CN107860925B - For detecting the ELISA detection kit of HuA21 antibody in serum - Google Patents
For detecting the ELISA detection kit of HuA21 antibody in serum Download PDFInfo
- Publication number
- CN107860925B CN107860925B CN201711069106.7A CN201711069106A CN107860925B CN 107860925 B CN107860925 B CN 107860925B CN 201711069106 A CN201711069106 A CN 201711069106A CN 107860925 B CN107860925 B CN 107860925B
- Authority
- CN
- China
- Prior art keywords
- antibody
- hua21
- humanization
- thr
- ser
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (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 for detecting the ELISA detection kit of HuA21 antibody in serum.The kit includes standard antigen, capture antibody and detection antibody；The standard antigen is humanization HuA21 antibody；The capture antibody is the antibody 1A4 of anti-humanization HuA21 antibody, and the detection antibody is the antibody 1C9 of the anti-humanization HuA21 antibody through biotin labeling.Enzyme linked immunological (ELISA) kit provided by the present invention based on humanization HuA21 antibody content in double antibody sandwich method detection serum can realize the accurate quantitative analysis detection to humanization HuA21 antibody content in serum, and stability is good, easy to operate, expense is also cheap compared with traditional detection method.Therefore there is important application value.
Description
Technical field
The invention belongs to biological medicines and technical field, and in particular to a kind of for detecting HuA21 antibody in serum
ELISA detection kit.
Background technique
HuA21 antibody (China Patent No. ZL201410489895.X) is a kind of monoclonal of the full humanization of high-affinity
Antibody, energy specific recognition simultaneously combines human tumor cells surface HER2 antigen, so that the highly expressed breast cancer cell of HER2 be made to increase
Reproductive growth is suppressed.The experimental results showed that relative to the Herceptin (trade name Herceptin, the English name that have listed
Trastuzumab/Herceptin), the anti-HER 2 humanized monoclonal antibody drug that Genetech company in the U.S. develops first,
HuA21 antibody can cause stronger antigen endocytosis, especially in the cell strain of Herceptin drug resistance, such as
In the BT474 breast carcinoma cell strain of Herceptin drug resistance, therefore (such as antibody-chemical drug is even in antibody target medicine by HuA21
Connection object ADC) exploitation in have a good application prospect.Moreover, HuA21 can also inhibit the phosphorylation of HER2, and HER2
Phosphorylation be and then to inhibit the downstream HER2 MAPK, PI3K-Akt signal path necessary to HER2 activation, this phenomenon exists
HuA21 and Herceptin combination is more obvious.Moreover, HuA21 and Herceptin combination can significantly inhibit body tumour
Development, this result is tested in vivo and experiment in vitro is all confirmed.Therefore, HuA21 expresses tumour for example in HER2 high
There is huge application potential in terms of the treatment of breast cancer.
Either in the pharmacological evaluation stage of preclinical study, the pharmacokinetics of antibody drug is studied, also
It is treatment stage in clinical stage and still later, determines that patient is rationally administered in the therapeutic window concentration of drug, accurately comment
Estimate concentration of the HuA21 in organism blood to be all extremely important.The method of traditional measurement blood concentration is main
There are gas chromatography (GC), gas phase-Mass Spectrometry (GC-MS), tablets by HPLC-MS (HPLC-MS) etc..
But these method prevailing prices are high, and need the equipment operator of profession, analysis time is also long, it is difficult to face
Carry out on bed.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of accurate, efficient, economy for detecting HuA21 in serum
The ELISA detection kit of antibody.
It is provided by the present invention for detecting the enzyme linked immunological kit of HuA21 antibody content in serum, specifically may include
Standard antigen, capture antibody and detection antibody；The standard antigen is humanization HuA21 antibody (China Patent No.
ZL201410489895.X)；The capture antibody is the antibody 1A4 (as coating antigen) of anti-humanization HuA21 antibody, the inspection
Survey the antibody 1C9 that antibody is the anti-humanization HuA21 antibody through biotin labeling.
In the kit, the amino acid sequence of the heavy chain of the antibody 1A4 of the anti-humanization HuA21 antibody is specifically such as
Shown in SEQ ID No.1；The amino acid sequence of the light chain of the antibody 1A4 of the anti-humanization HuA21 antibody is specifically such as SEQ ID
Shown in No.2.
In the kit, the amino acid sequence of the heavy chain of the antibody 1C9 of the anti-humanization HuA21 antibody is specifically such as
Shown in SEQ ID No.3；The amino acid sequence of the light chain of the antibody 1C9 of the anti-humanization HuA21 antibody is specifically such as SEQ ID
Shown in No.4.
Further, the antibody 1A4 of the anti-humanization HuA21 antibody is by by nucleotide sequence such as SEQ ID
The encoding gene of the heavy chain of the antibody 1A4 of anti-humanization HuA21 antibody shown in No.5 and nucleotide sequence such as SEQ ID No.6
Shown in the encoding gene of light chain of antibody 1A4 of anti-humanization HuA21 antibody expressed in recipient cell.Its
In, heavy chain encoding gene and light chain encoding gene can use for equimolar.
Further, the antibody 1C9 of the anti-humanization HuA21 antibody is by by nucleotide sequence such as SEQ ID
The encoding gene of the heavy chain of the antibody 1C9 of anti-humanization HuA21 antibody shown in No.7 and nucleotide sequence such as SEQ ID No.8
Shown in the encoding gene of light chain of antibody 1C9 of anti-humanization HuA21 antibody expressed in recipient cell.Its
In, heavy chain encoding gene and light chain encoding gene can use for equimolar.
Further, the encoding gene of the heavy chain of the antibody 1A4 of the anti-humanization HuA21 antibody is by recombination table
Import what the recipient cell obtained up to carrier A.The recombinant expression carrier A is specifically by DNA piece shown in SEQ ID No.5
The recombinant plasmid that section obtains after being inserted between restriction enzyme site BsrG I and the Hind III of pcDNA3.4 (Life company) carrier
(being named as pcDNA3.4-1A4-1).The encoding gene of the light chain of the antibody 1A4 of the anti-humanization HuA21 antibody is to pass through weight
Group expression vector B imports what the recipient cell obtained.The recombinant expression carrier B is specifically will be shown in SEQ ID No.6
The weight obtained after between restriction enzyme site Xba I and the Hind III that DNA fragmentation is inserted into pcDNA3.4 (Life company) carrier
Group plasmid (being named as pcDNA3.4-1A4-2).
Further, the encoding gene of the heavy chain of the antibody 1C9 of the anti-humanization HuA21 antibody is by recombination table
Import what the recipient cell obtained up to support C.The recombinant expression carrier C is specifically by DNA piece shown in SEQ ID No.7
Recombinant plasmid (the life that section obtains after being inserted between the restriction enzyme site BsrGI and HindIII of pcDNA3.4 (Life company) carrier
Entitled pcDNA3.4-1C9-1).The encoding gene of the light chain of the antibody 1C9 of the anti-humanization HuA21 antibody is to pass through recombination
Expression vector D imports what the recipient cell obtained.The recombinant expression carrier D is specifically by DNA shown in SEQ ID No.8
The recombinant plasmid obtained after between the restriction enzyme site XbaI and HindIII that segment is inserted into pcDNA3.4 (Life company) carrier
(being named as pcDNA3.4-1C9-2).
More specifically, the antibody 1A4 of the anti-humanization HuA21 antibody is to be prepared by a method comprising the following steps
: (1) by the encoding gene (SEQ ID No.5) of the heavy chain of the antibody 1A4 of the anti-humanization HuA21 antibody and described anti-human
After the encoding gene (SEQ ID No.6) of the light chain of the antibody 1A4 of source HuA21 antibody is cloned into respectively in pcDNA3.4 carrier
Two obtained recombinant plasmids；(2) it by recipient cell described in the resulting two recombinant plasmid cotransfections of step (1), is recombinated
Cell cultivates the recombinant cell, obtains the antibody 1A4 of the anti-humanization HuA21 antibody.In the method, parent can be used
The antibody 1A4 of the anti-humanization HuA21 antibody is separated and purified with the method for chromatography, it can be by institute using the method
The antibody 1A4 purifying for stating anti-humanization HuA21 antibody is substantially uniform substance, such as is single item on SDS-PAGE electrophoresis
Band.
More specifically, the antibody 1C9 of the anti-humanization HuA21 antibody is to be prepared by a method comprising the following steps
: (1) by the encoding gene (SEQ ID No.7) of the heavy chain of the antibody 1C9 of the anti-humanization HuA21 antibody and described anti-human
After the encoding gene (SEQ ID No.8) of the light chain of the antibody 1C9 of source HuA21 antibody is cloned into respectively in pcDNA3.4 carrier
Two obtained recombinant plasmids；(2) it by recipient cell described in the resulting two recombinant plasmid cotransfections of step (1), is recombinated
Cell cultivates the recombinant cell, obtains the antibody 1C9 of the anti-humanization HuA21 antibody.In the method, parent can be used
The antibody 1C9 of the anti-humanization HuA21 antibody is separated and purified with the method for chromatography, it can be by institute using the method
The antibody 1C9 purifying for stating anti-humanization HuA21 antibody is substantially uniform substance, such as is single item on SDS-PAGE electrophoresis
Band.
Wherein, the recipient cell can be mammalian cell or insect cell.In the present invention, the recipient cell
Specially mammalian cell is more specifically human renal epithelial cell line HEK293F.
In the present invention, the detection antibody specific is prepared according to the method included the following steps: will be described anti-
The antibody 1C9 of humanization HuA21 antibody is mixed with the biotin of activation according to molar ratio 1:30, and (20-25 DEG C) of room temperature is placed half
Hour, the unbonded biotin of subsequent over-molecular sieve column removal obtains the anti-of the anti-humanization HuA21 antibody through biotin labeling
Body 1C9 is the detection antibody.
It, can also be containing at least one of following in the enzyme linked immunological kit: ELISA Plate (such as 96 in addition to above-mentioned substance
Hole elisa Plates), Horseradish peroxidase-conjugated avidin, bed board buffer, cleaning solution, confining liquid, developing solution and terminate liquid.
Wherein, the Avidin is the substance with Avidin binding characteristic.Specifically, described there is Avidin to combine spy
The substance of property can be streptavidin, albumen avidin, yolk avidin or class avidin.In one embodiment of the present of invention
In, the Avidin is specially Streptavidin.
In the present invention, the bed board buffer is specially the bicarbonate buffer that pH is 9.6.The cleaning solution is specific
The phosphate buffer for being 7.4 for the pH only containing 0.05% volumn concentration Tween 20.The confining liquid is specially only
The phosphate buffer that pH containing 30g/L BSA is 7.4.The developing solution is specially tetramethyl benzidine.The terminate liquid
Specially 1M sulfuric acid.
Further, the solvent for the bicarbonate buffer that the pH is 9.6 is water, and solute and concentration are as follows:
Na2CO31.59g/L；NaHCO32.93g/L.The solvent for the phosphate buffer that the pH is 7.4 is water, solute be sodium chloride,
Potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate；Concentration of the sodium chloride in the phosphate buffer that the pH is 7.4 is
135mM, concentration of the potassium chloride in the phosphate buffer that the pH is 7.4 is 2.7mM, and the potassium dihydrogen phosphate is in institute
State the concentration 1.5mM in the phosphate buffer that pH is 7.4, the phosphate-buffered that the disodium hydrogen phosphate is 7.4 in the pH
Concentration in liquid is 8mM.
The antibody 1A4 of previously described anti-humanization HuA21 antibody and/or the anti-humanization HuA21 antibody it is anti-
Body 1C9 also belongs to protection scope of the present invention.
The antibody 1C9 of the antibody 1A4 of the anti-humanization HuA21 antibody and/or the anti-humanization HuA21 antibody is making
Application in the standby enzyme linked immunological kit also belongs to protection scope of the present invention.
The enzyme linked immunological kit or " the antibody 1A4 and/or the anti-humanization of the anti-humanization HuA21 antibody
Application of the antibody 1C9 " of HuA21 antibody in detection serum in HuA21 antibody content also belongs to protection scope of the present invention.
The enzyme linked immunological kit or " the antibody 1A4 and/or the anti-humanization of the anti-humanization HuA21 antibody
Application of the antibody 1C9 " of HuA21 antibody in following (A) or (B) also belongs to protection scope of the present invention:
(A) product for assessing concentration of the humanization HuA21 antibody in organism blood is prepared；
(B) for assessing concentration of the humanization HuA21 antibody in organism blood.
In the present invention, serum described previously is specially human serum.The organism is specifically people.
The present invention also protects the preparation method of the enzyme linked immunological kit.
The preparation method of the enzyme linked immunological kit, specifically may include following steps: by by nucleotide sequence such as SEQ
The encoding gene of the heavy chain of the antibody 1A4 of anti-humanization HuA21 antibody shown in ID No.5 and nucleotide sequence such as SEQ ID
The encoding gene of the light chain of the antibody 1A4 of anti-humanization HuA21 antibody shown in No.6 is expressed to obtain in recipient cell
The antibody 1A4 of the anti-humanization HuA21 antibody.By by nucleotide sequence anti-humanization as shown in SEQ ID No.7
Encoding gene and the nucleotide sequence anti-humanization as shown in SEQ ID No.8 of the heavy chain of the antibody 1C9 of HuA21 antibody
It is anti-that the encoding gene of the light chain of the antibody 1C9 of HuA21 antibody is expressed to obtain in recipient cell the anti-humanization HuA21
The antibody 1C9 of body；Wherein, heavy chain encoding gene and light chain encoding gene can use for equimolar.
The experimental results showed that provided by the present invention based on humanization HuA21 antibody in double antibody sandwich method detection serum
Enzyme linked immunological (ELISA) kit of content can realize the accurate quantitative analysis detection to humanization HuA21 antibody content in serum, and
Stability is good, easy to operate, and expense is also cheap compared with traditional detection method.Therefore there is important application value.
Detailed description of the invention
Fig. 1 is HuA21 standard curve schematic diagram.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Following embodiments do not include detailed descriptions of conventional methods, as those for gene magnification, construction of recombinant plasmid,
And by plasmid introduce host cell method.Such method is all described in many publications, including Sambrook,
J.,et al.(1989)Molecular Cloning:A Laboratory Manual,2nd edition,Cold Spring
Harbor Laboratory Press。
The recombinant expression carrier of the encoding gene of antibody 1A4 (abbreviation antibody 1A4) containing anti-humanization HuA21 antibody
PcDNA3.4-1A4-1, which is will be between the restriction enzyme site BsrGI and HindIII of pcDNA3.4 carrier (Life company)
Small fragment replaces with the recombinant plasmid obtained after the encoding gene of the heavy chain of antibody 1A4 shown in SEQ ID No.5, the plasmid energy
Enough express the heavy chain (SEQ ID No.1) of antibody 1A4.
The recombinant expression carrier of the encoding gene of antibody 1A4 (abbreviation antibody 1A4) containing anti-humanization HuA21 antibody
PcDNA3.4-1A4-2, which is will be between the restriction enzyme site XbaI and HindIII of pcDNA3.4 carrier (Life company)
Small fragment replaces with the recombinant plasmid obtained after the encoding gene of the light chain of antibody 1A4 shown in SEQ ID No.6, the plasmid energy
Enough express the light chain (SEQ ID No.2) of antibody 1A4.
The recombinant expression carrier of the encoding gene of antibody 1C9 (abbreviation antibody 1C9) containing anti-humanization HuA21 antibody
PcDNA3.4-1C9-1, which is will be between the restriction enzyme site BsrGI and HindIII of pcDNA3.4 carrier (Life company)
Small fragment replaces with the recombinant plasmid obtained after the encoding gene of the heavy chain of antibody 1C9 shown in SEQ ID No.7, the plasmid energy
Enough express the heavy chain (SEQ ID No.3) of antibody 1C9.
The recombinant expression carrier of the encoding gene of antibody 1C9 (abbreviation antibody 1C9) containing anti-humanization HuA21 antibody
PcDNA3.4-1C9-2, which is will be between the restriction enzyme site XbaI and HindIII of pcDNA3.4 carrier (Life company)
Small fragment replaces with the recombinant plasmid obtained after the encoding gene of the light chain of antibody 1C9 shown in SEQ ID No.8, the plasmid energy
Enough express the light chain (SEQ ID No.4) of antibody 1C9.
Embodiment 1 is used to detect the method for preparation and use of the enzyme linked immunological kit of HuA21 antibody content in serum
One, for detecting the preparation of the enzyme linked immunological kit of HuA21 antibody content in serum
Serum HuA21 monoclonal antibody ELISA detection kit provided by the present invention, comprising: standard antigen, capture are anti-
Body, detection antibody and ELISA Plate, bed board buffer, cleaning solution, confining liquid, the strepto- of horseradish peroxidase-labeled is affine
Plain (HRP-SA), developing solution and terminate liquid etc..Wherein, the standard antigen is humanization HuA21 antibody；The capture antibody is
The antibody 1A4 of anti-humanization HuA21 antibody, the detection antibody is the anti-of the anti-humanization HuA21 antibody through biotin labeling
Body 1C9.
(1) conventional equipment and reagent
1, elisa plate (96 hole elisa Plates), Nunc company).
2, bed board buffer: the bicarbonate buffer of pH9.6 needs to restore to room temperature before use.
3, cleaning solution: the phosphate buffer that the pH only containing 0.05% volumn concentration Tween 20 is 7.4 is (i.e.
PBST buffer).
4, confining liquid: the phosphate buffer that the pH only containing 30g/L BSA is 7.4.
5, the Streptavidin of horseradish peroxidase-labeled.
6, chromogenic substrate: tetramethyl benzidine (TMB) needs to restore to room temperature before use；
7, terminate liquid: 1M sulfuric acid needs to restore to room temperature before use.
Wherein, the solvent for the bicarbonate buffer that the pH is 9.6 is water, and solute and concentration are as follows: Na2CO31.59g/
L；NaHCO32.93g/L.The solvent for the phosphate buffer that the pH is 7.4 is water, and solute is sodium chloride, potassium chloride, phosphoric acid
Potassium dihydrogen, disodium hydrogen phosphate；Concentration of the sodium chloride in the phosphate buffer that the pH is 7.4 is 135mM, the chlorine
Changing concentration of the potassium in the phosphate buffer that the pH is 7.4 is 2.7mM, and the potassium dihydrogen phosphate is 7.4 in the pH
Concentration 1.5mM in phosphate buffer, concentration of the disodium hydrogen phosphate in the phosphate buffer that the pH is 7.4 are
8mM。
(2) preparation of standard antigen
Standard antigen (humanization HuA21 antibody) is according to preparation described in Chinese patent ZL201410489895.X
The method preparation of HuA21 antibody.
The preparation of (three) two kinds of anti-HuA21 antibody
By taking antibody 1A4 as an example, equimolar pcDNA3.4-1A4-1 and pcDNA3.4-1A4-2 is gone into people's kidney wink simultaneously
Epithelial cell line HEK293F (ATCC American Cell library), after culture three days, using Protein A affinity column from culture
Antibody purification albumen in clear.It is quantified by antibody protein of the BCA method to purifying.
The preparation method synantibody 1A4 of antibody 1C9.
(4) the biotinylation label of antibody is detected
By biotin (the EZ-Link NHS-PEG4-Biotin reagent of the antibody 1C9 of above-mentioned steps (three) preparation and activation
Box, Pierce company) it is mixed according to molar ratio 1:30, (20-25 DEG C) placement half an hour of room temperature, subsequent over-molecular sieve column removal is not
In conjunction with biotin (aforementioned EZ-Link NHS-PEG4-Biotin kit included molecular sieve column) to get to biology
The 1C9 of element label detects antibody.
Two, the application method of the enzyme linked immunological kit of HuA21 content in serum is detected
1, standard curve is made using standard antigen
Antibody 1A4 prepared by above-mentioned steps is diluted to 1 μ g/ml with bed board buffer (formula is seen above), every hole adds 100
μ l bed board, 37 DEG C are closed 1 hour.Three times, every hole adds 300 μ l confining liquids (formula is seen above) to board-washing, and 4 DEG C overnight.Use cleaning solution
Concentration is standard antigen (the humanization HuA21 antibody) BSA containing 10g/L of 22mg/ml three times by (formula is seen above) board-washing
Serum Special sample diluted then pressed to 62.5ng/ml, then with 6 Ep pipe doubling dilutions at gradient solution (table 1)
Two multiple holes of each concentration, every 100 μ l of hole are added in plate, vibrate 1 hour.Three times, added with cleaning solution (formula is seen above) board-washing
1% (10g/L) BSA (solvent is PBST buffer) is diluted to the antibody 1C9 through biotin labeling of 0.25 μ g/ml, every hole 100
μ l vibrates 1 hour.With cleaning solution (formula is seen above) board-washing three times, every hole add 1% (10g/L) BSA (solvent be PBST buffering
Liquid) 8000 times of 100 μ l of Horseradish peroxidase-conjugated avidin (ThermoFisher company) of dilution, vibrates 1 hour.With washing
Wash liquid (formula is seen above) board-washing three times, every hole adds tetramethyl benzidine (tetramethylbenzidine, TMB)
(ThermoFisher company) 100 μ l.It is protected from light colour developing 4 minutes, the sulfuric acid of 1M is added to terminate reaction.With BIO-TEK ELX-800 enzyme
Mark instrument read plate at 450 nm.With the logarithm (being bottom with 10) of the concentration of standard antigen (humanization HuA21 antibody) for abscissa,
Using the logarithm of OD450 value as ordinate, using GraphPad Prism software, made using 4 parameter Logistic approximating methods
It is as shown in Figure 1 to draw standard curve for figure.
The corresponding light absorption value (OD450) of standard antigen (humanization HuA21 antibody) of each concentration is as shown in table 1.It will be each dense
The light absorption value (OD450) of the standard antigen (humanization HuA21 antibody) of degree substitutes into above-mentioned standard curve, and (actually curve is quasi-
Close equation) to get corresponding apparent concentration under each standard antigen (humanization HuA21 antibody) concentration, it is more right than upper with apparent concentration
The actual concentrations answered are up to the rate of recovery.It is computed, the corresponding apparent concentration of standard antigen (humanization HuA21 antibody) of each concentration
And the rate of recovery is referring to table 1.
The light absorption value and its apparent concentration and the rate of recovery after conversion of 1 various concentration standard antigen of table
| Standard antigen | Concentration (ng/ml) | Light absorption value (OD450) | Apparent concentration (ng/ml) | The rate of recovery (%) |
| S1 | 62.5 | 1.978 | 59.17398 | 95 |
| S2 | 31.25 | 1.613 | 32.1717 | 103 |
| S3 | 15.625 | 0.972 | 14.99625 | 96 |
| S4 | 7.8125 | 0.589 | 8.148375 | 104 |
| S5 | 3.90625 | 0.331 | 4.089321 | 105 |
| S6 | 1.953125 | 0.173 | 1.715569 | 88 |
| S7 | 0.976563 | 0.1215 | 0.965808 | 99 |
| S8 | 0.0 | 0.057 | - | - |
2, in human serum sample to be measured HuA21 antibody content measurement
Standard antigen (humanization HuA21 antibody) in step 1 is replaced with into human serum sample to be measured, it will be resulting
OD450 value substitutes into the resulting standard curve of step 1, to obtain the content of HuA21 antibody in human serum sample to be measured.
The determination of recovery rates of the enzyme linked immunological kit of HuA21 antibody content in embodiment 2, detection human serum
The present inventor is further using the normal human serum conduct of addition standard antigen (humanization HuA21 antibody)
Sample to be tested determines time for being used to detect the enzyme linked immunological kit of HuA21 antibody content in human serum of the preparation of embodiment 1
Yield.
For totally 3 parts of normal human serum of examination, each blood serum sample represents one group.
Antibody 1A4 prepared by above-mentioned steps is diluted to 1 μ g/ml with bed board buffer (formula is seen above), every hole adds 100
μ l bed board, 37 DEG C are closed 1 hour.Three times, every hole adds 300 μ l confining liquids (formula is seen above) to board-washing, and 4 DEG C overnight.Use cleaning solution
(formula is seen above) board-washing is added to the serum Special sample dilution of BSA containing 10g/L according to 1:100 three times, by normal human serum
In, obtain three kinds of different serum Special sample dilutions.Then it is by concentration with three kinds of serum Special sample dilutions
The standard antigen (humanization HuA21 antibody) of 22mg/ml is diluted to 31.25ng/ml, 7.8125ng/ml and 1.953125ng/ml
Totally three concentration gradients, by two multiple holes of each sample, every 100 μ l of hole is added in plate, is vibrated 1 hour.With cleaning solution, (formula is shown in
Above) board-washing three times, add 1% (10g/L) BSA (solvent is PBST buffer) be diluted to 0.25 μ g/ml through biotin labeling
Antibody 1C9, every 100 μ l of hole, vibrate 1 hour.With cleaning solution (formula is seen above) board-washing three times, every hole adds 1% (10g/L)
BSA (solvent is PBST buffer) dilutes 8000 times of Horseradish peroxidase-conjugated avidin (ThermoFisher company)
100 μ l vibrate 1 hour.With cleaning solution (formula is seen above) board-washing three times, every hole adds tetramethyl benzidine
(tetramethylbenzidine, TMB) (ThermoFisher company) 100 μ l.It is protected from light colour developing 4 minutes, adds the sulfuric acid of 1M whole
Only react.With BIO-TEK ELX-800 microplate reader read plate at 450 nm.It is resulting that resulting OD450 value is substituted into embodiment 1
Standard curve (the actually equation of curve), to obtain the actual measurement content of HuA21 antibody in each sample.
It the results are shown in Table 2, average recovery rate is up to 93%.This is the result shows that using kit of the invention in sandwich method
The available satisfactory rate of recovery in ELISA detection.
The kit of the present invention of table 2 does serum antigen rate of recovery test result
| Blood serum sample title | It adds antigen concentration (ng/ml) | It surveys antigen concentration (ng/ml) | The rate of recovery (%) |
| Serum 1 | 31.25 | 29.90561 | 96 |
| Serum 1 | 7.8125 | 7.667601 | 98 |
| Serum 1 | 1.953125 | 1.823703 | 93 |
| Serum 2 | 31.25 | 32.88572 | 105 |
| Serum 2 | 7.8125 | 7.56085 | 97 |
| Serum 2 | 1.953125 | 1.636119 | 84 |
| Serum 3 | 31.25 | 27.37783 | 88 |
| Serum 3 | 7.8125 | 7.212758 | 92 |
| Serum 3 | 1.953125 | 1.707985 | 87 |
| Average recovery rate | 93 |
<110>the vast Ke Maibo Bioisystech Co., Ltd in Hefei
<120>for detecting the ELISA detection kit of HuA21 antibody in serum
<130> GNCLN171907
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 463
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 1
Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Glu Gln Leu Glu Glu Ser Gly Gly Asp Leu Val Lys
20 25 30
Pro Glu Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe
35 40 45
Ser Ser Met Phe Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly
50 55 60
Leu Glu Trp Val Ala Cys Ile Gly Gly Gly Asp Gly Thr Thr Asn Tyr
65 70 75 80
Ala Ser Trp Ala Thr Gly Arg Phe Thr Ile Thr Arg Ser Ser Ser Leu
85 90 95
Asn Thr Val Thr Leu Arg Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala
100 105 110
Thr Tyr Phe Cys Ala Arg Ala Ala Asp Gly Val Ala Thr Asp Phe Ser
115 120 125
Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys
130 135 140
Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser
145 150 155 160
Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro
165 170 175
Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr
180 185 190
Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
195 200 205
Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His
210 215 220
Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys
225 230 235 240
Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val
245 250 255
Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
260 265 270
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu
275 280 285
Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg
290 295 300
Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser
305 310 315 320
Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys
325 330 335
Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
340 345 350
Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly
355 360 365
Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met
370 375 380
Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn
385 390 395 400
Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser
405 410 415
Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu
420 425 430
Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu
435 440 445
His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455 460
<210> 2
<211> 236
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 2
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala Ser Ser
20 25 30
Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser
35 40 45
Pro Asn Val Tyr Gly Gly Asn Arg Leu Ser Trp Phe Gln Lys Lys Pro
50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser
65 70 75 80
Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr
85 90 95
Leu Thr Ile Ser Glu Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Gln Gly Tyr Phe Phe Gly Asp Ile Trp Ala Phe Gly Gly Gly Thr Glu
115 120 125
Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro
130 135 140
Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val
145 150 155 160
Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly
165 170 175
Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser
180 185 190
Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr
195 200 205
Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr
210 215 220
Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235
<210> 3
<211> 459
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 3
Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser
35 40 45
Ser Tyr Thr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln
50 55 60
Tyr Ile Gly Ile Ile Thr Gly Ser Asn Asn Pro Tyr Tyr Ala Ser Trp
65 70 75 80
Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95
Lys Ile Thr Ser Pro Thr Thr Ala Asp Thr Ala Thr Tyr Phe Cys Ala
100 105 110
Arg Gly Arg Asp Gly Thr Ile Ser Gly Asp Tyr Gly Leu Trp Gly Gln
115 120 125
Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val
130 135 140
Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr
145 150 155 160
Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr
165 170 175
Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val
180 185 190
Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr
195 200 205
Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn
210 215 220
Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Met
225 230 235 240
Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr
275 280 285
Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg
290 295 300
Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
305 310 315 320
Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg
340 345 350
Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu
355 360 365
Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
385 390 395 400
Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly Ser Tyr
405 410 415
Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
420 425 430
Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455
<210> 4
<211> 236
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 4
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser
20 25 30
Val Ser Glu Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser
35 40 45
Glu Ser Ile Tyr Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60
Pro Pro Lys Leu Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
85 90 95
Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
Gly Tyr His Met Tyr Asn Ile Asp Asn Val Phe Gly Gly Gly Thr Glu
115 120 125
Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro
130 135 140
Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val
145 150 155 160
Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly
165 170 175
Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser
180 185 190
Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr
195 200 205
Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr
210 215 220
Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235
<210> 5
<211> 1392
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 5
atggagacag gactgagatg gctgctgctg gtggctgtgc tgaagggtgt acagtgccag 60
gagcagctgg aggagtctgg aggagacctg gtgaagcctg agggatctct gacactgaca 120
tgcacagctt ctggattctc tttctctagc atgttctgga tctgctgggt gagacaggct 180
cctggcaagg gactggagtg ggtggcttgc atcggaggag gagacggcac aacaaactac 240
gcttcttggg ctacaggcag attcacaatc acaagatctt cttctctgaa cacagtgaca 300
ctgagactga actctctgac agctgctgac acagctacat acttctgcgc tagagctgct 360
gacggagtgg ctacagactt ctctctgtgg ggacctggaa cactggtgac agtctcgagc 420
ggacagccta aggctccttc tgtgttcccg ctagctcctt gctgcggaga cacaccttct 480
tctactgtga cactgggctg cctggtgaag ggatacctgc ctgagcctgt gacagtgaca 540
tggaactctg gaacactgac aaacggagtg agaacattcc cttctgtgag acagtcttct 600
ggactgtact ctctgtcctc tgtggtgtct gtgacatcta gctctcagcc tgtgacatgc 660
aacgtggctc accctgctac aaacacaaag gtggacaaga cagtggctcc ttccacatgc 720
tctaagccta tgtgccctcc tcctgagctg cctggaggac cttctgtgtt catcttccct 780
cctaagccta aggacacact gatgatctcc agaacacctg aggtgacatg cgtggtggtg 840
gacgtgtctc aggacgaccc tgaggtgcag ttcacatggt acatcaacaa cgagcaggtg 900
agaacagcta gacctcctct gagagagcag cagttcaact ccacaatcag agtggtgtct 960
acactgccta tcgctcacca ggactggctg agaggaaagg agttcaagtg caaggtgcac 1020
aacaaggctc tgcctgctcc tatcgagaag acaatctcca aggctagagg acagcctctg 1080
gagcctaagg tgtatacaat gggacctcct agagaggagc tgtctagcag atctgtgtct 1140
ctgacatgca tgatcaacgg attctaccct tctgacatct ctgtggagtg ggagaagaac 1200
ggaaaggctg aggacaacta caagacaaca cctacagtgc tggactctga cggatcttac 1260
ttcctgtact ctaagctgtc tgtgcctaca tctgagtggc agagaggaga cgtgttcaca 1320
tgctctgtga tgcacgaggc tctgcacaac cactacacac agaagtctat ctccagatct 1380
cctggcaagt ga 1392
<210> 6
<211> 711
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 6
atggacacac gcgctcctac acagctgctg ggactgctgc tgctgtggct gcctggcgct 60
acattcgctc aggtgctgac acagacagct tcttctgtgt ctgctgctgt gggcggaaca 120
gtgaccatct cttgccagtc ttctcctaac gtgtacggag gaaacagact gtcttggttc 180
cagaagaagc ctggccagcc tcctaagctg ctgatctact acgcttctac actggcttct 240
ggcgtgccta gcagattcaa gggctctggc tctggaacag agttcacact gacaatctct 300
gaggtgcagt gcgacgacgc tgctacatac tactgccagg gctacttctt cggcgacatc 360
tgggctttcg gaggaggtac cgaggtggtg gtgaagggag accctgtggc tcctacagtg 420
ctgatcttcc ctcctgctgc tgaccaggtg gctacaggaa cagtgacaat cgtgtgcgtg 480
gctaacaagt acttccctga cgtgacagtg acatgggagg tggacggaac cacacagacc 540
acaggaatcg agaactccaa gacacctcag aactctgctg actgcaccta caacctgagc 600
agcacactga cactgaccag cacacagtac aacagccaca aggagtacac atgcaaggtg 660
acacagggaa caacatctgt ggtgcagtcc ttcaacagag gagactgctg a 711
<210> 7
<211> 1380
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 7
atggagacag gactgagatg gctgctgctg gtggctgtgc tgaagggtgt acagtgccag 60
tctctggagg agtctggagg aagactggtg acacctggaa cacctctgac actgacatgc 120
acagtgtctg gaatcgacct gtcttcttac accatgatct gggtgagaca ggctcctgga 180
aagggactgc agtacatcgg catcatcaca ggctccaaca acccttacta cgcttcctgg 240
gctaagggca gattcaccat ctctaagaca tctacaaccg tggacctgaa gatcacatct 300
cctacaacag ctgacacagc tacatacttc tgcgctagag gcagagacgg cacaatctct 360
ggagactacg gactgtgggg acagggaaca ctggtgacag tctcgagcgg acagcctaag 420
gctccttctg tgttcccgct agctccttgc tgcggagaca caccttcttc tactgtgaca 480
ctgggctgcc tggtgaaggg atacctgcct gagcctgtga cagtgacatg gaactctgga 540
acactgacaa acggagtgag aacattccct tctgtgagac agtcttctgg actgtactct 600
ctgtcctctg tggtgtctgt gacatctagc tctcagcctg tgacatgcaa cgtggctcac 660
cctgctacaa acacaaaggt ggacaagaca gtggctcctt ccacatgctc taagcctatg 720
tgccctcctc ctgagctgcc tggaggacct tctgtgttca tcttccctcc taagcctaag 780
gacacactga tgatctccag aacacctgag gtgacatgcg tggtggtgga cgtgtctcag 840
gacgaccctg aggtgcagtt cacatggtac atcaacaacg agcaggtgag aacagctaga 900
cctcctctga gagagcagca gttcaactcc acaatcagag tggtgtctac actgcctatc 960
gctcaccagg actggctgag aggaaaggag ttcaagtgca aggtgcacaa caaggctctg 1020
cctgctccta tcgagaagac aatctccaag gctagaggac agcctctgga gcctaaggtg 1080
tatacaatgg gacctcctag agaggagctg tctagcagat ctgtgtctct gacatgcatg 1140
atcaacggat tctacccttc tgacatctct gtggagtggg agaagaacgg aaaggctgag 1200
gacaactaca agacaacacc tacagtgctg gactctgacg gatcttactt cctgtactct 1260
aagctgtctg tgcctacatc tgagtggcag agaggagacg tgttcacatg ctctgtgatg 1320
cacgaggctc tgcacaacca ctacacacag aagtctatct ccagatctcc tggcaagtga 1380
<210> 8
<211> 711
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 8
atggacacaa gagcacctac acagctgctg ggactgctgc tgctgtggct gcctggagct 60
agatgcgctt acgacatgac acagacacct gcttctgtgt ctgagcctgt gggaggaaca 120
gtgacaatca agtgccaggc ttctgagtct atctactctt ggctggcttg gtatcagcag 180
aagcctggac agcctcctaa gctgctgatc tacgacgctt ctaagctggc ttctggagtg 240
cctagcagat tcaagggatc tggatctgga acagagttca cactgacaat ctctgacctg 300
gagtgcgacg acgctgctac ctactactgc cagcagggct accacatgta taacatcgac 360
aacgtgttcg gaggaggtac cgaggtggtg gtgaagggag accctgtggc tcctacagtg 420
ctgatcttcc ctcctgctgc tgaccaggtg gctacaggaa cagtgacaat cgtgtgcgtg 480
gctaacaagt acttccctga cgtgacagtg acatgggagg tggacggaac cacacagacc 540
acaggaatcg agaactccaa gacacctcag aactctgctg actgcaccta caacctgagc 600
agcacactga cactgaccag cacacagtac aacagccaca aggagtacac atgcaaggtg 660
acacagggaa caacatctgt ggtgcagtcc ttcaacagag gagactgctg a 711
Claims (10)
1. a kind of for detecting the enzyme linked immunological kit of HuA21 antibody content in serum, including standard antigen, capture antibody and
Detect antibody；The standard antigen is humanization HuA21 antibody；The capture antibody is the antibody of anti-humanization HuA21 antibody
1A4, the detection antibody are the antibody 1C9 of the anti-humanization HuA21 antibody through biotin labeling；
The amino acid sequence of the heavy chain of the antibody 1A4 of the anti-humanization HuA21 antibody is as shown in SEQ ID No.1；It is described anti-
The amino acid sequence of the light chain of the antibody 1A4 of humanization HuA21 antibody is as shown in SEQ ID No.2；
The amino acid sequence of the heavy chain of the antibody 1C9 of the anti-humanization HuA21 antibody is as shown in SEQ ID No.3；It is described anti-
The amino acid sequence of the light chain of the antibody 1C9 of humanization HuA21 antibody is as shown in SEQ ID No.4.
2. enzyme linked immunological kit according to claim 1, it is characterised in that: the anti-humanization HuA21 antibody resists
Body 1A4 is by will be nucleotide sequence heavy chain of the antibody 1A4 of anti-humanization HuA21 antibody as shown in SEQ ID No.5
The coding of encoding gene and the nucleotide sequence light chain of the antibody 1A4 of anti-humanization HuA21 antibody as shown in SEQ ID No.6
What gene was expressed in recipient cell.
3. enzyme linked immunological kit according to claim 1, it is characterised in that: the anti-humanization HuA21 antibody resists
Body 1C9 is by will be nucleotide sequence heavy chain of the antibody 1C9 of anti-humanization HuA21 antibody as shown in SEQ ID No.7
The coding of encoding gene and the nucleotide sequence light chain of the antibody 1C9 of anti-humanization HuA21 antibody as shown in SEQ ID No.8
What gene was expressed in recipient cell.
4. enzyme linked immunological kit according to claim 1 to 3, it is characterised in that: the enzyme linked immunological kit
In also containing at least one of following: ELISA Plate, the Avidin of horseradish peroxidase-labeled, bed board buffer, cleaning solution,
Confining liquid, developing solution and terminate liquid.
5. enzyme linked immunological kit according to claim 4, it is characterised in that: it is 9.6 that the bed board buffer, which is pH,
Bicarbonate buffer；
The cleaning solution is the phosphate buffer that the pH only containing 0.05% volumn concentration Tween20 is 7.4；
The confining liquid is the phosphate buffer that the pH only containing 30g/L BSA is 7.4；
The developing solution is tetramethyl benzidine；
The terminate liquid is 1M sulfuric acid.
6. antibody, be the anti-humanization HuA21 antibody described in claim 1-5 is any antibody 1A4 and the anti-human source
Change the antibody 1C9 of HuA21 antibody.
7. application of the antibody as claimed in claim 6 in the enzyme linked immunological kit described in preparation claim 1-5.
8. enzyme linked immunological kit as claimed in any one of claims 1 to 5 or antibody as claimed in claim 6 are in detection serum
Application in HuA21 antibody content.
9. enzyme linked immunological kit as claimed in any one of claims 1 to 5 or antibody as claimed in claim 6 at following (A) or
(B) application in:
(A) product for assessing concentration of the HuA21 antibody in organism blood is prepared；
(B) for assessing concentration of the HuA21 antibody in organism blood.
10. the preparation method of enzyme linked immunological kit as claimed in any one of claims 1 to 5, includes the following steps:
By by the coding of nucleotide sequence heavy chain of the antibody 1A4 of anti-humanization HuA21 antibody as shown in SEQ ID No.5
The encoding gene of gene and the nucleotide sequence light chain of the antibody 1A4 of anti-humanization HuA21 antibody as shown in SEQ ID No.6
It is expressed to obtain the antibody 1A4 of the anti-humanization HuA21 antibody in recipient cell；
By by the coding of nucleotide sequence heavy chain of the antibody 1C9 of anti-humanization HuA21 antibody as shown in SEQ ID No.7
The encoding gene of gene and the nucleotide sequence light chain of the antibody 1C9 of anti-humanization HuA21 antibody as shown in SEQ ID No.8
It is expressed to obtain the antibody 1C9 of the anti-humanization HuA21 antibody in recipient cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711069106.7A CN107860925B (en) | 2017-11-03 | 2017-11-03 | For detecting the ELISA detection kit of HuA21 antibody in serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711069106.7A CN107860925B (en) | 2017-11-03 | 2017-11-03 | For detecting the ELISA detection kit of HuA21 antibody in serum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107860925A CN107860925A (en) | 2018-03-30 |
| CN107860925B true CN107860925B (en) | 2019-07-12 |
Family
ID=61700523
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711069106.7A Active CN107860925B (en) | 2017-11-03 | 2017-11-03 | For detecting the ELISA detection kit of HuA21 antibody in serum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107860925B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023086901A1 (en) * | 2021-11-12 | 2023-05-19 | Virginia Tech Intellectual Properties, Inc. | Borrelia burgdorferi peptidoglycan detection methods |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2849508C (en) * | 2011-09-30 | 2020-12-22 | Regeneron Pharmaceuticals, Inc. | Anti-erbb3 antibodies and uses thereof |
| US9803245B2 (en) * | 2012-03-14 | 2017-10-31 | University Health Network | Signature for predicting clinical outcome in human HER2+ breast cancer |
| NZ715896A (en) * | 2013-07-05 | 2022-02-25 | Genmab As | Humanized or chimeric cd3 antibodies |
| CN104225594B (en) * | 2014-09-23 | 2016-03-23 | 合肥瀚科迈博生物技术有限公司 | Anti-HER 2 humanized antibody and relevant anti-tumor compositions thereof |
| CN106248971B (en) * | 2016-08-19 | 2017-10-03 | 合肥瀚科迈博生物技术有限公司 | For detecting the ELISA kit of HER2 contents, application method and purposes in human serum |
-
2017
- 2017-11-03 CN CN201711069106.7A patent/CN107860925B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107860925A (en) | 2018-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111303279B (en) | Single-domain antibody for novel coronavirus and application thereof | |
| CN112250763B (en) | Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use | |
| WO2022007304A1 (en) | Iga antibody specifically recognizing rbd protein and testing kit | |
| Forrest et al. | Structure-function analysis of reovirus binding to junctional adhesion molecule 1: implications for the mechanism of reovirus attachment | |
| Leonard et al. | High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100 | |
| CN105793439B (en) | Assays with improved performance against IGFBP7 with biological samples | |
| Hastie et al. | Convergent structures illuminate features for germline antibody binding and pan-Lassa virus neutralization | |
| CN110684740B (en) | Monoclonal antibody of anti-human ubiquitin carboxyl terminal hydrolase-1 (UCH-L1) and application thereof | |
| CN107344968B (en) | Time-resolved fluorescence immunoassay method for detecting avian influenza virus H7N9 | |
| Gaiotto et al. | Cross-neutralising nanobodies bind to a conserved pocket in the hemagglutinin stem region identified using yeast display and deep mutational scanning | |
| Dacon et al. | Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses | |
| CN112111011B (en) | Variable region sequence of specific anti-thiamethoxam antibody and recombinant full-length antibody thereof | |
| Rosenfeld et al. | Combinatorial and computational approaches to identify interactions of macrophage colony-stimulating factor (M-CSF) and its receptor c-FMS | |
| CN108383907A (en) | Nano antibody for Procalcitonin and its application | |
| CN114702578B (en) | Novel coronavirus Omicron mutant strain specific antibody and application thereof | |
| Hunt et al. | Multivalent designed proteins protect against SARS-CoV-2 variants of concern | |
| Togtema et al. | Single-domain antibodies represent novel alternatives to monoclonal antibodies as targeting agents against the human papillomavirus 16 E6 protein | |
| CN107860925B (en) | For detecting the ELISA detection kit of HuA21 antibody in serum | |
| Pedrazzi et al. | Affinity and folding properties both influence the selection of antibodies with the selectively infective phage (SIP) methodology | |
| Qin et al. | Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b | |
| Penfold et al. | Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells | |
| CN112794910B (en) | anti-PD-1 nano antibody and application thereof | |
| Owens et al. | Screening Random Peptide Libraries with Subacute Sclerosing PanencephalitisBrain-Derived Recombinant Antibodies Identifies Multiple Epitopes in the C-Terminal Region of the Measles Virus Nucleocapsid Protein | |
| CN112041341B (en) | Antibodies and assays for CCL14 | |
| Ishi et al. | A facile method to screen inhibitors of protein–protein interactions including MDM2–p53 displayed on T7 phage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |