CN104777311B - The monoclonal antibody of anti human nerve somatomedin hNGF and the immue quantitative detection reagent box of hNGF - Google Patents
The monoclonal antibody of anti human nerve somatomedin hNGF and the immue quantitative detection reagent box of hNGF Download PDFInfo
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Abstract
The present invention provides monoclonal antibody and the immue quantitative detection reagent box of hNGF of a kind of anti human nerve somatomedin hNGF, belongs to immunology and biological technical field.Described monoclonal antibody is by secreted by the hybridoma that preserving number is CGMCC No.8771.According to its specificity, the present invention also provides for the immue quantitative detection reagent box of a kind of growth factor of human nerve hNGF, including ELISA Plate, the micropore of described ELISA Plate is coated with the monoclonal antibody of the present invention, there is the highest sensitivity and specificity, with mNGF no cross reaction, it is possible to calculating the content of hNGF albumen in testing sample accurately and fast, operation is simple.
Description
Technical field
The present invention relates to monoclonal antibody and the hNGF immue quantitative detection reagent box of a kind of anti human nerve somatomedin hNGF, belong to immunology and biological technical field.
Background technology
Nerve growth factor (Nerve growth factor, NGF) it is the target tissue synthesis arranged by its effector neuron and secretion, it affects periphery and the survival of some neuron of central nervous system and differentiation, has important function in neural growth and damage and reparation.
The method of detection by quantitative growth factor of human nerve hNGF has the DuoSet ELISA kit (R&D Systems, DY256) utilizing R&D to detect at present, is carried out the hNGF in sample quantitatively by enzyme linked immunoassay.R&D test kit is with the recombinant human nerve growth factor of insect cell expression as standard substance, it is coated elisa plate with coated antibody, the antigen captured with biotinylated detection antibodies after adding antigen, then adds the reaction of streptavidin-HRP conjugate, develops the color after washing.Owing to this test kit have employed biotin-streptavidin amplification system on the basis of double-antibody sandwich elisa, although sensitivity is high, but result also in complex operation, and detection process is the longest, an experimentation needs to complete for 6 hours.It is additionally, since its standard curve range at 31.25-2000pg/ml, therefore needs dilution for many times during detection enriched sample, thus result in testing result error the biggest.
Accordingly, it would be desirable to develop a kind of fast and convenient, sensitivity is moderate and the hNGF quick detection kit of high specificity to solve available reagent box exist problem, promote the research about growth factor of human nerve, the exploitation of medicine and clinical practice.
Summary of the invention
According to the needs in above-mentioned field, the monoclonal antibody of the anti-hNGF that we are prepared for high specificity, sensitivity is high, develop a kind of hNGF immue quantitative detection reagent box, solve quick, the problem of accurate quantitative analysis of hNGF.
The technical scheme that the present invention is claimed is as follows:
The monoclonal antibody of anti human nerve somatomedin, secreted by the hybridoma that preserving number is CGMCC No.8771.
The hybridoma cell line of the monoclonal antibody of secretion anti human nerve somatomedin, its preserving number is CGMCC No.8771.
The immue quantitative detection reagent box of growth factor of human nerve hNGF, it is characterised in that: include ELISA Plate, the micropore of described ELISA Plate is coated with said monoclonal antibody.
Also include enzyme labelled antibody, protein standard substance and auxiliary reagent；Described enzyme labelled antibody is the said monoclonal antibody of horseradish peroxidase HRP labelling.
Described auxiliary reagent includes substrate chromophoric solution, reaction terminating liquid and cleaning buffer solution；Described substrate chromophoric solution is substrate solution A:pH5.0, contains the hydrogenperoxide steam generator of final concentration of volumn concentration 3% in 50mmol/L phosphoric acid-citrate buffer solution；With substrate solution B: tetramethyl benzidine methanol solution, concentration is 0.l mg/ml；Described reaction terminating liquid is 2mol/L sulphuric acid；Described cleaning buffer solution is: containing the polysorbas20 solution that volumn concentration is 0.05% in pH7.4,20mmol/L PBS.
A kind of method detecting hNGF protein content, it is characterised in that using mentioned reagent box, its step is as follows:
(1) antigen-antibody reaction: be separately added into testing sample in the micropore of described ELISA Plate, then every hole adds enzyme labelled antibody, mixing, hatches 30 minutes for 37 DEG C, repeats to wash plate 4 times；
(2) chromogenic reaction: every hole is sequentially added into substrate chromophoric solution, adds reaction terminating liquid and terminates reaction after colour developing；
(3) colorimetric: return to zero with the light absorption value of blank control wells, measure OD value record under 450/630nm dual wavelength by microplate reader；
(4) obtaining standard curve and curvilinear equation thereof: the abscissa value of described standard curve is the concentration value of the protein standard substance of gradient concentration, vertical coordinate is the OD value of the protein standard substance of the gradient concentration using the method for step (1)-(3) to record；
(5) result judges: brings the OD value recorded into described curvilinear equation, is calculated the concentration of hNGF in each sample.
According to said method,
The gradient concentration of described protein standard substance is: 0,7.8125ng/ml, 15.625ng/ml, 31.25ng/ml, 62.5ng/ml and 125ng/ml；
Described substrate chromophoric solution is substrate solution A:pH5.0, contains the hydrogenperoxide steam generator of final concentration of volumn concentration 3% in 50mmol/L phosphoric acid-citrate buffer solution；With substrate solution B: tetramethyl benzidine methanol solution, concentration is 0.l mg/ml；
Described reaction terminating liquid is 2mol/L sulphuric acid；
Described cleaning buffer solution is: containing the polysorbas20 solution that volumn concentration is 0.05% in pH7.4,20mmol/L PBS；
Testing sample addition is 25 μ l, and enzyme labelled antibody addition is 75 μ l, and substrate chromophoric solution addition is each 50 μ l of substrate solution A and B, and reaction terminating liquid is 50 μ l.
The present invention provides the monoclonal antibody of a kind of anti human nerve somatomedin, and animal immune, screening hybridoma are cloned and obtained by using recombinant human nerve growth factor proteantigen by described monoclonal antibody.The advantage that this monoclonal antibody has high specificity, sensitivity is high, can use in the multiple method such as SABC, ELISA.
The present invention also provides for secreting efficiently and stably the hybridoma cell line of above-mentioned anti human nerve growth factor monoclonal antibody, and described cell line was preserved in China General Microbiological DSMZ with preserving number for CGMCC No.8771 on 01 16th, 2014.
By research further, the present invention also provides for the immue quantitative detection reagent box of a kind of growth factor of human nerve hNGF, including ELISA Plate, is coated with the monoclonal antibody of the present invention in the micropore of described ELISA Plate.Described test kit has the advantage that 1) using said monoclonal antibody as capture monoclonal antibody, have higher sensitivity and specificity, and mNGF no cross reaction, quantitatively accurate to the hNGF in sample, reproducible；2) test kit matched rhNGF protein standard substance, quantitative scoring accurately and fast can calculate the content of hNGF albumen in testing sample, whole experimentation only needs to complete for 1.5 hours, compared with existing hNGF detection kit, substantially reduce detection time-consuming, save time cost.
Test kit of the present invention also includes that enzyme labelled antibody, protein standard substance and auxiliary reagent, described auxiliary reagent include substrate chromophoric solution, reaction terminating liquid and cleaning buffer solution.ELISA Plate in test kit of the present invention is that flat board is coated and prepares by the mouse-anti hNGF monoclonal antibody obtained mouse immune by rhNGF albumen sterling.Described flat board can be selected for commercially available ELISA Plate, and specification can be 96 hole flat boards or the removable batten of 12X8,12X4.
In a preferred embodiment, the auxiliary reagent in test kit of the present invention is: a) 3% hydrogenperoxide steam generator that substrate chromophoric solution A:50mmol/L phosphoric acid-citrate buffer solution (pH5.0) is prepared；B: tetramethyl benzidine (TMB) methanol solution, concentration is 0.l mg/ml；B) reaction terminating liquid: 2mol/L sulphuric acid；C) cleaning buffer solution (20 times of concentrated solutions, 20X): 20mmol/L PBS (pH7.4) the 0.05% polysorbas20 solution prepared；D) Sample dilution: 2%BSA (bSA).
The present invention also provides for a kind of method detecting hNGF protein content, the mentioned reagent box that described method uses the present invention to provide is quantitative to the hNGF in sample, the most simple to operate, and highly sensitive, specificity good, it is possible to calculate the content of hNGF in testing sample rapidly and accurately.In actual applications, this method can be used to calculate the content of the rhNGF collecting liquid in the expression of rhNGF in fermentation liquid and rhNGF purge process, reflect the existence level of rhNGF in fermentation liquid and rhNGF purification of samples truly, cross reaction is not had with mNGF, it is easy to quality control, thus promotes the clinical research of hNGF.
Biological deposits information:
Biomaterial title: 6G3
Classification And Nomenclature: mouse hybridoma cell
Preservation date: on January 16th, 2014
Preserving number: CGMCC No.8771
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
Accompanying drawing explanation
Fig. 1. immunized mice serum titer testing result
Fig. 2. the SDS-PAGE of the monoclonal antibody-purified effect of anti-hNGF identifies
Fig. 3. the Western Blot result of anti-hNGF monoclonal antibody protein
Fig. 4. the standard curve of growth factor of human nerve immue quantitative detection reagent box
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, it should be understood that following embodiment is only used for explaining the present invention rather than limitation of the present invention.
BALB/c mouse: provided by Department Of Medicine, Peking University's Experimental Animal Center.
Recombined human NGF goods and rhNGF protein standard substance: expressed by our unit, purification prepare (happy big, Peng Lujia, history authority, etc. the high efficient expression of recombinant human nerve growth factor and evaluated biological activity research. Chinese Pharmaceutical Affairs .2014,28 (6): 601-606.).
The not specified experiment reagent of the present invention is this area conventional reagent, can be by being purchased or use this area conventional method to be prepared, and specification is for testing pure level.
Embodiment 1, the preparation of anti-hNGF monoclonal antibody
1) BALB/c mouse immunity
Choose 5 female BAl BIc/c mice, by recombinant human nerve growth factor (Liu Hezhong, happy big, history authority, Deng. the Chinese hamster ovary celI strain of efficiently expressing recombinant human nerve growth factor and construction method thereof. the patent No.: ZL 201110004328.7) antigen and Freund's complete adjuvant (SIGMA, F5881) mixing, subcutaneous injection after emulsifying completely, the immunizing dose of every mice is 80 μ g.After first immunisation, being spaced 2 weeks and 3 weeks, after not formula Freund's incomplete adjuvant (SIGMA, F5506) carries out emulsifying, rhNGF antigen is repeated immunity twice, mouse tail takes blood, surveys serum titer, chooses the high mice of titer and carry out test for fusion.Merge first 3 days, abdominal cavity booster immunization 1 time.
2) cell merges and filtering hybridoma
Splenocyte suspension is made in the spleen grinding taking immunity BALB/c mouse in super-clean bench under gnotobasis, washs 2 times by RPMI 1640 culture medium, collects 1 × 108Splenocyte and 2-5 × 107Myeloma cell SP2/0 is mixed in a 50ml fusion pipe, adds RPMI 1640 culture medium to 30ml, fully mixes.1000r/min is centrifuged 10 minutes, by supernatant exhaustion as far as possible.Palm touches at the bottom of fusion pipe, make sedimentation cell loose uniformly.Added 50%PEG 2000 (pH 8.0) 1ml being preheated to 37 DEG C at about 1 minute with 1ml suction pipe, limit edged rotates gently, has granule to occur seen from naked eyes, is slowly added to RPMI 1640 culture medium to 20ml.1000rpm is centrifuged 6 minutes, supernatant discarded.Within first 10 days, cultivate in HAT selective medium, afterwards until cloning for the first time selects HT culture medium culturing before completing.With the positive rate of indirect enzyme-linked immunosorbent assay (ELISA method) detection fusion cell after 2 weeks, select the hole that positive value is higher, through limiting dilution, the positive hybridoma cell detected is cloned, after the positive rate that continuously cloning makes positive colony hole 3 times reaches twice 100%, select the high hole of positive value and turn hole amplification culture frozen.Obtaining efficient stable and secrete the hybridoma cell strain 6G3 of anti-hNGF monoclonal antibody, be preserved in China General Microbiological DSMZ on 01 16th, 2014, preserving number is CGMCC No.8771.
3) preparation of anti-hNGF monoclonal antibody ascites and antibody purification
The internal method that induces is used to prepare monoclonal antibody in a large number.Take the healthy Balb/c female mice of 6-8 week old, lumbar injection paraffin (0.5ml/ is only).After 1 week, hybridoma centrifuge washing, adjust cell number to 1 × 10 with PBS6Individual/ml, every injected in mice 0.5ml.After 5～7d, treat that mouse web portion increases, gather ascites.Ascites collected after centrifugation supernatant, with HITRAP Protein A HP prepacked column (GE Healthcare, CAS:17-0402-01) antibody purification, in-70 DEG C of preservations after subpackage.
Embodiment 2, anti-hNGF monoclonal antibody Property Identification
1) anti-hNGF monoclonal antibody subgroup identification
Use Rapid ELISA Mouse mAB Isotyping Kit (PIERCE, Cat.No.:37503) to identify class and the subclass of monoclonal antibody, operate according to test kit description.Result shows, the IgG subclass of cell strain 6G3 is IgG1 type, and light chain is Kappa.
2) anti-hNGF monoclonal antibody affinity constant measures
The method using capture antibody, detects affinity and the binding kinetics of anti-hNGF monoclonal antibody with GE company biomolecular interaction analysis instrument Biacore 3000.
Table 1 Biacore detects the affinity of anti-hNGF monoclonal antibody and rhNGF antigen
3) anti-hNGF monoclonal antibody specificity is identified
Western Blot is used to identify the specificity of monoclonal antibody.The purification effect SDS-PAGE of anti-hNGF monoclonal antibody protein identifies (Fig. 2), shows by the result of Gel-Pro software analysis electrophoretic band, and antibody purity is all not less than 90%.RhNGF and mNGF carrying out SDS-PAGE electrophoresis, after electricity transfer, hatches with the monoclonal antibody (1 μ g/ml) prepared, the sheep anti-mouse igg of AP labelling, as two anti-(1: 5000 dilutions), is operated by the general step of Western Blot.The Western Blot experimental result of anti-hNGF monoclonal antibody protein is as shown in Figure 3, owing between the NGF and mice NGF of people, homology is up to 89.1%, and there are data to show and between people and mice NGF, there is immunological cross-reaction, therefore in Western qualification process, have also been obtained identical result.So our Western Blot testing result shows there is faint cross reaction with mNGF.Embodiment 3, the preparation of enzyme linked immunological kit of detection by quantitative hNGF protein content
Test kit entrust Beijing Hotgen Biotechnology Co., Ltd. to develop and assemble (woods is green for a long time, monoclonal antibody, its preparation method and the application of a kind of anti-GP73 albumen. number of patent application: 200810181016.1；Woods is green for a long time. hepatitis B virus large protein pre-S 1 antigen detection reagent kit. and number of patent application: 200510077451.6), method is summarized as follows:
1) ELISA Plate is coated: by anti-hNGF monoclonal antibody 50mmol/L, each hole of ELISA Plate is added after the carbonic acid buffer dilution of pH9.5, every hole 100 μ l, absorption is overnight, plate is washed with tween monophosphate buffer, again with tween monophosphate buffer blind overnight, dry after drying, i.e. obtain the coated ELISA Plate of monoclonal antibody.
2) monoclonal antibody linked with peroxidase in test kit can be prepared by horseradish peroxidase (HRP) labelling anti-hNGF monoclonal antibody.Preparation method is as follows:
A) with NaIO4-glycol method carries out the oxidation of HRP, reaches final concentration 10mg/ml.
B) at alkaline carbonic acid salt buffer (50mmol/L, the carbonate buffer solution of pH9.5) in dialysis 6 hours, realizing the labelling of HRP antagonism hNGF monoclonal antibody, reaction terminates reacting with freshly prepared 1mg NaBH4 solution after terminating, then to PBS overnight.
C) with saturated ammonium sulphate, it is thus achieved that the HRP enzyme mark anti-hNGF monoclonal antibody of purification.
3), in enzyme linked immunological kit, auxiliary reagent can include integrated enzyme reaction substrate solution, nitrite ion, reaction terminating liquid and cleaning buffer solution.A kind of auxiliary reagent that can be used for mentioned reagent box is as follows:
A) substrate chromophoric solution:
3% hydrogenperoxide steam generator that substrate solution A:50mmol/L phosphoric acid-citrate buffer solution (pH5.0) is prepared；
Substrate solution B: tetramethyl benzidine (TMB) methanol solution, concentration is 0.l mg/ml；
B) reaction terminating liquid: 2mol/L sulphuric acid；
C) cleaning buffer solution (20 times of concentrated solutions, 20X): 20mmol/L PBS (pH7.4) the 0.05% polysorbas20 solution prepared；
D) Sample dilution: 2%BSA (bSA).
The method of embodiment 4, enzyme linked immunological kit detection hNGF protein content
Recombined human NGF standard substance are by our unit's expression, purification and prepare (history authority, happy big, Huo Lihong, Deng. recombinant human nerve growth factor purification process based on expressing cho cell system. the patent No.: ZL 201210206171.0), the step of detection hNGF protein content is as follows:
Prepare standard curve:
1) antigen-antibody reaction: be separately added into 25 μ l variable concentrations (0 in the micropore of the ELISA Plate of test kit offer, 7.8125ng/ml, 15.625ng/ml, 31.25ng/ml, 62.5ng/ml, rhNGF protein standard substance 125ng/ml), then every hole adds 75 μ l enzyme marking reagent mixings, hatches 30 minutes for 37 DEG C.Repeat to wash plate to operate 4 times.
2) chromogenic reaction: every hole is sequentially added into each 50 μ l of substrate solution A, nitrite ion B, develops the color 10 minutes, every hole adds 50 μ l reaction terminating liquids and terminates reaction.
3) colorimetric: return to zero with the light absorption value of blank control wells, measure OD value record under 450/630nm dual wavelength by microplate reader.
4) use straight linear recurrence to fit mode and carry out data process.Deducting the value after the OD value of blank with the OD value of each standard substance as vertical coordinate (Y-axis), the concentration of each standard substance is abscissa (X-axis) mapping, obtains standard curve, as shown in Figure 4.
Sample detection: identical with standard substance detection (1)-(3), after the OD value of detection sample deducts the OD value of blank, brings into and obtains sample NGF concentration in standard curve equation.Whole detection can complete at about 1 hour.
Embodiment 5, the evaluation of enzyme-linked immunologic detecting kit of detection by quantitative hNGF content
1) accuracy: take the rhNGF protein solution of our unit 20120814 crowdes of concentration known 110 μ g/ml of self-control, in prescribed limit, be at least evaluated by 25 measurement results.Take each 5 parts of the need testing solution that diluted protein content is tri-concentration of 25ng/ml, 50ng/ml, 75ng/ml respectively, carry out 5 independent trialss by ELISA detection kit detecting step.With being 25ng/ml by concentration, the standard substance of 50ng/ml, 75ng/ml are detected the absorbance obtained and return the value that standard curve obtains and carry out the response rate and compare, and the response rate is between 88-105%.
2) precision: randomly draw 2 pieces be coated after plank, take our unit self-control 20120814 crowdes of concentration known 110 μ g/ml rhNGF protein solution, in prescribed limit, be at least evaluated by 25 measurement results.Take each 5 parts of the need testing solution that diluted protein content is tri-concentration of 25ng/ml, 50ng/ml, 75ng/ml respectively, carry out 5 independent trialss by ELISA detection kit detecting step.Calculate each measurement result, obtain average, standard deviation SD and relative standard deviation RSD (i.e. coefficient of variation CV).Adding up 25ng/ml, 50ng/ml, 75ng/ml 25 testing results of tri-concentration, average is respectively 21.87ng/ml, 52.10ng/ml and 76.95ng/ml；Standard deviation SD is respectively 2.86,3.91 and 3.66；Relative standard deviation RSD (i.e. coefficient of variation CV) is respectively 13.06%, 7.51% and 4.76%.Precision result display relative standard deviation RSD (i.e. coefficient of variation CV)≤15%.
3) detection sensitivity: adding up according to above-mentioned experimental implementation result, the detection sensitivity of this test kit is 0.63ng/ml.
4) specificity: take concentration known 15 μ g/ml mNGF standard solution (Soviet Union's peptide is raw, SHUTAISHEN pharmaceutcal corporation, Ltd, lot number: 201211148), in prescribed limit, be at least evaluated by 25 measurement results.Take the need testing solution 5 parts of diluted protein content 50ng/ml, carry out 5 independent trialss by ELISA detection kit detecting step.Taking 25 parts of mNGF protein solution samples to detect, equal no cross reaction occurs.
The above, be only the preferred embodiments of the present invention, and the present invention not does any pro forma restriction.Any those skilled in the art; in the range of technical scheme; all utilize technology contents disclosed above to make a little to change or modify; should be regarded as the Equivalent embodiments of equivalent variations; in every case it is without departing from technical solution of the present invention content; any simple modification, equivalent variations and the modification made above example according to the technical spirit of the present invention, all still falls within the scope of protection of the invention.
Claims (7)
1. the monoclonal antibody of anti human nerve somatomedin, secreted by the hybridoma that preserving number is CGMCC No.8771.
2. the hybridoma cell line of the monoclonal antibody of secretion anti human nerve somatomedin, its preserving number is CGMCC No.8771.
3. the immue quantitative detection reagent box of growth factor of human nerve hNGF, it is characterised in that: include ELISA Plate, the micropore of described ELISA Plate
In be coated with the monoclonal antibody described in claim 1.
Test kit the most according to claim 3, also includes enzyme labelled antibody, protein standard substance and auxiliary reagent；Described enzyme labelled antibody
For the monoclonal antibody described in the claim 1 of horseradish peroxidase HRP labelling.
Test kit the most according to claim 4, described auxiliary reagent includes substrate chromophoric solution, reaction terminating liquid and cleaning buffering
Liquid；Described substrate chromophoric solution is substrate solution A:pH5.0, containing final concentration of body in 50mmol/L phosphoric acid-citrate buffer solution
The hydrogenperoxide steam generator of long-pending percentage composition 3%；With substrate solution B: tetramethyl benzidine methanol solution, concentration is 0.l mg/ml；
Described reaction terminating liquid is 2mol/L sulphuric acid；Described cleaning buffer solution is: contain containing volume basis in pH7.4,20mmol/L PBS
Amount is the polysorbas20 solution of 0.05%.
6. the method detecting hNGF protein content, it is characterised in that using the test kit described in claim 5, its step is as follows:
(1) antigen-antibody reaction: be separately added into testing sample in the micropore of described ELISA Plate, then every hole adds enzyme labelled antibody,
Mixing, hatches 30 minutes for 37 DEG C, repeats to wash plate 4 times；
(2) chromogenic reaction: every hole is sequentially added into substrate chromophoric solution, adds reaction terminating liquid and terminates reaction after colour developing；
(3) colorimetric: return to zero with the light absorption value of blank control wells, measure OD value record under 450/630nm dual wavelength by microplate reader；
(4) standard curve and curvilinear equation thereof are obtained: the abscissa value of described standard curve is the concentration of the protein standard substance of gradient concentration
Value, vertical coordinate is the OD value of the protein standard substance of the gradient concentration using the method for step (1)-(3) to record；
(5) result judges: brings the OD value recorded into described curvilinear equation, is calculated the concentration of hNGF in each sample.
Method the most according to claim 6,
The gradient concentration of described protein standard substance is: 0,7.8125ng/ml, 15.625ng/ml, 31.25ng/ml, 62.5ng/ml
And 125ng/ml；
Described substrate chromophoric solution is substrate solution A:pH5.0, containing final concentration of body in 50mmol/L phosphoric acid-citrate buffer solution
The hydrogenperoxide steam generator of long-pending percentage composition 3%；With substrate solution B: tetramethyl benzidine methanol solution, concentration is 0.l mg/ml；
Described reaction terminating liquid is 2mol/L sulphuric acid；
Described cleaning buffer solution is: containing the polysorbas20 solution that volumn concentration is 0.05% in pH7.4,20mmol/L PBS；
Testing sample addition is 25 μ l, and enzyme labelled antibody addition is 75 μ l, and substrate chromophoric solution addition is substrate solution A and B
Each 50 μ l, reaction terminating liquid is 50 μ l.
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