CN104849462B - High-sensitivity anti-CD20 monoclonal antibody and applications thereof - Google Patents
High-sensitivity anti-CD20 monoclonal antibody and applications thereof Download PDFInfo
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Abstract
The present invention provides a high-sensitivity anti-CD20 monoclonal antibody and applications thereof, particularly a polypeptide, which is characterized in that the polypeptide has a structure represented by a formula I X0-Cys-Pro-Tyr-Ala-Asn-Pro-Ser-Leu-Cys-X1-COOH, wherein X0 is no or acetyl (CH3-CO-) and other protection groups, X1 is no or a peptide fragment comprising 1-10 amino acids, and the polypeptide has an anti-CD20 monoclonal antibody binding activity. According to the present invention, the polypeptide can specifically bind with anti-CD20 monoclonal antibodies so as to accurately determine the extremely trace rituximab and other anti-CD20 monoclonal antibodies in the sample through ELISA, and the method has characteristics of simpleness, economy, strong specificity, and high sensitivity.
Description
Technical field
The present invention relates to detection of biological samples field, in particular it relates to the anti-cd20 such as Rituximab (rituximab) is mono-
The detection of clonal antibody.
Background technology
Existing multiple methods are used for detecting the recombined human such as rituximab-Mus inosculating antibody cd20 monoclonal antibody at present.As utilized
The monoclonal antibody of Rituximab or polyclonal antibody are detected with elisa method.Have been reported that using unlabelled goat-anti rituximab
Monoclonal antibody polyclonal antibody as being coated thing, anti-set up elisa and test to detect rituximab list as two by hrp- goat-anti people's fc fragment
Anti-.Also someone devises a kind of Mus anti-Rituximab mb2a4, and with this antibody as being coated thing, hrp- goat-anti people's fc fragment is made
Test detecting Rituximab for the two anti-elisa that set up.Can also be made using the unlabelled goat-anti people that monkey serum adsorbed
For being coated thing, hrp- goat-anti people that monkey serum adsorbed anti-set up elisa and tests to detect Rituximab as two.These sides
Method is all the conventional elisa method based on Ag-Ab.
These above-mentioned methods still suffer from some shortcomings.Such as polyclonal antibody specificity is not strong, monoclonal antibody cost
Higher, and all have using the conventional elisa method of antigen and antibodies principle design that background is higher and a certain degree of intersection
Reaction, often especially prominent, the detection of other compositions severe jamming Rituximab in blood in the clinical blood sample of detection.
Therefore, this area is low, single-minded in the urgent need to setting up a kind of new background for detecting anti-cd20 monoclonal antibody
Property strong, sensitivity is high, the elisa method of low cost, for use in detection biological sample in cd20 monoclonal antibody.
Content of the invention
The invention provides the polypeptide of the anti-cd20 monoclonal antibody of detection of a kind of high specific and sensitivity and its application.
A kind of a first aspect of the present invention, there is provided polypeptide, has a structure as formula i:
x0-cys-pro-tyr-ala-asn-pro-ser-leu-cys-x1
Formula i；
Wherein, x0 is no, or protection group, or the peptide fragment of 1-10 Amino acid profile；X1 is no, or 1-10 aminoacid structure
The peptide fragment becoming；
And described polypeptide has the activity being combined with anti-cd20 monoclonal antibody.
In another preference, described anti-cd20 monoclonal antibody includes chimeric antibody, humanized antibody, non-humanization
Antibody, single-chain antibody, antibody fragment.
In another preference, described anti-cd20 monoclonal antibody is Chimeric antibody.
In another preference, described anti-cd20 monoclonal antibody derives from mammal, preferably source and people or
Rodent, more preferably, from people, mice or rat.
In another preference, the least concentration rank of described anti-cd20 monoclonal antibody is 1 × 10-1μg/ml.
In another preference, described protection group includes acetyl group (ch3-co-) or tertbutyloxycarbonyl (boc)；And/or
X0 is 8-9,6-7 is individual, 4-5 is individual or 2-3 is individual, the peptide fragment of more preferably 1-2 Amino acid profile；And/or
X1 is 8-9,6-7 is individual, 4-5 is individual or 2-3 is individual, the peptide fragment of more preferably 1-2 Amino acid profile.
In another preference, x1 is acetyl group.
In another preference, described anti-cd20 monoclonal antibody includes Rituximab (rituximab).
In another preference, between two cys in formula i polypeptide, form intrachain disulfide bond.
In another preference, described polypeptide be not incorporated into the fusion protein such as ibi302 or vid,OrDeng monoclonal antibody.
In another preference, x0 or x1 does not contain cys residue.
A kind of second aspect present invention, there is provided conjugate, described conjugate is polypeptide described in first aspect present invention
The conjugate being formed with carrier protein couplet.
In another preference, described coupling is to be coupled by the c-terminuses of formula i polypeptide.
In another preference, described carrier protein includes bovine serum albumin (bsa), hemocyanin (klh), egg white
Albumen or gamma Globulin.
In another preference, described protein carrier is hemocyanin or bovine serum albumin.
Third aspect present invention, there is provided even described in polypeptide described in a kind of first aspect present invention or second aspect present invention
The purposes of connection thing, for preparing the reagent of the anti-cd20 such as Rituximab monoclonal antibody, detection plate or reagent in detection sample
Box.
In another preference, described sample is included for blood sample, blood serum sample, tissue fluid sample or monoclonal antibody medicine sample
Product.
Fourth aspect present invention, there is provided whether contain the anti-cd20 monoclonal anti such as Rituximab in a kind of detection sample
The method of body, including step:
A sample is contacted by () with the polypeptide described in first aspect present invention or the conjugate described in second aspect present invention；
B () detects whether to form antigen-antibody complex, wherein form complex and mean that to there is anti-cd20 in sample mono-
Clonal antibody.
Fifth aspect present invention, there is provided a kind of method described in fourth aspect present invention is it is characterised in that in step
In (a), sample is contacted with the polypeptide described in first aspect present invention or the conjugate described in second aspect present invention, and
Detected by elisa method in step (b).
A kind of sixth aspect present invention, there is provided detection plate, described detection plate includes substrate (gripper shoe) and test strip,
Described test strip contains the polypeptide described in first aspect present invention or the conjugate described in second aspect present invention.
In another preference, described test strip also contains antibody spot sample area.
In another preference, described test strip is by filtering sample paper, chromatographic material, nitrocellulose filter and absorbent paper successively
Overlap joint composition.
A kind of seventh aspect present invention, there is provided test kit, described test kit contains a container and is located in container
The polypeptide described in first aspect present invention or the conjugate described in second aspect present invention, or described test kit contains this
Invent the detection plate described in the 6th aspect.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, thus constituting new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description
Fig. 1 shows the structure chart of cd20-acp peptide.
Fig. 2 shows preparation and the COUPLING PROCEDURE flow process of cd20-acp peptide.
Fig. 3 shows that cd20-acp-bsa-elisa method measures the Choice tests result of Rituximab.By cd20-
Acp-bsa is coated, and concentration is 100 μ g/ holes.With ibi302, avastin, vid, humira containing 10% human serum and rituximab
5 kinds of monoclonal antibody drugs such as monoclonal antibody (rituximab) (concentration be 100 μ g/ml) do by sample product, and hrp- sheep anti mouse is as two
Anti- survey od450nm absorption value.Result shows, with cd20-acp-bsa for being coated thing, has for Rituximab monoclonal antibody
High degree of specificity, is not substantially specifically bound with the antibody in other people sources, not the obvious cross reaction of generation anti-with two, to people
Serum has very strong capacity of resisting disturbance.
Fig. 4 shows that cd20-acp-bsa-elisa method measures the range of linearity result of the test of Rituximab.By cd20-
Acp-bsa is coated, and concentration is 1 μ g/ hole.Prepare the standard sample of Rituximab monoclonal antibody, concentration respectively 0.122,
0.244th, 0.488,0.976,1.95,3.9,7.8,15.6,31.25,62.5,125 and 250 μ g/ml.Result shows,
In the range of 0.122-250 μ g/ml, the logarithm value of the concentration of Rituximab monoclonal antibody and immunoreation absorbance value
(od450nm value) is linear.
Specific embodiment
The present inventor, through extensively in-depth study, has synthesized a kind of highly sensitive, high specific combination life first
The polypeptide of anti-cd20 monoclonal antibody in thing sample, polypeptide of the present invention and its elisa method stability are strong, and sensitivity is high, specifically
Property strong, can record 1 × 10 in sample-1The other anti-cd20 monoclonal antibody of μ g/ml concentration level, as anti-in elisa reaction
Former substrate, thus overcome conventional elisa method background high, cross reaction is big, the shortcoming of high cost, strengthen and detect anti-cd20 Dan Ke
The specificity of grand antibody, and pass through its highly sensitive characteristic, it is able to detect that micro anti-cd20 monoclonal antibody in sample,
Thus realizing the application in Clinical detection.On this basis, complete the present invention.
Active polypeptide
In the present invention, term " polypeptide of the present invention ", " cd20-acp ", " present invention anti-cd20 monoclonal antibody binding peptide "
It is used interchangeably, the polypeptide referring both to containing formula i structure and being combined with anti-cd20 monoclonal antibody specificity, a kind of preferred
Polypeptide of the present invention is as shown in seq id no.:1.
Additionally, described term also include having with anti-cd20 monoclonal antibody combined function, seq id no:1 sequence
Variant form.It is that 8-9 is individual, 6-7 is individual, 4-5 is individual or 2-3 is individual, more preferably 1-2 that these variant forms include (but being not limited to): x0
The peptide fragment of individual Amino acid profile；And/or x1 is that 8-9 is individual, 6-7 is individual, 4-5 is individual or 2-3 is individual, more preferably 1-2 Amino acid profile
Peptide fragment.It should be understood that generally, add in c end and/or n end or disappearance one or several aminoacid generally will not change albumen
The 26S Proteasome Structure and Function of matter.1-10 amino is carried out respectively to the aminoterminal of polypeptide or c-terminuses shown in seq id no.:1 of the present invention
The interpolation of acid, can't affect the basic function of polypeptide of the present invention.
An intrachain disulfide bond is also included, it is formed by natural or alpha-non-natural amino acid, preferably chain in polypeptide of the present invention
Interior two cys are formed under suitable condition.The condition forming disulfide bond between cys is condition generally in the art.
In actual applications, also polypeptide of the present invention can be modified further to strengthen its stability.Preferably example
Add blocking group including to described polypeptide, such as acetyl group, tertbutyloxycarbonyl etc..
Present invention additionally comprises polypeptide that additional aminoacid sequence is blended in this peptide sequence and is formed (with targeting sequencing,
The derived protein that the sequence label such as secretion sequence or 6his merges and formed).According to teaching herein, these fragments, derivant
Belong to scope known to those skilled in the art with analog.
Another aspect of the present invention, also includes the protein conjugate forming albumen of the present invention with carrier protein couplet.
As used herein, " protein carrier " or " carrier protein " in the present invention refer to any in immunology be subjected to
The protein for forming complete antigen, it can be for for example, hemocyanin, bovine serum albumin, ovalbumin or γ ball egg
White etc..The carrier protein of the present invention generally may connect to the c-terminuses of polypeptide of the present invention.
Invention also provides the analog of cd20-acp polypeptide.These analog can be ammonia with the difference of cd20-acp polypeptide
Difference on base acid sequence or the difference not affecting on the modified forms of sequence, or have both at the same time.Analog also wraps
Include the analog with the residue (as d- aminoacid) different from natural l- aminoacid, and there is non-naturally occurring or synthesis
Aminoacid (as β, gamma-amino acid) analog.It should be understood that the polypeptide of the present invention be not limited to above-mentioned enumerate representational
Polypeptide.
(generally the not changing primary structure) form of modification includes: the chemically derived form such as acetyl of inner or in vitro polypeptide
Change or carboxylated.Modify and also include glycosylation, such as those are carried out in the synthesis and processing of polypeptide or in further processing step
Glycosylation modified and produce polypeptide.This modification can carry out glycosylated enzyme (as mammal by being exposed to polypeptide
Glycosylase or deglycosylating enzyme) and complete.Modified forms are also included with phosphorylated amino acid residue (as phosphoric acid cheese ammonia
Acid, phosphoserine, phosphothreonine) sequence.Also include being modified thus improve its anti-Proteolytic enzyme performance or optimization
The polypeptide of solubility property.
Polypeptide of the present invention can also with by pharmaceutically or physiology acceptable acid or derived from alkali salt form use.These
Salt is including but not limited to and the salt that formed of following acid: hydrochloric acid, hydrobromic acid, sulphuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid,
Acetone acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethyl sulfonic acid, benzenesulfonic acid or hydroxyl second sulphur
Acid.Other salt include: the salt being formed with alkali metal or alkaline-earth metal (as sodium, potassium, calcium or magnesium), and with ester, carbamate
Or the form of " prodrug " of other routines.
Anti- cd20 monoclonal antibody
Cd20 is nonglycosylated cross-film Phospoprotein, and molecular mass is 35kd, and expression is in most of maturation b cell tables
Face, after being divided into plasma cell, cd20 expression disappears.Cd20 may participate in the regulation of b cell maturation and differentiation, leads to as calcium ion
Road plays some biological actions.What is more important, more than 95% b cell lymphoma expression cd20, and no notable internalization and
Come off.These features become the preferable target antigen of monoclonal antibody therapy.Clinical test results show, anti-cd20 monoclonal
Antibody both can be used alone, and can act also as the carrier of radiosiotope or cellulotoxic preparation, and it is evident in efficacy, and using peace
Entirely, side reaction is little.
In order to evaluate pharmacokineticss and the pharmacodynamics of anti-cd20 monoclonal antibody further, extensively carry out antagonism at present
The mensure research of cd20 monoclonal antibody, needs more preferably high specific, a highly sensitive method.
It is an object of the invention to have the polypeptide of function of specific connecting by preparation and anti-cd20 monoclonal antibody, from
And reach in the case of extremely micro it is also possible to measure anti-cd20 monoclonal antibody exactly.
In the present invention, term " anti-cd20 monoclonal antibody " refers to specifically bind to cell surface especially b cell table
The monoclonal antibody of face cd20 antigen.The anti-cd20 monoclonal antibody being combined with polypeptide of the present invention includes chimeric antibody, Ren Yuan
Change antibody, non-humanized antibody, single-chain antibody, antibody fragment.The preferably anti-cd20 monoclonal antibody behaviour Mus of one kind are fitted together to
Cd20 monoclonal antibody, such as Rituximab.
Preparation method
Polypeptide of the present invention can be recombinant polypeptide or synthesis polypeptide.The polypeptide of the present invention can be chemosynthesis, or weight
Group.Correspondingly, polypeptide of the present invention can be with conventional method synthetic it is also possible to recombination method produces.
A kind of it is preferable that using liquid phase synthesis techniques or solid phase synthesis technique, such as boc solid phase method, fmoc solid phase method
Or two methods are used in combination.Solid phase synthesis can quickly obtain sample, and it is suitable to be selected according to the sequence signature of purpose peptide
Resin carrier and synthesis system.For example, in fmoc system, preferred solid phase carrier is such as connected with the wang tree of c terminal amino acid in peptide
Fat, wang resin structure is the arm between polystyrene, and aminoacid is 4- alkoxyl benzylalcohol；With 25% hexahydropyridine/dimethyl
Methanamide room temperature treatment 20 minutes, to remove fmoc blocking group, and according to given aminoacid sequence from c end one by one to n end
Extend.After the completion of synthesis, with the trifluoroacetic acid containing 4% p-methyl phenol, the proinsulin related peptides synthesizing are cut from resin
Get off and remove protection group, may filter that except ether precipitate and separate obtains thick peptide after resin.After the solution lyophilizing of products therefrom, use
Peptide needed for gel filtration and reverse phase HPLC method purification.When carrying out solid phase synthesis using boc system, preferred resin
For being connected with the pam resin of c terminal amino acid in peptide, pam resin structure is the arm between polystyrene, and aminoacid is 4- hydroxyl first
Base phenyl acetamide；In boc synthesis system, in deprotection, neutralization, the circulation of coupling, removed with tfa/ dichloromethane (dcm)
Blocking group boc and with diisopropylethylamine (diea/ dichloromethane neutralize.After the completion of peptide chain condensation, with containing p-cresol (5-
10%) fluohydric acid gas (hf), processes 1 hour at 0 DEG C, peptide chain is cut from resin, removes blocking group simultaneously.With 50-
80% acetic acid (containing a small amount of mercaptoethanol) extracts peptide, is divided with molecular sieve sephadex g10 or tsk-40f further after solution lyophilizing
From purification, then obtain required peptide through high-pressure liquid phase purification again.Can be using known various coupling agents in chemistry of peptides field
It is coupled each amino acid residue with coupling method, for example, can use dicyclohexylcarbodiimide (dcc), hydroxyl benzotriazole
Or 1,1,3,3- tetra- urea hexafluorophosphoric acid ester (hbtu) is directly coupled (hobt).For the small peptide that obtains of synthesis, its purity with
Structure can be confirmed with RP-HPLC and mass spectral analyses.
In a preference, polypeptide cd20-acp of the present invention, by its sequence, using the method preparation of solid phase synthesis, through height
Effect liquid phase chromatogram purification, obtains high-purity purpose peptide freeze-dried powder, -20 DEG C of storages.
It is of course also possible to wait routine techniquess means to obtain polypeptide of the present invention using recombinant expressed.By conventional restructuring dna
Technology, the polynucleotide of the available present invention are used for expressing or produce the cd20-acp polypeptide of restructuring.In general there is following step
Rapid:
(1) polynucleotide (or variant) of the coding cd20-acp polypeptide of the present invention are used, or with containing this polynucleotide
Recombinant expression carrier conversion or transduce suitable host cell；
(2) host cell cultivated in suitable culture medium；
(3) separation, protein purification from culture medium or cell.
Recombinant polypeptide can be expressed in the cell or on cell membrane or is secreted into extracellular.If necessary, it can be utilized
Physics, chemistry and other characteristics separated by various separation methods and purification of Recombinant albumen.These methods are this areas
Known to technical staff.The example of these methods includes but is not limited to: conventional renaturation process, is processed with protein precipitant
(salting-out method), centrifugation, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion exchange
The combination of chromatography, high performance liquid chroma- tography (hplc) and other various liquid chromatography (LC) technology and these methods.
Because polypeptide of the present invention is shorter, it can be considered to multiple polypeptides are cascaded, recombinant expressed rear acquisition table
Reach product, then required small peptide is formed by methods such as enzyme action.
Enzyme-linked immunosorbent assay elisa
Enzyme-linked immunosorbent assay elisa(enzyme-linked immunosorbent assay): the base of elisa
Plinth is antigen or the immobilization of antibody and the enzyme labelling of antigen or antibody.Still protect in conjunction with the antigen of surface of solid phase carriers or antibody
Hold its immunologic competence, the antigen of enzyme labelling or antibody had both retained its immunologic competence, retain the activity of enzyme again.When measuring,
Reacted with the antigen of surface of solid phase carriers or antibody by inspection specimen (measuring antibody therein or antigen).Made with the method for washing
Antigen antibody complex and other materials in liquid of being formed on solid phase carrier separate.Add the antigen of enzyme labelling or anti-
Body, combines on solid phase carrier also by reaction.Now the enzyme amount in solid phase and the amount of tested substance in specimen are in certain
Ratio.After adding the substrate of enzyme reaction, substrate is become color products by enzyme catalysiss, the amount of tested substance in the amount of product and specimen
Directly related, therefore qualitative or quantitative analysis can be carried out according to the depth of colour generation.Because the catalytic efficiency of enzyme is very high, indirectly amplify
The result of immunoreation, makes the assay method reach very high sensitivity.
In the present invention, polypeptide of the present invention can as the antigen being fixed on surface of solid phase carriers, and with sample in cd20
Monoclonal antibody contacts, thus forming antigen-antibody complex.
A kind of anti-sheep anti mouse including horseradish peroxidase (hrp) labelling of preferred enzyme labelling two or goat-anti people two resist.
In a preference, when generating antigen-antibody complex and after add hrp, the substrate tmb(3 of hrp can be added, 3 ', 5,5 '-
Tetramethyl benzidine) colour developing.Hrp enzyme amount positive correlation in the intensity of colour developing and solid phase carrier, also just inspected anti-with specimen
The indirect positive correlation of amount of cd20 monoclonal antibody.
Detection plate and its material
The detection plate of the present invention can be using detection panel material commonly used in the art, using conventional detection plate preparation method system
Become.
Additionally, the present invention detects the detection plate of anti-cd20 monoclonal antibody, may also include containing by detectable label in advance
The detection plate of the polypeptide of the present invention processing, its gripper shoe including test strip and supporting test strip, such as can adopt pvc polyester offset plate
Deng；Described test strip is formed by filtering sample paper, chromatographic material, nitrocellulose filter and absorbent paper and overlap successively, and overlapping part can
So that using conventional method, such as adhesive tape etc. is fixedly connected；Wherein: the pre-coated colloid gold label of chromatographic material or coloured label anti-
Cd20 monoclonal antibody, preferably by the anti-cd20 monoclonal antibody of colloid gold label, on nitrocellulose filter absorption detection line and
Nature controlling line.
Detection method and result judgement
Keep flat detection plate, sample is dropped on filter sample paper, in sample about 120 μ l, 3～5min, observes tomographic results.According to
The fringe position occurring carrys out judged result.
Negative: obvious colour band all in quality control region, detection zone, it is shown as negative；
Positive: only in quality control region, obvious colour band to occur, and in detection zone no colour band, be shown as positive；
Invalid: the no any colour band of quality control region, detection zone or in quality control region, colour band does not occur and in detection zone, colour band occurs, table
Bright detection method mistake or detection plate go bad or lost efficacy, and should again exchange detection plate detection for.
Test kit
Present invention also offers a kind of examination of the detection plate referring to the anti-cd20 monoclonal antibody containing the present invention or the present invention
Agent box, in a preference of the present invention, described test kit also includes the immunity of container, operation instructions, buffer agent etc.
Detection plate, including the gripper shoe of test strip and support test strip, such as can adopt pvc polyester offset plate etc.；Described test strip is by filtering
Sample paper, chromatographic material, nitrocellulose filter and absorbent paper overlap composition successively, and overlapping part can be using conventional method, such as
Adhesive tape etc. is fixedly connected；Wherein: the anti-cd20 monoclonal antibody of the pre-coated colloid gold label of chromatographic material or coloured label, preferably
By the anti-cd20 monoclonal antibody of colloid gold label, absorption detection line and nature controlling line on nitrocellulose filter.
In a preferred scheme: on chromatographic material, the anti-cd20 monoclonal antibody of pre-coated colloid gold label is to adopt
Concentration be 0.5-1.5mg/ml colloid gold label anti-cd20 monoclonal antibody solution carry out pre-coated, package amount be 50 μ l/
cm2；Preferably concentration is 0.5 or 1.5mg/ml, 50 μ l/cm2.
Detection method and result judgement
Keep flat detection plate, sample is dropped on filter sample paper, in sample about 50-200 μ l, 3～5min, observes tomographic results.Root
Carry out judged result according to the fringe position occurring.
Negative: obvious colour band all in quality control region, detection zone, it is shown as negative；
Positive: only in quality control region, obvious colour band to occur, and in detection zone no colour band, be shown as positive；
Invalid: the no any colour band of quality control region, detection zone or in quality control region, colour band does not occur and in detection zone, colour band occurs, table
Bright detection method mistake or detection plate go bad or lost efficacy, and should again exchange detection plate detection for.
In the prior art, existing multiple detectable detecting anti-cd20 monoclonal antibody, its minimal detectable concentration is about
For 50 μ g/ml about.But clinically, in sample, the concentration of anti-cd20 monoclonal antibody is often far below this concentration limit, because
Even if this causes, specificity is very high, also cannot measure the cd20 monoclonal antibody of low concentration.
Therefore, the present invention, based on the polypeptide described in formula i, can carry out the detection of the anti-cd20 monoclonal antibody of low concentration.
It is demonstrated experimentally that polypeptide of the present invention can detect and minimum reach 1 × 10-1The anti-cd20 monoclonal antibody of μ g/ml, therefore can be clinically
Promotion and application.
Test kit
Present invention also offers a kind of test kit containing polypeptide of the present invention or the detection plate of the present invention for the finger, the present invention's
In one preference, described test kit also includes container, operation instructions, buffer agent etc..
The invention has the advantages that:
(1) sensitivity is high: the combinative anti-cd20 monoclonal antibody least concentration of polypeptide of the present invention reaches 1 × 10-1μg/
Ml, and in 0.122-250 μ g/ml detection range, there is good linear relationship, can delicately record denier in sample
Anti- cd20 monoclonal antibody, overcome in prior art cannot accurate measuring trace quantity antibody defect such that it is able to be able to reality
Application.Carrying out, the polypeptide of the present invention after acetylation closing etc. optimizes is better.
(2) background is extremely low, and cd20-acp has very strong specificity to make recombined human-Mus inosculating antibody cd20 monoclonal antibody
With eliminating the interference of foreign protein, the especially interference of serum.Because the peptide of this method synthesis is very short, substantially not in serum
Other protein produce adsorption.Conventional method is typically using monoclonal antibody or polyclonal antibody as being coated thing.Anti-
Body is usually had powerful connections interference.
(3) selectivity is strong, to fusion protein such as ibi302 or vid,WithDeng Clinical practice
Monoclonal antibody medicine does not have cross reaction.The polypeptide of this method synthesis does not produce non-specific adsorption effect substantially to other antibody.And it is normal
The method of rule is typically using monoclonal antibody or polyclonal antibody as being coated thing.This macro-molecular protein antibody detects to other
Thing protein unavoidably has different degrees of non-specific adsorption.
(4) with low cost, conventional method prepares polyclonal antibody or monoclonal antibody, and the cycle is long, high cost.This method letter
Single, quickly, with low cost.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as sambrook et al., molecular cloning: laboratory manual (new york:cold spring harbor
Laboratory press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, no
Then percentage ratio and number are percentage by weight and parts by weight.
The synthesis of embodiment 1cd20-acp
Cd20-acp polypeptide has been synthesized using commercially available Peptide synthesizer, its sequence as shown in seq id no.:1,
(cpyanpslc).
The acetyl group closing of embodiment 2cd20-acp, cyclization identification and the coupling with carrier protein
2.1 acetyl group closings
When Solid-phase synthesis peptides are to n end amino acid, n- acetyl-aminoacid is used as raw material, the so polypeptide of synthesis
N end just carried acetyl group.
2.2 cyclization identifications
After Peptide systhesis, aoxidized in the solution of ph8-9, formed disulfide bond.Ellman method dtnb [5,5- bis- sulfur
Base-bis- (2- nitrobenzoic acid)] reagent measures the method for free sulfydryl and can identify whether cyclization.Dtnb does not have in 412nm
Absorb, after reacting with free sulfydryl, generate 2- nitro -5- mercaptobenzoic acid (tnb).Tnb has very strong absorption in 412nm,
Therefore free sulfydryl can be quantitative determined.
2.3 with the coupling of carrier protein (bsa)
Coupling reaction is carried out using edc.A certain amount of synthetic peptide with carboxyl adds dmf dissolving, then plus dec, ultrasonic shake
After swinging 1h, centrifuging and taking supernatant, it is slowly added in the phosphate buffer of bsa, at room temperature ultrasonic agitation, reaction is overnight
Dialysed 24h with phosphate buffer afterwards, remove the peptide not being coupled and unnecessary reagent.
Embodiment 3 measures the specificity of cd20-acp-bsa and Rituximab by elisa method
Cd20-acp-bsa is coated elisa orifice plate, concentration is 100 μ g/ holes.With the ibi302 containing 10% human serum,
5 kinds of monoclonal antibody drugs such as avastin, vid, humira and Rituximab (concentration is 100 μ g/ml) do by sample product,
Hrp- sheep anti mouse is as two anti-survey od450nm absorption values.
Result is shown in Fig. 3, and the od450nm absorption value of Rituximab is 6-10 times of other several monoclonal antibody drugs.
Conclusion: with cd20-acp-bsa for being coated thing, have high degree of specificity for Rituximab monoclonal antibody, with it
The antibody in its people source does not substantially specifically bind, not the obvious cross reaction of generation anti-with two, has very strong resisting to human serum
Interference performance.
Embodiment 4 measures the range of linearity of cd20-acp-bsa and Rituximab by elisa method
Cd20-acp-bsa is coated elisa orifice plate, concentration is 1 μ g/ hole.Prepare the mark of Rituximab monoclonal antibody
Quasi- sample, concentration is respectively 0.122,0.244,0.488,0.976,1.95,3.9,7.8,15.6,31.25,62.5,125 and
250μg/ml.
Result shows (Fig. 4), in the range of 0.122-250 μ g/ml, the logarithm of the concentration of Rituximab monoclonal antibody
Value is linear with immunoreation absorbance value (od450nm value).
As can be seen here, polypeptide of the present invention low can reach 0.122 μ g/ml to the minimum detection value of monoclonal antibody, far below existing
There is the minimum detection value of 50 μ g/ml in technology, Clinical Laboratory can be fully applicable to.
Embodiment 5 goes out the detection of Rituximab monoclonal antibody according to the standard curve regression equation calculation of embodiment 4
Amount
Monoclonal antibody Rituximab made 10 with the diluent containing 10% human serum by the method according to embodiment 4,
2.5th, the quality-control sample of 3 Concentraton gradient such as 0.625 μ g/ml being measured, is repeated 5 times experiment, by the standard on same plate
The concentration that Regression Equations calculate is the detected level of Rituximab monoclonal antibody.The response rate is used for verifying this elisa
The recall rate of method, also demonstrates the repeatability of the method simultaneously.
From table 1, the response rate that quality-control sample detects according to the method described above, in 95.3%～113.9% scope, becomes
Different coefficient range is between 4.3%～8.6%.
Table 1.cd20-acp-bsa-elisa method measures Rituximab sample result
Embodiment 6cd20-acp derives preparation and the identification of polypeptide
According to the method in embodiment 1, the synthetically prepared derivative polypeptide of cd20-acp.Its sequence is as follows:
| Polypeptide title | seq id no.: | Sequence |
| cd20-acp1 | 2 | fpcpyanpslcna |
| cd20-acp2 | 3 | aalcpyanpslcyppn |
| cd20-acp3 | 4 | valsscpyanpslc |
| cd20-acp4 | 5 | fnkvvicpyanpslc |
| cd20-acp5 | 6 | cpyanpslcsppfnkry |
Embodiment 7 derives modification and identification and the coupling of polypeptide
In 7.1 pairs of embodiments 6, the polypeptide of the seq id no.:2-6 of synthesis is modified, further wherein to seq id
The polypeptide of no.:2-4 carries out acetyl group closing, carries out boc modification (under alkaline environment) to the polypeptide of seq id no.:5-6.
7.2 carry out cyclization identification according to the method in embodiment 2.2 to the polypeptide of seq id no.:2-6.
7.3 carry out the coupling with carrier protein according to the method in embodiment 2.3 to the polypeptide of seq id no.:2-6.Its
The polypeptide of middle seq id no.:2-4 is coupled to klh, and the polypeptide of seq id no.:5-6 is coupled to bsa.
The binding specificity that embodiment 8 derives polypeptide protein conjugate measures
According to embodiment 3, the special of anti-cd20 monoclonal antibody is carried out to the derivative polypeptide-protein conjugate being obtained in embodiment 7
Property measure, wherein, the od450 value after the polypeptide-protein conjugate of seq id no.:2-6 is combined with Rituximab can
Reach more than 0.5, and be above seq id no.:2-6 polypeptide-protein conjugate and other monoclonal antibodies (avastin, humira)
Adhesion (od450 value is 0.1-0.2).
Embodiment 9 derives the combination sensitivity determination of polypeptide protein conjugate
According to the method for embodiment 4, the derivative polypeptide-protein conjugate being obtained in embodiment 7 is coated elisa orifice plate,
Concentration is 1 μ g/ hole.Prepare the standard sample of rituximab monoclonal antibody.
Result represents, in the range of 0.100-250 μ g/ml, the logarithm value of the concentration of rituximab monoclonal antibody with immunity
Reaction absorbance value (od450nm value) also can be in good linear relationship.
As can be seen here, although the length of seq id these polypeptides of no.:2-6 extends through certain, but as long as be have as
The derivative polypeptide of polypeptide shown in seq id no.:1, polypeptide-protein conjugate that it is formed also can detect suitable low concentration
Monoclonal antibody.
The all documents referring in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (25)
1. a kind of polypeptide is it is characterised in that have the structure as formula i:
x0-cys-pro-tyr-ala-asn-pro-ser-leu-cys-x1
Formula i；
Wherein, x0 is protection group；X1 is no, or the peptide fragment of 1-10 Amino acid profile；
And described polypeptide has the activity being combined with anti-cd20 monoclonal antibody；
Described protection group includes acetyl group (ch3-co-) or tertbutyloxycarbonyl (boc)；
Form intrachain disulfide bond between two cys in formula i polypeptide.
2. polypeptide as claimed in claim 1 is it is characterised in that described anti-cd20 monoclonal antibody includes chimeric antibody, people
Source antibody, non-humanized antibody, single-chain antibody, antibody fragment.
3. polypeptide as claimed in claim 1 is it is characterised in that described anti-cd20 monoclonal antibody is Chimeric antibody.
4. polypeptide as claimed in claim 1 is it is characterised in that described anti-cd20 monoclonal antibody derives from mammal.
5. polypeptide as claimed in claim 4 is it is characterised in that described anti-cd20 monoclonal antibody derives from people or grinding tooth moves
Thing.
6. polypeptide as claimed in claim 4 it is characterised in that described anti-cd20 monoclonal antibody derive from people, mice or
Rat.
7. polypeptide as claimed in claim 1 is it is characterised in that the least concentration rank of described anti-cd20 monoclonal antibody is 1
×10-1μg/ml.
8. polypeptide as claimed in claim 1 is it is characterised in that described protection group is acetyl group (ch3-co-)；And/or
X1 is 8-9,6-7 is individual, 4-5 is individual, 2-3 is individual or the peptide fragment of 1-2 Amino acid profile.
9. polypeptide as claimed in claim 8 is it is characterised in that x0 is acetyl group.
10. polypeptide as claimed in claim 1 is it is characterised in that described anti-cd20 monoclonal antibody includes Rituximab
(rituximab).
11. polypeptides as claimed in claim 1 are it is characterised in that x1 is no.
12. polypeptides as claimed in claim 1 it is characterised in that described polypeptide be not incorporated into ibi302 or vid fusion protein,OrMonoclonal antibody.
A kind of 13. conjugates are it is characterised in that described conjugate is polypeptide described in claim 1 and carrier protein couplet shape
The conjugate becoming.
14. conjugates as claimed in claim 13 are it is characterised in that described coupling is even by the c-terminuses of formula i polypeptide
Connection.
15. conjugates as claimed in claim 13 are it is characterised in that described carrier protein includes bovine serum albumin
(bsa), hemocyanin (klh), ovalbumin or gamma Globulin.
16. conjugates as claimed in claim 13 are it is characterised in that described carrier protein is hemocyanin or bovine serum albumin
In vain.
The purposes of polypeptide described in 17. claim 1 or conjugate described in claim 13 is it is characterised in that detect for preparation
The reagent of anti-cd20 monoclonal antibody, detection plate or test kit in sample.
18. purposes as claimed in claim 17 are it is characterised in that described sample is blood sample, tissue fluid sample or monoclonal antibody
Drug sample.
19. purposes as claimed in claim 17 are it is characterised in that described sample is blood serum sample.
The method of anti-cd20 monoclonal antibody whether is contained it is characterised in that including step in a kind of 20. detection samples:
A sample is contacted by () with the polypeptide described in claim 1 or the conjugate described in claim 13；
B () detects whether to form antigen-antibody complex, wherein form complex and mean that there is anti-cd20 monoclonal in sample
Antibody.
21. methods as claimed in claim 20 it is characterised in that in step (a), by sample with described in claim 1
Conjugate contact described in polypeptide or claim 13, and detected by elisa method in step (b).
A kind of 22. detection plates, described detection plate includes substrate and test strip, and described test strip contains described in claim 1
Polypeptide or claim 13 described in conjugate.
23. detection plates as claimed in claim 22 are it is characterised in that described test strip also contains antibody spot sample area.
24. detection plates as claimed in claim 22 are it is characterised in that described test strip is by filtering sample paper, chromatographic material, nitric acid
Cellulose membrane and absorbent paper overlap composition successively.
A kind of 25. test kits are it is characterised in that described test kit contains a container and the claim 1 being located in container
Conjugate described in described polypeptide or claim 13, or described test kit contains the detection described in claim 22
Plate.
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| Title |
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| Identification of an Antigenic and Immunogenic Motif Expressed by Two 7-Mer Rituximab-Specific Cyclic Peptide Mimotopes: Implication for Peptide-Based Active Immunotherapy;Federico Perosa 等;《The Journal of Immunology》;20071231;第179卷;第7967-7974页 * |
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