CN107012127B - Monoclonal antibody, its purposes and the hybridoma for secreting the monoclonal antibody of anti-hepatitis B virus core antigen - Google Patents

Monoclonal antibody, its purposes and the hybridoma for secreting the monoclonal antibody of anti-hepatitis B virus core antigen Download PDF

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CN107012127B
CN107012127B CN201710213149.1A CN201710213149A CN107012127B CN 107012127 B CN107012127 B CN 107012127B CN 201710213149 A CN201710213149 A CN 201710213149A CN 107012127 B CN107012127 B CN 107012127B
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monoclonal antibody
hepatitis
antibody
virus core
sample
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CN107012127A (en
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黄家菊
王磊
何涛
舒川
龙腾镶
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Sichuan ankerei New Material Technology Co.,Ltd.
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Sichuan Maccura Biological New Material Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G01N33/5762—Hepatitis B core antigen
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

The present invention relates to the hybridomas of the monoclonal antibody of anti-hepatitis B virus core antigen, its purposes and the secretion monoclonal antibody.The invention further relates to the purposes that the monoclonal antibody is used to prepare the reagent of hepatitis B virus core antibody in detection sample, include the test strip of the monoclonal antibody.The monoclonal antibody specificity of the present invention is strong, high sensitivity, stability are good, available for the hepatitis B virus core antibody in quickly and accurately sample survey.

Description

Described in the monoclonal antibody of anti-hepatitis B virus core antigen, its purposes and secretion The hybridoma of monoclonal antibody
Technical field
The present invention relates to the monoclonal antibodies of anti-hepatitis B virus core antigen, secrete the hybridization of the monoclonal antibody The purposes of oncocyte and the monoclonal antibody in the detection of hepatitis B virus core antibody.
Background technology
Hepatitis B virus (Hepatitis virus B, HBV) belongs to DNA virus, is a kind of Hepadna Virus, is tunicary Partially double stranded cyclic DNA virus.The spread path of Type B virus hepatitis is mainly by transfusing blood, injecting and mother-to-baby transmission.Type B liver Scorching virus (HBV) infection is global problem, and chronic infection up to 400,000,000 in global range, developing country is viral hepatitis type b Severely afflicated area.Viral hepatitis type b would generally cause chronic infection, can progress to hepatic sclerosis or even hepatocellular carcinoma, have to human health huge Big threat.
Virus covalently closed circular DNA (covalenfly closed circular DNA, that is, cccDNA) is B Hepatitis virus (HBV) replicate in important link, be that the synthesis template and HBV of mRNA and pregenome RNA are difficult to control More recurrence has important relationship after the chronicity and antiviral therapy of key factor, long-term existence and viral hepatitis type b.Therefore it does The detection work of good cccDNA is for understanding conditions of patients variation, assessment therapeutic effect is of great significance to.But liver group Knitting the detection of interior cccDNA needs to do liver impedance rheograph, and patient is often difficult to receive, so finding a kind of easy to operate, sensitivity High serologic marker is necessary.
With going deep into hepatitis B virus blood serum designated object research, it was recognized that viral hepatitis type b disease in detection serum Malicious core antibody (Hepatitis B core Antibody, that is, HBcAb) possessed weight in the monitoring and treatment of HBV infection Want clinical meaning.Hepatitis B core antibody (HBcAb) is the correspondence antibody of hepatitis B virus core antigen, it is not protection antibody, Its presence is one of index encroached on by hepatitis B instead.The presence of HBcAb can be used as hepatitis B infectiousness activity venereal disease The mark of change, while contribute to the judgement of the hepatitis B state of an illness and prognosis.
But existing HBcAb detection methods are mainly chemiluminescence particulate immunodetection (CMIA), enzyme-linked exempt from Epidemic disease Competitive assays one-step method (ELISA) and colloidal gold method.No matter using which kind of detection method, the quality of testing result is with measuring Used in the sensitivity of monoclonal antibody, specificity and stability it is closely related.Therefore, in order to obtain better HBcAb Detection method finds sensitivity, the mesh that specificity is pursued always with all good HBcAg monoclonal antibodies of stability as people Mark.In addition, since detection reagent, the preservation condition of test strips sometimes can not be fully up to expectations, so it is often desirable to detecting Reagent can also keep the stability and confidence level of height with test strips after changing by rugged environment.
Invention content
The present invention is prepared for the good anti-hepatitis B virus core antigen monoclonal of high specificity, high sensitivity, stability and resists The hybridoma of body and the secretion antibody.
The present invention is using classical authentic monoclonal antibody technology.With Sichuan mikey biology new material technology Co., Ltd 2 Balb/C mouse are immunized in self-produced rHBcAg lot number (150827), preferably small with antibody titer is chosen after serum screening Mouse is used for cell fusion, filters out the hybridoma cell strain (12E4) of 1 plant of energy stably excreting antibody after fusion through limiting dilution assay, The cell strain is with preserving number CCTCC NO：C201747 is preserved in China typical culture collection center.Inventor then prepares Antibody after purification detects its potency through indirect ELISA, uses Shanghai Kehua Bio-engineering Co., Ltd's viral hepatitis type b disease Malicious core antibody detection kit detection activity, and detection kit detection clinical sample is prepared into, by multigelation and heat Continue to be prepared into detection kit detection clinical sample after acceleration processing, it was confirmed that we prepared secretes anti-viral hepatitis type b disease The knot of specificity can occur with destination protein for the monoclonal antibody secreted by the hybridoma of malicious core antigen monoclonal antibody Close, the high-titer, high sensitivity, high specific, high stability monoclonal antibody can be at colloidal gold platform (A competitive inhibition method) Quickly and accurately detect HBcAb in serum.
Description of the drawings
Fig. 1 illustrates the monoclonal antibody A of the present invention and two kinds of anti-hepatitis B virus core antigen monoclonals of commercialization resist The result of the antibody titer detection of body B and C.Using LOG (dilution) as abscissa, antibody OD values are as ordinate.
Fig. 2 illustrates the colloidal gold test of the anti-hepatitis B virus core antigen monoclonal antibody using the present invention Schematic diagram.1-PVC bottom plates, 2- sample pads, 3- colloidal gold pads, 4- detection zones (T lines), 5- quality control regions (C lines), 6- water absorption pads, 7- coated films.
Embodiment
Include following embodiment herein, for becoming apparent from, being explicitly described the technical side of the present invention in an exemplary manner Case.Those skilled in the art should be appreciated that according to disclosure herein to be permitted in disclosed specific embodiment It is change but still obtain similar or similar as a result, without departing from thought and range of the invention more.The specific reality of the present invention The mode of applying is only used for explaining the present invention, and is not intended to limit the present invention by any mode.
Embodiment 1
The preparation and screening of hybridoma cell strain
(1) mouse is immunized in rHBcAg (Sichuan mikey biology new material technology Co., Ltd, lot number 150827)
By the HBcAg recombinant proteins normal saline dilution to 2.0mg/ml of Bacillus coli expression, with Freund's complete adjuvant (Sigma companies, article No. SLBF-9338V) is mixed in equal volume, is oil emulsion with the emulsification of 1ml syringes, until instilling in water Oil emulsion does not disperse to stop emulsifying.The lotion is subcutaneously applied to BALB/c mouse with the dosage four limbs armpits of 150 μ l/ only (Chengdu reaches large Experimental Animal Center, and 6 week old are female, 2).Enhance after 14 days immune for the first time and be immunized, take antigen and Freund not Freund's complete adjuvant (Sigma companies, article No. SLBM9367V) is emulsified after mixing in equal volume, immunizing dose for 75 μ l/ only, later every Enhancing in one week is immune primary.It is immune every time to adopt tail blood before, serum is detached, potency is measured with indirect elisa method.After 8 times immune, All mice serum potency are more than 1：106, you can for merging.3 days before fusion, take HBcAg recombinant protein physiological saline dilute 2.0mg/ml is released, then mixes tail vein supplementary immunization in equal volume with physiological saline, dosage is 50 μ l/.
(2) the preparation of hybridoma cell line
1. the preparation of feeder cells
Feeder cells are made with normal 10 week old BALB/c mouse peritoneal macrophages.1 day before fusion, BALB/c takes eye Blood draws neck to put to death, and 0.1% bromogeramine impregnates 1 minute, is transferred to 75% alcohol and impregnates 1 minute, is used under sterile working in super-clean bench Scissors abdominal cut skin, exposure peritonaeum, with syringe Intraperitoneal injection RPMI1640 basic culture solution 3ml, after taken out with dropper, 7mlRPMI1640 basic culture solutions are added to rinse repeatedly.Flushing liquor is recycled, 1000rpm is centrifuged and stayed precipitation in 5 minutes, with having added The RPMI1640 culture solutions for entering 20% newborn bovine serum are resuspended, and adjustment cell concentration is 2-5 × 105A/ml adds in 96 orifice plates, 100 μ l/ holes, 37 DEG C, 5%CO2Culture.
2. the preparation of immune spleen cell
After mouse supplementary immunization three days, spleen is aseptically taken out, is placed in plate, the culture of RPMI1640 bases Liquid rinses primary, shred, grind, filter after the splenocyte that is disperseed, 1000rpm, which is centrifuged, stays precipitation centrifugation for 5 minutes, RPMI1640 basic culture solutions are resuspended, and the dilution of 3% acetic acid counts.
3. the preparation of myeloma cell
Murine myeloma cell Sp2/0 (being purchased from Cell Bank of Chinese Academy of Sciences) is cultivated after 8-anaguanine screens to right In number growth period, take 6 big bottle (75cm2) cell suspension is made, 1000rpm is centrifuged 5 minutes and is stayed precipitation, is trained with RPMI1640 bases Nutrient solution is resuspended, counts, by 0.5-2 × 105The cell concentration of a/ml carries out sub-bottle culture, and (ordinary circumstance replaced one per 1-2 days Secondary complete 1640 culture mediums of 15-30ml).
4. cell fusion and HAT selection culture hybridomas
Myeloma cell and immune spleen cell are pressed 1:5 ratio mixing, RPMI1640 is used in 50ml conical centrifuge tubes Basic culture solution is washed 1 time, and 1000rpm is centrifuged 5 minutes and stayed precipitation.By cell mixing, it is slowly added to the PEG4000 of 1.2ml50% Fusion, fusion adds in 30ml RPMI1640 basic culture solutions after 1 minute terminate cell fusion.700rpm/min is centrifuged 5 minutes It is resuspended in afterwards in 1640 culture mediums containing 1%HAT and 20% newborn bovine serum, averagely instills 20 set of 96 porocyte culture plates. 37 DEG C, 5%CO2Culture, next day is added to be expired containing 1%HAT and 1640 culture mediums to the hole of 20% newborn bovine serum.5 days later half Culture medium is changed, culture medium is partly changed again after 7 days.
5. the screening of positive cell strain
With 0.06M pH9.6 carbonate buffer solutions dilution rHBcAg (osmanthus health biology) to 2.5 μ g/ml, 96 hole enzyme marks 100 μ l are coated in plate per hole, for detecting cells and supernatant.It is positioned in refrigerator and stays overnight for 2-8 DEG C.Abandon liquid in hole within second day Body, ELISA washing lotions board-washing three times, pat dry, with the PBS of the 0.01M pH7.2 containing 10% calf serum, 150 μ l/ holes, 37 DEG C of envelopes It closes 2 hours, pats dry, Vacuum Package is for use.The 9th day after splenocyte fusion, 100 μ l of cell conditioned medium are taken in above-mentioned 96 hole detection plate In, 37 DEG C are incubated 40 minutes, and ELISA board-washings add in the goat-anti of 12000 times of diluted horseradish peroxidase labels after machine-washing five times 100 μ L of mouse IgG (Sichuan new material bio tech ltd), after 37 DEG C of incubations are ibid washed for 30 minutes, 100 μ L are added in per hole Containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 10 minutes, often Hole adds in 50 μ L 2M sulfuric acid solutions and terminates reaction, surveys 450nm absorption values.Mice serum is diluted to 100 times as the positive during merging Control, for 1640 complete culture solutions of RPMI as negative control, negative control OD value ＜ 0.2, positive control OD values ＞ 1.8 are inspection Examining system is effective, for the positive, otherwise is feminine gender during sample OD value >=2 × negative control OD value.Secretory antibody positive cell hole It is cloned on 96 well culture plates with limiting dilution assay with 1 cells/well, method continuously clone four times, make it on screening positive Kong Yi Reach 100% monoclonal, be transferred to 24 holes and continue to cultivate, after cell bottle expansion culture is transferred to when cell covers with 80%, treat cell Sub-bottle passes on when covering with cell bottle 80%, during cell growth to the logarithmic phase of passage, with appropriate serum-free RPMI-1640 culture mediums Cell dispersion bottle inner cell collects cell suspension in conical centrifuge tube, records cell suspension volume V, take appropriate cell suspension Cell count is carried out, cell suspension density (a/ml) is obtained, supernatant is abandoned after remaining 1000rpm centrifugations 5min, it is close according to cell Degree and centrifugation precursor calculate the cell number of precipitation, added in into cell precipitation appropriate frozen stock solution adjust cell density to 4-8 × 106A/ml (it takes and carries out counting confirmation in right amount, if cell number not in the range of, is centrifuged again according to cell count total amount, Appropriate frozen stock solution is rejoined, finally makes cell concentration in 4-8 × 106A/ml), it is then sub-packed in sterile cryopreservation tube, every Cryopreservation tube refinement cytosol 0.5ml.Cell fusion obtains 1 plant of energy anti-hepatitis B virus core antigen monoclonal of stably excreting and resists altogether The hybridoma HBcAb-12E4 of body was deposited in Sichuan mikey biology new material technology Co., Ltd on the 1st in August in 2015, And on March 30th, 2017 with preserving number CCTCC NO：C201747 is preserved in China typical culture collection center, and (China is military Chinese Wuhan University).
Embodiment 2
The preparation of monoclonal antibody
Select the BALB/c mouse of 6-8 weeks health, every mouse peritoneal injection 0.5mL atoleines (Tianjin Ke Miou), 7 days Every mouse peritoneal injection 0.5~1 × 10 afterwards6A hybridoma.Inoculating cell can generate ascites, close observation after 7-10 days The health status of animal and sign of ascites as, before treating that ascites is as more as possible, and mouse is dying, execution mouse, with dropper by ascites It sucks in test tube, a mouse can obtain 1-5mL ascites.The ascites centrifuging and taking supernatant of collection takes sample to be put in -20 DEG C of refrigerators and protects It deposits.After ascites is precipitated with sulfate of ammoniac saturation, then purified with Protein A affinity chromatography, SDS- PAGE detect list produced by the present invention Clonal antibody purity is more than 90%.
Embodiment 3
(1) Hybridoma Cell Culture supernatant bioactivity
With 0.06M pH9.6 carbonate buffer solutions dilution rHBcAg (osmanthus health biology) to 2.5 μ g/ml, 96 hole enzyme marks 100 μ l are coated in plate per hole.It being positioned in refrigerator and stays overnight for 2-8 DEG C, abandon within second day liquid in hole, ELISA board-washings are machine-washed three times, It pats dry, with the PBS of the 0.01M pH7.2 containing 10% calf serum, 150 μ l/ holes, 37 DEG C of closings abandon liquid in 2 hours, pat dry, and use In detection Hybridoma Cell Culture supernatant, ascites and antibody titer.The first hole of Hybridoma Cell Culture supernatant bioactivity is original Times supernatant culture solution, dilutes, the 5th hole to step by step again from the second hole to the 4th hole with the PBS buffer solution 10 of 0.01M pH7.2 Ten holes are diluted step by step with 2 times.Mice serum is diluted to 100 times and makees positive control when 11-holes are to merge, the 12nd hole with 1640 complete culture solutions of RPMI make negative control, are 100 μ l per hole sample volume.37 DEG C are incubated 40 minutes, ELISA board-washing machines It washes after five times and adds in sheep anti-mouse igg (the limited public affairs of Sichuan new material biotechnology of 12000 times of diluted horseradish peroxidase labels Department) 100 μ L, after 37 DEG C of incubations are ibid washed for 30 minutes, 100 μ L are added in per hole containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) Hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 10 minutes, and 50 μ L 2M sulfuric acid solutions are added in per hole and terminate reaction, Survey 450nm absorption values.Negative control OD value ＜ 0.2, positive control OD values ＞ 1.8 for detecting system it is effective, when OD value >=2 × It is on the contrary for feminine gender for the positive during negative control OD value.Dilution ratio corresponding to the minimum positive hole of detected value is that hybridoma is thin Born of the same parents' culture supernatant potency, this Hybridoma Cell Culture supernatant antibody titer are more than 1： 2000.
(2) titer of ascites detects
(1) ELISA detection method is same as above.Dilution process is different, specially：First hole is former times ascites, with 0.01M The PBS buffer solution of pH7.2 dilutes step by step again from the second hole to seven apertures in the human head 10, is diluted step by step with 2 times from octal to 11-holes. Dilution ratio corresponding to the minimum positive hole of detected value is titer of ascites, and the titer of ascites prepared by this hybridoma is more than 1：106。
(3) antibody titer detects
First by the present invention monoclonal antibody A 1mg/ml is uniformly diluted to the PBS buffer solution of 0.01M pH7.2, after again Initial first hole of 100 times of conducts is diluted, make 5 times to the tenth hole since the second hole dilutes step by step, small when 11-holes are to merge Mouse serum is diluted to 100 times and makees positive control, and negative control is made in the 12nd hole with PBS, is 100 μ l per hole sample volume.37℃ It is incubated 50 minutes, ELISA board-washings add in the sheep anti-mouse igg of 12000 times of diluted horseradish peroxidase labels after machine-washing five times (Sichuan new material bio tech ltd) 100 μ L, after 37 DEG C of incubation 1h are ibid washed, 100 μ L are added in per hole and contain 0.1% (M/ V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C are incubated 15 minutes, and 50 μ L are added in per hole 2M sulfuric acid solutions terminate reaction, detect 450nm absorption values.Negative control OD value ＜ 0.2, positive control OD values ＞ 1.8 are detection System is effective.
Antibody titer criterion：With LOG (dilution) for abscissa, make curve, curve by ordinate of antibody OD values Equation is y=min+ (max-min)/(1+10^ ((logEC50-x) × Hillslope)), passes through sigmaplot data processings Software matched curve takes middle titer=10logEC50.As a result show monoclonal antibody A of the present invention middle titer be more than 8 × 104.In kind two kinds of anti-hepatitis B virus core antigen monoclonal antibody B (Xiamen City Bo Shengsheng of commercialization of control test Object Technology Co., Ltd.) and C (one hundred Tai Ke Bioisystech Co., Ltd of Luoyang City), by fitting, middle titer is below 8 × 104, less than the monoclonal antibody A of this hybridoma acquisition.Table 1 and Fig. 1 lists titration as a result, it is evident that originally The antibody titer of invention is far above the potency of commercially available monoclonal antibody B and C.
Table 1：Antibody titer detects
Embodiment 4
Antibody activity measures：Using Shanghai Kehua Bio-engineering Co., Ltd's hepatitis B virus core antibody detection Kit detects the antibody secreted by hybridoma of the present invention.By the monoclonal antibody A of the present invention with being commercialized anti-viral hepatitis type b Virus core antigen monoclonal antibody B and C antibody is diluted with the PBS buffer solution 10 of 0.01M pH7.2 step by step again as sample, Detect 3 antibody competitions.As a result show that the monoclonal antibody A of the present invention still can detect that the positive after diluting 10000 times, OD values are 0.497 (Cut Off=feminine genders mean value × 0.3=0.698 is the positive less than 0.698), and B antibody is diluted to 1000 times After show that weakly positive, C antibody show the positive after being diluted to 100 times.These are statistics indicate that the monoclonal antibody and business of the present invention Reagent has stronger competition in the Shanghai Kehua Bio-engineering Co., Ltd B hepatovirus core antibody detection kits of change Property, activity is more than commercialized monoclonal antibody B and C.Testing result is listed in table 2.
Table 2：Antibody activity measures
Embodiment 5
Use the test strips and its stability of monoclonal antibody of the present invention
(1) test strips are prepared using monoclonal antibody of the present invention
The anti-hepatitis B virus core antigen monoclonal antibody energy of the present invention should be in colloidal gold platform, so as to fast qualitative Detect the hepatitis B virus core antibody in serum, blood plasma.
Sample pad of the test strip by PVC bottom plates and thereon, colloidal gold pad, coated film, water absorption pad form, the colloid Containing rHBcAg colloidal gold composite and rabbit igg colloidal gold composite in gold pad, along from sample pad to water suction on coated film The quality control region (C lines) being spaced from and detection zone (T lines) is sequentially distributed in the direction of pad on membrane body, and C, T spacing are fixed For 6mm (silk-screen has been fixed position on card, and so there is no need to optimize spacing).C lines are coated with anti-rabbit IgG antibody, and T lines are coated with anti-B Hepatitis virus core antigen monoclonal antibody, that is, following mouse Anti-HBc Serum Ag monoclonal antibodies.Fig. 2 is the schematic diagram of the test strips, 1-PVC bottom plates, 2- sample pads, 3- colloidal gold pads, 4- detection zones (T lines), 5- quality control regions (C lines), 6- water absorption pads, 7- coated films. Coating preferred embodiment is shown in Table 3 and table 4.
Table 3 is coated with concentration
Table 4 is coated with OD
When it is positive to detect sample, sample first is combined to form compound with colloid gold label rHBcAg compound, with Sidestream chromatography effect moves on coated film (nitrocellulose filter), and rHBcAg colloidal gold composite (is not detected with T lines Line) it combines, colour developing is assembled only at C lines (nature controlling line).When detecting sample and be negative then detection line and nature controlling line all assemble it is aobvious Color, colour developing result is in judgement in 15-20 minutes.
(2) above-mentioned ELISA test strip clinical sample is used
100 positive samples and 100 negative samples are examined using above-mentioned test strips.Lower Table A is the examination that this antibody is prepared Agent, B are commercialization reagent (Ying Kexin creates Science and Technology Ltd.), and inspection result shows what is prepared with the monoclonal antibody of the present invention Colloidal gold strip sensitivity and specificity are above commercialization reagent, list testing result in table 5.
Table 5 is positive to be examined with negative sample
(3) the stability of monoclonal antibody of the present invention in the presence of a harsh environment
The monoclonal antibody A of the present invention is handled under the following conditions：A-20 DEG C of multigelation 2 times；B-20 DEG C freezes repeatedly Melt 3 times；C-20 DEG C of multigelation 4 times；D-20 DEG C of multigelation 5 times；E-20 DEG C preserves 7 months；37 DEG C of f thermal accelerations 3 days；g 37 DEG C of thermal accelerations 7 days, other conditions are constant, are prepared into detection reagent as stated above and examine 15 parts of positive reference product and 15 respectively Part negative reference product, coincidence rate is 100%.Show that this Antibody stability is good, and utilize the detection reagent of this Antibody preparation Accuracy is good.It chooses potency and activity is preferably commercialized anti-hepatitis B virus core antigen monoclonal antibody B (Xiamen City Bo Sheng Bioisystech Co., Ltd), it is prepared into detection reagent after being handled with above-mentioned similarity condition and detects 15 parts of positive references respectively Product and 15 parts of negative reference product, as a result show its multigelation 3 to 4 times or -20 DEG C preserve 7 months or thermal acceleration and opened after 3 days Initial portion is degraded, inspection result and reference material not in full conformity with.Testing result is listed in table 6.
6 reference material of table detects
It can be seen that the monoclonal antibody stability of the present invention is high, the hepatitis B virus core antibody detection being made from it Reagent and test strips can also keep very high Stability and dependability under the conditions of more rugged environment, this point clinic with And in laboratory applications it is very valuable progress.

Claims (7)

1. a kind of hybridoma for secreting anti-hepatitis B virus core antigen monoclonal antibody, preserving number are CCTCCNO：C201747.
2. anti-hepatitis B virus core antigen monoclonal antibody, is secreted by hybridoma according to claim 1.
3. monoclonal antibody according to claim 2 is used to prepare the examination of hepatitis B virus core antibody in detection sample The purposes of agent.
4. for detecting the test strip of hepatitis B virus core antibody in sample, it includes according to claim 2 Monoclonal antibody.
5. test strip according to claim 4 is colloid metal type test strips.
6. test strip according to claim 5, the sample that sequentially fixed and end interconnects on bottom plate Pad, colloidal gold pad, coated film, water absorption pad contain rHBcAg colloidal gold composite and rabbit igg colloid in the colloidal gold pad Au composite is sequentially distributed on the coated film on membrane body along the direction from sample pad to water absorption pad and is spaced from Quality control region C and detection zone T.
7. test strip according to claim 6, wherein the quality control region C is coated with anti-rabbit IgG antibody, the detection Area T is coated with the monoclonal antibody described in claim 2.
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