CN104076146A - ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody - Google Patents
ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody Download PDFInfo
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- G—PHYSICS
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Abstract
The invention relates to an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody and a detecting method thereof. The kit comprises a capture antibody plate and a detection antibody plate. The method adopts the steps of capturing and eluting target antibody to remove the interference of intrinsic protein in a detection sample so as to reduce the probability of nonspecific binding during ELISA detection. The kit can be used for qualitatively or quantitatively detecting the human anti-rabies virus antibody in a human blood specimen, the detecting method is high in recovery rate, precision and linearity when the concentration of a sample is 12.7ng/ml -225ng/ml, and the kit can be used for accurately detecting samples with high specificity and high sensitivity.
Description
Technical field
The present invention relates to technical field of biological, and be particularly related to for the detection comprising blood of human body sample antibody.
Background technology
The people such as the Engvall of Sweden in 1971 using cellulose and poly-the third ethene test tube as solid phase carrier adsorption antigen/antibody, have set up enzyme linked immunosorbent assay (Enzyme-linked Immunosorbent Assay is called for short ELISA) respectively.The people such as Voller in 1974 use polystyrene micro-reaction plate instead as solid-phase immunity absorption carrier, ELISA method is applied, make to develop into the assay method of liquid sample micro substance for the enzyme-labelled antibody technique of antigen location, and become gradually a kind of method the most conventional in antigen and antibody.It combines the immune response of enzyme labeling thing synantigen antibody complex with the catalytic amplification of enzyme, both kept the susceptibility of enzymic catalytic reaction, has kept again the specificity of antigen-antibody reaction, thereby has improved greatly sensitivity.It is again a kind of heterogeneous immune analysis method simultaneously, has washing process in each step of reaction, thereby has removed unreacted reactant and other interfering materials.Due to ELISA method have highly sensitive, high specificity, easy and simple to handle, detect rapidly and on-radiation and the plurality of advantages such as can measure in batches, make ELISA method obtain application more and more widely.
In detection human blood, the ELISA method of IgA, IgG and IgM antibody mostly is double antibody sandwich method at present, because coated antibody and enzyme labelled antibody are specific antibody entirely, can reduce nonspecific reaction, thereby the detection sensitivity that makes double antibody sandwich method antagonist is high, recall rate is high, high specificity, current commercial detection kit adopts this kind of ELISA reaction method more, as human interleukin ELISA kit.
In human blood, the main interference factors of antibody test is the endogenous protein being subject in sample product, generally comprises the nonspecific immunity globulin, heterophile antibody, some autoantibody of rheumatoid factor, complement, high concentration, because using dynamics, the cross reacting material of mouse Antybody therapy or diagnosis induction all can cause that testing result background raises or false positive.In the time of haemolysis, the haemoglobin in red blood cell is discharged in serum, and haemoglobin has the character of peroxidase, when it passes through, after absorption or " P-P-effect " (phenomenon of mutually adsorbing between protein) combination, can develop the color and cause false positive by catalysis TMB.In daily clinical serum (slurry) sample, have significant proportion in various degree contain above-mentioned various interfering material, thereby cause ELISA detect in non-specific binding serious, detect negative background and raise, accuracy, sensitivity are low.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, a kind of ELISA kit that detects human rabies viruses resisting antibody is provided, this kit can be got rid of the interference that detects endogenous protein in sample, reduce the OD value of antibody test negative serum in human blood, improved the accuracy, sensitivity specificity and the repeatability that detect.
To achieve these goals, technical scheme provided by the invention is: a kind of ELISA kit that detects human rabies viruses resisting antibody, described kit comprises: the antibody capture plate of coated human rabies viruses resisting antibody mouse source monoclonal antibody, antibody test plate, antibody elution liquid, antibody neutralizer, the mouse source primary antibodie of coated goat anti-human igg-Fc, wash that plate liquid, sheep anti-mouse igg-HRP bis-are anti-, substrate solution and stop buffer.
Further described ELISA kit, the light chain variable region amino acid sequence of the mouse resource monoclonal antibody in antibody capture plate is as shown in SEQ ID NO:l in sequence table, weight chain variable region amino acid sequence is as shown in SEQ ID NO:2 in sequence table, its light chain variable region nucleotide sequence is as shown in SEQ ID NO:3 in sequence table, and weight chain variable region nucleotide sequence is as shown in SEQ ID NO:4 in sequence table; The light chain variable region amino acid sequence of mouse source primary antibodie is as shown in SEQ ID NO:5 in sequence table, and weight chain variable region amino acid sequence is as shown in SEQ ID NO:6 in sequence table, and its light chain variable region nucleotide sequence is as shown in SEQ ID NO:7 in sequence table; Weight chain variable region nucleotide sequence is as shown in SEQ ID NO:8 in sequence table.
Further described ELISA kit, described antibody elution liquid is 300mmol/L acetic acid solution; Described antibody neutralizer is 1mol/L Tris-HCl damping fluid, and pH8.0, comprises 2% BSA.
Further described ELISA kit, described in to wash plate liquid be PBST cleansing solution, described stop buffer is 2mol/L H
2sO
4, described substrate solution is TMB nitrite ion.
The present invention also provides the preparation method of this kit, and described preparation method comprises the following steps:
(1) preparation of antibody in capture antibody plate: the nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4 in artificial synthesized sequence table, nucleotide sequence shown in SEQ ID NO:7 and SEQ ID NO:8, build prokaryotic expression carrier, high efficient expression in genetic engineering bacterium, and by the recombinant protein purification of expressing; (2) recombinant protein step (1) being obtained is coated in enzyme reaction plate, obtains capture antibody plate;
(3) goat anti-human igg-Fc is coated in to enzyme reaction plate, obtains antibody test plate;
(4) capture antibody plate, antibody test plate and antibody elution liquid, antibody neutralizer, the mouse source primary antibodie prepared by step (1), (2), (3), wash that plate liquid, sample diluting liquid, sheep anti-mouse igg-HRP bis-are anti-, substrate solution and stop buffer be assembled into kit.
Further ELISA kit is in the application detecting in human rabies viruses resisting antibody.
Adopt after technique scheme, the invention has the beneficial effects as follows: detect in sample and have a large amount of endogenous proteins, if direct-detection can cause negative background to raise and accuracy, sensitivity is low, because the present invention has adopted antibody capture plate, the eluent taking object antibody as master under wash-out from antibody capture plate, the acidity of eluent has been destroyed the endogenous protein of most of non-specific binding, use again in antibody test plate and absorb-elute liquid in antibody, use again the mouse resource monoclonal antibody of object antibody, specific detection object antibody, get rid of the interference of endogenous protein in human blood, thereby reduce the non-specific binding of ELISA, improve its accuracy, sensitivity and repeatability.
Owing to the invention provides the ELISA kit that detects human rabies viruses resisting antibody, detection sensitivity is high, high specificity, and experimental implementation is simple, and error is little, has reduced the interference that false positive or false negative phenomenon are brought clinical diagnosis.
brief description of the drawings
Fig. 1 detects the quantitative criterion curve of NM57 monoclonal antibody blood concentration.
Specific embodiment
Below in conjunction with embodiment, the present invention is described further, its effect is understood to explaination of the present invention but not to any type of restriction of the present invention.Wherein goat anti-human igg-Fc antibody (SouthernBiotech company), sheep anti-mouse igg-HRP(sigma company), normal human serum (Zhong Kechen space bio tech ltd), ELISA Plate be 96 hole ELISA Plate (U.S. corning/Costar), TMB A, B nitrite ion (Beijing safe Milky Way Bioisystech Co., Ltd).
The preparation of the ELISA kit of embodiment 1, detection human rabies viruses resisting antibody
(1) preparation of reagent
Coated damping fluid: 0.1M carbonate buffer solution, pH9.6, takes Na
2cO
33.18g, NaHCO
35.88g, is dissolved to 1000ml with ultrapure water.
PBS damping fluid (pH7.4): 0.02M PBS damping fluid, pH7.4, takes Na
2hPO
412H
2o 5.16g, NaH
2pO
42H
2o 0.87g, NaCl 7.6g, is dissolved to 1000ml with ultrapure water.
Wash plate liquid (PBST): containing the PBS damping fluid (pH7.4) of 0.05%Tween-20.
Confining liquid: containing the PBS damping fluid (pH7.4) of 1%BSA.
Eluent: 300 mmol/L acetic acid solutions.
Neutralizer: containing the 1mol/L Tris-HCl damping fluid (pH8.0) of 2%BSA.
Stop buffer: 2 mol/L H
2sO
4solution.
Substrate: TMB A, B nitrite ion.
(2) preparation method of NM57 mouse source monoclonal antibody S-230, S-271
S-230 light chain variable region amino acid sequence is as shown in SEQ ID NO:l in sequence table, weight chain variable region amino acid sequence is as shown in SEQ ID NO:2 in sequence table, its light chain variable region nucleotide sequence is as shown in SEQ ID NO:3 in sequence table, and weight chain variable region nucleotide sequence is as shown in SEQ ID NO:4 in sequence table; The light chain variable region amino acid sequence of mouse source primary antibodie is as shown in SEQ ID NO:5 in sequence table, and weight chain variable region amino acid sequence is as shown in SEQ ID NO:6 in sequence table, and its light chain variable region nucleotide sequence is as shown in SEQ ID NO:7 in sequence table; Weight chain variable region nucleotide sequence is as shown in SEQ ID NO:8 in sequence table.After gene student on commission work bioengineering (Shanghai) incorporated company is synthetic, build prokaryotic expression carrier, high efficient expression in genetic engineering bacterium, and by the recombinant protein purification of expressing.
(3) preparation of capture board and check-out console
1. coated capture board: with coating buffer damping fluid dilution mouse-anti NM57 monoclonal antibody S-230, for catching coating buffer, in ELISA Plate, every hole adds 100 μ l and catches coating buffer to 10 μ g/ml, and 4 DEG C of hold over night, as capture board.
2. be coated with check-out console: be detection coating buffer with being coated with damping fluid by 500 times of goat anti-human igg-Fc antibody dilutions, in ELISA Plate, every hole adds 100 μ l detection coating buffers, and 4 DEG C of hold over night, as check-out console.
3. sealing: wash respectively capture board and check-out console with washing plate liquid (PBST), discard the liquid in plate hole, pat dry, every hole adds washes plate liquid 380 μ l, left standstill for 10 seconds, discarded cleansing solution, patted dry, and washed so 2 times again, washed altogether 3 times.Every hole adds 100 μ l confining liquids to seal excessive binding site, 37 DEG C or incubated at room 2 hours.
4. dry: to discard respectively the confining liquid in capture board and check-out console, dry 48 hours, complete the preparation of capture board and check-out console.
(4) the capture antibody plate of preparation, antibody test plate and antibody elution liquid, antibody neutralizer, mouse source primary antibodie, sample diluting liquid, sheep anti-mouse igg-HRP bis-are resisted, washed plate liquid, substrate solution and stop buffer and be assembled into kit.
Embodiment 2, use kit detect the blood concentration of human anti-rabies NM57 monoclonal antibody
(1) the NM57 monoclonal antibody in catch-wash-out serum (slurry) sample
1. catch object antibody: add confining liquid 40 μ l to every hole in antibody capture plate, add afterwards reference standard S1～S6, high, normal, basic concentration quality-control product, test serum sample, 37 DEG C of oscillation incubations 2 hours.The preparation method of reference standard S1～S6 is S1 for diluting NM57 to 300 ng/ml with normal human serum, obtain S2(150 ng/ml with normal human serum doubling dilution again), doubling dilution is S3(75ng/ml successively), S4(37.5ng/ml), S5(18.8ng/ml), S6(9.38ng/ml); The preparation method of high, normal, basic concentration quality-control product is high concentration quality-control product X1 for diluting NM57 to 225 ng/ml with normal human serum, again with 4 times of normal human serum dilutions to 56.25 ng/ml be middle concentration quality-control product X2, then with 4 times of normal human serum dilutions to 14.06ng/ml be low concentration quality-control product X3.NM57 preparation method is referring to Chinese patent " preparation method of recombinant human anti-rabies monoclonal antibodies ", publication number CN101100663A.
2. wash-out: antibody capture plate is taken out, discard liquid in hole, wash plate 2 times with washing plate liquid, then use the every hole of PBS damping fluid (pH7.4) to add 380 μ l washing 1 time, discard PBS, pat dry.Add 60 μ l eluents (300mmol/L acetic acid solution) to every hole in antibody capture plate, room temperature vibration 5 minutes.
(2) neutralization-testing goal antibody
1. in and NM57 monoclonal antibody: when capture board carries out wash-out, every hole in antibody test plate is added to 50 μ l neutralizers (1mol/l Tris-HCl pH of buffer 8.0 comprises 2% BSA), after plate wash-out to be captured, from capture board, 50 μ l eluents are drawn out in every hole, join in the corresponding plate hole of check-out console, room temperature vibration 5 minutes, hatches 1 hour for 37 DEG C.Discard capture board.
2. add mouse-anti NM57 monoclonal antibody S-271: check-out console is taken out, discard liquid in hole, wash plate 3 times with washing plate liquid.With confining liquid dilution mouse-anti NM57 monoclonal antibody S-271 to 1 μ g/ml, in check-out console, every hole adds 100 μ l, hatches 1 hour for 37 DEG C.
3. add ELIAS secondary antibody: check-out console is taken out, discard liquid in hole, wash plate 3 times with washing plate liquid.With the sheep anti-mouse igg-HRP of confining liquid 1:2000 dilution human serum adsorption treatment, in check-out console, every hole adds 100 μ l, hatches 1 hour for 37 DEG C.
4. colour developing: check-out console is taken out, discard liquid in hole, wash plate 3 times with washing plate liquid.In check-out console, every hole adds TMB nitrite ion 100 μ l, and 37 DEG C are developed the color 30 minutes.Every hole adds stop buffer 50 μ l, cessation reaction.Taking 450nm as detecting wavelength, 630 nm are reference wavelength, measure the every hole of ELISA Plate OD value.
5. typical curve: get NM57 monoclonal antibody, be diluted to the reference standard that NM57 concentration is 300,150,75,37.5,18.8,9.4ng/ml with normal human serum (Zhong Kechenyu Beijing bio tech ltd).Undertaken quantitatively, taking the logarithm as ordinate taking OD value by the method that the present invention sets up, take the logarithm as horizontal ordinate taking NM57 reference standard concentration, drawing standard curve, is shown in Fig. 1.According to the NM57 content of typical curve calculation sample.Assay example is in table 1.
OD value is absorbance, and SD is sample standard deviation, and CV is coefficient of standard deviation
Embodiment 3, kit test method are learned the result
(1) accuracy:
High, medium and low sample (X1, X2, X3) is carried out to 6 batches of mensuration, the statistics recovery.
Testing result demonstration, the recovery of high, medium and low concentration standard is 90%～110%.
(2) degree of accuracy (repeatability)
In the mensuration of one batch, high, medium and low sample (X1, X2, X3) is carried out to the mensuration of 6 repeated sample, statistics CV% value.
Testing result demonstration, in the plate of high, medium and low concentration standard, repeatability (within-assay) is 5.2%～6.4%.
(3) degree of accuracy (middle precision)
High, medium and low sample (X1, X2, X3) is carried out to 3 batches of mensuration, statistics CV% value.
Testing result demonstration, between the plate of high, medium and low concentration standard, repeatability (between-batch precision) is 4.05%～15.5%.
Above result shows, concentration has reappearance in good plate and between plate within the scope of 12.7ng/ml ~ 225ng/ml.
(4) specificity
In high, medium and low sample (X1, X2, X3), add rabies human immunoglobulin (Wuhan Biological Products Inst., the accurate word S10940014 of traditional Chinese medicines) 0.5IU/ml, make three samples of repetition, add up its recovery.
Testing result shows, contains when being different from NM57 concentration and being the human rabies viruses resisting antibody immunoglobulin (Ig) of 0.5IU/ml in serum, and the NM57 recovery in serum is not affected.
(5) detection limit:
In the mensuration of method validation test, blank multiple hole is set, add up the SD value of this batch of dummy OD value and the slope of typical curve.
Quantitatively limit (LOQ) computing formula is LOQ=10* σ/S
σ: the standard deviation of dummy OD value
The slope of the typical curve of S:Linear-Linear fitting a straight line
Detection limit (LOD) computing formula is LOD=3* σ/S
σ: the standard deviation of dummy OD value
The slope of the typical curve of S:Linear-Linear fitting a straight line
LOD=3*0.002317/0.00183=3.80ng/ml
LOQ=10*0.002317/0.00183=12.7ng/ml
Testing result shows, quantitative limit of the present invention is 12.7ng/ml, and detection limit is 3.8ng/ml.
(6) linearity:
As shown in the quantitative criterion curve that Fig. 1 detects NM57 monoclonal antibody blood concentration, this invention is between sample NM57 content 12.7ng/ml ~ 225ng/ml time, and linear relationship is good.
(7) scope
This invention has the good recovery, precision and linearity in the time of sample NM57 content 12.7ng/ml ~ 225ng/ml.
The above results shows, high specificity of the present invention is highly sensitive, and accuracy and favorable repeatability meet clinical request for utilization.
Although, above with a general description of the specific embodiments the present invention being carried out to detailed description, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore,, not departing from modifications or improvements in the spirit and scope of the present invention, all should belong to the scope of protection of present invention.
Claims (6)
1. one kind is detected the ELISA kit of human rabies viruses resisting antibody, it is characterized in that, described kit comprises: the antibody capture plate of coated human rabies viruses resisting antibody mouse source monoclonal antibody, antibody test plate, antibody elution liquid, antibody neutralizer, mouse source primary antibodie, the sheep anti-mouse igg-HRP bis-of coated goat anti-human igg-Fc resist, wash plate liquid, substrate solution and stop buffer.
2. ELISA kit according to claim 1, the light chain variable region amino acid sequence of the mouse resource monoclonal antibody in antibody capture plate is as shown in SEQ ID NO:l in sequence table, weight chain variable region amino acid sequence is as shown in SEQ ID NO:2 in sequence table, its light chain variable region nucleotide sequence is as shown in SEQ ID NO:3 in sequence table, and weight chain variable region nucleotide sequence is as shown in SEQ ID NO:4 in sequence table; The light chain variable region amino acid sequence of mouse source primary antibodie is as shown in SEQ ID NO:5 in sequence table, and weight chain variable region amino acid sequence is as shown in SEQ ID NO:6 in sequence table, and its light chain variable region nucleotide sequence is as shown in SEQ ID NO:7 in sequence table; Weight chain variable region nucleotide sequence is as shown in SEQ ID NO:8 in sequence table.
3. according to the ELISA kit described in claim 1 to 2, described antibody elution liquid is 300mmol/L acetic acid solution; Described antibody neutralizer is 1mol/L Tris-HCl damping fluid, and pH8.0, comprises 2% BSA.
4. ELISA kit according to claim 3, described in to wash plate liquid be PBST cleansing solution, described stop buffer is 2mol/L H
2sO
4, described substrate solution is TMB nitrite ion.
5. a preparation method who detects the ELISA kit of human rabies viruses resisting antibody, described preparation method comprises the following steps:
(1) preparation of antibody in capture antibody plate: the nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4 in artificial synthesized sequence table, nucleotide sequence shown in SEQ ID NO:7 and SEQ ID NO:8, build prokaryotic expression carrier, high efficient expression in genetic engineering bacterium, and by the recombinant protein purification of expressing;
(2) recombinant protein step (1) being obtained is coated in enzyme reaction plate, obtains antibody capture plate;
(3) goat anti-human igg-Fc is coated in to enzyme reaction plate, obtains antibody test plate;
(4) capture antibody plate, antibody test plate and the antibody elution liquid, antibody neutralizer, mouse source primary antibodie, sample diluting liquid, the sheep anti-mouse igg-HRP bis-that prepared by step (1), (2), (3) resist, wash plate liquid, substrate solution and stop buffer and be assembled into kit.
6. the arbitrary described ELISA kit of claim 1-5 is in the application detecting in human rabies viruses resisting antibody.
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| CN104034896A (en) * | 2014-06-19 | 2014-09-10 | 华北制药集团新药研究开发有限责任公司 | Double-antibody sandwiched ELISA (enzyme-linked immunosorbent assay) detection method |
| CN107436350A (en) * | 2017-07-31 | 2017-12-05 | 云南沃森生物技术股份有限公司 | A kind of separation method that each composition in mixture is carried out using ELISA Plate and monoclonal antibody |
| CN109580944A (en) * | 2018-12-07 | 2019-04-05 | 潍坊医学院 | A kind of human cytomegalovirus Test paper and its manufacturing method |
| CN109738631A (en) * | 2019-01-21 | 2019-05-10 | 潍坊医学院 | A kind of preparation method of human cytomegalovirus IgM antibody Test paper |
| CN110305874A (en) * | 2019-06-19 | 2019-10-08 | 浙江省肿瘤医院 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
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Cited By (6)
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| CN104034896A (en) * | 2014-06-19 | 2014-09-10 | 华北制药集团新药研究开发有限责任公司 | Double-antibody sandwiched ELISA (enzyme-linked immunosorbent assay) detection method |
| CN107436350A (en) * | 2017-07-31 | 2017-12-05 | 云南沃森生物技术股份有限公司 | A kind of separation method that each composition in mixture is carried out using ELISA Plate and monoclonal antibody |
| CN107436350B (en) * | 2017-07-31 | 2019-05-24 | 云南沃森生物技术股份有限公司 | A kind of separation method carrying out each ingredient in mixture using ELISA Plate and monoclonal antibody |
| CN109580944A (en) * | 2018-12-07 | 2019-04-05 | 潍坊医学院 | A kind of human cytomegalovirus Test paper and its manufacturing method |
| CN109738631A (en) * | 2019-01-21 | 2019-05-10 | 潍坊医学院 | A kind of preparation method of human cytomegalovirus IgM antibody Test paper |
| CN110305874A (en) * | 2019-06-19 | 2019-10-08 | 浙江省肿瘤医院 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
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