CN108998421A - Hybridoma cell strain A4-1B1 and its Procalcitonin monoclonal antibody and application of generation - Google Patents
Hybridoma cell strain A4-1B1 and its Procalcitonin monoclonal antibody and application of generation Download PDFInfo
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Abstract
The present invention provides hybridoma cell strain A4-1B1 and its Procalcitonin monoclonal antibodies and application of generation.Hybridoma cell strain A4-1B1 is preserved in China typical culture collection center, and deposit number is CCTCC NO:C2017144.Above-mentioned Procalcitonin monoclonal antibody can be applicable in method of immunity.The present invention also provides one to assemble to antibody, antibody caused by the antibody caused by hybridoma cell strain A4-1B1 and hybridoma cell strain A4-6A2 forms, the hybridoma cell strain A4-6A2 is preserved in China typical culture collection center, and deposit number is CCTCC NO:C2017143.Antibody specificity provided by the invention is high, is quick on the draw, at low cost, can reflect the active state of infection.Anti- PCT monoclonal antibody 1B1 and 6A2 provided by the invention are the optimal pairing antibody filtered out through double antibody sandwich method.
Description
Technical field
The present invention relates to monoclonal antibody art more particularly to hybridoma cell strain A4-1B1 and its calcitonin of generation
Former monoclonal antibody and application.
Background technique
Procalcitonin PCT is a kind of protein, when serious bacterium, fungi, helminth or pyemia and occur more for body
When Organ Failure, level of the PCT in blood plasma is significant to be increased, and PCT has become clinical systemic bacterial infections diagnosis and treatment at present
One of essential detection project in the process.Under normal circumstances, the neuroendocrine cell of primary limitation Thyreoidine and lung
Calcitonin C T is expressed and is cracked into, septicopyemia and pro-inflammatory cytokine can induce the various tissue multiple types cells of whole body
Expression and release PCT simultaneously enter blood circulation system.PCT under the induction of toxin, can increase sharply for 2-6 hours in the cell,
Reach peak value within 12-48 hours, there is high degree of specificity and sensitivity to bacterium infection, and not by disease of immune system and change
Treatment or the influence of immunosuppressor used, it may be said that be current best " pyemia index ".PCT is in serum, whole blood and blood
Highly stable in slurry, to diagnosis of sepsis, prognosis evaluation and Treatment monitoring, PCT embodies most excellent performance.PCT with
The inflammatory parameters such as traditional CRP, IL-6 compare the diagnostic sensitivity and specificity for the more advantage that embodies.
Accurately PCT methods for clinical diagnosis has the chemoluminescence method of Roche at present, high sensitivity, high specificity, but its at
This height only obtains more application in Grade A hospital, and middle and small hospital is difficult to popularize at present.Research shows that fluorescence immune chromatography method ratio
Enzyme-linked immunization, which detects PCT, has higher specificity and sensitivity, and analysis performance is able to satisfy clinical detection demand substantially, and
Its is easy to operate, without expensive instrument and equipment, is not necessarily to professional, time-consuming short, is the ideal chose of clinical PCT detection.This
Effective, specific binding PCT antibody that invention provides can be used in fluorescence immune chromatography platform, quickly examine to realization PCT
It surveys and bedside detection is of great significance.
Procalcitonin PCT has become effective and reliable marker of septicemia detection, and the application of PCT detection at present is more next
It is more extensive.PCT antigen contains 116 amino acid, encodes three albumen: amino Procalcitonin (N-proCT), calcitonin
(calcitonin, CT) and katacalcin.Under normal circumstances, PCT level is very low in human serum, is lower than 0.1ng/ml mostly, is good for
There is also the CT of certain level in Kang Renti.But the PCT antibody on the market in detection kit is directed to entire PCT at present
Antigen, obtained PCT antibody may be in conjunction with the CT in serum so as to cause the generation of false positive, and existing antibody test is clever
Sensitivity is low, these influence clinical detection level.
Summary of the invention
In order to solve the above technical problems, the present invention provides hybridoma cell strain A4-1B1, and generated by the cell strain
Procalcitonin monoclonal antibody and application.Antibody specificity provided by the invention is high, is quick on the draw, at low cost, is suitble to big
Sizable application is in clinical detection reagent box.
Hybridoma cell strain A4-1B1 provided by the invention, is preserved in China typical culture collection center, deposit number
For CCTCC NO:C2017144, the preservation time is on September 21st, 2017, and preservation place is Wuhan City, Hubei Province Wuchang Luo Ka
Mountain Wuhan University.
The present invention also provides a kind of anti-Procalcitonin monoclonal antibody of mouse, the antibody is by deposit number
The hybridoma cell strain A4-1B1 of CCTCC NO:C2017144, which secretes, to be generated, and identification antigen is PCT antigen.
The application that the present invention also provides Procalcitonin monoclonal antibodies in method of immunity, is included in fluidic cell
Technology, ELISA immunoassay, immunochormatography, immunocytochemical stain measuring method and immunohistochemical staining are surveyed
Determine the application of method etc..
The present invention also provides a kind of kit, the kit includes above-mentioned Procalcitonin monoclonal antibody.
The present invention also provides one to assemble to antibody, and the pairing antibody is as produced by above-mentioned hybridoma cell strain A4-1B1
Antibody and hybridoma cell strain A4-6A2 caused by antibody composition, the hybridoma cell strain A4-6A2 is preserved in China
Type Tissue Collection, deposit number are CCTCC NO:C2017143, and the preservation time is on September 21st, 2017, preservation
Place is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University.
Above-mentioned pairing antibody can be applied in method of immunity, and the method for immunity includes fluidic cell skill
Art, ELISA immunoassay, immunochormatography, immunocytochemical stain measuring method and immunohistochemical staining measurement
Method.
The present invention also provides a kind of kit, the kit includes above-mentioned pairing antibody.
High specific provided by the invention identifies Procalcitonin monoclonal antibody, applies in immunofluorescence and fluidic cell skill
In terms of art, infection cell can be detected, and can reflect the active state of infection.PCT monoclonal antibody in the present invention
Acquisition provides important specific raw material, the exploitation to PCT fluorescence immune chromatography kit for fluorescence immunoassay diagnostic method
Have great importance.Anti- PCT monoclonal antibody 1B1 and 6A2 provided by the invention are filtered out most through double antibody sandwich method
Excellent pairing antibody has higher sensitivity compared with the commercialization monoclonal antibody of purchase.It is expected in ELISA, chemiluminescence, is immunized
Chromatographic technique platform is applied, and lays a good foundation for the exploitation of the detection kit.
Detailed description of the invention
Fig. 1 is the agretope point diagram of the PCT antibody of Western Blot identification hybridoma cell strain 1B1 secretion；
Fig. 2 be coated antibody is done with 6A2 antibody, the antibody that made marks with 1B1 antibody, the fluorescence immune chromatography being prepared
The equation of linear regression figure of the detection data of immue quantitative detection reagent box；
Fig. 3 is the PCT immue quantitative detection reagent box of 6A2 antibody and 1B1 Antibody preparation and the correlation analysis that ten thousand inspire confidence in kit
Figure；
Fig. 4 is single-chain antibody heavy chain and light-chain variable region gene electrophoretogram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that the described embodiment is only a part of the embodiment of the present invention, rather than whole embodiments.Based on this hair
Embodiment in bright, all other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.The terms such as involved virus, cell strain and reagent are said in this specification embodiment
It is bright as follows:
The acquisition of embodiment 1:PCT monoclonal antibody
Anti- PCT monoclonal antibody is prepared to carry out as follows:
1. the preparation of immunogene: obtaining the protein sequence of PCT from NCBI, synthesize base after prokaryotic expression codon optimization
It because of sequence, is connected in prokaryotic expression carrier pet41a, through IPTG inducing expression.Thallus centrifuging and taking supernatant after ultrasonication,
Nickel affinity column, GST column are crossed, the albumen purified is as immunogen immune mouse.
2. mice immunized with antigen prepares anti-PCT monoclonal antibody, specific embodiment is as follows: with the albumen of above-mentioned purifying
Mouse is immunized in PCT, prepares monoclonal antibody.Specific step is as follows: with immunogene after purification to mouse carry out initial immunity and
Booster immunization twice, prepares Mouse spleen cells, and by its with the murine myeloma cell SP2/0 in growth logarithmic phase into
Row fusion, obtains hybridoma.Above-mentioned gained hybridoma carries out monoclonal by limiting dilution assay, then by indirect
ELISA filters out positive monoclonal cell strain.Cell infusion after monoclonal enters incomplete Freund's adjuvant pretreatment of mice abdomen
Chamber, induction generate ascites.127 plants of cell strain of monoclonal antibody are obtained altogether.
3. indirect ELISA surveys gained titer of ascites
3.1 titration
Antigen PCT is coated with 96 orifice plates after being diluted with carbonate coating buffer, and the hole 100ng/, 37 DEG C of heat incubate 2h, after incubation
PBS-T board-washing 1 time.Monoclonal antibody, 37 DEG C of incubation 30min, setting negative control hole, Positive control wells (3A2) and sky is added
White control (antibody diluent).After incubation, PBS-T board-washing 5 times.Enzyme mark dilution is by HRP labelled antibody (sheep anti mouse
IgG-HRP 1000 times) are diluted, every 100 μ l of hole.37 DEG C of incubation 30min.After incubation, PBS-T board-washing 5 times.Develop the color A liquid:
Develop the color B liquid=1:1, ready-to-use.Every hole 100 μ l, 37 DEG C of incubation 15min；Addition terminate liquid, 50 holes μ l/, color development stopping are anti-
It answers, detects absorbance, the 12 plants of titer of ascites measurement results screened are as follows, and gained titer of ascites about 46 the results are shown in Table
1, table 1 is the 12 plants of titer of ascites measurement results screened.Ascites is diluted through 4,000,000 times it can be seen from data in table 1
Substantially eliminated nonspecific reaction afterwards, as the result is shown ten thousand times of 1:400 dilution after the antibody 1B1 reacted with antigen PCT according to
Higher absorbance value is so presented, shows antibody sensitivity and specificity with higher, see Table 1 for details for specific value.
Table 1
| A4 ascites dilution | 1: 1 ten thousand | 1: 5 ten thousand | 1: 10 ten thousand | 1: 50 ten thousand | 1: 100 ten thousand | 1: 200 ten thousand | 1: 400 ten thousand | Blank |
| 1B1 | 2.277 | 2.067 | 1.901 | 1.734 | 1.513 | 1.21 | 0.908 | 0.009 |
| 9C2 | 2.195 | 1.981 | 1.818 | 1.638 | 1.425 | 1.168 | 0.801 | 0.004 |
| 1A2 | 1.276 | 1.185 | 0.799 | 0.609 | 0.406 | 0.248 | 0.156 | 0.005 |
| 3A2 | 1.323 | 1.143 | 0.914 | 0.664 | 0.463 | 0.282 | 0.155 | 0.006 |
| 8C3 | 1.414 | 1.297 | 1.193 | 1.118 | 0.837 | 0.668 | 0.421 | 0.008 |
| 6A2 | 2.293 | 2.033 | 1.929 | 1.843 | 1.694 | 1.48 | 1.211 | 0.004 |
| 1A3 | 1.383 | 1.286 | 1.23 | 1.08 | 0.929 | 0.724 | 0.503 | 0.008 |
| 3A6 | 1.21 | 1.036 | 0.83 | 0.528 | 0.354 | 0.254 | 0.179 | 0.007 |
| 9B1 | 1.013 | 0.783 | 0.538 | 0.336 | 0.191 | 0.103 | 0.075 | 0.002 |
3.2 sensitivity determination
12 plants of ascites concentration of screening are adjusted to 1mg/ml, (Chinese mugwort enables in Chongqing with commercial antibody (being lmg/m1) A
Up to)/B (Hytest Ltd)/C (Shanghai raw work) does indirect ELISA detection sensitivity to different diluted concentration antigens simultaneously.
Concrete operations are as follows: antigen PCT is coated with 96 orifice plates after being diluted with carbonate coating buffer, and 5 ladders are made in the hole 100ng/
Degree dilution, 37 DEG C of heat incubate 2h, PBS-T board-washing 1 time after incubation.Monoclonal antibody, 37 DEG C of incubation 30min, setting sky is added
White control (antibody diluent).After incubation, PBS-T board-washing 5 times.Enzyme mark dilution is by HRP labelled antibody (sheep anti mouse
IgG-HRP 1000 times) are diluted, every 100 μ l of hole.37 DEG C of incubation 30min.After incubation, PBS-T board-washing 5 times.Develop the color A liquid:
Develop the color B liquid=1:1, ready-to-use.Every hole 100 μ l, 37 DEG C of incubation 15min；Addition terminate liquid, 50 holes μ l/, color development stopping are anti-
It answers, detects absorbance, the results are shown in Table 2, table 2 is the result table of absorbance after chromogenic reaction, as can be seen from the results 1B1 and 6A2
Ascites high sensitivity in the commercialization monoclonal antibody of purchase.
Table 2
| Antigen letter is than dilution | The hole 100ng/ | The hole 50ng/ | The hole 25ng/ | The hole 12.5ng/ | 6.25ng/ hole | 3.125ng/ hole | Blank |
| A(1mg/ml) | 1.112 | 0.848 | 0.571 | 0.428 | 0.321 | 0.178 | 0.005 |
| B(1mg/ml) | 1.26 | 1.102 | 0.852 | 0.617 | 0.422 | 0.258 | 0.005 |
| C(1mg/ml) | 1.058 | 0.883 | 0.484 | 0.341 | 0.206 | 0.141 | 0.002 |
| 181 | 1.569 | 1.404 | 1.277 | 1.217 | 1.144 | 1.003 | 0.005 |
| 9C2 | 1.558 | 1.401 | 1.294 | 1.104 | 0.965 | 0.748 | 0.005 |
| 1A2 | 1.238 | 0.946 | 0.674 | 0.404 | 0.224 | 0.106 | 0 |
| 3A2 | 1.205 | 1.039 | 0.836 | 0.539 | 0.354 | 0.254 | 0.009 |
| 8C3 | 1.49 | 1.344 | 1.282 | 1.037 | 0.901 | 0.637 | 0.021 |
| 6A2 | 1.635 | 1.516 | 1.37 | 1.241 | 1.162 | 0.931 | 0.008 |
| 1A3 | 1.276 | 1.178 | 0.795 | 0.604 | 0.404 | 0.243 | 0.006 |
| 3A6 | 1.319 | 1.143 | 0.918 | 0.669 | 0.468 | 0.28 | 0.006 |
| 9B1 | 1.415 | 1.293 | 1.187 | 1.116 | 0.844 | 0.669 | 0.004 |
3.3 specific detection
It is coated with PCT and CT antigen respectively, the cross reaction using Inhibition ELISA detection 1B1 and 6A2 antibody to CT, with
This detection antibody specificity.
Concrete operations are as follows: antigen is coated with 96 orifice plates, the every hole 100/50ng after being diluted with carbonate coating buffer, and 37 DEG C of heat are incubated
2h, PBS-T board-washing 1 time after incubation.Monoclonal antibody, 37 DEG C of incubation 30min, blank control (antibody diluent) is added.
After incubation, PBS-T board-washing 5 times.HRP labelled antibody (sheep anti-mouse igg-HRP) is diluted 1000 times by enzyme mark dilution, often
100 μ l of hole.37 DEG C of incubation 30min.After incubation, PBS-T board-washing 5 times.Develop the color A liquid: develop the color B liquid=1:1, ready-to-use.
Every hole 100 μ l, 37 DEG C of incubation 15min；Terminate liquid, 50 holes μ l/ is added, color development stopping reaction detects absorbance, the results are shown in Table
3, table 3 is the result that Inhibition ELISA detects 1B1 antibody specificity.The result shows that three strain antibody such as 1B1 and 6A2 is not sent out with CT
Raw cross reaction, only specificity is directed to the both ends antigen sequence of PCT.It therefore, in clinical application can be anti-with CT to avoid antibody
It answers, reduces false positive results.
Table 3
Embodiment 2: double-antibody method screens optimal PCT pairing antibody and compares with import antibody
127 plants of cell strain of monoclonal antibody that we obtain us are recovered, and monoclonal antibody is prepared, and sad method is pure
Change, HRP label, screens pairing antibody with chessboard interior extrapolation method, be detection antigen with clinical PCT positive pooled serum, filter out most
Good pairing antibody pair.
Concrete operations are as follows: coated antibody 6A2 and labelled antibody 1B1.With commercial antibody to 16B3 (coating)/42 (mark
Note) (be purchased from Hytest Ltd), GRL1-2 (is purchased from Growth hormone secretagogue), and NEM1-2 (being purchased from norman) compares: 100 μ l/ of coated antibody
Hole, package amount 100ng, 37 DEG C of incubation 2h, PBS-T board-washing are primary；It is pressed after clinical PCT positive pooled serum doubling dilution
The hole 100ul/ is added, and blank control wells, 37 DEG C of incubation 30min, PBS-T board-washing 5 times after incubation are arranged.It is added through enzyme mark
1000 times of diluted of HRP labelled antibody 1B1-HRP and 42-HRP, the hole 100ul/, 37 DEG C of incubation 30min, board-washing 5 times.
Colour developing: developing solution A:B is ready-to-use by 1:1, and 100 holes μ l/ are added, and 37 DEG C of incubation 30min are added after incubation by 50 holes μ l/
Enter terminate liquid.It is placed in microplate reader and detects absorbance, testing result is shown in Table 4, and table 4 is that double-antibody method screens optimal PCT pairing
Antibody and comparing result with commercial antibody.As can be seen from Table 4 under similarity condition 6A2/1B1 than existing three kinds of commodity
Change pairing PCT antibody (control) and all has higher sensitivity.
Table 4
Embodiment 3:Western Blot identifies the antigen recognition site of the antibody
PCT is divided into 3 sections of PCT-1/2/3, corresponding amino acid sequence is as follows:
PCT-1:APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGS SLDSPRSKR
PCT-2, CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP
PCT-3:GKKRDMSSDLERDHRPHVSMPQNAN
Hybridoma cell strain 6A2 secretory antibody identifies PCT-1 sections of antigen recognition sequences, and hybridoma cell strain 1B1 secretion is anti-
Body identifies PCT-3 sections of antigen recognition sequences.
PCT3 sections of PCT-1/2/3 are respectively connected to pet41a, filter out recombinant plasmid pet41a/PCT-1, pet41a/
PCT-2, pet41a/PCT-3, and conversion BL21 (DE3) gold carries out prokaryotic expression respectively；3ml expression bacterium is cracked with 600 μ l
Liquid (20mM PB, pH7.8-8.0) is resuspended, ice bath 10min；0.3cm Probe Ultrasonic Searching wave breaks bacterium, and 8 times (ultrasonic 2s+ stops circulation
2s), above-mentioned circulating repetition 4 times；Maximum speed 10000rpm is centrifuged 10min, shifts supernatant into a clean centrifuge tube, point
Not Qu Yang (every kind of 2 hole of point sample) run SDS-PAGE electrophoresis, transferring film；37 DEG C of closing half an hour, film is cut into item, using respectively will be single
Anti- 1B1 is incubated for 2 hours at 37 DEG C, and TBS-T is washed 15 minutes, is during which changed liquid 3 times, and sheep anti-mouse igg-HRP is incubated in 37 DEG C
1.5 hours, TBS-T was washed 15 minutes, was during which changed liquid 2-3 times；Development: film is placed in gel imager, then chemiluminescence is shown
Toner A+B mixed liquor is added in film surface, takes pictures, WB the result is shown in Figure 1, and Fig. 1 is Western Blot identification hybridoma cell strain
The agretope point diagram of the PCT antibody of 1B1 secretion.Monoclonal antibody 6A2 specific recognition antigen fragment PCT- as can be seen from Figure 1
1, monoclonal antibody 1B1 specific recognition antigen fragment PCT-3.This experiment further proves that cross reaction does not occur for the antibody and CT,
Only specificity is directed to the both ends antigen sequence of PCT, can react in clinical application to avoid antibody with CT, reduces false positive knot
Fruit.
Embodiment 4: the kit made of 6A2 and 1B1 antibody is to clinical sample testing experiment
Coated antibody is done with above-mentioned 6A2 antibody, and 1B1 antibody makes marks antibody, is prepared into fluorescence immune chromatography quantitative detection
Kit detects clinical sample in terms of these in minimum detectability, accuracy, linear, repeatability and difference between batch；Simultaneously
Inspire confidence in that the same type fluorescence immune chromatography immue quantitative detection reagent box progress of detection is parallel to be compared with ten thousand, the results showed that with two kinds of the present invention
The Procalcitonin fluorescence immune chromatography standard measure detection kit and ten thousand of monoclonal antibody preparation inspires confidence in similar-type products testing result consistency
It is good；And testing result shows its high sensitivity, minimum detectability≤0.05ng/ml；Accuracy is preferable, and relative deviation all exists
In ± 15%；Linear effects in the detection range of kit mark the results show that have good linear gradient relationship (R=
0.999)；Reproducible, three kinds of concentration reference material coefficient of variation are respectively 6.73%, 12.5% and 9.38%, all≤15%.
It can thus be seen that homemade Procalcitonin fluorescence immune chromatography immue quantitative detection reagent box performance meets associated specifications.Specifically such as
Under:
1. minimum detectability
20 parts of the reagent and reference material matrix (human serum without Procalcitonin) for taking same lot number, respectively take 20 μ l, are added to
In sample treatment liquid, piping and druming is mixed, and stands 1min, draws 75 μ l mixed liquors, and loading reacts 15min, upper machine testing.It collects simultaneously
Determine as a result, reference material measurement result mean value and standard deviation S D are calculated, wherein the corresponding concentration of the result of (+2SD) is minimum
Detection limit.Table 5 is the kit minimum detectability of present invention pairing Antibody preparation as a result, being calculated according to detection data in table 5
Signal mean value is 0.110, and standard deviation 0.0110 obtains (X+2SD)=0.132, is substituted into matched curve and is returned
Return, calculating concentration value is 0.01ng/ml, meets the technical requirements of Procalcitonin Fluorescence kit sensitivity；Table 6 be other three
The kit minimum detectability of a producer pairing Antibody preparation as a result, by table 5 and table 6 result, it can be seen that and similarity condition
Kit (Medix Biochemica, luxuriant and rich with fragrance roc biology, Hangzhou Hua Kui of than three pairs commercialization pairing Antibody preparations of its lower detection limit
Biology) detection limit it is high.
Table 5
Table 6
2. accuracy detects
0.5ng/ml, 10.0ng/ml, 50.0ng/ml, the reference material of 3 concentration are taken, liquid-transfering gun draws reference to be detected
20 μ l of product, is added in sample treatment liquid, and piping and druming mixes, and stands 1min, draws 75ul mixed liquor, and loading reacts 15min, upper machine
Detection, each sample replication 3 times, test result is denoted as (Xi), calculates separately relative deviation (Bi), the results are shown in Table 7, table 7
For the detailed detection data of kit accuracy.By table 7 it can be concluded that, in the relative deviation Bi of 9 test strips all ± 15%,
Meet the accuracy technical requirements of Procalcitonin Fluorescence kit.
Table 7
3. linear result
Take 5 kinds of concentration of 0.5ng/ml, 2.0ng/ml, 5.0ng/ml, 10.0ng/ml, 50.0ng/ml, 100.0ng/ml
Reference material, liquid-transfering gun draws 20 μ l of reference material to be detected, is added in sample treatment liquid, and piping and druming mixes, and stands 1min, inhales
75ul mixed liquor is taken, loading reacts 15min, upper machine testing, and each concentration level repeats detection 3 times, takes the equal of 3 results
Value, does the logarithm scatter plot of both theoretical concentration value and actually detected value, makees matched curve, calculate linear equation y=ax+
B and correlation coefficient r, experimental result are shown in Table 8, table 8 be coated antibody is done with 6A2 antibody, the antibody that made marks with 1B1 antibody,
The result of linear detection for the fluorescence immune chromatography immue quantitative detection reagent box being prepared, be calculated coefficient R=
0.9999, meet linear requirements.Fig. 2 is done coated antibody with 6A2 antibody, the antibody that made marks with 1B1 antibody, is prepared
The equation of linear regression figure of the detection data of fluorescence immune chromatography immue quantitative detection reagent box.Antibody system of the invention as the result is shown
Standby kit has good linear as a result, illustrating that the kit of the Antibody preparation is with good stability.
Table 8
4. Precision Experiment
The reference material of 3 kinds of concentration of 0.5ng/ml, 2.0ng/ml, 10.0ng/ml is taken, liquid-transfering gun draws reference to be detected
20 μ l of product, is added in sample treatment liquid, and piping and druming mixes, and stands 1min, draws 75 μ l mixed liquors, and loading reacts 15min, on
Machine testing, each concentration level repeat detection 10 times, take the mean value and standard deviation SD of 10 results, calculate the change of three kinds of concentration
Different coefficient CV≤15%, meets the technical requirements of kit precision, and see Table 9 for details, and table 9 is repeatability in kit batch
Detailed detection data.
Table 9
5. kit comparison result
Respectively using 0.5ng/ml and 2.0ng/ml as critical value, two groups of detection datas are arranged, such as table 10 and table 11, table 10
The consistent data of two kinds of kits when for critical value being 0.5ng/ml, table 11 is critical value two kinds of kits when being 2ng/ml
Consistent data, analysis acquire P < 0.05, it was demonstrated that self-control Procalcitonin fluorescence immune chromatography immue quantitative detection reagent box and ten thousand
The correlation for inspiring confidence in Procalcitonin fluorescence immune chromatography immue quantitative detection reagent box is good.Simultaneously critical value be respectively 0.5ng/ml and
When 2.0ng/ml, the inspection of the two all calculates Kappa=0.899, it was demonstrated that the kit and ten thousand of the monoclonal antibody preparation inspires confidence in reagent
Box consistency is good.Through counting, the equations of linear regression of two kinds of kits is y=1.0669x-0.1264, and R=0.9953, such as
Fig. 3, Fig. 3 are the correlation analysis for inspiring confidence in kit by the PCT immue quantitative detection reagent box of 6A2 antibody and 1B1 Antibody preparation and ten thousand
Figure.The result shows that the Procalcitonin kit of described two monoclonal antibody preparations has higher correlation with the kit that ten thousand inspire confidence in.
Table 10
Table 11
By above-mentioned experiment, can obtain, by the sensitivity of 6A2 antibody and the PCT immue quantitative detection reagent box of 1B1 Antibody preparation,
The all requirements such as accuracy, linear requirements, repeatability reach kit preparation standard.
Embodiment 5: single-chain antibody variable region gene extracts
1. hybridoma RNA is extracted and cDNA synthesis: collecting the secretion monoclonal antibody 1B1 that fresh 15ml grows to logarithmic phase
Cell total rna is extracted with Trizol method after serum free medium washs with the hybridoma of 6A2；Again using total serum IgE as mould
Plate forms cDNA through M-MLV reverse transcriptase using oligo dT and random primer as primer.
2. the amplification of single-chain antibody heavy chain and light-chain variable region gene: using above-mentioned cDNA as template, respectively with this laboratory
VH-F/R, VL-F/R mixture of preservation are that primer amplification obtains the heavy chain of variable region of mab and light chain gene PCR is produced
Object is shown in that Fig. 4, Fig. 4 are single-chain antibody heavy chain and light-chain variable region gene electrophoretogram.Reaction condition is 94 DEG C, initial denaturation 5min,
94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle, last 72 DEG C of 10min.Above-mentioned PCR product is returned
Receipts are connected in carrier T, and sequencing obtains heavy chain and light chain gene sequence.It is online by EXPASY that resulting gene order is sequenced
Translate into amino acid sequence, Blast p comparison result show its for mouse single-chain antibody gene, and described two monoclonal antibodies
Other source of mouse monoclonal antibody variable region amino acid comparing results of heavy chain and chain variable region amino acid sequence and this room show that homology is equal
Lower than 67%, the results showed that there is no pollutions for the described two monoclonal antibody genes expanded, are newfound source of mouse single-chain antibody
Variable region fragment.The cell of secrete monoclonal antibody only generates a type of antibody molecule in principle, strictly speaking only generates
Containing a kind of heavy chain variable region and a kind of antibody molecule of light chain variable region, therefore obtained gene should say to be exactly the PCT
The corresponding two kinds of source of mouse antibody of antigen.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principle of the present invention, several improvement and deformations can also be made, these improvement and deformations also regard
For protection scope of the present invention.
Claims (9)
1. hybridoma cell strain A4-1B1, which is characterized in that the hybridoma cell strain is preserved in China typical culture collection
Center, deposit number are CCTCC NO:C2017144.
2. a kind of anti-Procalcitonin monoclonal antibody of mouse, which is characterized in that the antibody is CCTCC NO by deposit number:
The hybridoma cell strain A4-1B1 of C2017144, which secretes, to be generated.
3. application of the anti-Procalcitonin monoclonal antibody as claimed in claim 2 in method of immunity.
4. application according to claim 3, which is characterized in that the method for immunity include Flow Cytometry,
ELISA immunoassay, immunochormatography, immunocytochemical stain measuring method and immunohistochemical staining measuring method.
5. a kind of kit, which is characterized in that the kit includes Procalcitonin monoclonal antibody as claimed in claim 2.
6. assembling to antibody, which is characterized in that the pairing antibody is by hybridoma cell strain A4-1B1 described in claim 1
The composition of antibody caused by generated antibody and hybridoma cell strain A4-6A2, the hybridoma cell strain A4-6A2 are preserved in
China typical culture collection center, deposit number are CCTCC NO:C2017143.
7. application of the pairing antibody as claimed in claim 6 in method of immunity.
8. application according to claim 7, which is characterized in that the method for immunity include Flow Cytometry,
ELISA immunoassay, immunochormatography, immunocytochemical stain measuring method and immunohistochemical staining measuring method.
9. a kind of kit, which is characterized in that the kit includes pairing antibody as claimed in claim 6.
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| CN110747171A (en) * | 2019-09-17 | 2020-02-04 | 华南理工大学 | A pair of hybridoma cell strains capable of secreting anti-procalcitonin monoclonal antibody, anti-procalcitonin monoclonal antibody and application thereof |
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