CN104360057B - A kind of ELISA reaction system detecting anti-CD 52 antibody and method - Google Patents
A kind of ELISA reaction system detecting anti-CD 52 antibody and method Download PDFInfo
- Publication number
- CN104360057B CN104360057B CN201410566000.8A CN201410566000A CN104360057B CN 104360057 B CN104360057 B CN 104360057B CN 201410566000 A CN201410566000 A CN 201410566000A CN 104360057 B CN104360057 B CN 104360057B
- Authority
- CN
- China
- Prior art keywords
- antibody
- elisa
- detection method
- polypeptide
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of ELISA reaction system detecting anti-CD 52 antibody and method, the method includes building CD52 antigen polypeptide and modifying this antigen, this antigen is carried out wrapper sheet and catches anti-CD52 monoclonal antibody in sample, again by further combined with enzyme len antibody, and develop the color by adding suitable substrates, again by the contrast with concentration known standard substance, thus measure the content of antibody in sample.ELISA reaction system and the method for detecting anti-CD 52 antibody that the present invention provides, this reaction system and method can be used for detecting the content of anti-CD 52 antibody in culture supernatant and biological specimen, this method has height specificity, and the detection sensitivity in biological specimen can reach 0.1ug/ml.
Description
Technical field
The present invention relates to a kind of ELISA reaction system for detecting anti-CD 52 antibody and method.This reaction system and side
Method can be used for detecting the content of anti-CD 52 antibody in culture supernatant and biological specimen, and this method has height specificity, biological
Detection sensitivity in sample can reach 0.1ug/ml.
Background technology
Alemuzumab(anti-CD 52 antibody, Campath-1H) it is the anti-CD52 people of the expressing cho cell of recombinant DNA transfection
The monoclonal antibody in source, has the IgG1 of the complementarity-determining region (CDR) in people's antibody framework and constant region and Mus source
Kappa antibody.Antibody construction is by the weight of antihuman CD 52 Mus resource monoclonal antibody (rat YTH 34.5HL, Campath-1G)
The CDR of chain and light chain is inserted respectively into IgG1 heavy chain and the variable region of light chain of people, replaces the CDR of human antibody.Alemtuzumab
It is approved for the treatment of chronic lymphocytic leukemia the earliest, there is significant therapeutic effect.Due to the effect machine that it is special
Reason, follow-up has extensively carried out the clinical trial that multinomial autoimmune is relevant, including multiple sclerosis, GVHD etc..Wherein
Alemtuzumab is ratified by the regulator of the country such as European Union, Canada for the treatment of multiple sclerosis.
Alemtuzumab can specific recognition cell surface CD52 antigen, during lymphocyte differentiation, lymphocyte
Surface expression CD52 antigen, about 5x10 can be expressed in individual cells surface5CD52 molecule, constitutes antibody attack lymphocyte fine
Target spot.CD52 is made up of 12 amino acid residues, and Asn-3 is that N-connects glycosylation site, antigen and cell membrane binding site
It is C-end Ser-12, by the bonded cell membrane of glycolipid.Antigen component inconsistent (glycosylation) its molecular weight 21-of purification
After 28kD, N-Glycanase or Pronase process, decrease in molecular weight to 6kD.CD52 antigen molecule is the least, and has glycolipid
Property, antigenic determinant is near cell membrane, and these structures shape CD52 antigens are good drug targets.
CD52 antigen molecule is less, it is difficult to be directly used in ELISA detection.Additionally Alemtuzumab molecule mainly comprises portion
It is divided into human IgG1 region, and between each mammalian species, the homology of IgG1 is the highest, therefore in biological specimen
The specific detection of Alemtuzumab is short of specificity always.Rebello P. etc. are at Pharmacokinetics of
CAMPATH-1H:assay development and validation. J Immunol Methods 2002. describes
A kind of detection method based on HUT-78 cell line, but detection error based on cell is bigger, it is considered that can reach ±
25%, and this method operation complexity, time is longer.Iman Jilani etc. describes a kind of based on Alemtuzumab
The ELISA detection method Alemtuzumab:validation of a sensitive and simple of middle mouse CDR region
enzyme-linked immunosorbent assay. Leukemia Research 2004.The method carries to a certain extent
The high sensitivity of detection, but less stable.
Summary of the invention
The present invention establishes ELISA reaction system and the method for a set of detection anti-CD 52 antibody, including building CD52 polypeptide
This antigen is also modified by antigen, this antigen carries out wrapper sheet and catches anti-CD 52 antibody in sample, then by further combined with
Traget antibody such as enzyme len antibody, and develop the color by adding suitable substrates, by the contrast with concentration known standard substance, thus survey
Go out the content of antibody in sample.The method detection limit value is 0.1ug/ml, detection range 0.1 ~ 30ug/ml.
A kind of ELISA detection method detecting anti-CD 52 antibody of the present invention, generally specifically includes that synthesis CD52 polypeptide resists
Former and this polypeptide antigen is modified, this polypeptide antigen is configured to antigenic solution, is coated in ELISA Plate；Use afterwards and wash
Wash liquid and wash plate, add confining liquid after drying, wash plate with cleaning mixture the most again；Use testing sample buffer such as PBST or corresponding blood
Clear diluting biological specimen to be measured, diluted concentration is chosen according to biological specimen to be measured and is regulated, such as 10 ~ 50 times of dilutions, afterwards
Sample-adding, adds the ELISA Plate standing and reacting of sample, abandons liquid afterwards, wash plate with cleaning mixture；Add traget antibody such as enzyme len antibody to enter
Row reaction, washes plate afterwards；Be subsequently adding nitrite ion, ELISA Plate is put to microplate reader after terminating reaction by stop buffer, and OD450 reads
Number.Then by the contrast with concentration known standard substance, thus the content of anti-CD 52 antibody in sample is measured.
The pith of this ELISA detection method is synthesis and the modification of CD52 polypeptide antigen.CD52 sequence is (1-61):
MKRFLFLLLT ISLLVMVQIQ TGLSGQNDTS QTSSPSASSN ISGGIFLFFV ANAIIHLFCFS, described CD52 is many
The synthesis of peptide antigen, its aminoacid sequence is the polypeptide or protein sequence comprising XTSQTX or XQTSSX, and wherein X represents arbitrarily
Aminoacid sequence, particularly GQNDTSQTSSPS, 12 amino acid whose antibody recognition districts for choosing, the 25th to 36 amino
Acid: GQNDTSQTSSPS, carries out synthetic to it.Antigenic domains molecular weight due to synthetic, it is difficult to wrap
Plate, therefore the antigen to this synthetic carries out coupling and contains the label protein such as bovine serum albumin of-OH base-SH group
(BSA) modify.Bovine serum albumin (BSA), also known as BSA, is a kind of albumin in Ox blood serum, comprises 583 amino
Acid residue, molecular weight is 66.430 kDa, and isoelectric point, IP is 4.7.Bovine serum albumin is widely used in biochemical test, example
As in ELISA etc. in western blot as Blocking agent；BSA is added, by improving in endonuclease reaction buffer
The concentration of Proteins In Aqueous Solutions, shields to enzyme；Prevent decomposition and the non-specific adsorption of enzyme, the change of some enzyme can be alleviated
Property, some adverse environmental factors such as heating, the degeneration that surface tension and chemical factor cause can be alleviated.BSA is more stable, conventional
In various biologicallies, as protein protective agent and carrier.In addition to using bovine serum albumin, it is possible to use other are even
Connection thing is chemically modified, and is for example and without limitation to BSA, the label egg containing-OH base-SH group such as PEG, PEI, GST, MBP
In vain, straight or branched starchy carbohydrate, liposome, the hydrophobic carrier such as agarose.
With the contrast of concentration known standard substance for contrast with concentration known standard substance curve in the present invention, preferably pass through
Anti-CD 52 antibody standard substance and biological specimen to be measured is used to carry out parallel test, it is thus achieved that concentration known standard substance curve.Specifically,
By using containing the blank control sample gradient dilution CD52 antibody standard substance of PBST, and be loaded, the ELISA Plate after sample-adding according to
Above-mentioned same step carries out operation, and OD450 reading, obtains standard curve according to all readings by logistic fit.Should
Standard curve requires R2 > 0.98.Wherein, the dilution of CD52 antibody standard substance, generally by such as Gradient: start altogether from 40ug/ml
Do 9 gradients, successively 3 times of dilutions.
Testing sample buffer is conventional buffer, such as phosphate buffer etc., or with the replacement of corresponding serum, corresponding serum
For the serum of biological specimen to be measured, such as monkey, people, rat etc.；
Described biological specimen, can be people or animal serum (blood plasma), fermentation culture supernatant etc..
Described it is coated in the wrapper sheet condition that enzyme is put on, for using the CD52 polypeptide antigen solution of 2.5 μ g/ml, and in 4 DEG C of ice
Case stands overnight.
The described cleaning mixture washing plate, for sodium ascorbyl phosphate (pH 7.4 ± 0.2), washes plate number of times typically 2-6 time.
Described confining liquid is: 5% defatted milk powder or BSA or other conventional confining liquids.
Sample-adding process after described dilution step, during sample-adding, each sample is 2 multiple holes, every hole 100 μ l.
Described traget antibody is the human IgG antibody of luminescent substance labelling, and its label includes but not limited to Radix Cochleariae officinalis peroxidating
Thing enzyme (HRP), biotin (Biotin), Fluorescein isothiocyanate (FITC), alkali phosphatase (AP) etc., its test format is permissible
It is chemiluminescence, fluorescence, conventional visible etc..The mammal that anti-human IgG antibodies can commonly use prepares antibody, as anti-in rabbit, Mus
Anti-, goat-anti etc., it is also possible to be the IgG antibody of restructuring.One non-limitative example is such as: described traget antibody is enzyme len antibody,
Specifically it is for example and without limitation to the rabbit anti-human igg into enzyme connection labelling.Additionally, described enzyme len antibody also can multiple with other labellings
Anti-human igg.
The standing and reacting of the described ELISA Plate adding sample is antibody antigen association reaction, and its reaction temperature is 31-37 DEG C, time
Between be typically 1-4 hour.
Described nitrite ion is TMB(3,3', 5,5'-tetramethyl benzidine), the chromogenic reaction time is 5-30min, reaction
Temperature is 31-37 DEG C；Described stop buffer is 95% concentrated sulphuric acid.
A kind of ELISA reaction system for detecting anti-CD 52 antibody, mainly includes following components: synthetic also passes through
The CD52 polypeptide antigen of chemical modification, traget antibody, it is coated liquid, confining liquid, cleaning mixture, ELISA Plate, TMB(3,3', 5,5'-tetra-
Methyl biphenyl amine) nitrite ion, stop buffer, testing sample buffer or corresponding serum, biological specimen to be measured.
The liquid that is coated of this reaction system is phosphate buffer, and this phosphate buffer can be prepared and obtain, a non-limit
Property example processed is such as: weighing sodium carbonate 1.272g during preparation, sodium bicarbonate 2.344g, adds deionized water to 30ml, then adjusts pH extremely
9.4, add deionized water and be settled to 40ml.It is stored in 4 DEG C of refrigerators, dilutes 10 times of uses.
The confining liquid of this reaction system is defatted milk powder or BSA or other conventional confining liquids.
Described cleaning mixture is PBST(sodium ascorbyl phosphate) cleaning mixture.
The traget antibody of this reaction system is the human IgG antibody of luminescent substance labelling, and its label includes but not limited to peppery
Root peroxidase (HRP), biotin (Biotin), Fluorescein isothiocyanate (FITC), alkali phosphatase (AP) etc., its detection
Form can be chemiluminescence, fluorescence, conventional visible etc..The mammal that anti-human IgG antibodies can commonly use prepares antibody, as
Rabbit is anti-, mouse-anti, goat-anti etc., it is also possible to be the IgG antibody of restructuring.One non-limitative example is such as: 10ul HRP can be used to mark
Rabbit anti-human igg's stock solution of note joins concussion in 90ul PBS and mixes to obtain 1:10 enzyme len antibody.
The stop buffer purposes of this reaction system is color development stopping reaction, and improves the stability of colour developing, it is simple to detection.Example
If the stop buffer of HRP color development system can be 2M sulphuric acid, AP colour developing stop buffer can be NaOH.
Compared with prior art, beneficial effects of the present invention is as follows:
The invention provides a kind of ELISA reaction system for detecting anti-CD 52 antibody and method, this reaction system with
Method can be used for detecting the content of anti-CD 52 antibody in culture supernatant and biological specimen, and this method has height specificity, raw
Detection sensitivity in thing sample can reach 0.1ug/ml.
Certainly, the arbitrary product implementing the present invention it is not absolutely required to reach all the above advantage simultaneously.
Accompanying drawing explanation
Fig. 1 is the standard curve of the embodiment of the present invention 1；
Fig. 2 is the sample test curve of the embodiment of the present invention 1.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this
Bright, rather than limit protection scope of the present invention.Those skilled in the art are according to changing that the present invention makes in actual applications
Enter and adjust, still falling within protection scope of the present invention.
Embodiment 1
The present embodiment is said as a example by anti-CD 52 antibody in rabbit anti-human igg's enzyme len antibody detection machin serum of HRP labelling
The ELISA reaction system of the bright present invention and method.
The method specifically comprises the following steps that
The first step, wrapper sheet (Coating): take the synthetic of 25 μ l 1mg/ml and through the CD52 polypeptide of chemical modification
Antigen, joins 10ml and is coated in liquid mixing, is made into the antigenic solution that concentration is 2.5 μ g/ml, is coated in ELISA Plate, 100 μ
L/ hole, stands overnight in 4 DEG C of refrigerators.Described synthetic also comprises for choosing through the CD52 polypeptide antigen of chemical modification
The polypeptide of XTSQTX or XQTSSX or protein sequence, wherein X is the amino acid whose antibody recognition districts of 12 chosen, the 25th to 36
Individual aminoacid: GQNDTSQTSSPS, carries out synthetic, and carries out what coupling bovine serum albumin (BSA) modification obtained.Described
Being coated liquid is phosphate buffer.
Second step, closes (Blocking): discard liquid in hole, uses cleaning mixture to wash plate 3 times, and each 150rpm shakes
1min, adds the confining liquid in 250 μ l/ holes after drying, put 37 DEG C of incubators, acts on 3 hours, then washes 3 times with cleaning mixture, every time
150rpm shakes 1min.Described cleaning mixture is PBST(sodium ascorbyl phosphate) cleaning mixture.Described confining liquid be 5% defatted milk powder or BSA or
Other conventional confining liquids.
3rd step, the dilution of standard substance: simulating testing sample environment with the PBST containing 2% ~ 10% machin serum, gradient is dilute
Release anti-CD 52 antibody standard substance: take 8ul 2mg/ml CD52 antibody standard substance and join concentration in the PBST of 400ul 10% serum
For 40ug/ml, take 110ul 40ug/ml anti-CD 52 antibody and join in the PBST of 220ul 10% serum and be diluted to 13.33ug/
Ml, successively 3 times of dilutions.Start to do 9 gradients altogether from 40ug/ml.Each concentration does 2 multiple holes.
4th step, diluted sample: with PBST by biological sample to be measured 10 ~ 50 times dilution, during sample-adding, each sample does 2 again
Hole, every hole 100 μ l.
5th step, reaction: the ELISA Plate adding sample puts 37 DEG C of incubators, stands and carries out antigen-antibody binding reaction in 2 hours.
Abandoning liquid afterwards, wash plate 3 times with cleaning mixture, each 150rpm shakes 1min.Take 1:100 enzyme len antibody 20ul, dilute 500 with PBST
Times, after mixing, every hole 100 μ l adds in ELISA Plate, puts 37 DEG C of incubators, reacts 1 hour.Abandon liquid, wash plate 3 times with cleaning mixture, often
Secondary 150rpm shakes 1min.Clean absorbent paper pats dry.Described enzyme len antibody is rabbit anti-human igg's enzyme len antibody of HRP labelling.
6th step, develops the color, terminates, reading: every hole adds TMB 100 μ l, puts 37 DEG C of incubators, reaction 10min colour developing.It
Rear every hole adds stop buffer 50 μ l, terminates reaction.ELISA Plate is put to microplate reader, OD450 reading.Described stop buffer is 95%
Concentrated sulphuric acid.
Carry out anti-CD 52 antibody detection in machin serum according to the method described above, obtain anti-CD 52 antibody in machin serum
The OD value such as table 1 below of standard curve and testing sample.
Table 1
Wherein go up in table 1,
A1, A2 standard concentration 40ug/ml；
B1, B2 standard concentration 13.3ug/ml；
C1, C2 standard concentration 4.4ug/ml；
D1, D2 standard concentration 3.46ug/ml；
E1, E2 standard concentration 1.15ug/ml；
F1, F2 standard concentration 383.3ng/ml；
G1, G2 standard concentration 127.8ng/ml；
H1, H2 standard concentration 42.6ng/ml；
A3, A4 standard concentration 14.2ng/ml；
B3, B4 standard concentration 0 ng/ml；
C3, C4, D3, D4, E3, E4, F3, F4, G3, G4, H3, H4 are biological sample to be measured.
First pass through four parameter logistic fit and obtain standard curve such as Fig. 1, dense for machin serum anti-CD 52 antibody blood medicine
Degree monitoring standard curve.Wherein R2Being 0.997, detection limit is 0.1ug/ml-30ug/ml.
The detection of example 1 machin anti-CD 52 antibody pharmacokinetic trial blood drug level
Take 4 machins, be divided into 2 groups, often group 2.With 1mg/kg and 3mg/kg dosage injection anti-CD 52 antibody lotA and
LotB, intravenous injection 2 hours.Starting to inject latter 1 hour, after injection terminates, different time points takes Virus monitory blood drug level.
Operating procedure is ibid.The C3 that gained OD value is as shown in table 1, C4, D3, D4, E3, E4, F3, F4, G3, G4, H3, H4.This numerical value is tied
Fruit has difference in relatively low blank interference and group, shows that this method has good specificity and sensitivity.
Test result such as Fig. 2, for variable concentrations anti-CD 52 antibody machin therapeutic drug monitoring result.Fig. 2 can see
Going out, different antibodies lotA of injection same dose and the plasma concentration curve of lotB essentially coincide, and show that the method should in reality
The concentration of anti-CD 52 antibody in serum can be detected in accurately, it is possible to be widely used in anti-CD 52 antibody production technology,
In the researchs such as medicine generation, poison generation.
The method that the present invention provides can be used for detecting the anti-CD 52 antibody concentration in fermented liquid supernatant, it is possible to the biological sample of detection
Anti-CD 52 antibody concentration in Ben, as preclinical study pharmacokinetic test monkey serum antibody concentration, poison generation test
Anti-CD 52 antibody concentration in patients serum in Serum antibody concentrations, clinical trial.These are only that the present invention detects machin blood
The method of clear anti-CD 52 antibody concentration, but the present invention is not limited to this, any for anti-CD 52 antibody based on this theory
The change made, all should be within the scope of the present invention.
Under the teaching of the present invention and above-described embodiment, those skilled in the art are easy to it is envisioned that cited by the present invention
Or each raw material or its equivalent alterations, each processing method or its equivalent alterations enumerated can realize the present invention and each former
Material and the parameter bound value of processing method, interval value can realize the present invention, embodiment numerous to list herein.
Claims (11)
1. the ELISA detection method detecting anti-CD 52 antibody, it is characterised in that include synthesizing CD52 polypeptide antigen and to this
CD52 polypeptide antigen is modified, and the CD52 polypeptide antigen after this modification carries out wrapper sheet and catches anti-CD 52 antibody in sample, then
Further combined with traget antibody, and by add nitrite ion, stop buffer and develop the color, then right by with concentration known standard substance
Ratio, measures the content of anti-CD 52 antibody in sample；Wherein,
The synthesis of described CD52 polypeptide antigen, its aminoacid sequence is the polypeptide comprising XTSQTX or XQTSSX or albumen sequence
Row, wherein X is the amino acid whose antibody recognition districts of 12 chosen, and it is entered by the 25th to 36 aminoacid: GQNDTSQTSSPS
Row synthetic.
2. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 1, it is characterised in that described polypeptide is resisted
The former method that carries out modifying is: described polypeptide antigen carries out the modification that coupling contains the label protein of-OH base-SH group.
3. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 2, it is characterised in that described containing-OH
The label protein of base-SH group is selected from bovine serum albumin, Polyethylene Glycol, polymine, glutathione sulfydryl transferase, wheat
Bud carbohydrate-binding protein, straight or branched starchy carbohydrate, liposome, one or more of agarose.
4. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 1, it is characterised in that after modifying
CD52 polypeptide antigen carries out the method for anti-CD 52 antibody in wrapper sheet seizure sample: CD52 polypeptide antigen is configured to CD52 polypeptide
Antigenic solution, is coated in ELISA Plate；Use cleaning mixture to wash plate, after drying, add confining liquid；Use testing sample buffer or
Corresponding serum-dilution biological specimen to be measured, and be loaded；After adding the ELISA Plate standing and reacting of sample, abandon liquid, wash plate with cleaning mixture.
5. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 4, it is characterised in that described standing and reacting
For antibody antigen association reaction, its reaction temperature is 31-37 DEG C, and the time is 1-4 hour.
6. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 1, it is characterised in that described traget antibody
Human IgG antibody for luminescent substance labelling.
7. the ELISA detection method of the detection anti-CD 52 antibody as described in claim 1 or 6, it is characterised in that described labelling resists
Body is the rabbit anti-human igg of enzyme connection labelling.
8. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 1, it is characterised in that described nitrite ion
For TMB(3,3', 5,5'-tetramethyl benzidine), the chromogenic reaction time is 5-30min, and reaction temperature is 31-37 DEG C；Described end
Only liquid is 95% concentrated sulphuric acid.
9. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 1, it is characterised in that described and known dense
The contrast of degree standard substance is the contrast with concentration known standard substance curve, the R of described concentration known standard substance curve2 > 0.98。
10. the ELISA detection method detecting anti-CD 52 antibody as claimed in claim 1, it is characterised in that described ELISA examines
The limit value of survey method is 0.1ug/ml, detection range 0.1 ~ 30ug/ml.
11. 1 kinds for using the ELISA reaction system of the detection method detection anti-CD 52 antibody described in claim 1, its spy
Levy and be, mainly include following components: synthetic the CD52 polypeptide antigen through chemical modification, traget antibody, be coated liquid,
Confining liquid, cleaning mixture, ELISA Plate, 3,3', 5,5'-tetramethyl benzidine nitrite ions, stop buffer, testing sample buffer or corresponding
Serum, biological specimen to be measured；Wherein, described synthetic the CD52 polypeptide antigen through chemical modification comprise for choosing
The polypeptide of XTSQTX or XQTSSX or protein sequence, wherein X is the amino acid whose antibody recognition districts of 12 chosen, the 25th to 36
Individual aminoacid: GQNDTSQTSSPS, carries out synthetic, and the label protein that coupling afterwards contains-OH base-SH group obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410566000.8A CN104360057B (en) | 2014-10-22 | 2014-10-22 | A kind of ELISA reaction system detecting anti-CD 52 antibody and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410566000.8A CN104360057B (en) | 2014-10-22 | 2014-10-22 | A kind of ELISA reaction system detecting anti-CD 52 antibody and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104360057A CN104360057A (en) | 2015-02-18 |
| CN104360057B true CN104360057B (en) | 2016-10-05 |
Family
ID=52527338
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410566000.8A Active CN104360057B (en) | 2014-10-22 | 2014-10-22 | A kind of ELISA reaction system detecting anti-CD 52 antibody and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104360057B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003024993A2 (en) * | 2001-09-20 | 2003-03-27 | Board Of Regents, The University Of Texas System | Measuring circulating therapeutic antibody, antigen and antigen/antibody complexes using elisa assays |
| CN101395475A (en) * | 2006-03-07 | 2009-03-25 | 拜耳先灵医药公司 | Novel assay for the detection of an antibody bound to a cell membrane receptor |
| WO2009121024A2 (en) * | 2008-03-27 | 2009-10-01 | The Regents Of The University Of California | Methods for detecting antibodies |
| CN101619305A (en) * | 2007-10-19 | 2010-01-06 | 协和干细胞基因工程有限公司 | Antihuman CD52 monoclonal antibody hybridoma cell line, monoclonal antibody, engineered antibody, carrier, reagent kit and application thereof |
| CN102079787A (en) * | 2010-12-09 | 2011-06-01 | 协和干细胞基因工程有限公司 | Antihuman CD52 engineering antibody, carrier, kit and application thereof |
-
2014
- 2014-10-22 CN CN201410566000.8A patent/CN104360057B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003024993A2 (en) * | 2001-09-20 | 2003-03-27 | Board Of Regents, The University Of Texas System | Measuring circulating therapeutic antibody, antigen and antigen/antibody complexes using elisa assays |
| CN101395475A (en) * | 2006-03-07 | 2009-03-25 | 拜耳先灵医药公司 | Novel assay for the detection of an antibody bound to a cell membrane receptor |
| CN101619305A (en) * | 2007-10-19 | 2010-01-06 | 协和干细胞基因工程有限公司 | Antihuman CD52 monoclonal antibody hybridoma cell line, monoclonal antibody, engineered antibody, carrier, reagent kit and application thereof |
| WO2009121024A2 (en) * | 2008-03-27 | 2009-10-01 | The Regents Of The University Of California | Methods for detecting antibodies |
| CN102079787A (en) * | 2010-12-09 | 2011-06-01 | 协和干细胞基因工程有限公司 | Antihuman CD52 engineering antibody, carrier, kit and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Epididymal secretion of CD52 as measured in human seminal plasma by a fluorescence immunoassay;Iman Jilani et al;《Leukemia Research》;20041231;第28卷(第12期);1255–1262 * |
| 鼠抗人CD52单克隆抗体制备;刘友;《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》;20111231;E059-83 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104360057A (en) | 2015-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111566214B (en) | Anti-human IgG4 monoclonal antibody and human IgG4 assay reagent using same | |
| CN103134931B (en) | Double antibody sandwich method of detecting staphylococcus aureus enterotoxin A of food | |
| CN104614511B (en) | Immune chromatography analysis method, immune chromatography analysis device, and immune chromatography analysis reagent box | |
| CN110167967A (en) | For the idiotype antibody and application thereof of anti-PD-L1 antibody | |
| CN102246039A (en) | Insulin measurement method | |
| CN103616507A (en) | Protein-free coated plate confining liquid | |
| US20130224884A1 (en) | Device and method for immunoassays | |
| CN109596839A (en) | People and peptide element fast quantitative measurement method for detecting and kit | |
| CN113683702A (en) | Preparation method and application of polyclonal antibody of striped bamboo shark single-domain antibody | |
| CN108918851A (en) | A kind of preparation method of Lamotrigine colloidal gold strip | |
| CN107119023B (en) | The monoclonal antibody of the anti-human cardic fatty acid binding protein of resistance to aliphatic acid interference and its application | |
| Manouvriez et al. | Isotyping of rat monoclonal antibodies | |
| CN103257226A (en) | Colloidal gold triple-test strip for ractopamine, salbutamol and cimaterol, and preparation method and application thereof | |
| CN104360057B (en) | A kind of ELISA reaction system detecting anti-CD 52 antibody and method | |
| CN107973855A (en) | Monoclonal antibody reacted with glycopeptide and application thereof | |
| CN107478848A (en) | Quantitatively detect people NT proBNP kit and preparation method thereof | |
| US20200017582A1 (en) | Immunoassay method using anti-human bnp fragment (4-32) antibody | |
| KR101329344B1 (en) | Antibody for Detecting Hemagglutinin of Swine Influenza and Use Thereof | |
| CN102621311B (en) | Colloidal gold chromatography anti-SSB antibody detection test paper and preparation method thereof | |
| CN108727478A (en) | A kind of IBV specific polypeptides antigen, antibody, ELISA detection kit and its application | |
| CN111273041B (en) | ELISA kit for detecting phalloidin and preparation and application thereof | |
| CN102936584A (en) | Semicarbazide derivative monoclonal antibody and applications thereof | |
| CN102621313B (en) | Colloidal gold chromatography anti-Sp100 antibody detection test paper and preparation method thereof | |
| CN110514821A (en) | A kind of protein immunoblotting film is lived again liquid and its application method | |
| CN202083693U (en) | Test paper for detecting anti-ribosomal P0 antibody by adopting colloidal gold chromatographic assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhuang Chao Inventor after: Zeng Chen Inventor after: Zheng Chen Inventor after: Qi Nianmin Inventor before: Qi Nianmin Inventor before: Wang Peng Inventor before: Zhuang Chao Inventor before: Zeng Chen |
|
| COR | Change of bibliographic data | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhuang Chao Inventor after: Zeng Chen Inventor after: Zheng Chen Inventor after: Qi Nianmin Inventor after: Wang Peng Inventor before: Zhuang Chao Inventor before: Zeng Chen Inventor before: Zheng Chen Inventor before: Qi Nianmin |
|
| COR | Change of bibliographic data | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |