CN102399752B - Heart type-fatty acid binding protein monoclonal antibody mcf1 and application thereof - Google Patents
Heart type-fatty acid binding protein monoclonal antibody mcf1 and application thereof Download PDFInfo
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- CN102399752B CN102399752B CN201110336190A CN201110336190A CN102399752B CN 102399752 B CN102399752 B CN 102399752B CN 201110336190 A CN201110336190 A CN 201110336190A CN 201110336190 A CN201110336190 A CN 201110336190A CN 102399752 B CN102399752 B CN 102399752B
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Abstract
The invention belongs to the technical field of biomedicine, and discloses a heart type-fatty binding protein monoclonal antibody mcf1 and application thereof. A hybridoma cell strain MCF1 for secreting heart type-fatty acid binding proteins is collected in the China Center for Type Culture Collection with the collection number of CCTCC NO:C201170 on September 6, 2011. The heart type-fatty acid binding protein monoclonal antibody mcf1 secreted by the hybridoma cell strain MCF1 has high monoclonal antibody titer and high specificity. The heart type-fatty acid binding protein monoclonal antibody mcf1 can be applied to the detection or the purification of human heart type-fatty binding proteins.
Description
Technical field
The invention belongs to biomedical sector, relate to heart fatty acid binding protein monoclonal antibody mcf1 and application thereof.
Background technology
Fatty acid binding protein (FABPs) is the small molecules intracellular protein of one group of polyphyly, and molecular weight is 12～16KDa, is distributed widely in the various kinds of cell such as mammiferous small intestine, liver, fat, the heart, brain, Skelettmuskel.Little visible peristalsis visible intestinal peristalsis (I-FABP), myocardium type (H-FABP), liver type (L-FABP), kidney type hypotypes such as (K-FABP) are arranged.The sequence of different shaped FABP has bigger homology.H-FABP (H-FABP) molecular weight is 14～15KDa; Iso-electric point (PI) is 5.1; Can lipid acid be transported to the position that esterification and oxidation take place from cytoplasmic membrane; Thereby get among the mitochondrial energy metabolism system, make lipid acid at this oxygenolysis and the final Triphosaden (ATP) that generates, for myocardial contraction provides energy.People H-FABP is made up of 132 amino-acid residues, and content is abundant in cardiac muscle, accounts for 4%～8% of the whole soluble proteinss of heart, contains H-FABP (0.52 ± 0.06) mg approximately in every gram weight in wet base cardiac muscle.Do not contain H-FABP or content is very few in normal people's blood plasma and the urine.But snap-out release is in blood and urine when the myocardial cell is impaired.Because various FABP has special separately antigenic determinant, can H-FABP be distinguished with its alloytype FABP mutually through immunological method.Along with the research and the Clinical Application of FABP measuring method, H-FABP has become the mark (Chinese experimental diagnostics, the 9th the 1st phase of volume of February in 2005) of early stage myocardial damage diagnosis.
Summary of the invention
The purpose of this invention is to provide the hybridoma cell strain of secreting heart fatty acid binding protein monoclonal antibody.
Another object of the present invention provides heart fatty acid binding protein monoclonal antibody.
Another purpose of the present invention provides the application of heart fatty acid binding protein monoclonal antibody.
The hybridoma cell strain MCF1 of one strain secretion H-FABP is preserved in Chinese typical culture collection center with on September 6th, 2011, and preserving number is CCTCC NO:C201170.
Described preserving number is the hybridoma cell strain MCF1 excretory heart fatty acid binding protein monoclonal antibody mcf1 of CCTCC NO:C201170.
Described preserving number is the application of hybridoma cell strain MCF1 excretory heart fatty acid binding protein monoclonal antibody mcf1 in detection or purifying human heart type fatty acid binding protein of CCTCC NO:C201170.
Described preserving number is the application of hybridoma cell strain MCF1 excretory heart fatty acid binding protein monoclonal antibody mcf1 in preparation human heart type fatty acid binding protein detection reagent of CCTCC NO:C201170.
Described human heart type fatty acid binding protein detection reagent preferred detection detects Western Blot reagent, ELISA detection reagent, the gold-immunochromatographyreagent reagent for assay of human heart type fatty acid binding protein.
A kind of colloidal gold strip that detects human heart type fatty acid binding protein; Described anti-human heart type fatty acid binding protein monoclonal antibody mcf1 does detection line antibody with claim 2; Sheep anti-mouse igg is as nature controlling line; The monoclonal antibody mcf2 of colloid gold label obtains according to ordinary method preparation assembling as colour developing antibody; Wherein said monoclonal antibody mcf2 is the anti-human heart type of the hybridoma cell strain excretory fatty acid binding protein monoclonal antibody of CCTCC NO.C201181 by preserving number.
A kind of double fastener heart ELISA test kit that detects human heart type fatty acid binding protein; The monoclonal antibody mcf2 that comprises horseradish peroxidase-labeled, the enzyme plate that anti-human heart type fatty acid binding protein monoclonal antibody mcf1 encapsulates, PBST, TMB colour developing liquid, 2mol/L H
2SO
4Wherein said monoclonal antibody mcf2 is the anti-human heart type of the hybridoma cell strain excretory fatty acid binding protein monoclonal antibody of CCTCC NO.C201181 by preserving number.
Beneficial effect of the present invention:
The present invention utilizes the people H-FABP (H-FABP) of purifying as immunogen; Through subcutaneous immunity, conventional cytogamy, screening and cloning, obtained the hybridoma cell strain MCF1 (CCTCC NO:C201170) that a strain can the anti-H-FABP monoclonal antibody of stably excreting.Identify that through methods such as ELISA, immunoblottings the secreted monoclonal antibody mcf1 of this hybridoma can specific recognition H-FABP (Hytest company), the height of tiring, high specificity.
The application of H-FABP monoclonal antibody mcf1 of the present invention is following:
(1) but use the H-FABP that this antibody Western Blot detects cardiac muscular tissue and express.
(2) use the level that this antibody can be set up the H-FABP of the various body fluid of gold-labeled kit rapid detection such as blood, blood plasma, serum, urine.
(3) use the level that this antibody can be set up the H-FABP of the various body fluid of ELISA detection kit detection by quantitative such as blood, blood plasma, serum, urine.
(4) use this Antibody Preparation immune affinity chromatographic column, come separation and purification H-FABP albumen.
(5) use this antibody and carry out H-FABP expressing quantity in the immunohistochemical methods detection cardiac muscular tissue.
The present invention has confirmed further that also described monoclonal antibody mcf1 is in the external expression that is used for detection bodies liquid or tissue H-FABP.For example be used to detect the level of the H-FABP of various body fluid such as blood, blood plasma, serum and urine; Can also measure the expression level of acute myocardial injury disease H-FABP such as various diseases such as acute myocardial infarction, as a mark of clinical diagnosis or auxiliary diagnosis.The people H-FABP monoclonal antibody mcf1 that the present invention makes is for fundamental research and the clinical study of H-FABP provides beneficial tools.
Description of drawings:
Fig. 1 cardiac muscle crude extract Sephadex G75 gel filtration chromatography elution curve.
Fig. 2 cardiac muscle crude extract Sephadex G75 each component (Tricine)-SDS-PAGE electrophoresis behind gel filtration chromatography,
A: No. 36 pipe starts from clear protein band occurring near the M15000,
B: the 41st pipe has clear protein band near all visible M15000 of 71 pipes,
C: near the protein band of M15000 obviously shoals after the 91st pipe.
Fig. 3 H-FABP QSepharose Fas t Flow anion exchange chromatography elution curve.
The H-FABP electrophoresis result of Fig. 4 purifying,
A: the H-FABP of preparation, B: behind Sephadex G75 gel filtration chromatography, collect liquid.
The H-FABP Western Blot of Fig. 5 preparation analyzes
A: behind Sephadex G75 gel filtration chromatography, collect liquid; B: the H-FABP of preparation.
Fig. 6 is the specific reaction of Western Blot detection mcf1 monoclonal antibody and H-FABP,
Wherein swimming lane 1 is the H-FABP 2 μ g albumen+mcf1 monoclonal antibodies of purifying,
Swimming lane 2 is H-FABP 2 μ g albumen+mcf1 monoclonal antibodies of Hytest,
Swimming lane 3 is brain tissue homogenate's liquid 8 μ g albumen+mcf1 monoclonal antibodies,
Swimming lane 4 is Skelettmuskel homogenate 8 μ g albumen+mcf1 monoclonal antibodies.
Fig. 7 is the specific reaction of Western Blot detection F2 monoclonal antibody and H-FABP,
Wherein swimming lane 1 is Skelettmuskel homogenate 8 μ g albumen+mcf 2 monoclonal antibodies,
Swimming lane 2 is brain tissue homogenate's liquid 8 μ g albumen+mcf 2 monoclonal antibodies,
Swimming lane 3 is H-FABP 2 μ g albumen+mcf 2 monoclonal antibodies of Hytest,
Swimming lane 4 is H-FABP 2 μ g albumen+mcf 2 monoclonal antibodies of purifying.
Fig. 8 detects Hytest-H-FABP albumen result for gold-labeled kit,
Wherein the 1st, 2 is the detection negative control,
Article 3,4, be detection 10ng/ml Hytest-H-FABP albumen,
Biomaterial preservation information
Hybridoma cell strain MCF1 is preserved in Chinese typical culture collection center on September 6th, 2011, be called for short CCTCC, and the preservation place is Chinese Wuhan Wuhan University, and preserving number is CCTCC NO:C201170.
Hybridoma cell strain MCF2 is preserved in Chinese typical culture collection center on September 6th, 2011, be called for short CCTCC, and the preservation place is Chinese Wuhan Wuhan University, and preserving number is CCTCC NO.C201181.
Embodiment
Embodiment 1.H-FABP purifying
H-FABP purifying: 80g people's left compartment muscle (coming from the Jiangsu Prov. People's Hospital organ donor) homogenate in 400mL buffer A (pH 7.0 for 15mmol/L phosphate buffered saline buffer, 10mmol/L beta-mercaptoethanol).4 ℃ of homogenates, the centrifugal 10min of 2600g precipitates resuspended with the 150mL buffer A.4 ℃, the centrifugal 10min of 2600g merges centrifugal back supernatant 2 times, and 4 ℃, the centrifugal 120min of 75600g, centrifugal back supernatant is with 400g/L (NH
4)
2SO
4Saltout, 4 ℃, 10000g, centrifugal 25min, supernatant are again with 800g/L (NH
4)
2SO
4Saltout, 4 ℃, 10000g, centrifugal 25min.Abandon supernatant, deposition is with the dissolving of 10mL buffer A, and 4 ℃ of dialysed overnight obtain the 10mL crude extract, protein quantification in buffer A.(chromatography among the 2.0cm * 100cm) is collected effluent to the 5mL crude extract, measures each pipe and collects liquid A 280 values, is lower than at 0.05 o'clock to A value and stops collection in using buffer A equilibrated SephadexG-75 post.To collect liquid and carry out protein electrophoresis, deposition condition is: the amino glycocoll (Tricine) of trishydroxymethyl-SDS-PAGE, and resolving gel concentration is 16%, and spacer gel concentration is 10%, and concentrated gum concentration is 4%, 30mA constant current electrophoresis 2h.Gel is with coomassie brilliant blue staining, and the Bio-Rad gel imaging system is analyzed.The result shows: the protein peak (see figure 1) occurs at the 31st～86 pipe; Its main protein band of electrophoresis showed is positioned at 10000～17000; The H-FABP relative molecular mass of present embodiment preparation is about 15000 (see figure 2)s; 31～86 pipes are merged, obtain collecting liquid 50mL (1.9g/L) altogether.
Q Sepharose Fas t Flow post (20mL) is with buffer B (5mmol/L imidazoles, 1mmol/L YD 30,2mmol/L beta-mercaptoethanol; PH 7.0) after the balance, with appearance on the above-mentioned collection liquid of 50mL, buffer B is washed post; With AKTA protein purification system monitoring stream fluid A280 value, behind the ready to balance, carry out gradient elution with buffer B and 20mmol/L NaCl; The wash-out total amount is 3 times of column volumes, flow velocity 0.5mL/min.Monitoring stream fluid A280 value merges protein peak pipe 1-101 (see figure 3).Amalgamation liquid with buffer B dialysis 12h, obtains H-FABP (1.8g/L) (see figure 4) of 6mL purifying with Mr 35000 polyoxyethylene glycol embedding row concentration altogether.
Western Blot detects: and reference literature (Wang Jiazheng, model is bright. protein TM [M]. and Beijing: Beijing Science Press, 2002,166-184) carry out.The H-FABP of purifying is through the Tricine-SDS-PAGE electrophoresis, with the 30mA constant current commentaries on classics film that spends the night, milk sealing.Add an anti-mouse-anti human heart type fatty acid binding protein antibody H86101M (BIODESIGN company) of being, with dilution in 1: 600, room temperature reaction 1h washed three times.Sheep anti-mouse igg (SIGMA company) the room temperature reaction 1h that adds horseradish peroxidase-labeled subsequently; Wash three times; After 1: 1 equal-volume of two kinds of substrates of ECL mixes it is covered the film surface and make it even, wrap film, in the darkroom, the X-ray sheet is covered above the film with preservative film; Develop photographic fixing.The result shows: there is specificity trace band (see figure 5) the position near Mr15000, and the prompting purified proteins is a H-FABP.
The foundation of embodiment 2. hybridoma cell lines
Select the Balb/c mouse (Shanghai Slac Experimental Animal Co., Ltd.) of 8-12 age in week and myeloma cell system of the same race; 120 μ l H-FABP (containing protein 100 μ g) and the abundant mixing of 120 μ l Fu Shi Freund's complete adjuvants (SIGMA company) with embodiment 1 purifying; In the injection mouse peritoneal; Whenever at a distance from 2 all 100 μ g/ H-FABP and abundant mixing of equivalent freund 's incomplete adjuvant (SIGMA company) only, inject booster immunization in the mouse peritoneal, totally 3～4 times.Adopt indirect elisa method to detect many anti-the tiring of H-FABP albumen in the immune serum, the high person that tires carries out next step cytogamy.
Step 2, cytogamy
Sterile preparation H-FABP mice immunized splenocyte suspension merges under PEG1450 (SIGMA company) effect with 6: 1 ratios with murine myeloma cell SP2/0 (deriving from Chinese Academy of Sciences's Shanghai cell bank), merges and presses conventional method (Nature.1975; 256:495-497); Use 1ml PEG, slowly add in 1 minute, the reaction times is 90 seconds; The DMEM of serum-free (Gibco BRL company) substratum termination reaction; The centrifugal 10min of 1000rpm, (H xanthoglobulin, A aminopterin, T thymidine, (Gibco BRL company) DMEM fully cultivates the keynote cell concn to 1x10 to HAT
6/ ml adds 96 well culture plates that are covered with feeder cell (peritoneal macrophage of Balb/c mouse) in advance, 100 μ l/ml, and in 37 ℃, 5%CO
2Cultivate in the incubator, whenever observed and changed liquid, change liquid with HT DMEM (Gibco BRL company) substratum after 10 days, change complete DMEM substratum after 1 week at a distance from 3 days.
Step 3, indirect enzyme-linked immunosorbent assay (ELISA) screening positive hybridoma cell
Encapsulate enzyme with purifying H-FABP (1 μ g/ml) and mark 96 orifice plates, 37 ℃ of reactions were spent the night in 2 hours or 4 ℃; 3% bovine serum albumin (BSA) sealing, 4 ℃ are spent the night, and add hybridoma to be checked (cell that above-mentioned steps 2 makes) culture supernatant, hatch 1 hour the negative contrast of SP2/0 culture supernatant for 37 ℃; The sheep anti-mouse igg of horseradish peroxidase-labeled (SIGMA company) was hatched 1 hour for 37 ℃; Tmb substrate liquid colour developing 10min, 2M H
2SO
4Termination reaction.Per step accomplishes all with the PBS thorough washing that contains 0.05%Tween20, and ((Winooski VT) surveys the OD450 value to Ultra Microplate Reader for EL808 Ultra Microplate Reader BIO-TEK Instruments, Inc..The clone that the OD450 that is surveyed is higher 10 times than negative control carries out subcloning, and it is frozen to increase.
The cloning of step 4, positive hybridoma cell-employing limiting dilution assay
The cell that above-mentioned steps 3 screenings are obtained is diluted to 1 cell in every hole with HT DMEM substratum, is laid on U type 96 porocyte culture plates, and scope was observed the actual cell count in every hole down in 4 hours; Write down single cell hole, treat that it grows up to the clone and ELISA identifies that antibody-secreting is positive, promptly obtain monoclonal antibody secretion strain; A large amount of amplifications are also frozen; Go down to posterity for a long time cultivate after, with the cloning evaluation once more of identical method, thereby obtained the hybridoma cell strain MCF1 and the MCF2 of stably excreting monoclonal antibody.Hybridoma cell strain MCF1 and MCF2 are deposited in Chinese typical culture collection center on September 6th, 2011, and preserving number is respectively CCTCC NO.C201170 and CCTCC NO.C201181.
Embodiment 3. extracorporeal culture-ings prepare monoclonal antibody
With positive hybridoma cell MCF1 that has set up (CCTCC NO.C201170) and MCF2 (CCTCC NO.C201181) difference enlarged culturing, treat that cell concn reaches 10
5Stop to change liquid during/ml, continue to cultivate until cell all dead.Collect culture supernatant, 1500rpm, centrifugal 10 minutes, supernatant contained high-caliber monoclonal antibody, and-20 ℃ of preservations are subsequent use.
Induce the ascites legal system in embodiment 4. bodies and be equipped with monoclonal antibody
Select multiparity Balb/c mouse, intraperitoneal injection 0.5ml whiteruss (SIGMA company), every the interior injection of mouse peritoneal 0.5ml contains 2 * 10 after 7 days
5MCF1 (CCTCC NO.C201170) or MCF2 (CCTCC NO.201181) hybridoma; Backsight mouse web portion degrees of expansion was collected ascites in 7-10 days; 4 ℃ centrifugal, and the centrifugal 20min of 2500rpm collects supernatant; Obtain monoclonal antibody mcf1 and monoclonal antibody mcf2 respectively, two kinds of monoclonal antibodies difference packing and-70 ℃ of preservations of placement are subsequent use.The CHARACTERISTICS IDENTIFICATION of embodiment 5 monoclonal antibodies
Get the mouse ascites of embodiment 4 preparations and do proportional diluted; With H-FABP (Hytest company) serves as to detect antigen; Indirect elisa method (is measured the monoclonal antibody mcf1 of embodiment 4 preparations and the titre of monoclonal antibody mcf2 with embodiment 2 step 3); And the negative contrast of mouse ascites that induces with SP2/0, the result that ELISA detects is 1: 10
6
Step 2, monoclonal antibody IgG subgroup identification
Get MCF1 and MCF2 Hybridoma Cell Culture supernatant, (SIGMA company) carries out with mouse monoclonal antibody somatotype reagent.
Concrete grammar is following:
(1) in the elisa plate that encapsulates H-FABP albumen (embodiment 1 preparation), adds MCF1 and MCF2 Hybridoma Cell Culture supernatant respectively, incubated at room 2 hours.
(2) wash plate three times with the PBS that contains 0.05%Tween 20.The IgG1 that adds sheep anti mouse respectively, IgG2a, IgG2b, each 100 μ l/ hole of IgG3 (SIGMA company) (with PBST dilution in 1: 2000), the two multiple holes of each sample.Incubated at room 30 minutes.
(3) wash plate three times with the PBS that contains 0.05%Tween 20.Add the HRP anti-sheep IgG of link coupled rabbit (SIGMA company) (doing dilution in 1: 20000), 100 μ l/ holes, incubated at room 30 minutes with PBST.
(4) wash plate three times with the PBS that contains 0.05%Tween 20, tmb substrate colour developing 10min.
(5) 2M H
2SO
4Termination reaction.
(6) ELISA Reader surveys the OD450 value, judges the monoclonal antibody IgG subclass.
The result shows that MCF1 and MCF2 hybridoma excretory monoclonal antibody mcf1 and mcf2 are IgG1 subclass Tegeline.
The purifying of step 3, monoclonal antibody
The two kinds of H-FABP odd contradictive hydroperitoneum mcf1 and the mcf2 of embodiment 4 preparation are used 0.01mol/L respectively, the PBS two-fold dilution of pH 7.4.Utilize method (by specification operation) the proteic monoclonal antibody of purifying H-FABP of Protein A (Amersham Pharmacia Biotech company) affinity chromatography.Antibody purified is carried out the SDS-PAGE electrophoresis, carry out (separation gel is 12.5%, and concentrated glue is 4.5%) by ordinary method.It is subsequent use that antibody purified is put-70 ℃ of preservations.
The mensuration of step 4, monoclonal antibody stability
The recovery hybridoma is collected the cell conditioned medium of 3,4,5 numbers that go down to posterity, and is detected it with the ELISA method and tire.The result shows that it is stable to tire, and shows the generation monoclonal antibody that the MCF1 (CCTCC NO.C201170) and the cell of MCF2 (CCTCC NO.201181) the different passage numbers of cell strain all can be stable.
(1) indirect ELISA method (detects the H-FABP specific reaction of Hytest of H-FABP and purchase of two kinds of monoclonal antibody mcf1 and the mcf2 and embodiment 1 purifying of gained with embodiment 2 step 3).Experimental result is seen table 1.The result shows; The H-FABP of monoclonal antibody mcf1 and monoclonal antibody mcf2 and embodiment 1 purifying and the H-FABP of Hytest company all have specific reaction; And the OD value is higher than 9F3 (Fatty Acid Binding Protein (FABP), catalog number: 4F29-9F3, Hytest company)
Table 1 ELISA detects the specific reaction of mcf1 and mcf2 monoclonal antibody and purifying H-FABP and Hytest-H-FABP
(2) Western Blot immunoblot experiment detects the H-FABP albumen (embodiment 1 preparation) and the reaction of Hytest H-FABP protein binding of monoclonal antibody of the present invention and purifying:
In Bio-Rad electrotransfer system, the gel protein band is transferred to (Millipore company) on the pvdf membrane by ordinary method, spend the night with 4 ℃ of sealings of 5% skim-milk, add the H-FABP monoclonal antibody by embodiment 4 preparations, room temperature reaction 1h washs three times.Sheep anti-mouse igg (SIGMA company) the room temperature reaction 1h that adds horseradish peroxidase-labeled subsequently; Wash three times; After 1: 1 equal-volume of two kinds of substrates of ECL mixes it is covered the film surface and make it even, wrap film, in the darkroom, the X-ray sheet is covered above the film with preservative film; Develop photographic fixing.The result shows: monoclonal antibody mcf1 and monoclonal antibody mcf2 and H-FABP albumen have stronger specific reaction; And with cerebral tissue, skeletal muscle tissue no cross reaction (Fig. 6, Fig. 7).But show that the present invention obtains, by the anti-people H-FABP of two strains monoclonal antibody mcf1 and mcf2 specificity and H-FABP molecule generation intensive association reaction that MCF1 and MCF2 cell strain produce, can be applicable to the detection of H-FABP.
Step 6 antibody titers is measured
With the H-FABP of Hytest serves as to detect antigen, indirect elisa method (measure the titre of the two kinds of monoclonal antibody mcf1 and the mcf2 of embodiment 5 step 3 purifying with embodiment 2 step 3), and with diluent as negative control, with the positive contrast of the H-FABP of Hytest company antibody.Experimental result shows: the antibody titers that indirect ELISA detects is 1: 10
6More than, and the antibody mcf1 of MCF1 and MCF2 preparation and the mcf2OD value H-FABP antibody 9F3 (seeing table 2) that is higher than Hytest company.
Table 2 indirect ELISA detects mcf1 and mcf2 monoclonal antibody titre
Embodiment 6, two strain monoclonal antibodies prepare the gold test strip bar and detect H-FABP albumen
The anti-people H-FABP monoclonal antibody mcf1 that gets hybridoma MCF1 (CCTCC NO.C201170) preparation does detection line antibody, is 1mg/mL with damping fluid (10% trehalose, 6% methyl alcohol) dilution; With drawing the film appearance antibody is drawn on nitrocellulose filter, concentration is that 1 μ l/cm forms detection line, vacuum-drying.
Step 2, nature controlling line preparation:
Sheep anti-mouse igg (SIGMA company) is 0.5mg/mL with damping fluid (10% trehalose, 6% methyl alcohol) dilution; With drawing the film appearance antibody is drawn on nitrocellulose filter, concentration is that 0.5 μ l/cm forms nature controlling line, vacuum-drying.
The golden mark of step 3, colour developing antibody:
Use 0.1M K
2CO
3Regulate nano-Au solution to pH 8.2.The monoclonal antibody mcf2 3mg that in the 100mL nano-Au solution, dropwise adds hybridoma MCF2 (CCTCC NO.C201181) preparation stirs 5min, leaves standstill 30min.Add 25mL 5%BSA+20mM Tris-HCl (pH 8.2) again, 4 ℃ of sealings are spent the night, and get reaction solution.Reaction solution is in 4 ℃, 4000g, and centrifugal 30min precipitates resuspendedly with 125mL 1%BSA+20mM Tris-HCl (pH 8.2), and repeated centrifugation twice, centrifugal speed are followed successively by 3000g, 2000g must precipitate.With the resuspended liquid of 1200 μ l (0.01%Tween-20,0.02%NaN3,20mM Tris-HCl, pH 8.2 for 30% trehalose, the 1%BSA) deposition that suspends, golden labeling antibody.
Step 4, the pad preparation of gold mark and spray pad:
Film treatment solution preparation: 0.25%Triton X-100,0.15mM NaCl, 0.05M Tris (pH 7.5); Trevira film (S&S company) soaks 1h, vacuum-drying in the film treatment solution; With 2 μ l/cm, golden labeling antibody spray printing is filled up at the gold mark.
Sample pad, gold mark pad, nitrocellulose filter and thieving paper are sticked on the plastic plate of single face pressure sensitive adhesive in order, on nitrocellulose filter, be provided with detection line antibody and nature controlling line antibody; Plastic plate after cutting 6cm width is pasted, the test card of packing into; Heat-sealing Fresco Bag with having siccative is packed room temperature preservation.
Step 6, detection:
Hytest-H-FABP is diluted to 0ng/ml with 1%BSA-PBST, 10ng/ml, 20ng/ml.100 μ l are added dropwise to well with sample, observe during 15min, and the standard of interpretation test strip detected result is: the positive, and all there is the colour developing band at detection line place and nature controlling line place; Feminine gender, the detection line place does not have the colour developing band, and there is the colour developing band at the nature controlling line place.Experimental result is seen (seeing accompanying drawing 8).The result shows that mcf1 and mcf2 monoclonal antibody can make up the gold test strip box and detect H-FABP albumen.
Embodiment 7, the two sandwich ELISA methods of two strain monoclonal antibodies detect H-FABP albumen
The monoclonal antibody mcf2 mark horseradish peroxidase of step 1, hybridoma MCF2 (CCTCC NO.C201181) preparation
Get 2mg monoclonal antibody mcf2 in 0.05mol/L carbonate buffer solution (pH 9.5), 4 ℃ the dialysis 12 hours, during change dialyzate three times.Get 2mg horseradish peroxidase (Roche company) and be dissolved in the 0.5ml deionized water, add 0.5ml 0.06mol/L sodium periodate (SIGMA company), 4 ℃ of lucifuges were reacted 30 minutes behind the mixing.Add 0.5ml 0.16mol/L terepthaloyl moietie (SIGMA company), put room temperature lucifuge reaction 30 minutes.To dialyse back antibody and horseradish peroxidase with the mixed of 1: 1 (mass ratio) after in 0.05mol/L carbonate buffer solution (pH 9.5) dialysed overnight, change dialyzate three times, taking out dialyses block in traget antibody; Adding 0.2ml concentration is 5mg/ml Peng Qinghuana (SIGMA company), places 3 hours for 4 ℃, adds the equal-volume saturated ammonium sulphate; Placed 1 hour for 4 ℃, centrifugal 30 minutes of 4 ℃ of whizzer 3000g abandon supernatant; Deposition adds 50% ammoniumsulphate soln washing, and centrifugal 30 minutes of 4 ℃ of whizzer 3000g abandon supernatant; Deposition adds 1ml PBS (0.15mol/L sodium-chlor, 0.02mol/L sodium phosphate buffer pH 7.4) dissolving, and the dialysis card (PIERCE company) of packing into is dialysed more than 20 hours in PBS; Change dialyzate 4 times, take out traget antibody in the dialysis card, 4 ℃ of whizzer 10000g are centrifugal 30 minutes; Abandon deposition; Get the OD value that supernatant measures wavelength 280nm and wavelength 403nm with ultraviolet spectrophotometer and be respectively OD280nm and OD403nm, calculate marker antibody amount (mg/ml)=(OD280nm-OD403nm * 0.3) * 0.62, affinity tag horseradish peroxidase amount (mg/ml)=OD403nm * 0.4 according to formula.
The monoclonal antibody mcf1 coated elisa plate of step 2, hybridoma MCF1 (CCTCC NO.C201170) preparation
The monoclonal antibody mcf1 that gets MCF1 (CCTCC NO.C201170) preparation be dissolved in the coating buffer (0.05mol/L carbonate buffer solution PH9.6) to final concentration be 10 μ g/ml, every hole 100 μ l add the removable enzyme plate in 96 holes, 4 ℃ are spent the night.Washings is washed 3 times, and every hole adds the PBST of 300 μ l, 1% bovine serum albumin, and 4 ℃ of sealings are spent the night.Washings is washed 3 times ,-20 ℃ of preservations.
Step 3, detection H-FABP albumen
Getting, coated antibody encapsulates plate.Washings is washed 3 times, and every hole adds 100 μ l and uses PBST to be diluted to that concentration is 1000,100,10, the H-FABP (Hytest company) of 0ng/ml was hatched 1 hour for 37 ℃.Washings is washed 3 times, and every hole adds the monoclonal antibody mcf2 of the horseradish peroxidase-labeled of 100 μ l dilution in 1: 2000, hatches 30 minutes for 37 ℃.Washings is washed 5 times, and every hole adds 100 μ l TMB colour developing liquid (KPL company), hatches 10 minutes for 37 ℃.Every hole adds 50 μ l 2mol/L H
2O
4Termination reaction, with ELIASA in dual wavelength 450nm reading.Experimental result is seen table 3.The result shows that mcf1 and mcf2 monoclonal antibody can be combined into double fastener heart ELISA and detect H-FABP albumen.
The two monoclonal antibody sandwich ELISAs of table 3 mcf1 and mcf2 detect Hytest-H-FABP result
Claims (6)
1. a hybridoma cell strain MCF1 who secretes H-FABP is preserved in Chinese typical culture collection center on September 6th, 2011, and preserving number is CCTCC NO:C201170.
2. the anti-human heart type of the described hybridoma cell strain MCF1 of claim 1 excretory fatty acid binding protein monoclonal antibody mcf1.
3. the application of the described heart fatty acid binding protein monoclonal antibody mcf1 of claim 2 in preparation human heart type fatty acid binding protein detection reagent.
4. application according to claim 3 is characterized in that described human heart type fatty acid binding protein detection reagent is for detecting Western Blot reagent, ELISA detection reagent or the gold-immunochromatographyreagent reagent for assay of human heart type fatty acid binding protein.
5. colloidal gold strip that detects human heart type fatty acid binding protein; It is characterized in that doing detection line antibody with the described anti-human heart type fatty acid binding protein monoclonal antibody mcf1 of claim 2; Sheep anti-mouse igg is as nature controlling line; The monoclonal antibody mcf2 of colloid gold label obtains according to ordinary method preparation assembling as colour developing antibody; Wherein said monoclonal antibody mcf2 is the anti-human heart type of the hybridoma cell strain excretory fatty acid binding protein monoclonal antibody of CCTCC NO.C201181 by preserving number.
6. double fastener heart ELISA test kit that detects human heart type fatty acid binding protein; It is characterized in that comprising the monoclonal antibody mcf2 of horseradish peroxidase-labeled, the enzyme plate that the described anti-human heart type fatty acid binding protein monoclonal antibody mcf1 of claim 2 encapsulates, PBST, TMB colour developing liquid, 2mol/L H
2SO
4Wherein said monoclonal antibody mcf2 is the anti-human heart type of the hybridoma cell strain excretory fatty acid binding protein monoclonal antibody of CCTCC NO.C201181 by preserving number.
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| CN102735849A (en) * | 2012-07-05 | 2012-10-17 | 北京源德生物医学工程有限公司 | Enzymatic chemiluminescence immunoassay method for human heart-type fatty acid binding protein and reagent kit |
| CN103045541A (en) * | 2012-10-31 | 2013-04-17 | 中国人民解放军第三军医大学 | Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same |
| WO2015142305A1 (en) * | 2014-03-20 | 2015-09-24 | S.K Teknoloji Arastirma Gelistirme San. Ve Tic. Ltd. Sti. | Monoclonal antibodies developed against h-fabp and the use thereof |
| CN103901216B (en) * | 2014-04-22 | 2015-10-21 | 上海科华生物工程股份有限公司 | People H-FABP colloidal gold test and preparation method thereof |
| CN107119023B (en) * | 2016-12-27 | 2018-01-12 | 南京诺唯赞医疗科技有限公司 | The monoclonal antibody of the anti-human cardic fatty acid binding protein of resistance to aliphatic acid interference and its application |
| CN111505303A (en) * | 2019-01-31 | 2020-08-07 | 艾维可生物科技有限公司 | Kit for detecting heart-type fatty acid binding protein by chemiluminescence method and use method thereof |
| WO2024000345A1 (en) * | 2022-06-30 | 2024-01-04 | 中国科学院深圳先进技术研究院 | A-fabp monoclonal antibody 2c6, preparation method therefor and use thereof |
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