio changes relevant medical diagnosis on disease mark and studies.

Description

The ELISA kit of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination
Technical field
The invention belongs to field of biological technology detection, analyze sheep, people, ox, horse, pig, mouse, the more objects of rabbit by homologous comparison The amino acid sequence of kind IL-1 β and IL-1Ra albumen, determines the homologous conservative IL-1 β of several species and IL-1Ra domain gene and egg White tiles section with prokaryotic expression system preparation IL-1 β and the homologous conserved domain genetic engineering recombinant protein of IL-1Ra, and is done with it For immunogene, preparing can tie respectively in combination with the polyclonal capture antibody in rabbit source of several species IL-1 β and IL-1Ra, specificity The polyclonal detection antibody in cavy source and the specific binding several species IL-1Ra cavy source for closing several species IL-1 β are polyclonal Antibody is used in detection.Two kinds of detection antibody and blue and green fluorescent microspheres are coupled respectively, foundation can measure several species simultaneously IL-1 β and the double fluorescence ELISA detection methods of IL-1Ra double-antibody sandwich, and it is assembled into the detection kit for being easy to apply.It can use IL-1 β and the IL-1Ra assay in the serum of sheep, people, ox, horse, pig, mouse, rabbit etc., blood plasma and other tissue samples, and Its IL-1Ra/IL-1 β ratio can be calculated, the synchronous detection of two kinds of objects reduces the error of ratio calculation.
Background technique
Interleukin 1 (Interleukin 1, IL-1) cytokine family includes 11 members, mainly there is interleukin-11 α (IL-1 α), interleukin-11 β (IL-1 β), interleukin 1 receptor antagonism factor (IL-1Ra) etc..IL-1 α and IL-1 β are IL-1 families Middle primary activation downstream signaling pathway is to play 2 members of biological function.Mainly by raising in conjunction with specific receptor Collect co-receptor, and signal in active cell, induction inflammatory reaction etc., plays biological function.Research finds that IL-1 β itself does not have There is signal peptide sequence, is secreted by unconventional approach.In addition, IL-1 β precursor does not have bioactivity, only in its N- end regions quilt It just can produce activity after caspase-1 cutting.IL-1 β activity by protein translation, processing, maturation, receptor combine and Receptor spontaneous emissions conduction depressant drug strict control, wherein IL-1Ra is main IL-1 family antagonist.The study found that IL-1 Unbalance between IL-1Ra will lead to excessive inflammatory reaction, and there are different differences in some diseases from IL-1Ra by IL-1 β Different expression pattern, ratio are also not quite similar in various disease.Therefore the two expression quantity ratio measures certain related diseases hairs It gives birth to, develop, lapsing to certain indicative significance.Currently, the equilibrium relation between IL-1 and IL-1Ra is in arthritis, inflammation Carry out research extensively in a variety of diseases such as disease property enteropathy (IBD).IL-1 local excessive generates and/or IL-1Ra generation is insufficient all easy In generation disease, and the therapeutic of IL-1Ra can effectively prevent tissue damage.The detection side of IL-1 β and IL-1Ra content Method mainly has intracellular cytokine detection method, immunological detection, bioassay and molecular biology method etc.. First three methods detection species are excessively single, and specificity is poor, and detected value confidence level is lower.And molecular biology method mainly collects In detection to nucleic acid, detection operation is relatively cumbersome, needs the step of sample pre-treatments nucleic acid extraction, and extraction efficiency directly affects Testing result.
Brucellosis (Brucellosis, referred to as " cloth disease ") is that one of zoonosis the most serious is endangered in the whole world. There is no the detection method of effective district tap kind of cloth disease vaccine animal and infected animal in the purification work of cloth disease, to slaughtering infected animal Resistance is brought, effective prevention and control of cloth disease are seriously affected.
Summary of the invention
The present invention provides the ELISA kit of a kind of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination, passes through one The concentration and its ratio of IL-1Ra and IL-1 β, solve independent measuring IL-1 β and IL- in secondary experiment while test sample The problem of 1Ra concentration proportion inaccuracy, while serum, blood plasma, Various Tissues sample can more sensitively be detected using fluorescence detection In IL-1 β and IL-1Ra concentration.The present invention by it is homologous compare design sheep, people, ox, horse, pig, mouse, rabbit IL-1 β with The homologous conserved domain of IL-1Ra and its fused in tandem gene, with its gene engineering expression albumen preparation antibody can with it is natural Sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck IL-1 β and IL-1Ra combine, can be used for detecting multiple species Serum, the ratio of IL-1 β and IL-1Ra content and IL-1Ra/IL-1 β in blood plasma and histoorgan sample.
The technical solution adopted by the present invention is that: it is pre-coated including the polyclonal capture antibody of anti-il-i-beta and IL-1Ra rabbit source ELISA Plate, IL-1 β and IL-1Ra recombinant protein standard items, dilution, 20 × PBS-T concentrated cleaning solution, blue-fluorescence microballoon mark The polyclonal detection of the anti-IL-1R in cavy source that the polyclonal detection antibody of the cavy source anti-il-i-beta of note, green fluorescent microspheres mark is used Antibody.
The present invention:
(1) preparation step of the polyclonal capture antibody pre-coated elisa plate of anti-il-i-beta and IL-1Ra rabbit source is as follows:
(1) it is coated with the polyclonal capture antibody of anti-il-i-beta and IL-1Ra rabbit source: by anti-il-i-beta and more grams of IL-1Ra rabbit source Grand capture antibody diluted is added in ELISA Plate, 100 holes μ L/, 4 DEG C were coated with to final concentration of 0.078 μ g/mL Night；
(2) to contain the PBS of 0.2% Tween-20 as PBS-T cleaning solution, board-washing 3-4 times, 200 holes μ L/, every time Then 1min is closed, 200 holes μ L/, 37 DEG C of 1h, draining liq with 0.1M ammonium chloride solution；
(2) the polyclonal detection antibody of the cavy source anti-il-i-beta of blue-fluorescence microballoon label, excitation wavelength 360nm, Launch wavelength 450nm；Coupling step is as follows: the MES buffer solution 90mL of pH 4.5-7.5 is added in 10 μ L blue-fluorescence microballoons, mixes Even, 15000 revs/min are centrifuged 15 minutes, abandon supernatant, collect microballoon precipitating, and repetition is washed twice；In 90 μ L 0.2M pH 4.5- In 7.5 MES buffer solution, 4.8 μ L EDC (1mg/mL) are added, activate 20 minutes；Then 5.6 μ L NHS, (1mg/ are added ML), mix 20 minutes；4 DEG C 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；With the MES of 90 μ L pH 4.5-7.5 Microballoon is resuspended in buffer solution, adds the 10 polyclonal detection antibody (1mg/mL) of μ L cavy source anti-il-i-beta, the concussion of room temperature vortex Reaction 4 hours；10 μ L confining liquids (10%BSA) are added, react at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon The MES buffer solution of 100 μ L pH 4.5-7.5 is added in supernatant, and 4 DEG C are kept in dark place；
(3) the polyclonal detection antibody of the anti-IL-1R in cavy source of green fluorescent microspheres label, excitation wavelength 480nm, Launch wavelength 520nm；The MES buffer solution 90mL of pH 4.5-7.5 is added in 10 μ L green fluorescent microspheres, mixes, 15000 revs/min Zhongli's heart 15 minutes, supernatant is abandoned, collects microballoon precipitating, repetition is washed twice；It is molten in the MES buffering of 90 μ L 0.2M pH 4.5-7.5 In liquid, 4.8 μ L EDC (1mg/mL) are added, activate 20 minutes；Then 5.6 μ L NHS (1mg/mL) are added, mix 20 minutes；4 DEG C 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；Microballoon is resuspended with the MES buffer solution of 90 μ L pH 4.5-7.5, Add the 10 polyclonal detection antibody (1mg/mL) of the μ L cavy anti-IL-1R in source, room temperature vortex concussion reaction 4 hours；10 μ are added L confining liquid (10%BSA) reacts at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon supernatant, and 100 μ L pH are added The MES buffer solution of 4.5-7.5,4 DEG C are kept in dark place.
Polyclonal capture in anti-il-i-beta and IL-1Ra rabbit source of the present invention is used and is resisted with antibody, the polyclonal detection of cavy source anti-il-i-beta The preparation step of the polyclonal detection antibody of body, the anti-IL-1R in cavy source is as follows:
(1) the IL-1 β and IL- of overall length sheep, people, ox, horse, pig, mouse, rabbit are downloaded in the GenBank database of NCBI 1Ra gene and protein sequence, using DNAMAN version 6 carry out sequence identity compare analysis, determine sheep, people, ox, Horse, pig, mouse, IL-1 β and the IL-1Ra albumen of rabbit and the homologous conserved region sequence of nucleic acid several species, and analyze IL-1 β and IL-1Ra Otherness between homologous conserved region；
(2) select the homologous conserved region IL-1 β and IL-1Ra between have differ greatly, two sections of sequences that sequence identity is low Column, are respectively designated as IL-1 β -1 and IL-1Ra-1, and prokaryotic expression recombinant protein is used as to prepare anti-il-i-beta detection and use respectively and resist Body and anti-IL-1Ra the detection immunogene of antibody, in addition select have partial order between the homologous conserved region IL-1 β and IL-1Ra Consistent two sections of sequences are arranged, are respectively designated as IL-1 β -2 and IL-1Ra-2, and IL-1 β -2 and IL-1Ra-2 are passed through into amino acid Sequence is the DNA linker connection of FFMSH, constructs the IL-1 β and homologous conserved region tandem gene IL-1 β -1Ra-2 of IL-1Ra, Prokaryotic expression recombinant protein is used as the immunogene of preparation while anti-il-i-beta and IL-1Ra capture antibody；
(3) the several species homologous sequence selection for being used to prepare immunogene requires: IL-1 β -2 and IL-1Ra-2 has partial order IL-1 β -2 and IL-1Ra-2 fused in tandem are obtained tandem gene IL-1 β -1Ra-2, with its prokaryotic expression gene by column consistency Engineering recombinant protein immunizing rabbit prepares rabbit source anti-il-i-beta and IL-1Ra capture antibody, can be in combination with IL-1 β and IL- Sequence identity is not low homologous between 1Ra, IL-1 β -1 and IL-1Ra, IL-1Ra-1 and IL-1 β so that respectively with IL-1 β -1 and The detection of cavy source anti-il-i-beta antibody and the anti-IL-1Ra in cavy source of IL-1Ra-1 genetic engineering recombinant protein expression product preparation Detection antibody, can specifically bind IL-1 β and IL-1Ra respectively；
(4) upstream and downstream primer a pair of IL-1 β -1-S and IL-1 β -1-A of design amplification IL-1 β -1, to include overall length The cDNA recombinant plasmid of IL-1 β is pcr template, using the DNA of routine PCR reaction condition amplification IL-1 β -1, uses DNA gel The DNA of QIAquick Gel Extraction Kit purifying IL-1 β -1；
(5) upstream and downstream primer a pair of the IL-1Ra-1-S and IL-1Ra-1-A of design amplification IL-1Ra-1, to include complete The cDNA recombinant plasmid of long IL-1Ra is pcr template, using the DNA of routine PCR reaction condition amplification IL-1Ra-1, uses DNA The DNA of Gel Extraction kit IL-1Ra-1；
(6) upstream and downstream primer a pair of IL-1 β -2-S and IL-1 β -2-A of design amplification IL-1 β -2, to include overall length The cDNA recombinant plasmid of IL-1 β is pcr template, using the DNA of routine PCR reaction condition amplification IL-1 β -2, uses DNA gel The DNA of QIAquick Gel Extraction Kit purifying IL-1 β -2；
(7) upstream and downstream primer a pair of the IL-1Ra-2-S and IL-1Ra-2-A of design amplification IL-1Ra-2, to include complete The cDNA recombinant plasmid of long IL-1Ra is pcr template, using the DNA of routine PCR reaction condition amplification IL-1Ra-2, uses DNA The DNA of Gel Extraction kit IL-1Ra-2；
(8) using upper used in design the downstream primer IL-1 β -2-A used in amplification IL-1 β -2 and amplification IL-1Ra-2 The linker sequence in primer I L-1Ra-2-S is swum, using conventional Overlap extension PCR method, with the DNA and IL- of IL-1 β -2 The DNA of 1Ra-2 is that template leads to IL-1 β -2 and IL-1Ra-2 gene using IL-1 β -2-S and IL-1Ra-2-A as upstream primer Linker series connection is crossed, IL-1 β -1Ra-2 fusion dna is expanded, uses DNA gel QIAquick Gel Extraction Kit purifying IL-1 β -1Ra-2's DNA；
(9) poly- with the T4 DNA of disconnected enzyme dependence respectively by the DNA fragmentation of the IL-1 β -1 of acquisition and IL-1Ra-1 Synthase method is cloned on pMCSG9 carrier, and DNA and the pET-30a carrier of IL-1 β -1Ra-2 is connect, building recombinant expression matter Grain pMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1 and pET30a-IL-1 β -1Ra-2；
(10) recombinant expression plasmid is converted into the inducing expression recombinant protein into e. coli bl21-Codonplus cell, Correct single colonie is identified in the sequencing of picking recombinant plasmid, is inoculated in the LB liquid medium of the benzyl resistance of ammonia containing 50mg/L, 37 DEG C Culture 2 hours, is then inoculated in the fresh liquid LB culture medium of the benzyl resistance of ammonia containing 50mg/L of 200mL in 1:500 ratio, 37 DEG C shaken cultivation about 1 hour, add the IPTG of 300 μ L 1mol/mL, after continuing shaken cultivation 5.5 hours through 4 DEG C, 10000rpm from It is big to harvest pMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1, the genetic engineering of pET30a-IL-1 β -1Ra-2 respectively by heart 5min Enterobacteria bacterial sediment after being resuspended with PBS, is cracked with Ultrasonic Cell Disruptor, power 40W, ultrasonic 10s, interval 10s, altogether 30min obtains supernatant and inclusion body precipitates in 12000 revs/min, centrifugation 30min；
(11) recombinant protein of E. coli system expression has merged His label, by the broken bacterium of gene engineering expression bacterium Body supernatant uses nickel column routinely nickel ion affinity chromatograph natural subsidence method purification of target antigen protein IL-1 β -1, IL-1 β -2 and IL-1 β -1Ra-2, using the recombinant protein of SDS-PAGE and Western-blot analysis verifying after purification, with retention point 4 DEG C of the bag filter dialysis of son amount 8000-14000Da, dialyzate are the PBS of 0.01M pH 8.0, and every 2h replaces a dialyzate, It replaces 5 times altogether, carries out concentration of instead dialysing with PEG20000, SDS-PAGE identifies purity, and it is spare that packing is put into -80 DEG C of refrigerators；
(12) animal immune
Immune IL-1 β -1, IL-1Ra-1 the and IL-1 β -1Ra-2 albumen for taking 1mg respectively for the first time, with isometric assistant completely Agent is mixed and is emulsified, and IL-1 β -1 and the preparation cavy source anti-il-i-beta detection of IL-1Ra-1 protein immunization cavy are used after emulsifying respectively The anti-IL-1Ra detection antibody of antibody and cavy source, prepared by the IL-1 β -1Ra-2 protein immunization new zealand white rabbit after emulsification Rabbit source anti-il-i-beta and IL-1Ra, which are captured, uses antibody, carries out dorsal sc multiple spot and is immunized, and 15 days are an immune period, for the second time Immune that Freund's incomplete adjuvant is begun to use to prepare emulsified immunogen for being immunized, method is same as above, and after third time is immune, is surveyed within every 7 days A fixed serum titer, potency carry out Culling heart blood up to 50000 or more, collect serum potency to be measured and antibody purification；
(13) antibody titer detection and purifying
It leaves and takes a small amount of serum before immune to ask as negative blood, the rabbit and cavy after being immunized acquire blood, incubate in 37 DEG C 0.5h, 4 DEG C of blood coagulations 2h, 3000 revs/min of centrifugation 5min are educated, serum is drawn, potency is detected using conventional ELISA method, uses AKATA protein purification system and routine protein G affinity chromatography method antibody purification；
(14) interference antibody removal
By interference antibody removal experiment, the non-specific binding of two kinds of cavy source detection antibody, specific steps are reduced As follows: conventional induction is transferred to the Bacillus coli expression bacterium of pMCSG9 empty carrier, takes 1mL-5mL Bacteria Culture fermentation liquid, 1300 turns/ It minute, centrifugation 1min, abandons supernatant and collects thallus, take two kinds of cavy source detection antibody, 100 μ L suspension thallus, 37 DEG C of incubations respectively 45-90min, 3000 revs/min of centrifugation 5min, taking supernatant is the detection antibody for removing interference antibody.
In the present invention:
IL-1 β -1 nucleotide sequence is as described in SEQ ID No.1；
IL-1 β -1 amino acid sequence is as described in SEQ ID No.2；
IL-1 β -2 nucleotide sequence is as described in SEQ ID No.3；
IL-1 β -2 amino acid sequence is as described in SEQ ID No.4；
IL-1Ra-1 nucleotide sequence is as described in SEQ ID No.5；
IL-1Ra-1 amino acid sequence is as described in SEQ ID No.6；
IL-1Ra-2 nucleotide sequence is as described in SEQ ID No.7；
IL-1Ra-2 amino acid sequence is as described in SEQ ID No.8；
IL-1 β -1Ra-2 nucleotide sequence is as described in SEQ ID No.9；
IL-1 β -1Ra-2 amino acid sequence is as described in SEQ ID No.10.
In the present invention:
(1) IL-1 β -1-S:5/-tacttccaatccaatgcCTATACCTGTCTTGTGTGACCCA-3/；
IL-1 β -1-A:5/-ttatccacttccaatgtcAGAAGACGAATCGCTTTTCC-3/；
(2) IL-1Ra-1-S:5/-tacttccaatccaatgcCAAGCCTTCAGGATCTGGG-3/；
IL-1Ra-1-A:5/-ttatccacttccaatgtcAACCTTGCAAGTATCCAGC-3/；
(3) IL-1 β -2-S:5/-ccgcatatgTACAAGACAGAAATCAAGAACACAGT-3/；
IL-1 β -2-A:5/-atgcatgctgaagaaTATATCCTGGCCACCTCTAAAAC-3/；
(4) IL-1Ra-2-S:5/-ttcttcagcatgcatGACAAGCGCTTCGCCTTCATC-3/；
IL-1Ra-2-A:5/-ccgctcgagATTGGTGAGGCCCACGGG-3/；
(5) the linker sequence of IL-1 β and the homologous conserved region tandem gene IL-1 β -1Ra-2 of IL-1Ra ttcttcagcatgcat。
IL-1 β recombinant protein standard items and IL-1Ra recombinant protein standard items of the present invention, are with large intestine bar respectively The IL-1 β recombinant protein and IL-1Ra recombinant protein of bacterium expression system gene engineering expression, the original of specific preparation method routinely The open reading frame of IL-1 β and IL-1Ra are inserted into prokaryotic expression carrier pET-30a, building fusion His respectively by nuclear expression technology Recombinant expression carrier the pET-30a-IL-1 β and pET-30a-IL-1Ra of label are obtained high with conventional inducing expression purification technique The genetic engineering IL-1 β recombinant protein and IL-1Ra recombinant protein of purity, prepare standard items albumen.
Dilution of the present invention is the 0.1M PBS solution of pH7.2-7.6.
The operating method of the ELISA kit of a kind of several species IL-1 β and IL-1Ra and its ratio Simultaneous Determination, step is such as Under:
(1) according to the quantity of sample to be examined, anti-il-i-beta and the IL-1Ra capture coated black flat-bottom enzyme mark of antibody are taken out Plate, setting standard sample wells, positive hole, negative hole, blank well and sample well, it is 100 μ L that amount of liquid, which is added, in every hole every time.Standard items Two kinds of standard items mixed solutions through dilution gradient dilution are added in hole, and the end of two kinds of standard items protein I L-1 β and IL-1Ra are dense Degree be 78.125ng/ml, 39.625ng/ml, 19.531ng/ml, 9.766ng/ml, 4.883ng/ml, 2.441ng/ml, 12 concentration ladders of 1.221ng/ml, 0.610ng/ml, 0.305ng/ml, 0.153ng/ml, 0.076ng/ml and 0.038ng/ml 2 times of diluted serum to be checked are added in degree, sample well, and PBS dilution is added in negative hole, and any liquid is not added in positive hole and blank well Body, 37 DEG C of incubation 1h are added 0.1%PBST board-washing 3 times, each 1min；
(2) 0.125 μ g/mL detection antibody is added in sample well, standard sample wells and negative hole, 100 μ L of every hole, 37 DEG C It is incubated for 1h, is added 0.1%PBST board-washing 3 times, each 1min；
(3) any liquid is not added in blank well, and it is 0.125 μ g/mL that 100 μ L, two kinds of fluorescent microsphere concentration, which are added, in positive hole 100 μ L dilutions are added in fluorescent microsphere mixed liquor, standard sample wells, negative hole, sample well,
(4) double fluorescence detection readings are carried out using fluorescence microplate detector, emitted in 360nm excitation wavelength and 450nm Wavelength detecting IL-1 β concentration detects IL-1Ra concentration in 480nm excitation wavelength and 520nm launch wavelength；
(5) using blank well fluorescent value B as instrument blank zeroing value, the interference of instrument noise is deducted, established standards sample wells is glimmering Light value is ST, and sample well fluorescent value is SA, and negative hole fluorescent value is N, and positive hole fluorescent value is P, using positive hole fluorescent value P as Denominator correction calculates standard sample wells and corrects fluorescent value STaFluorescent value SA is corrected with sample wella, i.e. STa=ST/P or SAa=SA/ P draws IL-1 β and IL-1Ra standard curve respectively, using standard items IL- β and IL-1Ra protein concentration logarithm as abscissa, with Standard sample wells corrects fluorescent value STaAs ordinate, standard curve is drawn, calculates calibration curve equation and coefficient R2, with sample Sample wells corrects fluorescent value SAaSubstitute into calibration curve equation, calculate log concentration X, 10XIL-1 β's and IL-1Ra is dense as in sample Degree, and calculate IL-1Ra/IL-1 β concentration proportion；
(6) positive hole and negative hole are set up, can be with the validity of Quality Control fluorescence detection, and repeated detection result is had Comparativity.
Antibody of the present invention can with sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck IL-1 β and IL- 1Ra occur specific binding reaction, for detect sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck serum, blood The ratio of IL-1 β and IL-1Ra content and IL-1Ra/IL-1 β in slurry and histoorgan sample.
The ratio I L-1Ra/ of double-antibody sandwich of the present invention double fluorescence ELISA Simultaneous Determination IL-1Ra and IL-1 β IL-1 β, being tested and analyzed by clinical practice sheep blood sample confirms, IL-1Ra/IL-1 β and cloth in brucellosis illness sheep blood serum IL-1Ra/IL-1 β difference is extremely significant in disease vaccine immune sheep and healthy sheep blood serum.Moreover, confirmed by statistics ROC analysis, IL-1Ra/IL-1 β is distinguishing brucellosis infection and is having significance of differential diagnosis in immune clinical examination in sheep blood serum.This hair It is bright to can be used for brucellosis infection and immune antidiastole, it can also be used to other examining with IL-1Ra/IL-1 ss related diseases Marked effect research disconnected and occurring, develop, lapsing to.
The beneficial effects of the invention are that: the present invention provides a kind of synchronous with IL-1Ra and its ratio for several species IL-1 β The double-antibody sandwich fluorescence ELISA detection method of measurement and the application side in brucellosis infection is diagnosed with Immune dctection Method, the fluorescence ELISA detection method established pass through once experiment and can while accurately obtain IL-1 β and IL-1Ra and its IL- 1Ra/IL-1 β ratio, have the operating time is short, high sensitivity, high specificity, double fluoremetries interference is small, measurement result is accurate, Detect the advantages that species range is wide.It can be used for the serum, blood plasma and other tissues of the several species such as sheep, people, ox, horse, pig, mouse, rabbit IL-1 β and IL-1Ra assay in sample.The synchronous detection of two kinds of objects and ratio calculated, reduce IL-1Ra/IL-1 β ratio The error of calculating, sensitivity reach pik (pg) grade, solve currently without accurate calculating IL-1Ra/IL-1 β ratio approach Problem.Meanwhile detection method of the invention has extensive species versatility, expands the application range of this method, Ke Yigeng Accurate comparative studies different plant species IL-1 β and IL-1Ra participate in organism physiology pathologic function.This method measures IL-1Ra/IL-1 β ratio can be used for brucellosis natural infection and vaccine immunity difference diagnosis and other diseases relevant with ratio variation Disease diagnosis.It solves currently without reliable difference brucellosis infection and Immune dctection diagnostic method, causes a large amount of cloth diseases Immune animal is taken as cloth disease infected animal to slaughter because detection serum cloth disease antibody response is positive, brings huge economic losses. The present invention can effectively assist the disease for carrying out brucellosis is strong group is divided to work, and reduce unnecessary animal husbandry economy damage It loses, provides valuable checkout and diagnosis tool for animal cloth disease prevention and control, provided for science cleaning cloth disease infected animal applicable Method.
Detailed description of the invention
Fig. 1 is the homologous conserved domain sequence comparative analysis figure of several species IL-1 β and IL-1Ra, in which:
The homologous conserved protein domain sequence alignment analysis figure of A:IL-1 β -1；The homologous conservative protein structure of B:IL-1Ra-1 Domain sequence alignment analysis figure；The homologous conserved protein domain sequence alignment analysis figure of C:IL-1 β -2；D:IL-1Ra-2 is homologous conservative Protein structure domain sequence alignment analysis figure；
Fig. 2 is IL-1 β -2 and IL-1Ra-2 gene tandem pcr amplification product electrophoretogram；
Fig. 3 is IL-1 β -1, IL-1Ra-1 and IL-1 β -1Ra-2 recombinant protein expression and purification SDS-PAGE electrophoretic analysis Figure, in which:
M:Marker；1: inducing expression IL-1 β -1；2: inducing expression IL-1Ra-1；3:IL-1 β -1 does not induce control；4: IL-1Ra-1 does not induce control；5:IL-1 β -1Ra-2 does not induce control；6: inducing expression IL-1 β -1Ra-2；7:pMCSG9 is unloaded Body does not induce control；The induction control of 8:pMCSG9 empty carrier；
Fig. 4 is that anti-il-i-beta detection antibody, anti-IL-1Ra detection antibody while anti-il-i-beta and IL-1Ra capture are used and resisted Body purifies SDS-PAGE analysis chart, in which:
M,Marker；1, antibody is used in anti-il-i-beta and IL-1Ra capture；2, antibody is used in anti-il-i-beta detection；3, anti-IL-1Ra inspection Survey antibody；
Fig. 5 is that capture antibody and detection antibody and gene engineering expression protein I L-1 β and IL-1Ra are specifically bound Western blot analysis chart, in which:
A: anti-il-i-beta and IL-1Ra capture antibody analysis figure；B: anti-il-i-beta detection antibody analysis figure；C: anti-IL- 1Ra detection antibody analysis figure；1: gene engineering expression protein I L-1 β；2: gene engineering expression protein I L-1Ra；
Fig. 6 be capture antibody with detection with antibody in conjunction with the natural IL-1 β of several species and IL-1Ra protein-specific Western blot analysis chart, in which:
A: anti-il-i-beta detection holds the opposite sex with the natural IL-1 β albumen of several species with antibody and is combined figure；B: anti-IL-1Ra detection is used Antibody is schemed in conjunction with the natural IL-1Ra protein-specific of several species；C: anti-il-i-beta -1 and IL-1Ra capture antibody and several species Natural IL-1 β and IL-1Ra protein-specific combines figure；
Fig. 7 is the UV scanning analysis chart of detection antibody coupling fluorescent microsphere, in which:
A: anti-il-i-beta detection antibody coupling blue-fluorescence microballoon；B: anti-IL-1Ra detection antibody coupling green fluorescence Microballoon；
Fig. 8 be determining optimum antibody to working concentration analysis chart；
A: capture antibody concentration and detection fluorescent value relationship；B: detection antibody concentration and fluorescent value relationship；
Fig. 9 is the canonical plotting of IL-1 β and IL-1Ra；
Figure 10 is detection fluorescence intensity attenuation analysis figure；
Figure 11 is brucellosis vaccine immunity sheep group, brucella disease vaccine epidemic disease sheep feminine gender group and infection cloth Lu Shi rather Bacterium sick sheep positive group clinical practice blood serum sample tests and analyzes figure, in which:
A: sheep blood serum sample IL-1 β Concentration Testing；B: sheep blood serum sample IL-1Ra Concentration Testing；C: in sheep blood serum sample IL-1Ra/IL-1 β ratio measurement；
Figure 12 be in sheep blood serum sample IL-1Ra/IL-1 β ratio in brucellosis infected group, vaccine immunity group, feminine gender Diagnostic significance statistics ROC curve analysis chart is distinguished in control group, in which:
A: not immune cloth disease vaccine feminine gender group and cloth disease vaccine immune group；B: not immune cloth disease vaccine feminine gender group and cloth disease sense Contaminate positive group；C: cloth disease vaccine immune group and cloth disease infect positive group.
Specific embodiment
The selection of embodiment 1IL-1 β and the homologous conserved sequence of IL-1Ra
The IL-1 β and IL-1Ra of downloading sheep, people, ox, horse, pig, mouse, rabbit are searched in the GenBank database of NCBI Gene and protein sequence, using DNAMAN version 6 carry out sequence identity compare analysis, obtain sheep, people, ox, horse, Pig, mouse, IL-1 β and the IL-1Ra albumen of rabbit and the homologous conserved region sequence of nucleic acid several species, and it is conservative to analyze IL-1 β and IL-1Ra Otherness between area；
The homologous conserved sequence to differ greatly between selection IL-1 β and IL-1Ra, is respectively designated as IL-1 β -1 and IL- 1Ra-1 is used to prepare the immunogene of detection antibody；Other two sections the IL-1 β and IL-1Ra for having part similitude are selected, respectively Be named as IL-1 β -2 and IL-1Ra-2, and by be between the two ttcttcagcatgcat with sequence linker (amino acid sequence It is classified as FFSMH) connection building fusion tandem gene, it is named as IL-1 β -1Ra-2, is used to prepare the immunogene of capture antibody.
The homologous conserved sequence selection of several species for being used to prepare immunogene requires: except IL-1 β -2 and IL-1Ra-2 has portion Outside sub-sequence consistency, sequence is dissimilar between any two by other IL-1 β -1, IL-1 β -2, IL-1Ra-1, IL-1Ra-2, homologous ratio Fig. 1 is shown in conserved sequence analysis selection result.
The preparation of 2 recombinant protein of embodiment
(1) primer that design synthesizes for expanding IL-1 β -1, IL-1 β -2, IL-1Ra-1, IL-1Ra-2DNA sequence is shown in Table 1:
Table 1: the present invention designs the primer of synthesis
Primer Primer sequence
IL-1β-1-S 5/-tacttccaatccaatgcCTATACCTGTCTTGTGTGACCCA-3/
IL-1β-1-A 5/-ttatccacttccaatgtcAGAAGACGAATCGCTTTTCC-3/
IL-1Ra-1-S 5/-tacttccaatccaatgcCAAGCCTTCAGGATCTGGG-3/
IL-1Ra-1-A 5/-ttatccacttccaatgtcAACCTTGCAAGTATCCAGC-3/
IL-1β-2-S 5/-ccgcatatgTACAAGACAGAAATCAAGAACACAGT-3/
IL-1β-2-A 5/-atgcatgctgaagaaTATATCCTGGCCACCTCTAAAAC-3/
IL-1Ra-2-S 5/-ttcttcagcatgcatGACAAGCGCTTCGCCTTCATC-3/
IL-1Ra-2-A 5/-ccgctcgagATTGGTGAGGCCCACGGG-3/
(2) target dna sequence of IL-1 β -1, IL-1Ra-1, IL-1 β -2, IL-1Ra-2 are expanded using conventional PCR method, PCR system is as follows: Es Taq:25 μ l；Upstream primer: 1 μ l；Downstream primer: 1 μ l；IL-1 β or IL-1Ra overall length plasmid template: 2 μl；ddH2O:21 μ l；Total:50 μ l；IL-1 β -1 and IL-1Ra-1 reaction condition are as follows: 94 DEG C of 3min；94 DEG C of 30s, 62 DEG C 30s, 72 DEG C of 90s, totally 32 recycle；72℃10min；IL-1 β -2 and IL-1Ra-2 reaction condition are as follows: 94 DEG C of 3min；94℃ 30s, 60 DEG C of 30s, 72 DEG C of 90s, totally 32 recycle；72℃10min；
(3) IL-1 β -2 and IL-1Ra-2 gene tandem: IL-1 β -2 downstream primer IL-1 β -2-A and IL-1Ra-2 draws upstream Contain one section of linker sequence ttcttcagcatgcat in object IL-1Ra-2-S, therefore, using IL-1 β -2-S as upstream primer, IL-1Ra-2-A is as downstream primer, using IL-1 β -2 and IL-1Ra-2 glue recovery product as template, carries out PCR amplification, expands item Part is as follows: 94 DEG C of 3min；94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 90s, totally 32 recycle；72℃10min；PCR system is as follows: Es Taq:25 μ l；IL-1 β -2-S:1 μ l；IL-1Ra-2-A:1 μ l；The PCR product of IL-1 β -2: 1 μ l；The PCR product of IL-1Ra-2: 1μl；ddH2O:21 μ l；Total:50 μ l, gene tandem PCR amplification electrophoresis result are shown in Fig. 2；
(4) by pMCSG9 carrier with I digestion of SSP processing, system is I enzyme of SSP, 4 μ l, pMCSG9 plasmid, 32 μ l, 10 × 4 μ l of Buffer overnight in 37 DEG C is recycled without glue；
(5) I digestion products of the SSP of pMCSG9 are attached with IL-1 β -1 and IL-1Ra-1 glue recovery product respectively, are pressed Table 2 constructs reaction system, and 70 DEG C of heat preservation 5min, move into 37 DEG C of thermostats after mixing, and 1 μ l T4DNA pol polymerization is added Enzyme, 10 μ l of final volume, in 37 DEG C of heat preservation 5min after mixing gently, by IL-1 β -1 or IL-1Ra-1 reaction system 2 μ l and pMCSG9 2 μ l of reaction system mix gently after in 22 DEG C of reaction 15min, later plus 2 μ l of 25mMEDTA, in 22 DEG C of reaction 10min, success Construct recombinant expression plasmid pMCSG9-IL-1 β -1 and pMCSG9-IL-1Ra-1；
Table 2；Reaction system
(6) pET-30a plasmid and IL-1 β -1Ra-2 glue recovery product are pressed respectively with restriction enzyme Nde I and Xho I Conventional method carries out double enzyme digestion reaction, and product after digestion is attached, and successfully constructs recombinant expression plasmid pET30a-IL-1 β- 1Ra-2；
(7) recombinant expression plasmid is transformed into E.coli BL21 (DE3) expression bacterium, carries out final concentration according to a conventional method The IPTG inducing expression of 0.4mM, His label-affinity chromatography purifying, respectively obtains prokaryotic expression genetic engineering recombinant protein IL-1 β -1, IL-1Ra-1 and IL-1 β -1Ra-2, are shown in Fig. 3；Specific step is as follows:
Recombinant expression plasmid is converted into the inducing expression recombinant protein into e. coli bl21-Codonplus cell, picking Correct single colonie is identified in recombinant plasmid sequencing, is inoculated in the LB liquid medium of the benzyl resistance of ammonia containing 50mg/L, 37 DEG C of cultures 2 Hour, it is then inoculated in the fresh liquid LB culture medium of the benzyl resistance of ammonia containing 50mg/L of 200mL in 1:500 ratio, 37 DEG C of vibrations Culture about 1 hour is swung, the IPTG of 300 μ L 1mol/mL is added, is centrifuged after continuing shaken cultivation 5.5 hours through 4 DEG C, 10000rpm 5min harvests the genetic engineering large intestine of pMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1, pET30a-IL-1 β -1Ra-2 respectively Bacillus bacterial sediment after being resuspended with PBS, is cracked with Ultrasonic Cell Disruptor, power 40W, ultrasonic 10s, interval 10s, altogether 30min obtains supernatant and inclusion body precipitates in 12000 revs/min, centrifugation 30min；
The recombinant protein of E. coli system expression has merged His label, will be on the broken thallus of gene engineering expression bacterium Clear liquid using nickel column routinely nickel ion affinity chromatograph natural subsidence method purification of target antigen protein IL-1 β -1, IL-1 β -2 and IL-1 β -1Ra-2 uses molecular cut off using the recombinant protein of SDS-PAGE and Western-blot analysis verifying after purification 4 DEG C of the bag filter dialysis of 8000-14000Da, dialyzate are the PBS of 0.01M pH 8.0, and every 2h replaces a dialyzate, altogether more It changes 5 times, carries out concentration of instead dialysing with PEG20000, SDS-PAGE identifies purity, and it is spare that packing is put into -80 DEG C of refrigerators.
3 anti-il-i-beta of embodiment detection antibody, anti-IL-1Ra detection antibody, simultaneously anti-il-i-beta and IL-1Ra capture are used The preparation of antibody
(1) immune for the first time, the IL-1 β -1, IL-1Ra-1 and IL-1 β -1Ra-2 albumen and isometric Buddhist of 1mg are taken respectively With dorsal sc multiple spot immunization ways globefish is immunized in IL-1 β -1, IL-1Ra-1 by the mixing of family name's Freund's complete adjuvant and sufficiently emulsification completely New zealand white rabbit is immunized in IL-1 β -1Ra-2 by mouse, and 15 days are an immune period, and second immune incomplete using Fo Shi Adjuvant and immunogene are mixed with emulsified immunogen, are immunized according to the above method, after third is immune, every 7 days measurement serum titers, Potency collects serum up to Culling heart blood after 50000；
(2) antibody titer detects: leaving and taking a small amount of serum before being immunized and asks as negative blood, takes a small amount of immune rear rabbit and globefish Mouse blood, later with 3000 revs/min of centrifugation 5min, draws serum, uses routine in 37 DEG C of incubations 0.5h, 4 DEG C of blood coagulation 2h ELISA method detects potency, after ELISA detection antibody titer reaches 50000, using AKATA protein purification system and often Protein g affinity chromatography method antibody purification is advised, sees Fig. 4；
(3) interference antibody removal: induction is transferred to E.coli BL21 (DE3) the expression bacterium of pMCSG9 empty carrier, takes respectively 1mL-5mL Bacteria Culture fermentation liquid, 1300 revs/min of centrifugation 1min abandon supernatant and obtain thallus；Two kinds of guinea pig antibodies are taken respectively 100 μ L and above-mentioned different amounts of thallus mix, and are incubated for 45min-90min respectively in 37 DEG C；After the completion of incubation 3000 revs/min from Heart 5min, taking supernatant is the detection antibody for removing interference antibody.
The capture antibody of embodiment 4 and detection Identification of the antibodies and analysis
(1) using the Blot method hybridization analysis rabbit source routine Western capture antibody and the detection of cavy source with antibody and The specificity and cross reaction characteristic that its target gene engineering recombinant protein IL-1 β and IL-1Ra are combined, are shown in Fig. 5；
(2) using the Blot method hybridization analysis rabbit source routine Western capture antibody and the detection of cavy source with antibody and The specificity and cross reaction characteristic that its target several species native protein IL-1 β and IL-1Ra are combined, native protein extract: utilizing Conventional RIPA lysate extracts histone, dilute to 100 times of 0.1M PMSF isopropanol storing liquid progress with conventional RIPA lysate It releases, is configured to complete RIPA lysate.Specific extraction step is as follows: the appropriate tissue block of clip is put into the flat grinding pipe of 2mL, by every Gram tissue be added 8mL complete RIPA crack liquid proportional, be added the complete RIPA lysate of proper volume, be fully ground homogenate 10min, 4 DEG C of 13000 revs/min of centrifugation 20min shift supernatant into 1.5mL centrifuge tube, total protein content are measured, using normal Advise Western blot method hybridization analysis capture antibody and detection antibody and several species native protein IL-1 β and IL-1Ra Binding characteristic is shown in Fig. 6.
The preparation of embodiment 5IL-1 β standard items and IL-1Ra standard items
Respectively with escherichia expression system routinely prokaryotic expression technology preparation genetic engineering IL-1 β recombinant protein and IL-1Ra recombinant protein is as standard items albumen of the invention.Specific preparation method respectively reads the opening of IL-1 β and IL-1Ra Frame is inserted into prokaryotic expression carrier pET-30a, recombinant expression carrier the pET-30a-IL-1 β and pET- of building fusion His label 30a-IL-1Ra obtains the genetic engineering IL-1 β recombinant protein and IL-1Ra weight of high-purity with conventional inducing expression purification technique Standard items albumen is made in histone.
The detection antibody coupling fluorescent microsphere of embodiment 6
Two kinds of polyclonal detections in cavy source are coupled with different colours fluorescent microsphere respectively with antibody, wherein anti-IL-1 Antibody coupling blue-fluorescence microballoon (excitation wavelength 360nm, launch wavelength 450nm) is used in β detection, anti-IL-1Ra detection antibody idol Join green fluorescent microspheres (excitation wavelength 480nm, launch wavelength 520nm), preparation has the detection antibody of fluorescent microsphere, can use In the direct detection of IL-1 β and IL-1Ra；Its specific coupling step is as follows: the MES buffering of pH 4.5-7.5 is added in 10 μ L microballoons Solution 90mL is mixed, and 15000 revs/min are centrifuged 15 minutes, abandon supernatant, collects microballoon precipitating, and repetition is washed twice；In 90 μ L In the MES buffer solution of 0.2M pH 4.5-7.5,4.8 μ L EDC (1mg/mL) are added, activate 20 minutes；Then 5.6 μ L are added NHS (1mg/mL) is mixed 20 minutes；4 DEG C 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；With 90 μ L pH 4.5- Microballoon is resuspended in 7.5 MES buffer solution, adds 10 μ L antibody (1mg/mL), room temperature vortex concussion reaction 4 hours；10 μ are added L confining liquid (10%BSA) reacts at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon supernatant, and 100 μ L pH are added The MES buffer solution of 4.5-7.5,4 DEG C are kept in dark place, and analyze coupling fluorescent microsphere antibody with ultraviolet full wavelength scanner, see Fig. 7.
7 chessboard method of embodiment determines optimum antibody to working concentration
Anti-il-i-beta and the source capture of IL-1Ra rabbit simultaneously will be coated on 96 hole black microwell plates with antibody, be added IL-1 β and IL-1Ra mixed protein standard items add the fluorescence detection antibody of IL-1 β and IL-1Ra, for detecting IL-1 β and IL- The content of 1Ra IL-1 β and IL-1Ra in mixed protein finally detect its fluorescent value using fluorescence microplate analyzer, specifically Operating procedure is as follows:
(1) it is coated with capture antibody: being coated with capture antibody using the 0.1M PBS of pH7.2-7.6 as dilution, will catch It obtains with antibody dilution doubling dilution to final concentration of 1.248 μ g/mL, 0.624 μ g/mL, 0.312 μ g/mL, 0.156 μ g/ ML, 0.078 μ g/mL, 0.039 μ g/mL are added in ELISA Plate, 100 holes μ L/, and each tandem is same concentration capture antibody, It is coated with overnight under the conditions of 4 DEG C；
(2) with the PBS containing 0.2% Tween-20 as PBS-T cleaning solution, board-washing 3-4 times, 200 holes μ L/, every time Then 1min is closed, 200 holes μ L/, 37 DEG C of 1h, draining liq with 0.1M ammonium chloride solution；
It (3) is 1 μ with diluted IL-1 β and IL-1Ra hybrid standard product albumen to IL-1 β and IL-1Ra final concentration G/mL is added in ELISA Plate, reacts 1h, replaces standard items as negative control with PBS dilution, and every group of antibody is to setting up Negative control, the blank well of any reagent not to be added as blank control；
(4) with PBS-T board-washing 3-4 times, it is glimmering that with antibody mixing is formed by IL-1 β -1 detection antibody and IL-1Ra-1 detection Light detection antibody with antibody to two kinds of detections with antibody final concentration is 2 μ g/ with dilution doubling dilution mixing fluorescence detection ML, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, 0.0625 μ g/mL, 37 DEG C of reaction 1h；
(5) with PBS-T board-washing 3-4 times, the naked fluorescent microsphere of not coupled antibody is also diluted into identical multiple, is individually set up One group of positive controls, using fluorescence microplate analyzer respectively at excitation wavelength 360nm, launch wavelength 450nm and excitation wave Long 480nm, launch wavelength 520nm measure fluorescence intensity value.
(6) established standards sample wells fluorescent value is S, and blank well fluorescent value is B, and negative hole fluorescent value is N, dense according to each antibody Corresponding fluorescent value power and variation tendency are spent, determines optimum antibody used in this method to concentration, capture antibody Optimal packet quilt 0.078 μ g/mL of working concentration, the detection optimal 0.125 μ g/mL of working concentration of antibody, is shown in Fig. 8.
Embodiment 8 determines that antibody action time is used in off-period, antigenic action time, detection
Tetra- time gradients of 0.5h, 1h, 1.5h, 2h, antigenic action time is arranged in 0.1M ammonium chloride confining liquid action time Setting five time gradients of 45min, 60min, 75min, 90min and 120min, detection antibody action time setting 30min, Five time gradients of 45min, 60min, 75min and 90min are 1 μ g/mL IL-1 β to concentration and final concentration with optimal antibody ELISA detection reaction is completed with IL-1R hybrid standard product albumen, fluorescent value is measured respectively, determines the best effort time.As a result it shows Show: sealing effect is not thorough before confining liquid action time 1h, and fluorescent value is steady after 1h, and sealing effect is almost the same, most preferably Off-period is 1h；The antigenic action time is smaller on ELISA detection fluorescent value influence, selects the antigenic action time for 30min；Inspection With antibody action time, the fluorescent value before 1h is more steady for survey, and fluorescent value starts to reduce after 1h, selects detection antibody The best use time be 1h.
The structure of embodiment 9IL-1 β and the double fluorescence ELISA detection method standard curves of IL-1Ra Simultaneous Determination double-antibody sandwich It builds
With 0.1M PBS diluted IL- β and IL-1Ra the standard items mixed liquor of pH7.2-7.6 to two kinds of standard items ends Concentration be 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL, 78.125ng/mL, 39.625ng/mL, 19.531ng/mL、9.766ng/mL、4.883ng/mL、2.441ng/mL、1.221ng/mL、0.610ng/mL、0.305ng/ mL,0.153ng/mL,0.076ng/mL,0.038ng/mL；According to determining optimal ELISA reaction condition, by the behaviour of embodiment 7 Make process, measure the fluorescence signal value of the standard items of 16 concentration, replaces standard items to be loaded as negative control, with not with PBS The blank well that any reagent is added is blank control, is 0.125 μ g/mL so that 100 μ L, two kinds of fluorescent microsphere concentration are only added Fluorescent microsphere mixed liquor is as positive control；Using blank well fluorescent value B as instrument blank zeroing value, it is dry to deduct instrument noise It disturbs.Established standards sample wells fluorescent value is ST, and negative hole fluorescent value is N, and positive hole fluorescent value is P；Using positive hole fluorescent value P as Denominator correction calculates standard sample wells and corrects fluorescent value STa, i.e. STa=ST/P.With standard items IL- β and IL-1Ra protein concentration pair Number is used as abscissa, corrects fluorescent value ST with standard sample wellsaAs ordinate, IL-1 β and IL-1Ra standard curve is drawn respectively, Calculate calibration curve equation and coefficient R2.The final range of linearity for determining IL-1 β and IL-1Ra is 76pg/mL- The calibration curve equation of 625ng/mL, IL-1 β are y=0.0076x-0.0058, R2It is 0.9937；The standard curve side of IL-1Ra Journey is y=0.0027x+0.0251, R2It is 0.9904.Calibration curve equation, calculating pair are substituted into 8 negative hole Mean Fluorescences The concentration value answered is the minimum detection limit of this method, and the minimum detection limit of IL-1 β and IL-1Ra are 11pg/mL, see Fig. 9.
The experiment of 10 rate of recovery of embodiment
IL-1 β standard items and IL-1Ra standard items are mixed in 1:3,1:2,1:1,2:1,3:1 ratio respectively, it is glimmering to detect its Light value, calculates corresponding protein concentration according to calibration curve equation, calculates the rate of recovery, IL-1 divided by known standard items protein concentration β rate of recovery range is 80.89%~101.13.52%, average recovery rate 87.842%；IL-1Ra rate of recovery range is 80.07%~114.18%, average recovery rate 93.93% is shown in Table 3；
Table 3: the rate of recovery analyzes result
The analysis of 11 fluorescent signal decay phase of embodiment
With PBS diluted IL-1 β and IL-1Ra standard items albumen to two kinds of final concentration of protein be 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL are examined with the double fluorescence ELISA methods of the double antibodies sandwich of foundation It surveys, carries out fluorescent value measurement after 0h, 0.5h, 1h, 2h is added in fluorescence detection antibody.As the result is shown: anti-fluorescence is added Within body 0.5h, fluorescence intensity value is without significant change；It is being added in fluorescence antibody 2h, can effectively detect object；Recommend Fluorescence antibody, which is added in 0.5h, completes fluorescence detection, sees Figure 10.
Embodiment 12 is a kind of for the double fluorescence of the double-antibody sandwich of several species IL-1 β and IL-1Ra and its ratio Simultaneous Determination The operating procedure of ELISA detection method
(1) according to the quantity of sample to be examined, anti-il-i-beta and the IL-1Ra capture coated black flat-bottom enzyme mark of antibody are taken out Plate, setting standard sample wells, positive hole, negative hole, blank well and sample well, it is 100 μ L that amount of liquid, which is added, in every hole every time.Standard items Two kinds of standard items mixed solutions through dilution gradient dilution are added in hole, and the end of two kinds of standard items protein I L-1 β and IL-1Ra are dense Degree be 78.125ng/ml, 39.625ng/ml, 19.531ng/ml, 9.766ng/ml, 4.883ng/ml, 2.441ng/ml, 12 concentration ladders of 1.221ng/ml, 0.610ng/ml, 0.305ng/ml, 0.153ng/ml, 0.076ng/ml and 0.038ng/ml 2 times of diluted serum to be checked are added in degree, sample well, and PBS dilution is added in negative hole, and any liquid is not added in positive hole and blank well Body, 37 DEG C of incubation 1h are added 0.1%PBS-T board-washing 3 times, each 1min；
(2) be added concentration be 0.125 μ g/mL two kinds of detection antibody in sample well, standard sample wells and negative hole, Every hole 100 μ L, 37 DEG C of incubation 1h are added 0.1%PBS-T board-washing 3 times, each 1min；
(3) any liquid is not added in blank well, and it is 0.125 μ g/mL that 100 μ L, two kinds of fluorescent microsphere concentration, which are added, in positive hole 100 μ L PBS dilutions are added in fluorescent microsphere mixed liquor, standard sample wells, negative hole, sample well；
(4) double fluorescence detection readings are carried out using fluorescence microplate analyzer.Emit in 360nm excitation wavelength and 450nm Wavelength detecting IL-1 β concentration detects IL-1Ra concentration in 480nm excitation wavelength and 520nm launch wavelength；
(5) using blank well fluorescent value B as instrument blank zeroing value, the interference of instrument noise is deducted.Established standards sample wells is glimmering Light value is ST, and sample well fluorescent value is SA, and negative hole fluorescent value is N, and positive hole fluorescent value is P, using positive hole fluorescent value P as Denominator correction calculates standard sample wells and corrects fluorescent value STaFluorescent value SA is corrected with sample wella, i.e. STa=ST/P or SAa=SA/ P draws IL-1 β and IL-1Ra standard curve respectively, using standard items IL- β and IL-1Ra protein concentration logarithm as abscissa, with Standard sample wells corrects fluorescent value STaAs ordinate, standard curve is drawn, calculates calibration curve equation and coefficient R2, with sample Sample wells corrects fluorescent value SAaSubstitute into calibration curve equation, calculate log concentration X, 10XIL-1 β's and IL-1Ra is dense as in sample Degree, and calculate IL-1Ra/IL-1 β concentration proportion；
(6) positive hole and negative hole are set up, can be with the validity of Quality Control fluorescence detection, and repeated detection result is had Comparativity.
Embodiment 13 is a kind of for the double fluorescence of the double-antibody sandwich of several species IL-1 β and IL-1Ra and its ratio Simultaneous Determination ELISA detection method is in brucellosis infection and the application in Immune dctection diagnosis
(1) 904 parts of sheep blood serums and plasma sample are acquired, is analyzed through brucellosis rose bengal precipitation test.It is not immunized 196 parts of sample of negative healthy group sheep blood serum of the cloth disease of cloth disease vaccine, rose bengal precipitation test result are negative；Immune brucella 592 parts of sample of the cloth disease immune group sheep blood serum of S2 vaccine, rose bengal precipitation test result are positive；Infect the cloth of brucellosis Sick positive 115 parts of sheep blood serum sample of group, rose bengal precipitation test result are positive.Exempted from based on antigen-antibody reaction at present Epidemiology detection method, as brucellosis natural infection and vaccine immunity cannot be distinguished in rose bengal precipitation test；
(2) the double fluorescence of double-antibody sandwich of the IL-1 β and IL-1Ra and its ratio Simultaneous Determination established using the present invention ELISA detection method measures the concentration of IL-1 β and IL-1Ra in serum, and calculates IL-1Ra/IL-1 β ratio.Cloth disease infected group IL-1 β concentration is apparently higher than cloth disease immune group and cloth disease negative healthy group；Cloth disease infected group IL-1Ra concentration is immune lower than cloth disease Group and cloth disease negative healthy group, cloth disease infected group IL-1Ra/IL-1 β ratio are significantly lower than negative healthy group and immune group, statistics Credit analysis difference is extremely significant, and P ﹤ 0.01 is shown in Figure 11；
(3) statistics ROC curve analyzes IL-1Ra/IL-1 β ratio in brucellosis infected group, vaccine immunity group, yin Diagnostic significance is distinguished in property control group.As the result is shown: the negative group of cloth disease and cloth disease immune group area under the curve AUC are 0.155 ﹤ 0.5, the negative group of cloth disease and the IL-1Ra/IL-1 β of cloth disease immune group are not different Identification Significance；The negative group of cloth disease is infected with cloth disease Group AUC and cloth disease immune group and cloth disease infected group AUC are 1.000 ﹥ 0.5, and IL-1Ra/IL-1 β ratio can be used for brucella The antidiastole of sick natural infection and vaccine immunity, is shown in Figure 12, according to statistics normal distribution method calculate separately cloth disease infection sheep, Cloth disease immune sheep, IL-1 β of cloth disease negative healthy sheep, IL-1Ra, IL-1Ra/IL-1 β ratio distribution, are shown in Table 4；
Table 4: IL-1 β, IL-1Ra, IL-1Ra/IL-1 β ratio statistical analysis in sheep blood serum
(4) natural infection of Brucella melitensis disease recommends operating instruction as follows with vaccine immunity antidiastole:
1. rose bengal precipitation test
Take 30 μ L sheep serum to be detected and the 30 red agglutination antigens of μ L tiger in mixing on glass plate.Result judgement: by inspection serum Between 5min, there is not agglutination phenomenon, reaction system is in uniform pink, then is the red negative of tiger, is determined as no cloth Disease infection sheep；There is naked eyes visible agglutination phenomenon, then it is positive for the red test of tiger, it is determined as the infection of cloth disease or immune sheep, it is pending Next step IL-1Ra/IL-1 β ratio measurement.
2. IL-1Ra/IL-1 β ratio measurement
Utilize the double detection sides fluorescence ELISA IL-1 β and IL-1Ra and its ratio Simultaneous Determination double-antibody sandwich of the invention Method, by 12 operating procedure of embodiment, IL-1Ra/IL-1 β ratio in the brave red test positive serum samples of measurement.Result judgement: IL- 1Ra/IL-1 β is then determined as brucella illness sheep within the scope of 44.42-53.93；IL-1Ra/IL-1 β is in 74.12- In 139.45 ranges, then it is judged to being not suffering from brucella sick sheep；IL-1Ra/IL-1 β, need to be in conjunction with clinic not in two above range Symptom or other further check analyses of cloth disease detection method.
Embodiment 14 is a kind of for the double fluorescence of the double-antibody sandwich of several species IL-1 β and IL-1Ra and its ratio Simultaneous Determination Detection application of the ELISA detection method in serum and plasma sample
The serum and plasma sample for acquiring 100 sheep simultaneously according to a conventional method, using IL-1 β of the invention and IL-1Ra and The double fluorescence ELISA detection methods of its ratio Simultaneous Determination double-antibody sandwich measure serum and blood plasma by 12 operating procedure of embodiment Middle IL-1 β, IL-1Ra, IL-1Ra/IL-1 β ratio, as the result is shown: IL-1 β, IL-1Ra, IL-1Ra/IL-1 in the serum of measurement There was no significant difference in β ratio and blood plasma, and method of the invention can effectively measure IL-1 β, IL- in serum and blood plasma 1Ra, IL-1Ra/IL-1 β ratio.
Embodiment 15 is a kind of for the double fluorescence of the double-antibody sandwich of several species IL-1 β and IL-1Ra and its ratio Simultaneous Determination Detection application of the ELISA detection method in healthy mice and human serum sample
50 parts of healthy mice serum and 57 parts of Healthy Human Serum samples are acquired according to a conventional method, utilize IL-1 β of the invention It is measured with IL-1Ra and its double fluorescence ELISA detection methods of ratio Simultaneous Determination double-antibody sandwich by 12 operating procedure of embodiment IL-1 β, IL-1Ra, IL-1Ra/IL-1 β ratio, calculate separately according to statistics normal distribution method in mice serum and human serum IL-1 β, IL-1Ra, IL-1Ra/IL-1 β ratio distribution, are shown in Table 5 in healthy mice and human serum；IL-1 β of the present invention with The double fluorescence ELISA detection methods of the double-antibody sandwich of IL-1Ra and its ratio Simultaneous Determination can be used for the IL-1 β and IL- of several species 1Ra and its ratio measurement.
Table 5: IL-1 β, IL-1Ra, IL-1Ra/IL-1 β ratio statistical analysis in mouse and human serum
The ratio I L-1Ra/ of double-antibody sandwich of the present invention double fluorescence ELISA Simultaneous Determination IL-1Ra and IL-1 β IL-1 β, being tested and analyzed by clinical practice sheep blood sample confirms, IL-1Ra/IL-1 β and cloth in brucellosis illness sheep blood serum IL-1Ra/IL-1 β difference is extremely significant in disease vaccine immune sheep and healthy sheep blood serum.Moreover, confirmed by statistics ROC analysis, IL-1Ra/IL-1 β is distinguishing brucellosis infection and is having significance of differential diagnosis in immune clinical examination in sheep blood serum.This hair It is bright to can be used for brucellosis infection and immune antidiastole, it can also be used to other examining with IL-1Ra/IL-1 ss related diseases Marked effect research disconnected and occurring, develop, lapsing to.
Sequence table
<110>Jilin University
<120>ELISA kit of several species IL-1 β and IL-1Ra and its ratio Simultaneous Determination
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 108
<212> DNA
<213>sheep (sheep)
<400> 1
ctatacctgt cttgtgtgac ccaaggtgat acaccgaccc tgcagctgga ggaagtagac 60
cccaaagtct accccaagag gaatatggaa aagcgattcg tcttctga 108
<210> 2
<211> 35
<212> PRT
<213>sheep (sheep)
<400> 2
Leu Tyr Leu Ser Cys Val Thr Gln Gly Asp Thr Pro Thr Leu Gln Leu
1 5 10 15
Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met Glu Lys Arg
20 25 30
Phe Val Phe
35
<210> 3
<211> 126
<212> DNA
<213>sheep (sheep)
<400> 3
tacaagacag aaatcaagaa cacagttgaa tttgagtctg tcctgtaccc taactggtac 60
atcagcactt ctcaaatcga agaaaagccc gtcttcctgg gacgttttag aggtggccag 120
gatata 126
<210> 4
<211> 42
<212> PRT
<213>sheep (sheep)
<400> 4
Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu Phe Glu Ser Val Leu Tyr
1 5 10 15
Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile Glu Glu Lys Pro Val Phe
20 25 30
Leu Gly Arg Phe Arg Gly Gly Gln Asp Ile
35 40
<210> 5
<211> 84
<212> DNA
<213>sheep (sheep)
<400> 5
caagccttca ggatctggga tgtcaaccag aagatcttct acctgaggaa taaccaacta 60
gttgctggat acttgcaagg ttga 84
<210> 6
<211> 27
<212> PRT
<213>sheep (sheep)
<400> 6
Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Ile Phe Tyr Leu Arg
1 5 10 15
Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly
20 25
<210> 7
<211> 123
<212> DNA
<213>sheep (sheep)
<400> 7
gacaagcgct tcgccttcat ccgctctgac aacggcccca ccaccagctt cgagtcggct 60
gcctgccccg gctggttcct ctgcacatca ctggaggccg accagcccgt gggcctcacc 120
aat 123
<210> 8
<211> 41
<212> PRT
<213>sheep (sheep)
<400> 8
Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Asn Gly Pro Thr Thr Ser
1 5 10 15
Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ser Leu Glu
20 25 30
Ala Asp Gln Pro Val Gly Leu Thr Asn
35 40
<210> 9
<211> 264
<212> DNA
<213>sheep (sheep)
<400> 9
tacaagacag aaatcaagaa cacagttgaa tttgagtctg tcctgtaccc taactggtac 60
atcagcactt ctcaaatcga agaaaagccc gtcttcctgg gacgttttag aggtggccag 120
gatatattct tcagcatgca tgacaagcgc ttcgccttca tccgctctga caacggcccc 180
accaccagct tcgagtcggc tgcctgcccc ggctggttcc tctgcacatc actggaggcc 240
gaccagcccg tgggcctcac caat 264
<210> 10
<211> 88
<212> PRT
<213>sheep (sheep)
<400> 10
Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu Phe Glu Ser Val Leu Tyr
1 5 10 15
Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile Glu Glu Lys Pro Val Phe
20 25 30
Leu Gly Arg Phe Arg Gly Gly Gln Asp Ile Phe Phe Ser Met His Asp
35 40 45
Lys Arg Phe Ala Phe Ile Arg Ser Asp Asn Gly Pro Thr Thr Ser Phe
50 55 60
Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ser Leu Glu Ala
65 70 75 80
Asp Gln Pro Val Gly Leu Thr Asn
85

Claims (6)

1. the ELISA kit of a kind of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination, it is characterised in that: including anti- The polyclonal capture antibody pre-coated elisa plate in IL-1 β and IL-1Ra rabbit source, IL-1 β recombinant protein standard items and IL-1Ra recombination The polyclonal inspection of cavy source anti-il-i-beta of protein standard substance, dilution, 20 × PBS-T concentrated cleaning solution, blue-fluorescence microballoon label The polyclonal detection antibody of the anti-IL-1Ra in cavy source of survey antibody, green fluorescent microspheres label；Wherein:
The polyclonal capture antibody of the anti-il-i-beta and IL-1Ra rabbit source, cavy source anti-il-i-beta polyclonal detection antibody, globefish The preparation step of the polyclonal detection antibody of the anti-IL-1Ra of source of mouse is as follows:
(1) IL-1 β and the IL-1Ra base of overall length sheep, people, ox, horse, pig, mouse, rabbit is downloaded in the GenBank database of NCBI Cause and protein sequence, using DNAMAN version 6 carry out sequence identity compare analysis, determine sheep, people, ox, horse, pig, Mouse, IL-1 β and the IL-1Ra albumen of rabbit and the homologous conserved region sequence of nucleic acid several species, and analyze the homologous guarantor of IL-1 β and IL-1Ra Otherness between defending zone；
(2) select the homologous conserved region IL-1 β and IL-1Ra between have differ greatly, two sections of sequences that sequence identity is low, point It is not named as IL-1 β -1 and IL-1Ra-1, IL-1 β -1 nucleotide sequence is as described in SEQ ID No.1, IL-1Ra-1 nucleotide Sequence is as described in SEQ ID No.5；Its prokaryotic expression recombinant protein is used separately as preparing anti-il-i-beta detection antibody and anti-IL- The 1Ra detection immunogene of antibody, wherein IL-1 β -1 amino acid sequence is as described in SEQ ID No.2, IL-1Ra-1 amino acid sequence Column are as described in SEQ ID No.6；In addition select that there are consistent two sections of partial sequence between the homologous conserved region IL-1 β and IL-1Ra Sequence is respectively designated as IL-1 β -2 and IL-1Ra-2, and IL-1 β -2 nucleotide sequence is as described in SEQ ID No.3, IL-1 β -2 Amino acid sequence is as described in SEQ ID No.4, and IL-1Ra-2 nucleotide sequence is as described in SEQ ID No.7, IL-1Ra-2 amino Acid sequence is as described in SEQ ID No.8；And IL-1 β -2 and IL-1Ra-2 is passed through into the DNA that amino acid sequence is FFMSH Linker connection, building IL-1 β and the homologous conserved region tandem gene IL-1 β -1Ra-2 of IL-1Ra, the IL-1 β -1Ra-2 nucleotide Sequence as described in SEQ ID No.9, IL-1 β -1Ra-2 amino acid sequence as described in SEQ ID No.10, prokaryotic expression recombination Albumen is used as the immunogene that antibody is used in preparation anti-il-i-beta and IL-1Ra capture simultaneously；
(3) the several species homologous sequence selection for being used to prepare immunogene requires: IL-1 β -2 and IL-1Ra-2 has partial sequence one IL-1 β -2 and IL-1Ra-2 fused in tandem are obtained tandem gene IL-1 β -1Ra-2, with its prokaryotic expression genetic engineering by cause property Recombinant protein immunizing rabbit prepares rabbit source anti-il-i-beta and IL-1Ra capture antibody, can be in combination with IL-1 β and IL-1Ra, IL- Sequence identity is not low homologous between 1 β -1 and IL-1Ra, IL-1Ra-1 and IL-1 β, so that respectively with IL-1 β -1 and IL-1Ra- The anti-IL-1Ra detection of the cavy source anti-il-i-beta detection antibody of 1 genetic engineering recombinant protein expression product preparation and cavy source is used Antibody can specifically bind IL-1 β and IL-1Ra respectively；
(4) upstream and downstream primer a pair of IL-1 β -1-S and IL-1 β -1-A of design amplification IL-1 β -1, to include overall length IL-1 β CDNA recombinant plasmid be pcr template, using PCR react amplification IL-1 β -1 DNA, purified using DNA gel QIAquick Gel Extraction Kit The DNA of IL-1 β -1；
(5) upstream and downstream primer a pair of the IL-1Ra-1-S and IL-1Ra-1-A of design amplification IL-1Ra-1, to include overall length IL- The cDNA recombinant plasmid of 1Ra is pcr template, and the DNA of amplification IL-1Ra-1 is reacted using PCR, uses DNA gel QIAquick Gel Extraction Kit Purify the DNA of IL-1Ra-1；
(6) upstream and downstream primer a pair of IL-1 β -2-S and IL-1 β -2-A of design amplification IL-1 β -2, to include overall length IL-1 β CDNA recombinant plasmid be pcr template, using PCR condition amplification IL-1 β -2 DNA, purified using DNA gel QIAquick Gel Extraction Kit The DNA of IL-1 β -2；
(7) upstream and downstream primer a pair of the IL-1Ra-2-S and IL-1Ra-2-A of design amplification IL-1Ra-2, to include overall length IL- The cDNA recombinant plasmid of 1Ra is pcr template, and the DNA of amplification IL-1Ra-2 is reacted using PCR, uses DNA gel QIAquick Gel Extraction Kit Purify the DNA of IL-1Ra-2；
(8) drawn using upstream used in design the downstream primer IL-1 β -2-A used in amplification IL-1 β -2 and amplification IL-1Ra-2 Linker sequence in object IL-1Ra-2-S, using Overlap extension PCR method, with the DNA of the DNA of IL-1 β -2 and IL-1Ra-2 IL-1 β -2 and IL-1Ra-2 gene are gone here and there by Linker using IL-1 β -2-S and IL-1Ra-2-A as upstream primer for template Connection expands IL-1 β -1Ra-2 fusion dna, uses the DNA of DNA gel QIAquick Gel Extraction Kit purifying IL-1 β -1Ra-2；
(9) respectively by the DNA fragmentation of the IL-1 β -1 of acquisition and IL-1Ra-1, with the T4 archaeal dna polymerase of disconnected enzyme dependence Method is cloned on pMCSG9 carrier, and DNA and the pET-30a carrier of IL-1 β -1Ra-2 is connect, and constructs recombinant expression plasmid PMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1 and pET30a-IL-1 β -1Ra-2；
(10) recombinant expression plasmid is converted into the inducing expression recombinant protein into e. coli bl21-Codonplus cell, picking Correct single colonie is identified in recombinant plasmid sequencing, is inoculated in the LB liquid medium of the benzyl resistance of ammonia containing 50mg/L, 37 DEG C of cultures 2 Hour, it is then inoculated in the fresh liquid LB culture medium of the benzyl resistance of ammonia containing 50mg/L of 200mL in 1:500 ratio, 37 DEG C of vibrations Culture 1 hour is swung, the IPTG of 300 μ L1mol/mL is added, is centrifuged 5min through 4 DEG C, 10000rpm after continuing shaken cultivation 5.5 hours, The gene engineering colibacillus bacterium of pMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1, pET30a-IL-1 β -1Ra-2 are harvested respectively Body precipitating, after being resuspended with PBS, is cracked, power 40W, ultrasonic 10s, interval 10s, total 30min with Ultrasonic Cell Disruptor, in 12000 revs/min, centrifugation 30min, obtain supernatant and inclusion body precipitates；
(11) recombinant protein of E. coli system expression has merged His label, will be on the broken thallus of gene engineering expression bacterium Clear liquid presses nickel ion affinity chromatograph natural subsidence method purification of target antigen protein IL-1 β -1, IL-1 β -2 and IL-1 using nickel column β -1Ra-2, using the recombinant protein of SDS-PAGE and Western-blot analysis verifying after purification, with molecular cut off 8000- 4 DEG C of the bag filter dialysis of 14000Da, dialyzate are the PBS of 0.01M pH 8.0, and every 2h replaces a dialyzate, replaces 5 altogether It is secondary, concentration of instead dialysing is carried out with PEG20000, SDS-PAGE identifies purity, and it is spare that packing is put into -80 DEG C of refrigerators；
(12) animal immune
Immune IL-1 β -1, IL-1Ra-1 the and IL-1 β -1Ra-2 albumen for taking 1mg respectively for the first time, it is mixed with isometric Freund's complete adjuvant Merge emulsification, IL-1 β -1 after emulsification and IL-1Ra-1 protein immunization cavy are prepared into cavy source anti-il-i-beta detection antibody respectively With the anti-IL-1Ra detection antibody in cavy source, the IL-1 β -1Ra-2 protein immunization new zealand white rabbit after emulsification is prepared into rabbit source Anti-il-i-beta and IL-1Ra capture antibody, carry out dorsal sc multiple spot and are immunized, and 15 days are an immune period, and second immune Freund's incomplete adjuvant is begun to use to prepare emulsified immunogen for being immunized, method is same as above, after third time is immune, measurement one in every 7 days Secondary serum titer, potency carry out Culling heart blood up to 50000 or more, collect serum potency to be measured and antibody purification；
(13) antibody titer detection and purifying
It leaves and takes a small amount of serum before immune to ask as negative blood, the rabbit and cavy after being immunized acquire blood, are incubated in 37 DEG C 0.5h, 4 DEG C of blood coagulations 2h, 3000 revs/min of centrifugation 5min draw serum, detect potency using ELISA method, use AKATA egg White purification system and protein g affinity chromatography method antibody purification；
(14) interference antibody removal
By interference antibody removal experiment, the non-specific binding of two kinds of cavy source detection antibody is reduced, the specific steps of which are as follows: Induction is transferred to the Bacillus coli expression bacterium of pMCSG9 empty carrier, takes 1mL-5mL Bacteria Culture fermentation liquid, 1300 revs/min, centrifugation 1min abandons supernatant and collects thallus, takes two kinds of cavy source detection antibody, 100 μ L suspension thallus respectively, 37 DEG C of incubation 45-90min, 3000 revs/min of centrifugation 5min, taking supernatant is the detection antibody for removing interference antibody.
2. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that:
(1) preparation step of the polyclonal capture antibody pre-coated elisa plate of anti-il-i-beta and IL-1Ra rabbit source is as follows:
(1) be coated with anti-il-i-beta and the polyclonal capture antibody in IL-1Ra rabbit source: by anti-il-i-beta and IL-1Ra rabbit source is polyclonal catches It obtains with antibody diluted to final concentration of 0.078 μ g/mL, is added in ELISA Plate, 100 holes μ L/, 4 DEG C of coatings are overnight；
(2) to contain the PBS of 0.2% Tween-20 as PBS-T cleaning solution, board-washing 3-4 times, 200 holes μ L/, each 1min, so It is closed afterwards with 0.1M ammonium chloride solution, 200 holes μ L/, 37 DEG C of 1h, draining liq；
(2) the polyclonal detection antibody of the cavy source anti-il-i-beta of blue-fluorescence microballoon label, excitation wavelength 360nm, transmitting Wavelength 450nm；Coupling step is as follows: the MES buffer solution 90mL of pH 4.5-7.5 is added in 10 μ L blue-fluorescence microballoons, mixes, 15000 revs/min are centrifuged 15 minutes, abandon supernatant, collect microballoon precipitating, and repetition is washed twice；In 90 μ L 0.2M pH 4.5-7.5 MES buffer solution in, be added 4.8 μ L 1mg/mL EDC, activate 20 minutes；Then 5.6 μ L 1mg/mL NHS are added, mix 20 minutes；4 DEG C 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；With the MES buffer solution of 90 μ L pH 4.5-7.5 Microballoon is resuspended, adds the 10 polyclonal detection antibody of μ L 1mg/mL cavy source anti-il-i-beta, room temperature vortex concussion reaction 4 is small When；10 μ L 10%BSA confining liquids are added, react at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon supernatant, are added The MES buffer solution of 100 μ L pH 4.5-7.5,4 DEG C are kept in dark place；
(3) the polyclonal detection antibody of the anti-IL-1Ra in cavy source of green fluorescent microspheres label, excitation wavelength 480nm, transmitting Wavelength 520nm；10 μ L green fluorescent microspheres be added pH 4.5-7.5 MES buffer solution 90mL, mix, 15000 revs/min from The heart 15 minutes, supernatant is abandoned, collects microballoon precipitating, repetition is washed twice；In the MES buffer solution of 90 μ L 0.2M pH 4.5-7.5 In, 4.8 μ L 1mg/mL EDC are added, activate 20 minutes；Then 5.6 μ L 1mg/mL NHS are added, mix 20 minutes；4℃ 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；Microballoon is resuspended with the MES buffer solution of 90 μ L pH 4.5-7.5, then The 10 polyclonal detection antibody of the μ L 1mg/mL cavy anti-IL-1Ra in source, room temperature vortex concussion reaction 4 hours is added；10 μ L are added 10%BSA confining liquid reacts at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon supernatant, and 100 μ L pH are added The MES buffer solution of 4.5-7.5,4 DEG C are kept in dark place.
3. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that:
(1) IL-1 β -1-S:5/-tacttccaatccaatgcCTATACCTGTCTTGTGTGACCCA-3/；
IL-1 β -1-A:5/-ttatccacttccaatgtcAGAAGACGAATCGCTTTTCC-3/；
(2) IL-1Ra-1-S:5/-tacttccaatccaatgcCAAGCCTTCAGGATCTGGG-3/；
IL-1Ra-1-A:5/-ttatccacttccaatgtcAACCTTGCAAGTATCCAGC-3/；
(3) IL-1 β -2-S:5/-ccgcatatgTACAAGACAGAAATCAAGAACACAGT-3/；
IL-1 β -2-A:5/-atgcatgctgaagaaTATATCCTGGCCACCTCTAAAAC-3/；
(4) IL-1Ra-2-S:5/-ttcttcagcatgcatGACAAGCGCTTCGCCTTCATC-3/；
IL-1Ra-2-A:5/-ccgctcgagATTGGTGAGGCCCACGGG-3/；
(5) the linker sequence ttcttcagcatgcat of IL-1 β and the homologous conserved region tandem gene IL-1 β -1Ra-2 of IL-1Ra.
4. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that: the IL-1 β recombinant protein standard items and IL-1Ra recombinant protein standard items, are with large intestine bar respectively The IL-1 β recombinant protein and IL-1Ra recombinant protein of bacterium expression system gene engineering expression, specific preparation method press prokaryotic expression The open reading frame of IL-1 β and IL-1Ra are inserted into prokaryotic expression carrier pET-30a respectively by technology, building fusion His label Recombinant expression carrier pET-30a-IL-1 β and pET-30a-IL-1Ra obtain the gene of high-purity with inducing expression purification technique Engineering IL-1 β recombinant protein and IL-1Ra recombinant protein, prepare standard items albumen.
5. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that: the dilution is the 0.1M PBS solution of pH7.2-7.6.
6. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that: the antibody can with sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck IL-1 β and IL- 1Ra occur specific binding reaction, for detect sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck serum, blood The ratio of IL-1 β and IL-1Ra content and IL-1Ra/IL-1 β in slurry and histoorgan sample.
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