CN104558167B - Detect I types shiga toxin double-antibody method enzyme linked immunological kit and its application - Google Patents
Detect I types shiga toxin double-antibody method enzyme linked immunological kit and its application Download PDFInfo
- Publication number
- CN104558167B CN104558167B CN201410827932.3A CN201410827932A CN104558167B CN 104558167 B CN104558167 B CN 104558167B CN 201410827932 A CN201410827932 A CN 201410827932A CN 104558167 B CN104558167 B CN 104558167B
- Authority
- CN
- China
- Prior art keywords
- seq
- sequence
- amino acid
- sequence table
- light chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to one kind detection I types shiga toxin (StxI) Double-antibody sandwich enzymelinked immunosorbent detection kit and its application.The present invention obtains 4 plants of monoclonal antibodies for being directed to StxI A subunits by hybridoma technology altogether, it is and 4 plants of monoclonal antibodies of preparation are paired with each other, therefrom filter out two plants of noncompetitive monoclonal antibodies, respectively 8E7 E6 and 2F6 F8, prepare DAS-ELISA kit, prepared kit can specific detection StxI, there is good clinical value.
Description
Technical field
The present invention relates to kit field, and in particular to I types shiga toxin (StxI) double antibodies sandwich is enzyme-linked exempts from for one kind detection
Epidemic disease detection kit and its application.
Background technology
Enterohemorrhagic Escherichia coli (EHEC) infection is a kind of borne Parasitic Encephalopathy communicable disease, and foreign countries are commonly called as " hamburger disease ", its disease
Originally it was enterohemorrhagic escherichia coli (Enterohemorrhagic E.coli, EHEC), its typical strain is EHEC O157:H7,
Main performance symptom is the serious gastrointestinal complication such as hemorrhagic colitis, appendicitis, lemostenosis and perforation of colon, in children
With can also cause hemolytic urinary tract syndrome (haemolytic uraemic syndrome, HUS) and thrombotic blood in the elderly
The systemic complications such as platelet purpura (thrombocytpenic purpura, TTP).The HUS and TTP state of an illness is dangerous, case fatality rate
It is high.It is reported that due to O157:H7 appeal is strong, acidproof, and 100-200 O157 bacterium is with regard to that can break through hydrochloric acid in gastric juice barrier, in enteron aisle
Amount reproduction, people is caused to infect this disease, and other enteropathogenic E. Colis need more than 1,000,000 bacteriums can just cause morbidity.
Because the bacterium is strong to the pathogenicity of people, can cause sequelae, poor prognosis and there is no specific short both at home and abroad at present and cause the world
The great attention of various countries.
Research shows that the principal causative mechanism of EHEC infection can be divided into two aspects, first, by phage gene group pathogenicity island
The floating mechanism of adhesion that a variety of virulence factors of LEE (Locus of Enterocyte Effacement) codings are mediated
(Attaching and Effacing, A/E), by A/E mechanism, bacterium can destroy enterocyte, adhere to and be colonized in intestines
Road；Second, secretion shiga toxin (Shiga toxin, Stx), the enterocyte that Stx can pass through breakage enters blood circulation,
Combined with its acceptor-triose acyl sphingosine Gb3 or tetrose acyl sphingosine Gb4, cause the organs such as enteron aisle, central nervous system
Damage, the particularly higher kidney of Gb3 content receptors, easily trigger HUS, threat to life after being damaged；The toxin be divided into StxI and
Two hypotypes of StxII.For the research of shiga toxin, it was concentrated mainly in the past on StxII, because clinically StxII is compared with StxI
HUS can more be caused.However, the HUS as caused by StxI is still often reported, antibody and detection kit prepared by the present invention are filled out
Blank is in this respect mended.
Clinically detect O157:H7 common method includes isolated culture, immunological method, PCR the amplification skill of bacterium
Art.Bacteria Culture develops to obtain comparative maturity, and because equipment requirement needed for it is not high, oneself turns into the most frequently used method of basic unit, but operates
It is complicated, time-consuming, it is impossible to accomplish to early diagnose.Although PCR is quick, Sensitivity and Specificity is high, specific gene is needed, is removed at present
RfbE gene specifics are higher outer, most of to have certain homology with other microorganisms, specific also undesirable.Immunology
Method is quick because of its sensitivity, specificity and the visual diagnosis and monitoring for being widely used in clinical disease, particularly ELISA detections
Perception is high, easy to operate, and the development of necessary instrument equipment makes operation sequence standardize and automate, and further increases stabilization
Property, it is widely used in detecting trace of albumin composition, cell factor in multiple pathogens antigen or antibody, blood and other body fluid
Deng.But the O157 of immunological method detection at present:H7 candidate antibodies are still undesirable.
In order to provide a kind of method that quick, easy-operating diagnosis EHEC using StxI as targeting infects, prepared by the present invention
The more plants of monoclonal antibodies for being directed to StxI, therefrom filter out two plants of noncompetitive monoclonal antibodies, respectively 8E7-E6 and 2F6-F8, make
Standby DAS-ELISA kit.DAS-ELISA kit prepared by the present invention being capable of specific detection
StxI, there is good clinical value.
The content of the invention
It is an object of the invention to provide the monoclonal antibody of anti-StxI a kind of or its fragment, including light chain CDR1-3 and again
Chain CDR1-3, it is characterised in that the amino acid sequence of the light chain CDR1-3 is：
CDR1：Such as SEQ ID NO in sequence table：1 or SEQ ID NO：Shown in 11；
CDR2：Such as SEQ ID NO in sequence table：2 or SEQ ID NO：Shown in 12；
CDR3：Such as SEQ ID NO in sequence table：3 or SEQ ID NO：Shown in 13；
Further, the nucleotides sequence of the light chain CDR1-3 is classified as：
CDR1：Such as SEQ ID NO in sequence table：46 or SEQ ID NO：Shown in 52；
CDR2：Such as SEQ ID NO in sequence table：47 or SEQ ID NO：Shown in 53；
CDR3：Such as SEQ ID NO in sequence table：48 or SEQ ID NO：Shown in 54；
The amino acid sequence of the heavy chain CDR1-3 is：
CDR1：Such as SEQ ID NO in sequence table：4 or SEQ ID NO：Shown in 14；
CDR2：Such as SEQ ID NO in sequence table：5 or SEQ ID NO：Shown in 15；
CDR3：Such as SEQ ID NO in sequence table：6 or SEQ ID NO：Shown in 16.
Further, the nucleotides sequence of the heavy chain CDR1-3 is classified as：
CDR1：Such as SEQ ID NO in sequence table：49 or SEQ ID NO：Shown in 55；
CDR2：Such as SEQ ID NO in sequence table：50 or SEQ ID NO：Shown in 56；
CDR3：Such as SEQ ID NO in sequence table：51 or SEQ ID NO：Shown in 57.
It is an object of the invention to provide a kind of anti-StxI monoclonal antibody 8E7-E6, described monoclonal antibody 8E7-
Complementary determining region CDR1, CDR2, CDR3 of E6 light chain protein matter molecule variable region amino acid sequence, respectively as in sequence table
SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：Shown in 3.The heavy chain protein matter molecule of the monoclonal antibody 8E7-E6
Complementary determining region CDR1, CDR2, CDR3 of variable region amino acid sequence are respectively such as SEQ ID NO in sequence table：4、SEQ ID
NO：5、SEQ ID NO：Shown in 6.
Further, the complementary determining region CDR1 of described monoclonal antibody 8E7-E6 light chain protein matter molecule variable region,
CDR2, CDR3 nucleotide sequence, respectively such as SEQ ID NO in sequence table：46、SEQ ID NO：47、SEQ ID NO：48 institutes
Show.Complementary determining region CDR1, CDR2, CDR3 of the heavy chain protein matter molecule variable region of the monoclonal antibody 8E7-E6 nucleosides
Acid sequence is respectively such as SEQ ID NO in sequence table：49、SEQ ID NO：50、SEQ ID NO：Shown in 51.
The monoclonal antibody 8E7-E6 includes light chain and heavy chain, and its amino acid variable region sequences is respectively as in sequence table
SEQ ID NO：7、SEQ ID NO：Shown in 8, its encoding gene is respectively such as SEQ ID NO in sequence table：9、SEQ ID NO：10
It is shown.
It is an object of the invention to provide a kind of anti-StxI monoclonal antibody 2F6-F8, described monoclonal antibody 2F6-
Complementary determining region CDR1, CDR2, CDR3 of F8 light chain protein matter molecule variable region amino acid sequence, respectively as in sequence table
SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：Shown in 13.The heavy chain protein matter point of the monoclonal antibody 2F6-F8
Complementary determining region CDR1, CDR2, CDR3 of sub- variable region amino acid sequence are respectively such as SEQ ID NO in sequence table：14、SEQ
ID NO：15、SEQ ID NO：Shown in 16.
Further, the complementary determining region CDR1 of described monoclonal antibody 2F6-F8 light chain protein matter molecule variable region,
CDR2, CDR3 nucleotide sequence, respectively such as SEQ ID NO in sequence table：52、SEQ ID NO：53、SEQ ID NO：54 institutes
Show.Complementary determining region CDR1, CDR2, CDR3 of the heavy chain protein matter molecule variable region of the monoclonal antibody 2F6-F8 nucleosides
Acid sequence is respectively such as SEQ ID NO in sequence table：55、SEQ ID NO：56、SEQ ID NO：Shown in 57.
The monoclonal antibody 2F6-F8 includes light chain and heavy chain, and its amino acid variable region sequences is respectively as in sequence table
SEQ ID NO：17、SEQ ID NO：Shown in 18, its encoding gene is respectively such as SEQ ID NO in sequence table：19、SEQ ID NO：
Shown in 20.
It is an object of the invention to provide a kind of ELISA kit of detection I type shiga toxins, it is characterised in that described
The amino acid sequence of antibody or its fragment light chain CDR1-3 in kit is selected from SEQ ID NO：1-3 or 11-13, heavy chain
CDR1-3 amino acid sequence is selected from SEQ ID NO：4-6 or 14-16.
Further, the ELISA kit is double-antibody method ELISA kit, and the coated antibody is that 8E7-E6 is mono-
Clonal antibody, the detection antibody is 2F6-F8 monoclonal antibodies.
Further, kit also includes the one or more in following reagent：Horseradish peroxidase substrate buffer solution, egg
White standard items StxI, negative control sample, substrate, the washing lotion of BSA, PBS+0.05%Tween 20, confining liquid, terminate liquid.
It is an object of the invention to provide said monoclonal antibody or its fragment, ELISA kit to prepare detection I type will
Congratulate the application in toxin.
It is an object of the invention to provide above-mentioned monoclonal antibody or its fragment to be drawn in preparation treatment I type shiga toxins
Play the application in the medicine of disease.
Advantages of the present invention：Antibody specificity target StxI provided by the invention, prepared double crush syndrome reagent
Box only identifies I type shiga toxins, and nonrecognition II type shiga toxins, has good clinical value.
Brief description of the drawings
The total serum IgE that Fig. 1 extract from hybridoma
The band of Fig. 2 .MHV11/MHCG1 primers combination amplification
Swimming lane 1 is Lane1vk；Swimming lane 2 is lane2vh；Swimming lane 3 is marker
The band of Fig. 3 .MHV2/MHCG1 primer sets amplification
Swimming lane 1 is Lane1vk；Swimming lane 2 is lane2vh；Swimming lane 3 is marker
Embodiment
With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention
So that these embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
The structure of the anti-StxI monoclonal antibody hybridoma cells strain of embodiment 1
Experimental method
1st, Balb/C mouse immunes
5 week old female Balb/c mouse are immunized in I type Shiga toxin A recombinant proteins (StxIA), and 100 μ g/ are only, first
Secondary immune, 100 μ g antigens mix with isometric Freund's complete adjuvant, intraperitoneal injection；With equivalent amount of antigen and not exclusively after 3rd week
Adjuvant mixing pneumoretroperitoneum is immunized；5th week the 3rd time immune, is not added with adjuvant.
2nd, splenocyte and myeloma cell are merged
Merge the last week, recovery murine myeloma cell Sp2/0 to OPTI-MEM culture mediums (hyclone for containing 10%),
37 DEG C are placed in, 5%CO2Cultivated in incubator, 3 days before fusion, by passage once.On the fusion same day, myeloma cell is harvested,
Count, 5.0 × 107Myeloma cell with serum free medium wash 2 times it is standby.Mouse is plucked 3-5 days after the 3rd time immune
Except eyeball bloodletting, put to death.Mouse spleen is taken out in sterile working, puts in sterilizing plates, separating Morr. cell, counts, standby.
The splenocyte for being equal to the spleen of mouse 1/2 is mixed with myeloma cell, 1300rpm centrifugation 5min, gone as far as possible
Except supernatant.1.5ml 50% PEG was added in 1.5 minutes, side edged shakes up；Then added 20ml's in 8.5 minutes
Serum free medium, side edged shake up.
The cell 1000rpm merged through PEG is centrifuged 5 minutes, removes supernatant, adds 150ml HAT Selective agar medium weights
It is outstanding, fused cell is seeded to the sterile μ l/ holes of 96 orifice plate 150, is placed in 37 DEG C, 5%CO2Cultivate 4 days in incubator, added per hole
100 μ l Selective agar mediums.
3rd, the screening of hybridoma and clone
10 days after fusion, 50 μ l supernatants are inhaled from every hole, are added to the 96 hole elisa plates for being coated with StxIA (with 1% BSA
Closing), it is incubated at room temperature 1.5 hours；Wash 2 times.Dilute horseradish peroxidase (Horseradish Peroxidase, HRP) mark
The goat anti-mouse 1 of note:2000,50 μ l are added per hole, are incubated at room temperature 1.5 hours；Washing 4 times.100 μ l HRP substrates are added per hole
(H2O2+ TMB), it is incubated at room temperature 0.5 hour, 100 μ l 2M H is added per hole2SO4, survey A450nm values.For positive colony, continue
Following experiment.
Positive hole cell is collected, is resuspended in HT Selective agar mediums, using limiting dilution assay diluting cells, and is planted in 96
In porocyte culture plates, observe, the hole of only one cell clone growth of pair determination, identified as ELISA after 5 days.To sun
Property hole cell carry out limiting dilution, by 3-4 time it is unicellular be separately cultured, until obtaining hybridoma cell clone stably.Greatly
Amount culture hybridoma, collects the culture supernatant containing antibody, is purified with protein G affinity columns, and dialyse
Into PBS, survey concentration, -20 DEG C freeze it is standby.
By above-mentioned hybridoma technology obtain altogether 4 plants be directed to StxIA subunits monoclonal antibodies, respectively 1H2-G7,
2H1-C12,8E7-E6 and 2F6-F8.
The selection optimum antibody pairing of the double-antibody sandwich elisa of embodiment 2
Using double antibody sandwich ELISA, 4 strain antibodies for allowing embodiment 1 to prepare are paired with each other, select best of breed with profit
Detected in toxin, specific method is as follows：
1st, the coating of monoclonal antibody
With 0.1M NaHCO3/Na2CO3Buffer solution (pH 9.6) dilutes antibody concentration to 10 μ g/ml, adds to 96 hole enzymes
Target, 100 μ l/ holes, 4 DEG C of coatings overnight, are washed 1 time with PBST (Tween-20 of addition 0.05% in PBS), add 5%
The closing of 4 DEG C of milk washes 1 time overnight, with PBST, -20 DEG C freeze it is standby.
2nd, the coupling of monoclonal antibody and horseradish peroxidase (Horseradish Peroxidase, HRP)
Monoclonal antibody is dialysed into PBS, regulation concentration to 1mg/ml.Using the production of Beijing Thailand day and bio tech ltd
Product activation horseradish peroxidase is coupled, and concrete operations are with reference to its company's specification.50% is added in HRP labelled antibodies
Glycerine, -20 DEG C freeze it is standby.
3rd, ELISA is operated
The StxI of purifying is diluted with the PBS containing 1% bovine serum albumin(BSA) (bovine serum albumin, BSA) extremely
Concentration is 10 μ g/ml, and 100 μ l are added per hole, is incubated 1 hour in 37 DEG C of water-baths, is washed 5 times with PBST.With containing 1%BSA's
PBS is according to 1:1000 dilution HRP enzyme labelled antibodies, according to 4 strain antibodies principle paired with each other, add 100 μ l enzymes in respective aperture
Labeling antibody, it is incubated 1 hour in 37 DEG C of water-baths, after washing 5 times with PBST, 100 μ l TMB+H is added in every hole2O2Substrate, room temperature are incubated
Educate 20 minutes, 100 μ l 2M sulfuric acid terminating reaction is added per hole, OD values are determined at 450nm.Concrete outcome is as shown in table 1.
Table 1
Result can be seen that only 8E7-E6/2F6-F8 combinations from table, and only when 8E7-E6 is anti-as coating
Body, and when 2F6-F8 is as detection antibody, it detects the OD value highests of toxin, reaches 1.435, and the OD values of other combinations compared with
It is low.Because the epitope of 4 plants of monoclonal antibodies of above-mentioned acquisition is respectively positioned on the A subunits of toxin, when two antibody simultaneously with the antigen binding
When, there is steric hindrance.And only when 8E7-E6 is used as detection antibody as the antibody and 2F6-F8 of capture toxin, can
Obstruction that can be between it is minimum, therefore the OD values shown also highest.
The monoclonal antibody 8E7-E6/2F6-F8 of embodiment 3 weights, the identification of light chain isotype (Isotype)
Use the SBA Cloning System/HRP isotype identification kits (5300- of SouthernBiotech companies
05), with PBS, pH 7.4, dilution goat anti-mouse (H+L) antibody to 10 μ g/ml, ELISA ELISA Plates are added to, per the μ l of hole 100,4
DEG C be incubated overnight.Coating buffer is abandoned, is washed 3 times with the PBS (PBST) containing 0.05%Tween 20, the 1% of 200 μ l are added per hole
BSA, 4 DEG C closing overnight.Washed 3 times with PBST, add 100 μ l Hybridoma culture supernatants per hole in 12 holes, be placed in
It is incubated 1 hour in 37 DEG C of water-baths, is washed 3 times with PBST.1 is pressed with the PBS containing 1%BSA:500 dilute the mountain of HRP marks respectively
Sheep anti-Mouse secondary antibody, its specificity are as follows：Heavy chain is IgG1, IgG2a, IgG2b, IgG3, light chain κ, λ, each specificity two
2 holes of anti-addition, per the μ l of hole 100, are placed in 37 DEG C of water-baths and are incubated 1 hour, washed 5 times with PBST.100 μ l substrates are added per hole
(TMB+H2O2), room temperature is placed 20 minutes, and 2M H are added per hole2SO4100 μ l terminating reactions, are placed under 450nm and measure absorbance.
Concrete outcome is shown in Table 2.
Table 2
| ELIAS secondary antibody | IgG1 | IgG2a | IgG2b | IgG3 | κ | λ |
| OD450nm(8E7-E6) | 1.353 | 0.095 | 0.065 | 0.087 | 1.900 | 0.075 |
| OD450nm(2F6-F8) | 1.367 | 0.099 | 0.085 | 0.091 | 1.675 | 0.086 |
The result listed from upper table can be seen that monoclonal antibody 8E7-E6/2F6-F8 weight, light chain isotype difference
For：The isotype of heavy chain is G1, and the isotype of light chain is κ.
The monoclonal antibody 8E7-E6/2F6-F8 of embodiment 4 weights, the clone of chain variable region gene
Take the logarithm the hybridoma in growth period, using the Trizol extracted total RNAs of Invitrogen companies, use oligo
(dT) 20 be primer, and reverse transcription generates cDNA.Then its heavy, chain variable region gene is expanded respectively using specific primer PCR.
PCR primer clones insertion pMD-18T carriers after Purified in electrophoresis, by TA, sequencing, carries out sequence analysis.Concrete operation step
It is as follows：
1st, total serum IgE extracts
Trizol extracted total RNA specification of the operating method with reference to Invitrogen companies.Electrophoresis is carried out after extraction total serum IgE
Detection, is as a result shown in Fig. 1.
Electrophoresis result shows that RNA does not degrade.
2nd, cDNA is synthesized
Using the total serum IgE of extracting as template, oligo (dT) 20 is primer, and reverse transcription synthesizes cDNA.Reaction system is as follows：
| Reagent | Dosage |
| oligo(dT)20(500μg/ml) | 1μl |
| RNA | 10μl |
| dNTPs mix(10mM each) | 1μl |
65 DEG C are incubated 5 minutes, are then quickly placed at 3 minutes on ice, add following component：
| Reagent | Dosage |
| 5×first-strand buffer | 4μl |
| DTT(0.1M) | 2μl |
| RNase inhibitor (40U/ μ l) | 1μl |
| Reverse transcriptase (200U/ μ l) | 1μl |
| ddH2O | Filling-in is to 20 μ l |
42 DEG C are incubated 50 minutes, and then 70 DEG C are incubated 15 minutes inactivation reverse transcriptases.
3rd, PCR expands monoclonal antibody weight, chain variable region gene VH and VL
Expanding VH primer is：
SEQ ID NO：21MHV1：5’ATGAAATGCAGCTGGGGCATSTTCTTC3’
SEQ ID NO：22MHV2：5’ATGGGATGGAGCTRTATCATSYTCTT3’
SEQ ID NO：23MHV3：5’ATGAAGWTGTGGTTAAACTGGGTTTTT3’
SEQ ID NO：24MHV4：5’ATGRACTTTGGGYTCAGCTTGRTTT3’
SEQ ID NO：25MHV5：5’ATGGACTCCAGGCTCAATTTAGTTTTCCTT3’
SEQ ID NO：26MHV6：5’ATGGCTTGTCYTRGSGCTRCTCTTCTGC 3’
SEQ ID NO：27MHV7：5’ATGGRATGGAGCKGGRTCTTTMTCTT 3’
SEQ ID NO：28MHV8：5’ATGAGAGTGCTGATTCTTTTGTG 3’
SEQ ID NO：29MHV9：5’ATGGMTTGGGTGTGGAMCTTGCTATTCCTG 3’
SEQ ID NO：30MHV10：5’ATGGGCAGACTTACATTCTCATTCCTG 3’
SEQ ID NO：31MHV11：5’ATGGATTTTGGGCTGATTTTTTTTATTG 3’
SEQ ID NO：32MHV12：5’ATGATGGTGTTAAGTCTTCTGTACCTG 3’
SEQ ID NO：33MHCG1：5’CAGTGGATAGACAGATGGGGG 3’
Expanding VL primer is：
SEQ ID NO：34MKV1：5’ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG 3’
SEQ ID NO：35MKV2：5’ATGGAGWCAGACACACTCCTGYTATGGGTG 3’
SEQ ID NO：36MKV3：5’ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 3’
SEQ ID NO：37MKV4：5’ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG 3’
SEQ ID NO：38MKV5：5’ATGGATTTWCAGGTGCAGATTWTCAGCTTC 3’
SEQ ID NO：39MKV6：5’ATGAGGTKCYYTGYTSAGYTYCTGRGG 3’
SEQ ID NO：40MKV7：5’ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 3’
SEQ ID NO：41MKV8：5’ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG 3’
SEQ ID NO：42MKV9：5’ATGGTRTCCWCASCTCAGTTCCTTG 3’
SEQ ID NO：43MKV10：5’ATGTATATATGTTTGTTGTCTATTTCT 3’
SEQ ID NO：44MKV11：5’ATGGAAGCCCCAGCTCAGCTTCTCTTCC 3’
SEQ ID NO：45MKC：5’ACTGGATGGTGGGAAGATGG 3’
Degenerate code：R=A or G Y=C or C M=A or C K=G or T S=C or G W=A or
T H=A or C or T B=C or G or T V=A or C or G D=A or G or T N=A or C or G
or T。
Reaction system is as follows：
| Reagent | Dosage |
| ExTaq(5U/μl) | 0.25μl |
| 10×buffer(Mg++free) | 5.0μl |
| MgCl2(25mM) | 4.0μl |
| dNTPs(2.5mM each) | 4.0μl |
| Sense primer (10 μM) | 1.0μl |
| Anti-sense primer (10 μM) | 1.0μl |
| cDNA | 2.0μl |
| ddH2O | Filling-in is to 50 μ l |
Amplification condition：94 DEG C of pre-degeneration 5min, then 94 DEG C are denatured 30sec；56 DEG C of annealing 30sec；72 DEG C of extension 1min,
Totally 30 circulations.
For monoclonal antibody 8E7-E6, the combination of weight chain variable district, only MHV11/MHCG1 primers can amplify positive band,
Positive band can be amplified for the combination of light chain variable district MKV5 MKC primers, stripe size is 450bp or so (as schemed
2).For monoclonal antibody 2F6-F8, the combination of weight chain variable district, only MHV2/MHCG1 primers can amplify positive band, be 450bp
Left and right, positive band can be amplified for the combination of light chain variable district MKV3 MKC primers, stripe size is 430bp or so
(such as Fig. 3).
4th, the sequencing of PCR primer
According to Dalian TAKARA company's T A Cloning Kit specifications, by monoclonal antibody 8E7-E6/2F6-F8 weights, light variable region base
Because PCR primer inserts pMD-18T carriers, company is sent to be sequenced.As a result show：8E7-E6VH nucleotide sequences are shown in sequence table SEQ ID
NO：10,8E7-E6VH amino acid sequences are shown in sequence table SEQ ID NO：8；8E7-E6VK nucleotide sequences are shown in sequence table SEQ ID
NO：9,8E7-E6VH amino acid sequences are shown in sequence table SEQ ID NO：7；2F6-F8Vh nucleotide sequences are shown in sequence table SEQ ID
NO：20,2F6-F8Vh amino acid sequences are shown in sequence table SEQ ID NO：18；2F6-F8Vk nucleotide sequences are shown in sequence table SEQ ID
NO：19,8E7-E6VH amino acid sequences are shown in sequence table SEQ ID NO：17.
Embodiment 5 prepares the DAS-ELISA kit of 8E7-E6/2F6-F8 compositions
1st, the coating of monoclonal antibody
With 0.1M NaHCO3/Na2CO3It is 10 μ g/ml that buffer solution (pH 9.6), which dilutes antibody 8E7-E6 to concentration, is added
To 96 hole elisa Plates, 100 μ l/ holes, 4 DEG C of coatings overnight, are washed 1 time with PBST (Tween-20 of addition 0.05% in PBS), added
Enter 5% 4 DEG C of milk closing overnight, 1 time washed with PBST, -20 DEG C freeze it is standby.
2nd, the coupling of monoclonal antibody 2F6-F8 and horseradish peroxidase (Horseradish Peroxidase, HRP)
Monoclonal antibody 2F6-F8 is dialysed into PBS, regulation concentration to 1mg/ml.Using Beijing Thailand day and the limited public affairs of biotechnology
The product activation horseradish peroxidase of department is coupled, and concrete operations are with reference to its company's specification.Add in HRP labelled antibodies
Enter 50% glycerine, -20 DEG C freeze it is standby.
Washing lotion：PBS+0.05%Tween 20
Substrate：TMB and H2O2, it is Thermo Products.
StxI materials in the DAS-ELISA kit detection sample that embodiment 68E7-E6/2F6-F8 is formed
Material：
Used EHEC bacterial strains list：
| Title | 00G097 | 99A021 | 99G143 | 23A2C | 85933 | 882364 |
| Bacterial strain | O157H7 | O157H7 | O157H7 | O157H7 | O157H7 | O157H7 |
| Source place | China | China | China | China | The U.S. | China |
| Hosts | HUMAN | PIG | HUMAN | HUMAN | HUMAN | HUMAN |
| StxI | - | - | + | + | + | + |
| StxII | + | + | + | + | + | - |
Method：
1st, the preparation of Stx crude extracts.For above-mentioned bacterial strains, respectively in the picking EHEC single bacterium colonies on LB flat boards, LB liquid is put
250rpm in body culture medium, 37 DEG C of overnight incubations.Next day overnight culture 1:100 are diluted to 2L, cultivate to 4 hours, add eventually
Concentration is 0.4mg/L mitomycin Cs (mitomycin C), then proceedes to culture 20 hours.4 DEG C of centrifugations of culture 16,000g
30 minutes, 0.45 μm of filtering with microporous membrane of supernatant, regulation pH value was to 7.5, packing, -20 DEG C freeze it is standby.
2nd, ELISA is detected.The DAS-ELISA reagent that the 8E7-E6/2F6-F8 prepared using embodiment 5 is formed
Box.
As a result：
Testing result is as follows：
As a result the reagent that StxI types bacterial strain (such as 23A2C, 85933,99G143,882364) can be prepared by the present invention is shown
Box detects that testing result is compared with a control with significant difference.When bacterial strain be StxII types bacterial strain (00G097,
99A021), testing result is compared with the control without statistical discrepancy.The kit that i.e. prepared by the present invention being capable of specific detection StxI types
Bacterial strain.
Claims (9)
1. a kind of anti-StxI monoclonal antibody or its fragment, including light chain CDR1-3 and heavy chain CDR1-3, it is characterised in that light
Chain CDR1-3 amino acid sequence is：Light chain CDR1 amino acid sequence is sequence table SEQ ID NO：1, light chain CDR2 amino
Acid sequence is sequence table SEQ ID NO：2, light chain CDR3 amino acid sequence are sequence table SEQ ID NO：3；Heavy chain CDR1-3
Amino acid sequence be：Heavy chain CDR1 amino acid sequence is sequence table SEQ ID NO：4, heavy chain CDR2 amino acid sequence is
Sequence table SEQ ID NO：5, heavy chain CDR3 amino acid sequence are sequence table SEQ ID NO：6.
2. a kind of anti-StxI monoclonal antibody or its fragment, including light chain CDR1-3 and heavy chain CDR1-3, it is characterised in that light
Chain CDR1-3 amino acid sequence is：Light chain CDR1 amino acid sequence is sequence table SEQ ID NO：11, light chain CDR2 ammonia
Base acid sequence is sequence table SEQ ID NO：12, light chain CDR3 amino acid sequence are sequence table SEQ ID NO：13；Heavy chain
CDR1-3 amino acid sequence is：Heavy chain CDR1 amino acid sequence is sequence table SEQ ID NO：14, heavy chain CDR2 amino
Acid sequence is sequence table SEQ ID NO：15, heavy chain CDR3 amino acid sequence are sequence table SEQ ID NO：16.
3. monoclonal antibody according to claim 1 or its fragment, it is characterised in that light chain CDR1-3 nucleotide sequence
For：Light chain CDR1 nucleotides sequence is classified as SEQ ID NO in sequence table：46, light chain CDR2 nucleotides sequence is classified as in sequence table
SEQ ID NO：47, light chain CDR3 nucleotides sequence are classified as SEQ ID NO in sequence table：48；Heavy chain CDR1-3 nucleotides sequence
It is classified as：Heavy chain CDR1 nucleotides sequence is classified as SEQ ID NO in sequence table：49, heavy chain CDR2 nucleotides sequence is classified as sequence table
Middle SEQ ID NO：50, heavy chain CDR3 nucleotides sequence are classified as SEQ ID NO in sequence table：51.
4. monoclonal antibody according to claim 2 or its fragment, it is characterised in that light chain CDR1-3 nucleotide sequence
For：Light chain CDR1 nucleotides sequence is classified as SEQ ID NO in sequence table：52, light chain CDR2 nucleotides sequence is classified as in sequence table
SEQ ID NO：53, light chain CDR3 nucleotides sequence are classified as SEQ ID NO in sequence table：54；Heavy chain CDR1-3 nucleotides sequence
It is classified as：Heavy chain CDR1 nucleotides sequence is classified as SEQ ID NO in sequence table：55, heavy chain CDR2 nucleotides sequence is classified as sequence table
Middle SEQ ID NO：56, heavy chain CDR3 nucleotides sequence are classified as SEQ ID NO in sequence table：57.
5. monoclonal antibody according to claim 1 or its fragment, it is characterised in that the amino acid of the light chain variable district
SEQ ID NO in sequence such as sequence table：Shown in 7, SEQ ID NO in the amino acid sequence of the weight chain variable district such as sequence table：8
It is shown.
6. monoclonal antibody according to claim 2 or its fragment, it is characterised in that the amino acid of the light chain variable district
SEQ ID NO in sequence such as sequence table：Shown in 17, SEQ ID NO in the amino acid sequence of the weight chain variable district such as sequence table：
Shown in 18.
7. a kind of ELISA kit of detection I type shiga toxins, it is characterised in that it is any one that kit includes claim 1-6
Monoclonal antibody or its fragment described in.
8. ELISA kit according to claim 7, it is characterised in that ELISA kit is double-antibody method ELISA
Kit, coated antibody will selected from right selected from the monoclonal antibody described in claim 1,3 or 5 any one, detection antibody
Seek the monoclonal antibody described in 2,4 or 6 any one.
9. the ELISA reagents described in monoclonal antibody or its fragment, claim 7-8 described in claim 1-6 any one
Box is preparing the application in detecting I type shiga toxin reagents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410827932.3A CN104558167B (en) | 2014-12-26 | 2014-12-26 | Detect I types shiga toxin double-antibody method enzyme linked immunological kit and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410827932.3A CN104558167B (en) | 2014-12-26 | 2014-12-26 | Detect I types shiga toxin double-antibody method enzyme linked immunological kit and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104558167A CN104558167A (en) | 2015-04-29 |
| CN104558167B true CN104558167B (en) | 2017-12-26 |
Family
ID=53075320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410827932.3A Active CN104558167B (en) | 2014-12-26 | 2014-12-26 | Detect I types shiga toxin double-antibody method enzyme linked immunological kit and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104558167B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115856296B (en) * | 2022-12-16 | 2023-07-25 | 华北理工大学 | Anti-shigella monoclonal antibody and its application in detection |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6541013B1 (en) * | 2000-07-13 | 2003-04-01 | Idaho Research Foundation, Inc. | Methods and compositions for suppressing bovine leukemia virus with a Shiga toxin polypeptide |
| EP2019684A2 (en) * | 2006-04-20 | 2009-02-04 | The Henry M. Jackson Foundation for the advancement of Military Medicine, Inc. | Methods and compositions based on shiga toxin type 1 protein |
| CN101434956A (en) * | 2008-12-02 | 2009-05-20 | 江苏省疾病预防控制中心 | Heavy and light chain variable region gene of monoclonal antibody, encoding polypeptide thereof and use |
| CN101570574A (en) * | 2009-06-10 | 2009-11-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Anti-I type Shiga toxin IgY antibody as well as preparation method and use thereof |
| CN102292098A (en) * | 2009-01-23 | 2011-12-21 | 杰克孙M.亨利基金会先进军事医学有限公司 | Methods and compositions based on Shiga toxin type 2 protein |
-
2014
- 2014-12-26 CN CN201410827932.3A patent/CN104558167B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6541013B1 (en) * | 2000-07-13 | 2003-04-01 | Idaho Research Foundation, Inc. | Methods and compositions for suppressing bovine leukemia virus with a Shiga toxin polypeptide |
| EP2019684A2 (en) * | 2006-04-20 | 2009-02-04 | The Henry M. Jackson Foundation for the advancement of Military Medicine, Inc. | Methods and compositions based on shiga toxin type 1 protein |
| CN101434956A (en) * | 2008-12-02 | 2009-05-20 | 江苏省疾病预防控制中心 | Heavy and light chain variable region gene of monoclonal antibody, encoding polypeptide thereof and use |
| CN102292098A (en) * | 2009-01-23 | 2011-12-21 | 杰克孙M.亨利基金会先进军事医学有限公司 | Methods and compositions based on Shiga toxin type 2 protein |
| CN101570574A (en) * | 2009-06-10 | 2009-11-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Anti-I type Shiga toxin IgY antibody as well as preparation method and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| Stx1-A亚单位的原核表达、复性及抗原性鉴定;葛以跃等;《医学分子生物学杂志》;20081231;第5卷(第4期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104558167A (en) | 2015-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103509756B (en) | Mycoplasma bovis monoclonal antibody, and preparation method and application thereof | |
| CN103073644A (en) | Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof | |
| CN104862283B (en) | The monoclonal antibody of a pair of of high specific high-affinity combination human muscle hemoglobin and its application | |
| US8940496B2 (en) | Method for detecting microorganisms belonging to Mycoplasma pneumoniae and/or Mycoplasma genitalium | |
| Gong et al. | Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library | |
| Richard et al. | Antibody binding to the O-specific antigen of Pseudomonas aeruginosa O6 inhibits cell growth | |
| CN104497142A (en) | Monoclonal antibody of CP4-EPSPS protein | |
| CN105001328B (en) | Anti- TTF-1 monoclonal antibodies and its application are secreted by hybridoma cell strain | |
| CN104558167B (en) | Detect I types shiga toxin double-antibody method enzyme linked immunological kit and its application | |
| CN101748136A (en) | Expression of cymbidium mosaic virus coat protein gene and preparation method for antibody of cymbidium mosaic virus coat protein gene | |
| CN106380510A (en) | Novel targets of acinetobacter baumannii | |
| CN109971726B (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and preparation method of antibody | |
| CN109777785B (en) | Hybridoma cell line and its application | |
| CN103837684A (en) | Antibody reagent for rapidly detecting salmonellas and detection method thereof | |
| CN106544324B (en) | Monoclonal antibody of mycoplasma hyopneumoniae and application thereof | |
| CN103319593B (en) | Anti StxII monoclonal antibody | |
| CN101535811A (en) | Method for in vitro diagnosis of PVL-producing staphylococcus aureus | |
| CN107099505A (en) | Anti- FtsH2 protein monoclonal antibodies and its application | |
| CN104450628B (en) | Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application | |
| CN101970468A (en) | Component protein pa1698 for type-iii secretion system of pseudomonas aeruginosa | |
| CN114539393A (en) | 2019-novel coronavirus N protein single domain antibody and application thereof | |
| KR101349413B1 (en) | Recombinant recombinant vector for producing antigen for Vibrio vulnificus diagnosis and Monoclonal antibody specific to Vibrio vulnificus RtxA1 | |
| Fasihi-Ramandi et al. | Production and characterization of monoclonal and polyclonal antibody against recombinant outer membrane protein | |
| CN115856296B (en) | Anti-shigella monoclonal antibody and its application in detection | |
| KR101402279B1 (en) | Rapid isolation method of enterohaemorrhagic Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |