CN108265032A - Anti- OmpC hybridoma cell strains and its monoclonal antibody of generation - Google Patents

Abstract

The invention discloses a kind of hybridoma cell strain, deposit number is CCTCC NO：The anti-OmpC monoclonal antibodies that C2016161 and thus hybridoma cell strain generate.The invention further relates to the engineered antibody based on this hybridoma cell strains and its applications in immune detection tool, and people source blood can be replaced as positive control by the use of this engineered antibody.

Description

Anti- OmpC hybridoma cell strains and its monoclonal antibody of generation

Technical field

The present invention relates to immunological technique field, more particularly to prepare it is a kind of prepare secrete anti-OmpC monoclonal antibody it is miscellaneous Hand over tumor cell strain and the application based on OmpC chimeric antibodies prepared by the hybridoma cell strain in inflammatory bowel disease detection.

Background technology

Inflammatory bowel disease (inflammatory bowel disease, IBD) is a kind of chronic nonspecific intestinal inflammatory Disease, including ulcerative colitis (ulcerative colitis, UC) and Crohn disease (Crohn ' s disease, CD), closely In China's incidence in gradually increasing trend over more than 10 years.The complicated clinical manifestation of IBD is various, not only there is symptom of digestive tract, also There can be parenteral performance.Since symptom is abdominal pain, diarrhea, non-specific enteritis performance, CD and UC and IBD and the tuberculosis such as have blood in stool Other enteron aisle chronic diseases such as property enteritis are difficult to discriminate between diagnosing.

It is micro- with resisting to be concentrated mainly on autoimmune antibody for the biomarker of discriminating IBD and other chronic gut inflammations at present Biological antibody.Escherichia coli are one of normal bacterias in human body intestinal canal, not pathogenic under normal circumstances.But in certain special circumstances Under, when certain albumen radical response of body to Escherichia coli, the enteritis symptom such as abdominal pain, diarrhea can occur.Research shows that IBD has the radical response of Escherichia coli outer membrane duct PROTEIN C (Outer membrane protein C, OmpC) with body It closes, the IgA or IgG antibody of the about 50% anti-OmpC of IBD patients serums are positive, therefore, by detecting serum moderate resistance The IgA and IgG antibody of OmpC can be with auxiliary diagnosis IBD.

Using immunoassay technology, such as antibody in the determination samples such as enzyme-linked immunization, chemoluminescence method, immunochromatographic method Concentration is common detection method.In such experiment, it is necessary to set up Positive control wells, the blood of positive control multipurpose clinical people Clear or other forms human serums.But humanized's blood product, it is likely that containing pathogenic infections substance, there are potential to operator Threat, while a large amount of serum or blood plasma are needed in positive control procedure is prepared, and serum or blood plasma of some diseases compare Seldom arrive.Therefore using people source blood as positive control, for preparing a large amount of detection kit products, raw material is by visitor The limitation of sight.So a kind of positive control substitute need to be sought applied to the IgA of OmpC or IgG antibody detection kit.

Invention content

The object of the present invention is to provide the monoclonals of a kind of anti-OmpC monoclonal antibody hybridoma cells strain and its secretion Antibody.

Another object of the present invention is：A kind of engineered antibody based on OmpC monoclonal antibody hybridoma cell strains is provided And its purposes in field of medical examination.

OmpC monoclonal antibody hybridoma cells strain provided by the invention, is preserved in China typical culture collection center (referred to as CCTCC), preservation date are August in 2016 12 days, and deposit number is CCTCC NO：C2016161.

The present invention provides following technical solutions：OmpC monoclonal antibody hybridomas are developed, based on this hybridoma Strain prepares humanized chimeric antibody, i.e., the light and heavy chain variable region gene of the monoclonal antibody of OmpC is inserted into the IgA of source containing someone Or in the expression vector of IgG antibody constant region genes, be transferred to the OmpC expressed in zooblast humanization IgA Fc or The chimeric antibody of humanization IgG Fc, both OmpC humanization IgA Fc or humanization IgG Fc chimeric antibodies can be respectively as Detect the IgA of anti-OmpC or the positive control of IgG antibody.

The present invention program includes the following steps：

(1) prepared by OmpC antigen proteins；

(2) BALB/c mouse, ELISA method detection immune serum potency is repeatedly immunized in OmpC albumen；

(3) BALB/c mouse spleen cell is immunized to be merged with SP2/0 cells,

(4) limiting dilution assay obtains monoclonal, ELISA method screening positive hybridoma cell；

(5) Identification of Monoclonal Antibodies of hybridoma：The hybridoma cell strain of anti-OmpC specific antibodies can be secreted by obtaining eventually, Hypotype is accredited as IgG1, and it is 1: 320000 that ELISA, which surveys potency,；

(6) based on OmpC monoclonal antibody hybridoma cell strains, by engineered antibody technology, with humanization IgA Fc or people Fc sections of the mouse source of the IgG Fc sections of replacement OmpC monoclonal antibody in source prepares the chimeric antibody of anti-OmpC；

(7) calibration object and its application that OmpC chimeric antibodies are prepared.

It is an advantage of the invention that：

(1) hybridoma cell strain chromosome stabilityX of the present invention, can steadily secretory antibody.

(2) antibody titer of hybridoma cell strain of the present invention secretion is high, and culture supernatant potency is up to 1: 32 ten thousand.

(3) it is positive right to be used as based on the alternative people source blood of chimeric antibody prepared by hybridoma cell strain of the present invention According to, for preparing a large amount of detection kit products, the objective limitation of human blood raw material sources is avoided, it is also safer.

Preservation information

Scientific description for the hybridoma cell strain of preservation is：Anti-human OmpC monoclonal antibody hybridoma cells strain HR002 13-5；

Depositary institution's full name：China typical culture collection center；

Depositary institution is referred to as：CCTCC；

Depositary institution address：Wuhan City, Hubei Province Wuchang District Luo Jia Shan road Wuhan University；

Preservation date：On August 12nd, 2016；

Deposit number：CCTCC NO：C2016161.

Description of the drawings

The electrophoretogram of Fig. 1 monoclonal antibodies after purification；

Fig. 2 OmpC antibody I gA fluorescent quantitation immunochromatography calibration curves.

Specific embodiment

With reference to specific embodiments and the drawings, the present invention is further explained.

Embodiment 1：It is prepared by OmpC antigen proteins

1. reagent and instrument

1) reagent

E. coli host bacteria DH5 α, BL21 (DE3), cloning vector pCR2.1 T-vector, expression plasmid pET28a (+), archaeal dna polymerase rTaq, T4 DNA ligase, archaeal dna polymerase rTaq, LA Taq and restriction enzyme BamH I, Hind III and EcoR I, BamH I, DL2000 DNA Marker, T4 DNA ligases, low molecular weight standard protein, DNA glue QIAquick Gel Extraction Kit, IPTG etc.

2) instrument

Common shaking table SCS-24；Water isolation type constant temperature electric heating incubator；Biophotometer spectrophotometers, desk-top freezing Centrifuge Centrifuge 5810R, desk centrifuge MiniSpin；High speed freezing centrifuge；Protein electrophorese instrument and gel Imaging system；PCR instrument；Ultrasound cracking instrument；Constant-temperature metal bath；HIS protein purification columns etc..

2 experimental methods

1) vector construction：

Design primer CATG ccatgg ATG CAT CAT CAT CAT CAC CAT (NcoI) and CGC ggatcc TTA GAA CTG GTA AAC CAG GCC (BamHI) PCR amplification from template DNA goes out OmpC segments, plastic recovery kit recycling piece PCR2.1 cloning vectors, which are connected to, after section carries out sequencing identification.It will identify that correct sequence is cloned into expression vector pET28a (+) In, restriction enzyme site is EcoR I, BamH I, while has 6 × HIS labels on carrier, convenient for subsequent protein purification.

2) it expresses

Obtained plasmid is converted to e. coli bl21 (DE3), passes through resistance screening positive expression bacterial strain.It will filter out The positive bacterium solution come is inoculated in 1: 1000 ratio in the LB culture mediums added with Kan resistances, and each two pipe of bacterium inoculation a, pipe is used for Induction, another pipe is used for non-induced control, while to be inoculated with a pipe empty plasmid bacterium and compare.37 DEG C are incubated overnight to OD600 values During about 0.4-0.6, induction pipe and empty control plasmid pipe add in IPTG to final concentration of 1mmol/L, and non-induced control is not added with, most Two high bacterium of selection ability to express eventually, for the expression of a large amount of albumen.And according to conventional SDS-PAGE methods to expression Product is identified.

3) it purifies

By the product of expression HIS protein purification column purification albumen, and dialysis desalting, protein concentration after purification after measured For 490mg/L.

Embodiment 2：The preparation of anti-OmpC hybridomas

The emulsification of 1.OmpC immunizing antigens

Head exempts from：OmpC antigens mix emulsification, booster immunization in equal volume with complete Freund's adjuvant：OmpC antigens with not exclusively not Family name's adjuvant mixes in equal volume, is emulsified with the mutual pushing manipulation of double syringe.

2. animal immune

6-8 week old BALB/c mouses are immunized using subcutaneous or intraperitoneal injection method, immunizing dose only, is spaced two for 50 μ g/ It carries out being immunized for second after week, be emulsified with incomplete Freund's adjuvant, immunizing dose is 50 μ g/.It is immune take afterwards twice tail blood with ELISA method gradient dilution measures serum titer；Booster immunization is determined whether according to result.Carry out booster immunization within 3 days before fusion, often The injection of mouse peritoneal is not added with 100 μ g antigens of adjuvant, and cell fusion is carried out after 3 days.

3. cell fusion

PEG1500 is placed in pre-temperature in 37 DEG C of incubators before fusion, draws 1 × 10⁷A SP2/0 myeloma cell's suspension and 5×10⁷It is a to derive from SPF grades of Balb/c mouse spleen bone-marrow-derived lymphocytes suspensions (cell number 1: 5) to a 50ml centrifuge tube, 30mlIMDM incomplete culture mediums, abundant mixing are added, 1500rpm centrifugation 5min abandon supernatant, flick tube bottom, make cell mass loose Paste is dissipated into, by 37 DEG C of water-baths of centrifuge tube, the 50%PEG1500 solution of 0.8ml pre-temperatures is drawn with dropper, from tube bottom about 2cm Place is slowly added into along tube wall in cell, and edged rotation centrifuge tube in side adds in 1min or so, then stands 90s, is added dropwise 37 The incomplete culture medium IMDM 8ml of DEG C pre-temperature terminate fusion, are added within 2min, and fast after speed is first slow, action is soft, will be from Heart pipe stands 5min in 37 DEG C of incubators, takes out centrifuge tube, and 1000rpm centrifugation 5min discard supernatant, add in 10ml HAT (SIGMA) cell is resuspended in culture medium, gently blows and beats, mixing, fused cell is seeded to the 96 hole cells for being covered with trophocyte Culture plate, by 100 μ l/ holes, every piece of culture plate stays 6 holes to be inoculated with SP2/0 cells, as the negative control of HAT selections, puts 37 DEG C, 5%CO₂It is cultivated in incubator.

4. screening and clone

The growing state of cell can be observed under inverted microscope, and add 100 μ l of HAT culture mediums within the 5th day after fusion, Hybridoma potency can be surveyed with indirect elisa method within 10th day, change within the 14th day HT (SIGMA) culture medium (containing feeder cells and 1%HT liquid Body) 96 orifice plates in.It is put into 5%CO₂Cell incubator.

One sieve：It chooses after cloning 3 days or so, when observation cell concentration accounts about floor space 2/3,100 μ L supernatants ELISA is taken to sieve Choosing.Positive colony carries out changing liquid, adds 200 μ L complete mediums (liquid containing 1%HT)；Two sieves：Step above-mentioned steps are repeated after 2 days Carry out postsearch screening.Positive strain is transferred to 24 orifice plates of the culture medium that is prepared in advance (containing feeder cells and 1%HT liquid).Three sieves：5 It after it, screens again, satisfactory clone is transferred to Tissue Culture Flask and expands culture.Through multiple colony screening, training is finally obtained The positive hybridoma cell strain for the anti-OmpC that supernatant antibody titer is 1: 32 ten thousand is supported, is named as HR002 13-5

5. hybridoma cell strain preserves

Satisfactory hybridoma cell strain is preserved in China typical culture collection center, address by inventor：Wuhan City Wuchang Luo Jia Shan Wuhan University, preserving number are CCTCC NO：C2016161.

Embodiment 3：It is CCTCC NO by preserving number：The hybridoma of C2016161 prepares anti-OmpC monoclonal antibodies

1. prepared by ascites

0.5ml atoleines are injected intraperitoneally in the male BALB/c mouse of 10-12 week old, and every mouse is noted with 1ml after a week The monoclonal cell suspension that emitter intraperitoneal injection is resuspended through PBS washings, cell dosage are 5 × 10⁶/ only.Treat that mouse ascites gather After collect ascites.

It is 2. monoclonal antibody-purified

By ascites under the conditions of 4 DEG C, 10000 revs/min centrifuge 10 minutes, remove lipid material.Supernatant is drawn after centrifugation, And with 0.45 μm of membrane filtration.Protein G are purified.Monoclonal antibody concentration mensuration after purification is dispensed, is frozen at -20 DEG C.

3.ELISA methods identify monoclonal antibody subclass

Coating sheep anti-mouse igg (Zhong Shan Golden Bridge) is diluted with 100mM PBS (pH7.4) and is diluted to 0.5 μ g/mL, adds 100 per hole μ L, are stayed overnight by 4 DEG C.Liquid is emptied, is washed 3 times with the PBS (PBST) containing 0.05%Tween, the 200 μ L confining liquids of addition per hole, 37 DEG C It is incubated 1 hour.Liquid is emptied, is cleaned 3 times with PBST.0.1mL hybridoma supematants are added in per hole, 37 DEG C are incubated 1 hour.It is emptied liquid Body is cleaned 3 times with PBST.Sheep anti mouse (κ, the λ) antibody or 1: 2000 dilution HRP labels of HRP labels are diluted with confining liquid 1: 1000 Sheep anti mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody 0.1mL per hole, be separately added into appropriate hole, 37 DEG C With incubation 1 hour.Liquid is emptied, is cleaned 3 times with PBS-T.Add 50 μ L substrate solutions per hole, 450nm wavelength is surveyed in 10-20 minutes Under OD values.

Experimental result shows that monoclonal antibody of the present invention is IgG1 type mouse resource monoclonal antibodies, is named as HR002 13-5.

Embodiment 4：It is prepared by OmpC engineered antibodies

Total serum IgE is extracted from the hybridoma cell strain (HR002 13-5) of anti-OmpC monoclonal antibodies with Trizol reagents, The DNA encoding area gene of mAb VH and VL are expanded by RT-PCR with a pair of of specific primer.According to sequence analysis as a result, setting Count the corresponding signal peptide sequence of primer amplification VH and VL gene.Using gene recombination technology, by VH, VL gene of HR002 13-5 And its corresponding signal peptide sequence and CH genes, the C κ chain genes of people IgA or IgG are spliced, and build Chimeric antibody gene Expression plasmid pIRES/HR002 13-5.It is conducted into 293T cell strains with liposome method and carries out transient expression, utilize stream Formula cell art identifies expression product.

NCBI gene databases Blast's the results show that gene order of clone meets mouse VH, VL gene and its signal The feature of peptide sequence.The structure of expression plasmid pIRES/HR002 13-5 is correct, and obtains transient expression in 293T cell strains. The Chimeric antibody and mAbHR002 13-5 of the anti-OmpC of expression has identical antigen binding site.

The anti-OmpC mAb V areas encoding gene of mouse is cloned, and the Chimeric antibody for realizing soluble anti-OmpC exists Transient expression in 293T cells.Further with the recombinant vector transfected Chinese hamster gonad cell (CHO), table can be stablized by obtaining Up to the Chimeric antibody IgA or IgG of anti-OmpC.

Embodiment 5：ELISA detects OmpC IgA antibodies

In the present embodiment, positive quality control, reference material and the moon are prepared respectively with the OmpC chimeric antibodies obtained in embodiment 4 Property control, homemade OmpC antigens are as envelope antigen.

The preparation and operation of OmpC external diagnosis reagent cases are as follows：

1. the preparation of various buffer solutions and reagent：

A is coated with buffer solution：The CB (carbonate buffer solution) of 0.050M, pH9.6；

B confining liquids：0.1M PBS+0.5%BSA+0.5%Tween-20+0.04%Proclin300+2% sucrose；

C sample/washing buffer：10 × PBS-Tween-20 of pH7.2；

D calibration object dilution pH7.6 0.1M PBS+20%FBS+0.04%Proclin300

E enzyme marker dilutions：PH7.4 0.1M PBS-Tween20+20%FCS+0.04%Proclin300

F TMB one-component developing solutions：It buys in Huzhou Ying Chuan biotech firms

2. it is coated with the preparation of plate：

OmpC antigens are dissolved in the carbonate buffer solution of the 0.05M of pH=9.6, pre-coated liquid are made, on ELISA Plate Per hole by 0.5 μ g/ holes add in 100 μ l, put 4 DEG C place 20 hours, take out, get rid of coating buffer, wash, through BSA close 16 hours, It is fitted into after being dried overnight in aluminide-coating bag and vacuumizes sealing, be placed in 4 DEG C of preservations.

The goat-anti people IgA of 3.HRP labels：

The goat-anti people IgA purchases of HRP labels are in ABCAM, and working solution is by 1: 2 ten thousand times of dilution.

4. the preparation of positive quality control product, critical reference product and negative quality-control product.

ELISA method detects 20 clinical negative samples simultaneously, calculates the OD average values of clinical negative sample, according to P/N >= 2.1, it may be determined that the critical OD values of yin and yang attribute.

The OmpC chimeric antibodies of detection negative sample and different dilutions in same ELISA Plate, according to the detection knot of table 1. Fruit, negative sample mean OD value are 0.432, and the OD values for calculating critical value reference material accordingly are 0.9, dilution 1: 8.5 ten thousand Times OmpC chimeric antibodies can be as the prepared and diluted degree of critical reference product, the concentration setting 100U/L of this critical value reference material.

The OD values of positive quality control product should be greater than 1.5 times of critical reference product OD values, and the OD values of negative quality-control product should approach the moon Property sample mean OD value, prepare positive quality control product, critical reference product and negative quality-control product respectively with calibration object dilution, experiment It the results are shown in Table 1.

Table 1.

	Negative sample mean value	Negative Quality Control	Critical reference product	Positive quality control
					Dilution	1∶200	1: 35 ten thousand	1: 8.5 ten thousand	1: 2 ten thousand
OD	0.428	0.406	0.91	2.204

5.ELISA operating procedures

Sample needs sample 100ul of 200 times of dilutions per hole after the negative Quality Control of addition, positive quality control and calibration object and dilution, It is diplopore, 37 DEG C are incubated 60 minutes, are washed 5 times, patted dry with washing buffer.The goat-anti people of HRP labels is added in each hole IgA, 100 μ l/ holes, 37 DEG C are incubated 30 minutes, are washed 5 times, patted dry with 1 × washing buffer.Enzyme-linked object is added in each hole again 100 μ l/ holes, 37 DEG C are incubated 30 minutes, are washed 5 times, patted dry with 1 × washing buffer.Tmb substrate liquid is added in, per each 100 μ in hole L, mixing, 37 DEG C are incubated 15 minutes.50 μ l/ holes of terminate liquid is added to terminate reaction, (450nm) detection absorbance value is detected with microplate reader (OD), detection OD values are shown in Table 2.

6. testing result

As a result it explains：

Critical value is 100U/L

When detecting numerical value ＜ 100U/L, it is judged as feminine gender；

When detecting numerical value 100U/L-120U/L, it is judged as weakly positive；

When detecting numerical value ＞ 120U/L, it is judged as strong positive.

As seen from the results in Table 2：

Patient's positive sample coincidence rate=(12/12) * 100%=100%,

Patient's negative sample coincidence rate=(15/15) * 100%=100%.

Table 2.

Detect test sample	OD	Detect test sample	OD	Detect test sample	OD
						Positive reference product	2.160	Negative sample 8	0.384	Positive sample 3	1.623
Negative reference product	0.418	Negative sample 9	0.391	Positive sample 4	2.070
						Critical value reference material	0.902	Negative sample 10	0.467	Positive sample 5	2.652
Negative sample 1	0.521	Negative sample 11	0.581	Positive sample 6	1.085
						Negative sample 2	0.406	Negative sample 12	0.406	Positive sample 7	2.422
Negative sample 3	0.530	Negative sample 13	0.536	Positive sample 8	2.114
						Negative sample 4	0.319	Negative sample 14	0.319	Positive sample 9	1.686
Negative sample 5	0.352	Negative sample 15	0.352	Positive sample 10	1.935
						Negative sample 6	0.448	Positive sample 1	1.860	Positive sample 11	1.243
Negative sample 7	0.362	Positive sample 2	2.362	Positive sample 12	2.226

Embodiment 6：Western blot detects OmpC IgG antibodies

1. prepare OmpC IgG immunoblotting test strips

Prepare OmpC IgG immunoblotting test strips, alkaline phosphatase (AP) label goat anti-human igg antibody, test strip packet By OmpC antigens, Quality Control band is coated with anti-AP antibody, is debugged according to the critical value reference material prepared by embodiment 6.

1. various buffers：

A. film buffer solution is drawn：10mM PBS PH7.4+1% trehaloses

B. confining liquid：0.1m PBS+1%BSA+0.1%Tween-20+0.04%Proclin300+5% sucrose

C. Generic buffer：0.01M PH7.2 PBS+0.1%BSA+0.1%Tween20

D. enzyme marker dilution：10 × PBS-Tween20+2%FCS+0.1% enzyme stabilizers of pH7.2,0.04% Proclin300

E.BCIP/NBT substrates：It buys in Huzhou Ying Chuan biotech firms

2. the preparation of film item：NC films are labelled to the formulation position of backing bottom plate, with drawing film buffer solution by OmpC antigen diluents To 1mg/ml, it is used to prepare detection line；It draws film buffer solution and anti-AP antibody is diluted to 0.5mg/ml, be used to prepare nature controlling line；By 1 μ l/cm draw liquid measure, mark the antigen after HM3035 metal spraying stroke film instrument dilutes above two by gold and antibody is uniformly drawn to NC Corresponding position on film；The NC films pulled are positioned in 37 DEG C of drying boxes, are dried overnight；After drying, by reagent strip entirety It is put into confining liquid and impregnates 30S, after complete moistening, take out into 37 DEG C of drying boxes, be dried overnight, it, will be big after dry Block plate cuts into the small reagent strips of 5mm wide with cutting machine, and dry environment is packed, for use.

3.AP marks goat anti-human igg：Purchased from ABCAM, working solution 1: 2.5 ten thousand dilutes.

4. prepared by yin and yang attribute quality-control product：With embodiment 6.

5. detect operating procedure：Film item to be measured is put into and is incubated in slot, adds in 1.0ml Generic buffers, shaking table is slow Speed is incubated 5min；Liquid is sucked, the undiluted samples of 1.0ml are added in slot incubating, 37 DEG C of shaking tables are incubated at a slow speed 1 hour；Instead Should after suck extra liquid, clean 5min with 1ml Generic buffers, repeated washing three times, each 5min；Suck liquid Body adds in 1.0ml enzyme conjugates incubating in slot, shaking table is incubated at a slow speed 1 hour；Surplus liquid is sucked, is added in incubating in slot 1.0mlBCIP/NBT substrates, shaking table are incubated at a slow speed 10min；Surplus liquid is sucked, film item is rinsed well with distilled water, then will Film item is fixed in result judgement template, judging result after air-drying.

6. testing result：Various concentration quality-control product with film item is tested, the results are shown in Table 3：

Table 3

	Negative sample mean value	Negative Quality Control	Critical reference product	Positive quality control	Strong positive Quality Control
						Dilution	/	1: 45 ten thousand	1: 9.2 ten thousand	1: 3 ten thousand	1: 1 ten thousand
Color signal	-	-	+	++	+++

Same concentration positive quality control is taken, continuous to survey 10, colour developing is ++, colour developing is uniform.

Embodiment 7：Latex immunochromatography detects OmpC IgA antibodies

1. prepare OmpC antibody I gA colour latex immuno-chromatographic test paper strips

Prepare OmpC antibody I gA colour latex immuno-chromatographic test paper strips, red latex microballoon mark respectively goat-anti people IgA and Goat-anti chicken IgY, detection band T line coating OmpC antigens, quality control band C line packet chicken IgY, the critical value ginseng according to prepared by embodiment 5 Product are examined to debug.

1) label of latex microsphere

Latex microsphere 1000ul (210nm) (1% stoste), 13000rpm are taken, 4 DEG C of centrifugation 10min abandon supernatant, add in 1000ul deionized water ultrasound 10S mixings；Supernatant is abandoned in centrifugation, adds in MES buffer (50mM, PH6.0) 1000ul, ultrasound 10S mixings；Supernatant is abandoned in centrifugation, adds in MES buffer1000ul, and ultrasonic mixing weighs 50mgEDC, is slowly added to microballoon and mixes It closes in liquid, mixing, part reunion is had in reaction, in time ultrasound mixing, reacted at room temperature 15 minutes；Centrifugation, abandons supernatant, adds boric acid Salt buffer (20mM, PH8.0) 1000ul, ultrasonic 10 seconds mixings；Centrifugation, abandons supernatant, adds borate buffer solution 1000ul, ultrasound 10 seconds mixings, add in labelled antibody 120ug (label concentration 120ug/ml), and room temperature shaker middling speed is reacted 2 hours；Centrifugation, is abandoned Clearly, 1000ul confining liquids, ice water ultrasound mixing are added in, room temperature shaker middling speed is reacted 1 hour；Supernatant is abandoned in centrifugation, adds in 1000ul Borate buffer solution (20mM PH8.0), 10-30 seconds mixings of ice water ultrasound carry out label, and 4 DEG C preserve for use.

2) processing of sample bonding pad

Buffer solution (formula of the glass fibre element film containing surfactant：100mM pH 7.4PB, wherein containing 2% NaCl, 2%BSA, 0.5% casein, 0.1% Tween-20,0.5%S9 and 5% sucrose) impregnate and close in advance after, 50 DEG C are dry It is dry overnight；The nozzle of HM3035 metal spraying stroke film instrument is marked by gold, the goat-anti people IgA of latex microsphere and goat-anti chicken IgY will be marked with Antibody is according to the amount ullrasonic spraying of 8 μ l/cm to width on the glass fibre element film of 1cm, 37 DEG C (humidity is less than 30%) are dry, do At least 3 hours dry time, sample bonding pad is prepared.

3) nitrocellulose filter (NC films) is handled

NC films are labelled to the formulation position of backing bottom plate, with 7.4 phosphate buffers of pH of 50mM by OmpC antigen diluents To 1mg/ml, it is used to prepare T lines；Chicken IgY antibody is diluted to 0.5mg/ml by 7.4 phosphate buffers of pH of 50mM, for making Standby C lines；Liquid measure is drawn by 1 μ l/cm, the antigen after HM3035 metal spraying stroke film instrument dilutes above two is marked by gold and antibody is uniform Draw to preparing T lines and C lines on NC films；The NC films pulled are positioned in 50 DEG C of drying boxes, are dried overnight.

4) it assembles

The sample bonding pad fixation that step 2) obtains is laminated on to the one end for the nitrocellulose filter that step 1) obtains, and will Absorbing membrane fixes the other end for being laminated on nitrocellulose filter, is cut with film instrument is cut out by the width of every 4mm, and be packed into layer It analyses in a housing to get finished product.

2. the preparation of positive quality control product, critical reference product and negative quality-control product

According to the method for embodiment 5, with CBirl chimeric antibodies prepare various concentration calibration object 500U/L, 300U/L, 150U/L、100U/L、50U/L。

3. detection

Linearity test：Prepared various concentration quality-control product is detected with colloidal gold strip, and knot is read after 15min Fruit, correspondence the results are shown in Table lattice 4, and from low to high, colored intensity gradually enhances concentration, and W-response is linearly good, and concentration is less than 100U/L displays are negative, and concentration is positive higher than 100U/L displays.

Table 4

Concentration	50U/L	100U/L	150U/L	200U/L	250U/L
						Colored intensity	-	-/+	+	++	+++

Yin and yang attribute coincidence rate：Prepare 50 various concentration positive quality control products (concentration ＞ 100U/L), 50 various concentration the moon Property quality-control product (concentration ＜ U/L), is tested, two kinds of colloid gold test papers of yin and yang attribute quality-control product with certain brand colloidal gold strip Item test coincidence rate is consistent, and yin and yang attribute coincidence rate is 100%, and 5. are shown in Table to testing result

Table 5

	My company's latex test strips	Certain brand kit
			50 negative quality-control product results	50 results are feminine gender	50 results are feminine gender
50 positive quality control product results	50 results are the positive	50 results are the positive

Embodiment 8：Fluorescence immune chromatography detects OmpC IgA antibodies

3. prepare OmpC antibody I gA fluorescence immune chromatography test paper bars

OmpC antibody I gA fluorescence immune chromatography test paper bars are prepared, fluorescent microsphere marks goat-anti people IgA and goat-anti chicken respectively IgY, detection band T lines coating OmpC antigens, quality control band C line packet chicken IgY, critical value reference material according to prepared by embodiment 5 come Debugging.

1) label of fluorescent microsphere

Fluorescent microsphere 1000ul (210nm) (1% stoste), 13000rpm are taken, 4 DEG C of centrifugation 10min abandon supernatant, add in 1000ul deionized water ultrasound 10S mixings；Supernatant is abandoned in centrifugation, adds in MES buffer (50mM, PH6.0) 1000ul, ultrasound 10S mixings；Supernatant is abandoned in centrifugation, adds in MES buffer1000ul, and ultrasonic mixing weighs 50mgEDC, is slowly added to microballoon and mixes It closes in liquid, mixing, part reunion is had in reaction, in time ultrasound mixing, reacted at room temperature 15 minutes；Centrifugation, abandons supernatant, adds boric acid Salt buffer (20mM, PH8.0) 1000ul, ultrasonic 10 seconds mixings；Centrifugation, abandons supernatant, adds borate buffer solution 1000ul, ultrasound 10 seconds mixings, add in labelled antibody 120ug (label concentration 120ug/ml), and room temperature shaker middling speed is reacted 2 hours；Centrifugation, is abandoned Clearly, 1000ul confining liquids, ice water ultrasound mixing are added in, room temperature shaker middling speed is reacted 1 hour；Supernatant is abandoned in centrifugation, adds in 1000ul Borate buffer solution (20mM PH8.0), 10-30 seconds mixings of ice water ultrasound carry out label, and 4 DEG C preserve for use.

2) processing of sample bonding pad

Buffer solution (formula of the glass fibre element film containing surfactant：100mM pH 7.4PB, wherein containing 2% NaCl, 2%BSA, 0.5% casein, 0.1% Tween-20,0.5%S9 and 5% sucrose) impregnate and close in advance after, 50 DEG C are dry It is dry overnight；The nozzle of HM3035 metal spraying stroke film instrument is marked by gold, the goat-anti people IgA of latex microsphere and goat-anti chicken IgY will be marked with Antibody is according to the amount ullrasonic spraying of 8 μ l/cm to width on the glass fibre element film of 1cm, 37 DEG C (humidity is less than 30%) are dry, do At least 3 hours dry time, sample bonding pad is prepared.

3) nitrocellulose filter (NC films) is handled

NC films are labelled to the formulation position of backing bottom plate, with 7.4 phosphate buffers of pH of 50mM by OmpC antigen diluents To 1mg/ml, it is used to prepare T lines；Chicken IgY antibody is diluted to 0.5mg/ml by 7.4 phosphate buffers of pH of 50mM, for making Standby C lines；Liquid measure is drawn by 1 μ l/cm, the antigen after HM3035 metal spraying stroke film instrument dilutes above two is marked by gold and antibody is uniform Draw to preparing T lines and C lines on NC films；The NC films pulled are positioned in 37 DEG C of drying boxes, are dried overnight.

4) it assembles

The sample bonding pad fixation that step 2) obtains is laminated on to the one end for the nitrocellulose filter that step 1) obtains, and will Absorbing membrane fixes the other end for being laminated on nitrocellulose filter, is cut with film instrument is cut out by the width of every 4mm, and be packed into layer It analyses in a housing to get finished product.

2. detection

2.1 standard curve making

2.1.1 positive calibration object in Example 6, a concentration of 400U/ml, and it is diluted with 6 alignment product of embodiment Gradient dilution, concentration value are shown in Table 6 to liquid successively.

Table 6

Number	C0	C1	C2	C3	C4	C5	C6
								Concentration (U/ml)	0	10	25	50	100	200	400

2.1.2 detection method

100 μ l of calibration object are taken, add in sample window in chromatography strip；After 10 minutes, determined with resolved fluorometric quantitative analysis instrument Amount detection luminous value.

Each calibration object detects 2 times, takes the average value of T/C values.Concrete outcome is shown in Table 7：

Table 7

2.1.3 prepared by standard curve

According to above-mentioned testing result, using the logarithm of T/C values as X-axis, linearly returned using the logarithm of concentration as Y-axis Return, obtain linear equation y=0.4262x+2.184, R²=0.9961, calibration curve is shown in Fig. 2.

2.2 precision are tested：

2.2.1 10 chromatography strips prepared are taken, the Ompc working calibration product of a concentration (100U/mL) are configured；

2.2.2 100 μ l calibration objects are taken, are added in reagent strip well；

2.2.3 after product chromatography 10min to be calibrated, analysis is scanned to result, and will be upper with quantitative fluorescence analysis instrument State equation input instrument, instrument according to equation measure 10 reagent strips as a result, result such as following table, when illustrating the Ompc of the present invention Between resolved fluorometric immunochromatographiassay assay quantitative detection test paper precision it is good.

Claims (6)

1. the anti-OmpC hybridoma cell strains HR002 13-5 that a kind of OmpC protein immunizations mouse of recombinant expression obtains, are preserved in China typical culture collection center, deposit number are CCTCC NO：C2016161.

2. a kind of monoclonal antibody of anti-I2 is secreted by hybridoma cell strain described in claim 1 and generated.

3. application of the monoclonal antibody in I2 immune detection tools are prepared described in claim 2.

4. the application described in claim 3, which is characterized in that the immune detection tool is reagent, kit or test strips.

5. the immune detection tool described in claim 3 or 4, which is characterized in that resisted with the monoclonal described in Claims 2 or 3 F (ab) sequences or hypervariable region sequence of body, the recombinant protein or humanized antibody formed by genetic engineering.

6. application of any immune detection tool of claim 3 to 5 in autoimmune enteropathy.