CN102226171A - Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof - Google Patents
Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof Download PDFInfo
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Abstract
A diagnostic kit containing an anti-dog hemoglobin monoclonal antibody and an application thereof. The invention relates to a monoclonal antibody, and particularly relates to an anti-dog hemoglobin monoclonal antibody. The invention provides a mouse hybridoma cell strain, which is a strain preserved in China general microbiological culture collection center with a preservation number of CGMCC No. 4619. The invention also provides a monoclonal antibody generated by the mouse hybridoma cell strain with a preservation number of CGMCC No. 4619, and a kit containing the monoclonal antibody. The kit provided by the invention has high sensitivity, strong specificity, good accuracy and precision, and good stability, can detect dog hemoglobin in a sample to be detected specifically and sensitively, can exactly evaluate the gastrointestinal toxicity, the hepatic toxicity, the hematopoietic system toxicity, and the like of a medicament, and provides more scientific and accurate evaluation of the nonclinical safety of a medicament.
Description
Technical field
The present invention relates to a kind of monoclonal antibody, particularly a kind of monoclonal antibody of anti-dog oxyphorase.
Background technology
Dog is one of laboratory animal the most frequently used during the non-clinical safety of medicine is estimated, the gastrointestinal toxicity of medicine, hepatotoxicity are the conventional indexs that non-clinical safety is estimated, and check that it is the gastrointestinal toxicity of judging medicine, the important method of hepatotoxicity that dog ight soil is occulted blood.
The detection reagent that the detection that present dog ight soil is occulted blood does not have the dog special use can only adopt humanized's reagent.Whether the detection that dog ight soil is occulted blood exists the dog oxyphorase to realize by detecting in the sample to be checked, but there are structural difference in dog oxyphorase and human hemoglobin, cause most the detection false negative result all to occur, had a strong impact on the scientific evaluation of drug toxicity.
Therefore, the specific detection reagent of preparation dog oxyphorase is a problem demanding prompt solution.
Summary of the invention
In order to address the above problem, the invention provides the specific detection reagent of a kind of dog oxyphorase.
The invention provides a strain mouse hybridoma cell strain, it is that the preserving number that is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center is the cell strain of CGMCC No.4619.
The present invention also provides a kind of monoclonal antibody, and it is to be that the mouse hybridoma cell strain of CGMCC No.4619 produces by preserving number.It belongs to IgG1 type antibody, and light chain is the kappa type.Monoclonal antibody of the present invention produces by mouse hybridoma cell strain of the present invention, therefore can from the nutrient solution of cultivating mouse hybridoma cell strain of the present invention, obtain, or with the mouse hybridoma cell strain cell seeding to laboratory animal abdominal cavity generation ascites and obtain.
The present invention also provides the purposes of said monoclonal antibody in preparation dog hemoglobin detection kit.
The present invention also provides a kind of test kit, and it comprises said monoclonal antibody.This test kit also comprises the anti-dog oxyphorase polyclonal antibody of enzyme labelling, and described enzyme is preferably horseradish peroxidase.This test kit is the ELISA test kit.Test kit provided by the invention can be used for detecting the dog oxyphorase.
The present invention provides a kind of method that detects the dog oxyphorase again, and this method is to utilize the aforementioned agents box to detect sample to be checked.
The present invention provides the aforementioned agents box whether to contain purposes in the dog oxyphorase detecting sample to be checked at last.
Test kit provided by the invention is highly sensitive, high specificity, accuracy and precision is good, stability is strong, can detect the dog oxyphorase in the sample to be checked specifically, delicately, accurately estimate gastrointestinal toxicity, the hepatotoxicity of medicine, make the non-clinical safety of medicine estimate science, accurate more.
Description of drawings
The anti-dog oxyphorase of Fig. 1 MONOCLONAL ANTIBODIES SPECIFIC FOR schema
The SDS-PAGE electrophoresis of Fig. 2 dog oxyphorase is identified figure, and the band that the 2nd road molecular weight is big is a dog oxyphorase β subunit, and the band that molecular weight is little is a dog hemoglobin alpha subunit
The Western of the anti-dog oxyphorase monoclonal antibody that Fig. 3 immune mouse produces identifies figure, and swimming lane 1 expression sample is the M4 serum of 200 times of dilutions; Swimming lane 2 expression samples are the M4 serum of 1000 times of dilutions; Swimming lane 3 expression samples are the M4 serum of 5000 times of dilutions; Swimming lane 4 expression samples are the M5 serum of 200 times of dilutions; Swimming lane 5 expression samples are the M5 serum of 1000 times of dilutions; Swimming lane 6 expression samples are the M5 serum of 5000 times of dilutions
Fig. 4 uses test kit of the present invention and method to detect the typical curve of the dog oxyphorase of 5～320ng/mL
Embodiment
The embodiment of form is described in further detail foregoing of the present invention by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Dictionary of abbreviations table: ELISA:Enzyme-Linked Immunosorbent Assay, enzyme linked immunosorbent assay; OD:Optical Density, optical density(OD); %RE:Relative Error, the relative error percentage; %CV:coefficient ofvariation, variation coefficient percentage; QC:quality control, quality control; R
2: Coefficient of Correlation, relation conefficient; SD:Standard Deviation, standard deviation; Hb:hemoglobin, oxyphorase.
The schema of anti-dog oxyphorase Monoclonal Antibody as shown in Figure 1.
1, antigen prepd
Separate the dog erythrocyte, and with its cracking, centrifugal removal cell debris is collected supernatant; Supernatant carries out separation and purification with the Q post, and the salt concn gradient elution is collected different components; Electrophoresis identifies, the result as shown in Figure 2, collect SDS-PAGE detect dog oxyphorase (dog Hb) purity greater than 85% part as immunity antigen.
2, hybridoma preparation
Immune animal: get six Bal b/c mouse (being respectively M1-M6), with phosphate buffered saline buffer (PBS) dilution immunogen, mix at 1: 1 with corresponding adjuvant then, antigen and adjuvant mix the stable emulsion of formation fully, this emulsion is carried out abdominal injection, adopt low dosage long-range immunization to carry out immunity, concrete grammar is:
The 0th week--pre-blood sampling: complete Freund's adjuvant (CFA) emulsive 1mg antigen immune mouse;
The 2nd week: incomplete Freund's adjuvant (IFA) emulsive 1mg antigen immune mouse;
The 4th week: incomplete Freund's adjuvant (IFA) blended 1mg antigen immune mouse;
The 6th week: incomplete Freund's adjuvant (IFA) blended 1mg antigen immune mouse;
The 7th week: mouse orbit blood sampling, separation of serum, the dog oxyphorase that obtains with step 1 is an antigen, (antigen is 10 μ g/mL by the ELISA method, every hole 100 μ 1) detect tiring of anti-dog hemoglobin antibodies in the serum, the results are shown in Table 1, (two is anti-for enzyme is marked the anti-mouse of donkey (1: 2000), luminous substrate is ECL-Plus by the Western method, time shutter is 1 minute) detect antibody concentration, detected result is seen Fig. 3.
Table 1ELISA method detects tiring of the anti-dog oxyphorase of anti-mice serum
| Extension rate | 1000 | 3000 | 9000 | 27000 | Negative mice serum (1: 1000) |
| Mouse-4 | 2.327 | 0.915 | 0.385 | 0.153 | 0.063 |
| Mouse-5 | 10995 | 0.728 | 0.284 | 0.114 | 0.071 |
By table 1 and Fig. 3 as can be known, the 3rd and the 4th mouse have all produced the higher anti-dog hemoglobin antibodies of tiring, wait to tire reach a certain height after, choose the highest mouse of antibody titer and put to death, and separate its splenocyte.
Hybridoma merges and screening: with the PEG method above-mentioned mouse boosting cell and myeloma cell SP2/0 are merged.The dog oxyphorase that obtains with step 1 is that antigen screens the hybridoma clone of merging, and the clone of ELISA tests positive carries out subclone 3-4 time.
3, Monoclonal Antibody
Monoclonal Antibody: the hybridoma clone of step 2 screening is increased, cell inoculation prepares ascites in the mouse peritoneal of injecting fluid paraffin in advance, with the Protein G post ascites is carried out purifying, average each clone can obtain 3-5mg antibody, and-20 degree are preserved.
4, the polyclonal antibody of HRP mark preparation
(1) Antiserum Preparation:
1) be 2mg/ml with PBS with the dilution of dog oxyphorase, packing is frozen in-20 degree refrigerators;
2) get 1ml antigen and add 1ml Fu Shi Freund's complete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection, 2 New Zealand white rabbit of immunity;
3) the 15th day, get 1ml antigen and add the 1ml freund 's incomplete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection, 2 New Zealand white rabbit of immunity;
4) the 29th day, get 1ml antigen and add the 1ml freund 's incomplete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection, 2 New Zealand white rabbit of immunity;
5) the 43rd day, get 1ml antigen and add the 1ml freund 's incomplete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection, 2 New Zealand white rabbit of immunity;
6) the 53rd day, carotid artery was got blood, and rabbit is put to death;
7) rabbit blood is placed at 4 degree and is spent the night, and centrifugal (4 degree 10000rpm) 30 minutes, are collected serum.
(2) antibody purification:
1) the dog oxyphorase be coupled on the affinity media (CNBr-activated Sepharose, GE company product, 17-0430-01), according to product description, preparation antigen affinity column;
2) rabbit anti-serum is by affinity column, and 10 times of column volume PBS wash;
3) with 0.1M glycine solution (pH2.7) wash-out, be in charge of the antibody of collecting wash-out;
4) antibody of wash-out is dialysed with PBS, promptly obtains polyclonal antibody, and-20 degree are preserved.
5, anti-dog oxyphorase monoclonal antibody-right screening of anti-dog oxyphorase polyclonal antibody
The reagent configuration:
1, PBS (phosphate-buffered salt) liquid:
1. PBS stock solution (10 *) is joined method:
Be dissolved in the 100ml deionized water, regulate pH value to 7.2.
2. PBS uses liquid to join method (1 *):
Get stock solution 50ml and add deionized water 450ml and get final product, put room temperature or 4 ℃ of preservations are standby.
2, antigen coated liquid (CBS, 1 *):
Take by weighing Na
2CO
31.59g, NaHCO
32.93g deionized water 950ml regulates pH value to 9.6, adds deionized water and is settled to 1000ml, 4 ℃ of preservations.
3, confining liquid
Take by weighing the 5g skim-milk, be dissolved among the 100mlPBS, mixing, 4 ℃ of preservations standby (4 ℃ preservation should not above 3 days).
4, washing lotion
0.5mlTWEEN-20 is added among the PBS of 1L, mixing, room temperature preservation is standby.
5, TMB colour developing liquid
1. TMB stock solution: take by weighing the TMB powder, be made into the stock solution of 1.5mg/ml with DMSO ,-20 ℃ of packing are preserved;
2. NaAc damping fluid: the NaAc solution of preparation 200mM, regulate pH value to 5.3 with HAc;
3. H
2O
2Solution: the H of preparation 0.03%
2O
2Solution;
When 4. using according to the TMB stock solution: NaAc damping fluid: H
2O
2The ratio preparation colour developing liquid of solution=1: 4: 5, now with the current.
6, stop buffer (2M sulfuric acid)
The 100ml vitriol oil is slowly added in the 900ml water, and room temperature preservation is standby.
(1) just join:
1), bag quilt: the anti-dog oxyphorase of difference monoclonal antibody is diluted to 2 μ g/ml with coating buffer, and 100 μ l/ holes add to enzyme plate, and 4 ℃ are spent the night;
2), sealing: get rid of coating buffer, pat dry, every hole adds confining liquid 200 μ l, places 1.5 hours for 37 ℃;
3), get rid of deblocking liquid, tap water flushing 10 times pats dry;
4), add sample: the dog oxyphorase is diluted to 1 μ g/ml with confining liquid adds enzyme plate, 1 hour (staying 1 hole only to add confining liquid, as negative control) placed for 37 ℃ in 100 μ l/ holes;
5), tap water flushing 10 times, pat dry;
6), add ELIAS secondary antibody: mark anti-dog oxyphorase for enzyme and resist more, with confining liquid dilution in 1: 1000, placed 0.5 hour for 37 ℃ in 100 μ l/ holes;
7), tap water flushing 10 times, pat dry;
8), add colour developing liquid TMB: instant joining, placed 15 minutes for 37 ℃ in 100 μ l/ holes;
9), termination reaction: in each reacting hole, add the sulfuric acid of 2M, 50 μ l/ holes;
10), microplate reader reading: the 450nm wavelength is measured;
11), select the highest monoclonal antibody of OD value to continue the pairing experiment;
12), detect tiring and the antibody type of the highest monoclonal antibody of OD value.Detected result sees Table 2:
The detected result of tiring of table 2 monoclonal antibody
| The monoclonal antibody extension rate | 2000 | 4000 | 8000 | 16000 | 32000 | 64000 | 128000 | 256000 | 512000 | 1024000 |
| OD 450Value | 2.168 | 1.915 | 1.947 | 1.657 | 1.372 | 0.897 | 0.5 | 0.259 | 0.136 | 0.095 |
As shown in Table 2, tiring of monoclonal antibody is 1024000.Identify the type and the hypotype of this antibody with Invitrogen company product (90-6550), detecting this antibody is IgG1 type antibody, and light chain is the kappa type.
Mouse hybridoma cell strain called after 016-2D3 with this monoclonal antibody of secretion, and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 3rd, 2011, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, be called for short CGMCC, deposit number is CGMCC No.4619.
(2) drip and join:
1) bag quilt: selected anti-dog oxyphorase monoclonal antibody is diluted to 2 μ g/ml and two concentration of 10 μ g/ml with coating buffer, the difference wrapper sheet, 100 μ l/ holes add to enzyme plate, and 4 ℃ are spent the night;
2) sealing: get rid of coating buffer, pat dry, every hole adds confining liquid 200 μ l, places 1.5 hours for 37 ℃; Get rid of deblocking liquid, tap water flushing 10 times pats dry;
3) add sample: the dog oxyphorase (diluting with confining liquid) of gradient dilution is added enzyme plate, and 1 hour (staying 1 hole only to add confining liquid, as negative control) placed for 37 ℃ in 100 μ l/ holes;
4) the tap water flushing is 10 times, pats dry;
5) add ELIAS secondary antibody: for the concentration of HRP mark is how anti-the anti-dog oxyphorase of 2.5mg/ml is, be diluted to 3 concentration (1: 500,1: 1000,1: 2000) with confining liquid, placed 0.5 hour for 37 ℃ in 100 μ l/ holes;
6) the tap water flushing is 10 times, pats dry;
7) add colour developing liquid TMB: instant joining, placed 15 minutes for 37 ℃ in 100 μ l/ holes;
8) termination reaction: in each reacting hole, add the sulfuric acid of 2M, 50 μ l/ holes;
9) microplate reader reading: the 450nm wavelength is measured, and the result is as shown in table 3:
Table 3: monoclonal antibody and the how anti-working concentration detected result of enzyme mark
As shown in table 3, under different monoclonal antibodies and how anti-concentration, detected result is linear good, and particularly the working concentration at the Sheet clonal antibody is 2 μ g/ml, and the extent of dilution of enzyme mark polyclonal antibody is 1: 1000 o'clock, and the detected result linearity of dog oxyphorase is best.
Experimental results show that anti-dog oxyphorase monoclonal antibody provided by the invention can be used to prepare dog oxyphorase test kit.
Embodiment 2ELISA test kit is formed and the detection step
1, test kit is formed
(1) anti-dog oxyphorase monoclonal antibody: prepare concentration 2.5mg/mL ,-18～-24 ℃ of storages as the embodiment of the invention 1.
(2) how anti-the anti-dog oxyphorase of HRP mark is: prepare concentration 2.5mg/mL ,-18～-24 ℃ of storages as the embodiment of the invention 1.
(3) coating buffer (carbonate buffer solution): Na
2CO
31.59g, NaHCO
32.93g be dissolved in surely in the 1000mL pure water, pH is 9.6～9.8.
(4) PBS damping fluid: NaCl 8.0g, KCl 0.2g, Na
2HPO
42.9g, KH
2PO
40.2g be dissolved in surely in the 1000mL pure water, pH is 7.2～7.4.
(5) washings: comprise the PBS damping fluid of 0.1%Tween20, pH is 7.2～7.4.
(6) confining liquid: skim-milk 5.00g adds in the 100mLPBS damping fluid, fully dissolving.
(7) standard substance diluent: skim-milk 2.00g adds in the 100mLPBS damping fluid, fully dissolving.
(8) sample diluting liquid: with the standard substance diluent.
(9) the anti-dog oxyphorase of HRP mark polyclonal antibody diluent: with the standard substance diluent.
(10) stop buffer: 2N H
2SO
4
2, detect step
(1) wrapper sheet: will resist dog oxyphorase monoclonal antibody to be diluted to 2 μ g/mL with carbonate buffer solution, 100 μ l/ holes join in the 96 hole enzyme plates, 4 ℃ of overnight incubation (16-20h).
(2) wash plate machine washing plate 5 times, washing lotion 400 μ l are annotated in each every hole, stop 20 seconds; On thieving paper, pat dry.
(3) sealing: add 5% skim-milk confining liquid, 1.5h is hatched for 37 ℃ in 200 μ l/ holes.
(4) wash plate machine washing plate 5 times, washing lotion 400 μ l are annotated in each every hole, stop 20 seconds; On thieving paper, pat dry.
(5) application of sample: add oxyphorase standard product, testing sample (diluent is 2% skim-milk), the diluent that does not add sample is as negative control, respectively 100 μ l/ holes.Cover the shrouding film and be placed on mixing on the vibrator, hatch 1h for 37 ℃.
(6) wash plate machine washing plate 5 times, washing lotion 400 μ l are annotated in each every hole, stop 20 seconds; On thieving paper, pat dry.
(7) add ELIAS secondary antibody: the anti-dog oxyphorase that adds with the dilution in 1: 10000 of 2% skim-milk resists-HRP more, and 100 μ l/ holes cover the shrouding film and are placed on mixing on the vibrator, hatch 0.5h for 37 ℃.
(8) wash plate machine washing plate 5 times, washing lotion 400 μ l are annotated in each every hole, stop 20 seconds; On thieving paper, pat dry.
(9) colour developing: every hole adds tmb substrate 100 μ l, 37 ℃ of colour developing 10min.
(10) stop: every hole adds 50 μ l 2N H
2SO
4Termination reaction.
(11) 30 minutes inherent 450nm (reference wavelength 630nm) locate to read each hole OD value.
The characteristic of embodiment 3 test kits of the present invention
1, typical curve and linearity range thereof checking
Dog oxyphorase standard product are diluted to a plurality of non-zero concentration point with standard substance diluent (2% skim-milk), be respectively 640ng/mL, 320ng/mL, 160ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, test kit and the method for using embodiment 2 to provide detect.Detected result as shown in Figure 4, relation conefficient is greater than 0.99, linear relationship is good.
According to typical curve shown in Figure 4, calculate the measured concentration of each group, detected result sees Table 4:
Table 4 linearity range checking result
| Graticule concentration (ng/mL) | 320 | 160 | 80 | 40 | 20 | 10 | 5 |
| First day | 367.440 | 138.721 | 85.807 | 40.242 | 19.192 | 9.878 | 5.380 |
| Second day | 317.800 | 161.295 | 79.599 | 40.190 | 19.737 | 10.361 | 4.794 |
| The 3rd day | 332.537 | 153.678 | 80.820 | 40.698 | 19.488 | 9.990 | 5.134 |
| Average | 339.259 | 151.231 | 82.075 | 40.377 | 19.472 | 10.076 | 5.103 |
| Average %RE (average relative error) | 6.018 | -5.480 | 2.594 | 0.942 | -2.638 | 0.763 | 2.053 |
| %CV (variation coefficient) | 7.514 | 7.594 | 4.007 | 0.692 | 1.401 | 2.509 | 5.767 |
As shown in Table 4, when content of hemoglobin was greater than 5ng/mL in sample, test kit of the present invention and method can accurately detect; 7 concentration point in 320ng/mL to 5ng/mL scope, the average %RE of each concentration point＜20%, %CV＜15%.Experiment shows the highly sensitive of anti-dog oxyphorase monoclonal antibody provided by the invention, test kit and detection method.
2, specificity checking
Design two groups of samples (all the hole is detected again), first group: concentration is monkey/rat hemoglobin of 100ng/mL, 50ng/mL, 10ng/mL; Second group: at final concentration is that to add final concentration in the dog oxyphorase of 160ng/mL, 40ng/mL, 10ng/mL be the rat hemoglobin of 50ng/mL, and perhaps adding final concentration in final concentration is the dog oxyphorase of 200ng/mL, 50ng/mL, 10ng/mL is the monkey oxyphorase of 50ng/mL.Test kit and the method for using embodiment 2 to provide detect.
Detected result sees Table 5 and table 6:
Table 5 monkey oxyphorase disturbs specificity checking result
Table 6 rat hemoglobin disturbs specificity checking result
By table 5 and table 6 as can be known, first group of concentration that detects less than monkey/rat hemoglobin, second group only detects the dog hemoglobin concentration, does not detect monkey/rat hemoglobin concentration.Originally experimental results show that anti-dog oxyphorase monoclonal antibody provided by the invention, test kit and method have good specificity, all do not exist cross reaction to rat and monkey oxyphorase.
3, accuracy and precision checking
The dog oxyphorase quality control product of high, medium and low three concentration is set, and concentration is respectively 160ng/mL, 40ng/mL, 10ng/mL, and each concentration is provided with 5 multiple holes, carries out 3 batches of detections in 3 days, adds up each concentration %CV and estimates %RE.The results are shown in Table 7 and table 8:
Precision and accuracy validation result in table 7 plate
Table 8 day to day precision and accuracy validation result
| Parameter | The high density Quality Control | Middle concentration Quality Control | The lower concentration Quality Control |
| (160ng/mL) | (40ng/mL) | (10ng/mL) | |
| First day | 178.185 | 40.514 | 10.617 |
| Second day | 169.208 | 43.072 | 11.075 |
| The 3rd day | 173.829 | 41.929 | 10.760 |
| Average %RE | 8.588 | 4.595 | 8.172 |
| ％CV | 6.393 | 3.863 | 2.929 |
| Average %RE+%CV | 14.981 | 8.458 | 11.101 |
By table 7 and table 8 as can be known, the average %RE of different concns＜20% illustrates that test kit provided by the invention and method accuracy are good; In each concentration plate and in the daytime %CV＜15% illustrates that test kit provided by the invention and method precision are good.
4, extension rate checking
Dog oxyphorase quality control product with 0.8 μ g/mL dilutes 10 times respectively, 100 times of the dog oxyphorase quality control product dilutions of 8 μ g/mL, and 1000 times of the dog oxyphorase quality control product dilutions of 20 μ g/mL, each concentration is provided with 3 multiple holes and detects.
The results are shown in Table 9:
Table 9 extension rate checking result
Average %RE after 10 times of the dog oxyphorase quality control product of the 8 μ g/mL dilutions be-8.733, and %CV is 14.147, dilute 100 times after on average %RE be-10.568, %CV is 11.012.Average %RE was-8.010 after the dog oxyphorase quality control product of 20 μ g/mL diluted 1000 times, and %CV is 5.674.
Average %RE＜20% of each extension rate, %CV＜15%, so the maximum dilution multiple of this detection method is 1000 times.
5, stability checking
1. room temperature stability checking
The dog oxyphorase quality control product of high, medium and low three concentration is set, and concentration is respectively 160ng/mL, 40ng/mL, 10ng/mL, and room temperature storage is after 4 hours and put into 2-8 ℃ of storage detection respectively after 24 hours, and multiple hole is set during detection.The results are shown in Table 10:
Table 10 room temperature stability checking result
| Quality control product concentration (ng/mL) | Behind the room temperature 4h (ng/mL) | ％RE | Behind the 2-8 ℃ of 24h (ng/mL) | ％RE |
| 160 | 159.674 | -0.204 | 169.991 | 6.244 |
| 40 | 41.305 | 3.263 | 43.346 | 8.365 |
| 10 | 10.140 | 1.400 | 10.437 | 4.370 |
Detect the average of each concentration determination value for twice and compare with the concentration theoretical value respectively, relative error all in 100 ± 10%, reaches 100 ± 30% requirement, and this sample room temperature is placed stable.
2. reagent stability checking
With before 1 month in advance coated elisa plate take out and detect, typical curve is provided with ditto, the results are shown in Table 11:
Table 11 reagent stability checking result
| Graticule concentration (ng/mL) | Graticule concentration (ng/mL) after January | ％RE |
| 320 | 352.039 | 10.12 |
| 160 | 145.834 | -8.854 |
| 80 | 83.932 | 4.915 |
| 40 | 39.852 | -0.370 |
| 20 | 19.476 | -2.620 |
| 10 | 10.355 | 3.550 |
| 5 | 4.939 | -1.220 |
Each concentration point measured value of coated elisa plate typical curve is compared with the concentration theoretical value in advance, relative error is all in 100 ± 10%, reach 100 ± 30% requirement, the typical curve linearly dependent coefficient reaches 0.998, shows that anti-dog oxyphorase monoclonal antibody provided by the invention has good stability.
The stability that experimental results show that anti-dog oxyphorase monoclonal antibody provided by the invention, test kit and method is strong.
To sum up, anti-dog oxyphorase monoclonal antibody provided by the invention tire high, highly sensitive, high specificity, highly sensitive, high specificity, the accuracy of test kit and precision is good, stability strong, can detect the dog oxyphorase in the sample to be checked specifically, delicately, accurately estimate gastrointestinal toxicity, the hepatotoxicity of medicine, make the non-clinical safety of medicine estimate science, accurate more.
Claims (10)
1. a strain mouse hybridoma cell strain is characterized in that: it is that the preserving number that is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center is the cell strain of CGMCC No.4619.
2. monoclonal antibody is characterized in that: it is produced by the described hybridoma cell strain of claim 1.
3. monoclonal antibody according to claim 2 is characterized in that: it belongs to IgG1 type antibody, and light chain is the kappa type.
4. claim 2 or the 3 described monoclonal antibodies purposes in preparation dog hemoglobin detection kit.
5. a test kit is characterized in that: comprise claim 2 or 3 described monoclonal antibodies.
6. test kit according to claim 5 is characterized in that: the anti-dog oxyphorase polyclonal antibody that also comprises enzyme labelling.
7. test kit according to claim 6 is characterized in that: described enzyme is a horseradish peroxidase.
8. test kit according to claim 5 is characterized in that: described test kit is the ELISA test kit.
9. method that detects the dog oxyphorase, it is characterized in that: this method is to utilize the described test kit of claim 5-8 to detect sample to be checked.
10. whether the described test kit of claim 5-8 contains purposes in the dog oxyphorase detecting sample to be checked.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104914250A (en) * | 2015-06-10 | 2015-09-16 | 长沙安迪生物科技有限公司 | Rapid mutton detection card and detection method thereof |
| CN109402063A (en) * | 2017-08-18 | 2019-03-01 | 河北省科学院生物研究所 | Anti- porcine hemoglobin hybridoma cell strain and its monoclonal antibody and application |
| CN113004402B (en) * | 2019-12-18 | 2022-11-04 | 东莞市朋志生物科技有限公司 | Binding protein containing hemoglobin antigen structural domain |
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|---|---|---|---|---|
| US4582811A (en) * | 1983-02-02 | 1986-04-15 | Australian Monoclonal Development Pty. Ltd. | Method and diagnostic aid for detecting occult faecal blood |
| CN1490407A (en) * | 2003-08-13 | 2004-04-21 | 公安部第二研究所 | Antihuman hemoglobin detection reagent strips and monoclone antibody contained therewith |
-
2011
- 2011-03-29 CN CN2011100764558A patent/CN102226171B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4582811A (en) * | 1983-02-02 | 1986-04-15 | Australian Monoclonal Development Pty. Ltd. | Method and diagnostic aid for detecting occult faecal blood |
| CN1490407A (en) * | 2003-08-13 | 2004-04-21 | 公安部第二研究所 | Antihuman hemoglobin detection reagent strips and monoclone antibody contained therewith |
Non-Patent Citations (2)
| Title |
|---|
| 《1. The Journal of Immunology》 19860601 LH Stanker et al Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin 4174-4180 1-10 第136卷, 第11期 * |
| 《Clinical Chemistry》 19891031 H Moscoso et al Monoclonal antibody to the gamma chain of human fetal hemoglobin used to develop an enzyme immunoassay 2066-2069 1-10 第35卷, 第10期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104914250A (en) * | 2015-06-10 | 2015-09-16 | 长沙安迪生物科技有限公司 | Rapid mutton detection card and detection method thereof |
| CN109402063A (en) * | 2017-08-18 | 2019-03-01 | 河北省科学院生物研究所 | Anti- porcine hemoglobin hybridoma cell strain and its monoclonal antibody and application |
| CN109402063B (en) * | 2017-08-18 | 2020-11-24 | 河北省科学院生物研究所 | Porcine hemoglobin-resistant hybridoma cell strain, monoclonal antibody and application thereof |
| CN113004402B (en) * | 2019-12-18 | 2022-11-04 | 东莞市朋志生物科技有限公司 | Binding protein containing hemoglobin antigen structural domain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102226171B (en) | 2012-08-15 |
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