CN101317089A - Detection or quantification of aggrecan and its fragments - Google Patents
Detection or quantification of aggrecan and its fragments Download PDFInfo
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Abstract
An immunoassay for aggrecan and/or aggrecan derived fragments comprises contacting a sample with an immunological binding partner which has specific binding affinity for the G2 domain of aggrecan at least when bearing keratan sulphate chains, and determining the existence or amount of specific binding of the immunological binding partner, which may be conducted as a sandwich assay using a first antibody that binds an N-terminal amino acid sequence comprising FFGVG.. and a second antibody that binds an N-terminal amino acid sequence comprising ARGS.
Description
The present invention relates to adopt the antibody of identification G2 domain or the determination method of the detection of other immunologic opsonin binding partners and/or quantitative aggrecan (Aggrecan) and fragment thereof.
Aggrecan is the principal ingredient of articular cartilage by cartilage cell synthetic (Archer and Francis, 2003), in articular cartilage with II Collagen Type VI and other substrate molecule systematisms (Hardingham and Fosang, 1995).
Aggrecan is a high glycosylation, comprises more than 2000 amino acid residue.Aggrecan structurally has three independent structures territory: G1, G2 and G3 (figure A).Have between G2 and the G3 domain-and between G1 and G2 domain, having-grow section high glycosylation zone than low degree ground, electronegative chondroitin sulfate and keratan sulfate oligosaccharide structure (Hardingham and Fosang, 1995) are contained in this zone.
The feature of osteoarthritis is the irreversible destruction of articular cartilage.The cartilage cell can comprise aggrecan by synthetic new matrix components, attempts to repair cartilage people such as (, 2000) Garnero of degeneration.
Need to provide the biochemical marker of cartilage metabolism procedural information.Biochemical marker is existing to be described, and to the also existing report of the mensuration of aggrecan and fragment thereof.
The antibody (IBEX PharmaceuticalsInc.) of the chondroitin sulfate side chain that identification combines with amino acid 846 between aggrecan molecule G2 and the G3 domain is adopted in CS 846 test.FA-846 sandwich immunoassay method also has report, and this method is to be used for improving one's methods of the quantitative CS of embryo's aggrecan 846 detections.
Also developed some other aggrecan detection method, and for example " aggrecan proteoglycans (Aggrecan Proteoglycan) " (Biosource, US).Yet the specificity of these antibody still awaits determining.
Other aggrecan determination method is the glycosaminoglycan zone at aggrecan, i.e. (Kongtawelert and Ghosh 1990 between G2 and the G3 domain; People such as Ratcliffe, 1993).
Developed antibody 1-C-6, this antibodies G1 and the non-G2 domain of sheltering (Fosang and Hardingham, 1991).Must adopt keratanase to remove the reaction that the keratan sulfate side chain is beneficial to 1-C-6 and G2 domain.Therefore 1-C-6 antibody is not suitable for using in the mensuration of aggrecan in detecting body fluid or bodily tissue or aggrecan fragment.
Some proteolysis cleavage sites (people such as Fosang, 2000 of aggrecan have been put down in writing; People such as Caterson, 2000), the main action site of metalloproteinases (MMP) is positioned at amino acid N
341And F
342Between ball intracellular domain (IGD) in.Developing a kind of monoclonal antibody in the past is AF28 (ATCC HB11671), it with contain the N end sequence
342FFGVG... the new epi-position of polypeptide (neo-epitope) specificity is in conjunction with people such as (, 1995) Fosang.AF28 antibody has been used for competitive ELISA and has detected aggrecan fragment in synovia and human serum people such as (, 1995) Fosang.Yet the coupling of this antibody and G2 domain antibodies does not still have report.
Aggrecan has mentioned in some patent publication us.Wherein there are several pieces to mention some peculiar fragment of measuring aggrecan or protein for the diagnostic purpose of estimating cartilage metabolism.US4704356 has disclosed that the abnormal level of keratan sulfate (KS) is the unusual indication of cartilage or chondroid tissue in the peripheral blood.Peripheral blood KS level raises and is considered to the indication of osteoarthritis.What is interesting is that the shortage of KS and very high KS level are all indicated muscular dystrophy and relevant disease in the discovery peripheral blood.Being used for the quantitative technology of peripheral blood KS is the immunoassays of adopting monoclonal antibody.
US5935796 has described other and has related to the diagnostic method and the composition of the proteoglycans albumen of cartilage decomposition.Described method is used for early diagnosis, monitoring and treatment osteoarthritis, wherein adopt the monoclonal antibody of the antigenic determinant on atypia chondroitin sulfate (CS)/dermatan sulfate glycosaminoglycan chains in specific recognition body tissue and the body fluid, these two kinds of compositions all are derived from the articular cartilage aggrecan.
The described method of US4779768 is used for detecting the carrying out property destruction of joint articular cartilage, more specifically, is used for the variation of determining that articular cartilage takes place.This method comprises that (a) carries out quantitatively the proteoglycans monomer in the synovia sample and/or its antigenicity fragment and (b) institute's value be associated with carrying out property destruction corresponding to the articular cartilage of this sample liquid.The proteoglycans fragment is measured by the method for immunity that adopts proteoglycans monomer specific antibody.The described determination method of this patent is identical with aforementioned polyclone HABr ELISA method.
The determination method that US5948692 describes is that the glycan that adopts the size separation method will have hyaluronic acid (HA) affinity separates with the proteoglycans that does not have this affinity.This assay method can be measured the proteoglycans that combines with HA, for example aggrecan.It is believed that this can realize the biochemical diagnosis to joint disease and RA, OA and other joint diseases of field of orthopedic surgery.This method also can be used for distinguishing natural joint and pathologic joint, provides the prognosis of progression of disease to determine, and the effect of monitor therapy intervention.
US5427954 has described and has adopted immunoassay to detect the aggrecan that has the new epi-position of ARGSVI.This is one piece that describes in the many pieces of publications that new epi-position diagnostic uses, and described new epi-position is produced by the aggrecan specific proteins hydrolysis of the proteinase mediation that participates in the joint disease pathologic process.
US5387504 has described by molten stromatin enzyme and has acted on site N
341-F
342The new epi-position of VDIPEN of locating and discharging, and use the RIA of the monoclonal antibody specific of this epi-position to measure.The use that the aggrecan fragment of the molten stromatin enzyme cutting of specificity generation is had specific monospecific antibody has mainly been described.Molten stromatin enzyme level raises and appears in osteoarthritis, rheumatoid arthritis, atherosclerotic lesion, gout, inflammatory bowel disease (IBD), idiopathic pulmonary fibrosis (IPF), part cancer, joint injury and the multiple inflammatory disease.Clinical value for these aggrecans " new epi-position " specific assay also is not very clear at present, and whether also indeterminate these fragments can be released in the blood circulation in a large number and they are the metabolism that how to be decomposed.
US5935796 relates to the method and composition of early diagnosis, monitoring and treatment cartilage degenerative disease, its adopt can discern since the cartilage aggrecan at N
341-F
342Site cutting and the antibody of the peptide that comprises the FFGVG sequence that produces.This epi-position is that molten stromatin enzyme acts on aggrecan and " other end " of the VDIPEN epi-position that discharges.Having recommended a kind of sandwich assay to increase the detection sensitivity of aggrecan FFGVG fragment in the literary composition, more specifically, is the sandwich assay with AF-28 and sulfuric-resisting keratan antibody such as 5-D-4 coupling.Yet until now, adopt AF-28 not succeed yet in practice as the trial that one of two kinds of antibody carry out gratifying sandwich assay.
US5185245 has described a kind of immunoassay of the proteoglycans in the synovia and monitoring is decomposed into the treatment of diseases situation of feature with proteoglycans method of detecting.The synovia test sample is undertaken quantitatively by the immunoassay of the antibody of use specific recognition proteoglycans, and wherein antibody is fixed on the solid support.In conjunction with proteoglycans contact with second specific antibody of reagent (as peroxidase) mark after testing again.Two kinds of antibody all have affinity to the glycosaminoglycan on the proteoglycans (GAG/CS) part.
US5354662 and US5217903 have mainly described the mensuration to " tissue decomposition product " in the body fluid, and carry out quantitatively connective tissue in the animal body fluid or musculature decomposition product for adopting the standard items that contain the isotope labeling decomposition product on its basis.Standard items should have known specific activity, and when therefore standard items being mixed with humoral sample, the specific activity that adopts RIA/IRMA type determination method to detect can measuring as decomposition product content in the sample.Also described the situation of estimating selected animal connective tissue in the animal body fluid or musculature method, estimate the lysis that comprises that specific connective tissue composition or musculature are destroyed method, estimate the method for the curative effect of the therapy that is used for the treatment of this lysis, comprise and measure the method step of organizing the decomposition product amount.
All adopt in the said method can be identified in keratanase handle before can in conjunction with the antibody that is positioned at the epi-position on the aggrecan G2 domain, the application of this antibody-like is not discussed yet.
The present invention is based on following discovery, promptly the antibody at aggrecan G2 domain can provide better application in the immunoassays of detection or quantitative aggrecan.
Discovery is at not had more advantage by the antibody of the G2 domain epi-position of natural cover in the immunoassays of measuring aggrecan.
Therefore, first aspect present invention provide a kind of detect or the quantitative measurement sample in the method for immunity of aggrecan and/or aggrecan derived fragment, comprise making the sample contact have the immune binding partners of specificity binding affinity, and detect generation or its amount of this immunity binding partners specificity combination the aggrecan G2 domain that has the keratan sulfate chain.
Randomly, when described G2 domain has the keratan sulfate chain, and when described G2 domain was expressed with recombinant forms thereby lacked the keratan sulfate chain, described immune binding partners all had the specificity binding affinity to the G2 domain of aggrecan.
Described method can be carried out according to the sandwich immunoassay method, adopts described immune binding partners as trapping agent or detection agent.As trapping agent, it can be fixed on the solid surface, and as detection agent, it can (for example) use suitably mark of enzyme labeling, radioactive label or fluorescence or other marks.In this sandwich assay, described binding partners can with other immunity aggrecans or the coupling of aggrecan fragment binding partners, be used separately as detection agent or trapping agent.Especially, described other binding partners can be antibody or the antibody fragment that the new epi-position of aggrecan is had binding affinity, and described new epi-position is possible between IGD domain or G2 and G3 or the N end at other positions or the terminal new epi-position of C.
Especially, described mensuration can be carried out according to the sandwich immunoassay method, adopts described immune binding partners as trapping agent and use described immune binding partners as detection agent.Suitably, described trapping agent is provided by the described immune binding partners that is fixed on the solid surface, and described detection agent is then provided by the described specific binding partner that has detectable label.
Alternatively, mensuration of the present invention can be carried out according to the competitive immunometric assay method, wherein (a) is fixed in described immune binding partners on the solid surface, and hatch with described sample and the competitive reagent of mark that comprises described G2 domain or its antibody-binding fraction, perhaps (b) will comprise described G2 domain or its paratope competitive immobilization of reagents on solid surface, and with described sample and the described immune binding partners of mark hatch.
One of the present invention particularly preferred aspect, described mensuration is carried out according to the sandwich immunoassay method, use (a) described immune binding partners and the immune binding partners that (b) the N terminal amino acid sequence that comprises FFGVG.. is had the specificity binding affinity, wherein (a) and (b) in any in described mensuration, be used as trapping agent, and (a) and (b) another is as detection agent.
Described immune binding partners (b) can be the AF-28 antibody that is produced by hybridoma cell line ATCCHB11671, and this hybridoma cell line is existing in the art to be described.
Suitably, described sample is synovia, serum or cultivates or the sample of the conditioned medium of cultured chondrocytes or contain their sample from the cartilage explant.
The present invention includes and a kind ofly cartilage in the sample is upgraded (turnover) mark detect or quantitative in-vitro method, comprise and make the sample contact when described G2 domain has the keratan sulfate chain and when described G2 domain is expressed with recombinant forms, all have the immune binding partners of specificity binding affinity and generation or its amount of measuring this immunity binding partners specificity combination aggrecan G2 domain.This method can be carried out according to above-mentioned random procedure.
Sample is preferably the sample that derives from the patient, described method may further include with measure in conjunction with level with corresponding to existing and/or not existing the calibration value of cartilage degradation disease illness to compare.
The present invention includes a kind of immune binding partners that the aggrecan G2 domain that has the keratan sulfate chain is had the specificity binding affinity.Preferably, when described G2 domain has the keratan sulfate chain and when described G2 domain is expressed with recombinant forms, this immune binding partners all has the specificity binding affinity to the G2 domain of aggrecan.This immunity binding partners can be the monoclonal antibody with specificity binding characteristic or the form of its fragment.
The present invention includes the hybridoma cell line of expressing aforesaid monoclonal antibody, also extend to these recombinant expressed immune binding partners.
The present invention includes a kind of immunoassay kit, it comprises immune binding partners of the present invention, and one or more following compositions:
Anti-aggrecan in addition or aggrecan fragment antibody; With the peptide competition reagent that combines with anti-aggrecan or aggrecan fragment antibody; With optional one or more: washing reagent, damping fluid, termination reagent, enzyme labeling thing, enzyme base number of a tender thing, anti-mouse antibodies, calibration criterion product and instructions.
Generally speaking, all known immunoassays forms all can be applied in the present invention, comprise heterogeneous and homogeneous phase form, sandwich assay, competitive assays, enzyme connection determination method, radioimmunoassay etc.
Determination method as herein described can be used for diagnosing disease of patient, comprises that osteoarthritis, rheumatoid arthritis and other influence the disease of cartilaginous tissue.In addition, these tests can be used for estimating the progress of disease, and monitoring is to the reaction of treatment.Also can be used for studying in for example generation of aggrecan or aggrecan fragment in cartilage or the chondrocyte culture of culture, and study the influence of different reagent, drug candidate and enzyme inhibitor these cultivating systems.Immune binding partners of the present invention also can be used for the material that contains the aggrecan fragment is carried out immunostaining.
Term used herein " immune binding partners " comprises polyclone and monoclonal antibody, also has the specificity binding fragment of antibody such as Fab or F (ab ')
2
The monoclonal antibody of identification aggrecan G2 domain can be prepared as follows: adopt the synthetic peptide immune mouse that derives from G2 domain amino acid sequence, splenocyte and the myeloma cell of selected mouse are merged, and detect the monoclonal antibody of secretion and combining of aggrecan.Importantly, also should estimate the binding ability of these antibody, for example estimate by hatching altogether with synovia or blood serum sample to natural aggrecan.
Alternatively, mouse can adopt the complete aggrecan of purifying to carry out immunity, selects monoclonal antibody according to the reactivity to the G2 domain.Specificity to the G2 domain can be guaranteed in the following manner: (1) requires purifying G2 domain and randomly in addition reorganization G2 or G1-G2 is had reactivity, perhaps (2) require (people such as Fosang to purifying G1-G2,1989) and the aggrecan fragment (the aggrecan fragment that contains new epi-position FFGVG and G2 of cutting corresponding to mmp enzyme) that contains FFGVG have reactivity and randomly reorganization G2 or G1-G2 had reactivity; Perhaps (3) require at least purifying G1-G2 is had reactivity, and lack the reactivity to reorganization G1 and synthetic IGD (the ball intracellular domain of separating G1 and G2).
One aspect of the present invention relates to aggrecan G2 domain is detected and/or quantitative methods.A kind of such method is to utilize the competitive immunometric assay method of the monoclonal antibody that combines with the G2 domain.Bag can be combined described monoclonal antibody with the sample competition by the synthetic peptide of the suitable selection on the microwell plate solid surface.On solid surface, also can use the natural aggrecan of purifying.Another selection is that monoclonal antibody is fixed in solid surface, hatches altogether with sample and with signaling molecule such as horseradish peroxidase or the suitable synthetic peptide that is connected of biotin again.
One aspect of the present invention relates to by the aggrecan G2 domain in immunoassay mensuration synovia and the blood serum sample.
The present invention relates to by immunoassay mensuration on the other hand from the G2 domain in the conditioned medium of the outer plant cultivation of cartilage, cultured chondrocytes.
The present invention relates to the kit that can be advantageously used in implementing said method on the other hand.This kit can comprise: the microwell plate of (1) synthetic peptide bag quilt; (2) monoclonal antibody of identification G2 domain; (3) the anti-mouse IgG immunoglobulin (Ig) of mark.Alternatively, this kit can comprise: the microwell plate of (1) purifying aggrecan bag quilt; (2) monoclonal antibody of identification G2 domain; (3) the anti-mouse IgG immunoglobulin (Ig) of mark.Alternatively, this kit can comprise: the microwell plate of (1) streptavidin bag quilt; (2) the synthetic peptide that is connected with biotin; (3) monoclonal antibody of identification G2 domain; (4) the anti-mouse IgG immunoglobulin (Ig) of mark.Kit as another kind of alternatives comprises: the microwell plate of (1) streptavidin bag quilt; (2) the synthetic peptide that is connected with biotin; (3) identification G2 domain and with the monoclonal antibody of horseradish peroxidase.
The present invention relate on the other hand utilization at the non-antibody of epi-position of sheltering among the G2 with the sandwich method for determining aggrecan.
The present invention relates to the antibody and the coupling that is identified in other antibody of the new epi-position that produces in the aggrecan proteolysis cutting process of identification G2 domain on the other hand.Surprisingly, the antibody coupling of G2 antibody and the new epi-position of Recognition Protein hydrolysis can improve the detection sensitivity to the aggrecan fragment that has new epi-position greatly.
One aspect of the present invention relates to G2 antibody and the use in conjunction of identification through the antibody of the new epi-position of matrix metalloproteinase (MMP) proteolysis cutting aggrecan generation.A preferred implementation is for adopting the antibody that is identified in the N end sequence FFGVG... that cutting produces between ball intracellular domain (IGD) amino acid 341 and 342.
The present invention relates to G2 antibody and the use in conjunction of identification through the antibody of aggrecan enzyme (aggrecanase) the proteolysis cutting new epi-position that aggrecan produced on the other hand.A preferred implementation is for adopting the antibody that is identified in the N end sequence ARGS... that cutting produces between ball intracellular domain (IGD) amino acid 373 and 374.The antibody that is fit to comprises the BC-3 antibody (ab3773) that can obtain and put down in writing from abcam in people Biochem J (1995) 305,799-804 such as Hughes, and people Osteoarthritis ﹠amp such as Pratta; The monoclonal antibody OA-1 of record among the cartilage March 2006.
The present invention relates to the exploitation and the application of used antibody in above-mentioned any determination method on the other hand, and described antibody can be discerned the non-epi-position of sheltering that is arranged in G2 and/or G1.G1 and G2 share the amino acid homology sequence that prolongs, and be therefore all available according to this antibody-like of the present invention.
Can know further feature of the present invention and advantage from description, following examples and the appended claims of accompanying drawing and preferred implementation.
In the accompanying drawings:
Fig. 1 shows the structure of aggrecan;
The pig G1-G2 that Fig. 2 shows purifying suppresses combining of anti-G2 antibody F78 and aggrecan in the mode of dose dependent;
Fig. 3 shows the typical curve that the immunoassays of embodiment 2 obtain;
Fig. 4 shows the typical curve that the immunoassays of embodiment 3 obtain;
Fig. 5 shows the result who adopts sandwich assay to obtain among the embodiment 4;
Fig. 6 shows the result who adopts the relative immunity determination method to obtain among the embodiment 5;
Fig. 7 shows the quantitative result that employing obtains as embodiment 6 described G2 sandwich assay; With
Fig. 8 shows the quantitative result that employing obtains as embodiment 7 described FFGVG-G2 sandwich assay.
Embodiment
The MONOCLONAL ANTIBODIES SPECIFIC FOR of identification G2 domain
(SIGMA is Denmark) with 1: 1 mixed liquor of incomplete Freund to give five the seven complete ox aggrecans of week female Balb/c mouse subcutaneous injection in age.Per two all immune animals once, the bimester of continuing (immunity 5 times), 4 all immunity are once at interval afterwards.Mouse is before the beginning immunity and the 5th immunity back one week blood sampling.Adopt complete ox aggrecan to spend the night the microwell plate (NUNC, Denmark) of bag quilt by ELISA assessment immune response.The mouse resisting anteserum of serial dilution was hatched one hour, and the washing microwell plate proves the antibody that combines by hatching with the sheep anti mouse IgG antibody of horseradish peroxidase.
Do not raise if in described shaker test, tire, then allow the mouse of selecting have a rest for 4 weeks, then with booster immunization in the 200 μ l immunogene peritonaeums that do not contain adjuvant.Take out spleen after three days, adopt standard technique and myeloma cell to merge.
The antibody that the hybridoma of growth produces adopts described ELISA to measure.Select hybridoma and adopt limiting dilution assay to clone, in culture flask, breed, adopt Protein G affinity chromatography monoclonal antibody purification.
At last, according to the reactivity of not sheltering epi-position that is arranged in the G2 domain is selected monoclonal antibody (term " do not shelter " when this is meant epi-position in natural the existence not can (for example) owing to exist keratan sulfate to stop it by antibodies, rather than refer to remove " covert " epi-position as keratan sulfate).This realizes by evaluating and testing with a series of immunoassays.With complete ox aggrecan bag by microwell plate.Combining of monoclonal antibody and solid surface by (Chemicon, US) competition is determined with the pig G1-G2 (Fosang and Hardingham, 1989) of purifying and synthetic IGD.For the monoclonal antibody that combines with G1-G2 but do not combine with IGD, the further binding ability of testing it and having the aggrecan fragment of the terminal FFGVG sequence of free C (the amino acid 342-346 in the IGD domain), this fragment is corresponding to the fragment (seeing below) of no G1 domain.
But Fig. 2 shows purifying pig G1-G2 dose dependent ground and suppresses monoclonal antibody F78 and bag combining by the aggrecan on the microwell plate solid surface.On the contrary, (Chemicon US) then can't replace antibody (data not shown) to synthetic IGD.
The sandwich immunoassay of G2 fragment
Adopt aforesaid monoclonal antibody to develop a kind of sandwich assay.The monoclonal antibody F78 of streptavidin plate and the anti-aggrecan G2 of 100 μ l 600ng/ml biotinylations domain was hatched 1 hour under 20 ℃, 300RPM.
After reaction plate hatched 1 hour, wash plate 5 times with lavation buffer solution (0.15mol/l NaCl, 0.05% (v/v) polysorbas20).(the ox aggrecan of purifying 0.05ng/ml-10ng/ml) or with the prediluted explant supernatant of PBS-BTB, is hatched plate 1 hour in 300RPM, 20 ℃ to add 100 μ l standard items afterwards.After hatching end, wash plate 5 times, add the F78 antibody of 500ng/ml horseradish peroxidase (POD) mark by described method.After 300RPM, 20 ℃ are hatched 1 hour, wash plate 5 times, add 100 μ l tmb substrates, reaction plate was in the dark hatched 15 minutes in 300RPM, 20 ℃, then add 150 μ l0.18M sulfuric acid.Measure absorbance immediately in the 450nm place.
Fig. 3 shows the typical curve that described sandwich assay records.
Right
342The immunoassays of FFGVG-G2
At
342The existing report of the monoclonal antibody of FFGVG amino acid sequence (US5935796).
It is anti-to add 100 μ l 1500ng/ml biotinylations in the streptavidin reaction plate
342The AF-28 monoclonal antibody of FFGVG-G2 epi-position was hatched 1 hour in 300RPM, 20 ℃.Antibody dilution adopts the PBS-BTB damping fluid to carry out.After reaction plate hatched 1 hour, wash plate 5 times with lavation buffer solution (0.15mol/l NaCl, 0.05% (v/v) polysorbas20).(the purifying pig aggrecan of MMP-13 digestion 47-3000ng/ml) or with the prediluted explant supernatant of PBS-BTB, was hatched 1 hour in 300RPM, 20 ℃ to add 50 μ l standard items afterwards.After hatching end, wash plate as stated above 5 times, add the F78 antibody of 500ng/ml horseradish peroxidase (POD) mark.After 300RPM, 20 ℃ are hatched 1 hour, wash plate 5 times, add 100 μ l tmb substrates, reaction plate was in the dark hatched 15 minutes in 300RPM, 20 ℃, then add 150 μ l 0.18M sulfuric acid.Measure absorbance immediately in the 450nm place.
Fig. 4 shows the typical curve that the said determination method is measured.Importantly,
342The detection limit that FFGVG-G2 measures is low to moderate 0.004pmol/mL (being equivalent to 10ng/ml), the detection limit of the competitive ELISA of the employing AF28 of report before this is significantly less than, i.e. 7pmol/mL (or 17500ng/ml) people such as (, 1995) Fosang.
G2 in the ox articular cartilage explant supernatant and
342The detection of FFGVG-G2 fragment
Above-mentioned G2 and
342The FFGVG-G2 test is adopted and is estimated from the conditioned medium of cartilage explant.The ox articular cartilage is taken from the heifer patella.(16 ± 4mg) place 96 well culture plates, in 37 ℃, 5%CO with the cartilage fritter
2And hatch under jolting (50rpm) condition.Use contains or the serum-free D-MEM nutrient culture media of factor-containing oncostatin M and tumor necrosis factor (TNF α) or MMP inhibitor GM6001 not.As negative control, cartilage is placed frozen pipe, freezing in liquid nitrogen, in 37 ℃ of water-baths, melt triplicate freeze thawing circulation then.The explant nutrient culture media was changed once in per three days, and supernatant is in-20 ℃ of preservations, up to further analysis.
The measured value of aggrecan amount in supernatant when Fig. 5 shows the different number of days that adopts two kinds of immunoassay quantitative measurements.As can be seen from the figure OSM and TNF α stimulated after the 12nd day
342FFGVG-G2 increases.When in the presence of GM6001, handling explant simultaneously,
342FFGVG-G2 discharges and is eliminated fully.Under same experimental conditions, the release of G2 then shows diverse curve.The release of G2 in supernatant is initial to be increased earlier, but then reduces gradually after the 5th day, reduces to background level after the 12nd day.Add GM6001 and do not suppress G2 release, show that G2 mensuration is different from
342FFGVG-G2, it measures the fragment that does not rely on the MMP activity.
The mensuration that CS846 discharges in the ox articular cartilage explant supernatant
Also the supernatant of above-mentioned detection has been measured CS846 (IBEX CS 846 competitive ELISA kits).Fig. 6 shows that OSM and TNF α do not stimulate the release of CS846 in supernatant.Yet during suppressing the MMP activity, observing when CS846 is released in beginning increases strongly.These data show that the analyte of measuring is destroyed by proteolytic activity in CS846 detects, this activity can be suppressed by GM6001.
The measured value of RA patient's G2 fragment reduces
Blood serum sample from 15 healthy persons and 15 RA patients adopts the G2 determination method to carry out quantitatively the results are shown among Fig. 7.Data show that compared with the control the measured value of the G2 molecule that discharges in the RA individuality significantly reduces, the synthetic minimizing of aggrecan among this explanation arthritic.
Embodiment 7
RA patient's
342The measured value of FFGVG-G2 fragment raises
The same individual group has also been carried out
342FFGVG-G2 measures, and is opposite with the G2 measurement result, discharges among the RA patient
342The FFGVG-G2 fragment the results are shown in Figure 8 than the remarkable increase of control group.This result is consistent with expection, because this determination method is to measure the degraded of aggrecan.
If no specified otherwise, mean the containing of word " perhaps " when any or both in the described condition satisfy then that operator returns true value in this instructions, and be different from the operator distance that only needs to satisfy a kind of condition.Word " comprises " that the meaning is " comprising ", but not " by ... form ".
List of references
Caterson?B.，Flannery?C.R.，Hughes?C.E.，Little?C.B.，Mechanisms?involved?in?Cartilage?Proteoglycan?Catabolism.Matrix?Biology?2000；Volume?19：333-344
Fosang?A.J.，Hardingham?T.E.，Isolation?of?the?N-terminalglobular?protein?domains?from?cartilage?proteoglycans，Biochemical?Journal?1989；Vol?261：801-809.
Fosang?A.J.，Hardingham?T.E.，1-C-6?Epitope?in?CartilageProteoglycan?G2?Domain?is?masked?by?Keratan?Sulphate.Biochemical?Journal?1991；Volume?273：369-373
Fosang?A.J.，Last?K.，Gardiner?P.，Jackson?D.C.，Brown?L.，Development?of?a?cleavage-site-specific?Monoclonal?Antibodyfor?detecting?metalloproteinase-derived?Aggrecan?Fragments?inHuman?Synovial?Fluid.Biochemical?Journal?1995；Volume?310：337-343
Fosang?A.J.，Last?K.，Maciewicz?R.A.，Aggrecan?is?degradedby?matrix?metalloproteinases?in?human?arthritis.Evidencethat?matrix?metalloproteinase?and?aggrecanase?activities?canbe?independent.J?Clin?Invest.1996；98?Nr.10：2292-9
Fosang?A.J.，Last?K.，Stanton?H.，Weeks?D.B.，Campbell?I.K.，Hardingham?T.E.，Hembry?R.M.，Generation?and?noveldistribution?of?matrix?metalloproteinase-derived?aggrecanfragments?in?porcine?cartilage?explants.The?Journal?ofBiological?Chemistry?2000；Volume?275?Nr.42：33027-33037
Garnero?P.，Rousseau?J.C.，Delmas?P.，Molecular?Basis?andClinical?use?of?Biochemical?Markers?of?Bone，Cartilage?andSynovium?in?Joint?Diseases.Arthritis?and?Rheumatism?2000；Volume?43?No.5：953-968
Hardingham?T.E.and?Fosang?A.J.，The?Structure?of?Aggrecanand?its?Turnover?in?Cartilage.The?Journal?of?Rheumatology1995；Volume?22：1?supplement?43：86-89
Kongtawelert?P.and?Ghosh?P.，A?new?Sandwich-ELISA?Method?forthe?Determination?of?Keratan?Sulphate?Peptides?in?BiologicalFluids?employing?a?Monoclonal?Antibody?and?labelled?avidinbiotin?Technique.Clinica?Chimica?Acta?1990；Volume?195：17-26
Ratcliffe?A.，Shurety?W.，Caterson?B.，The?quantitation?of?anative?chondroitin?sulfate?epitope?in?synovial?fluid?lavagesand?articular?cartilage?from?canine?experimentalosteoarthritis?and?disuse?atrophy.Arthritis?and?Rheumatism1993；Volume?36?No.4：543-51
Claims (17)
- One kind detect or the quantitative measurement sample in the method for immunity of aggrecan and/or aggrecan derived fragment, comprise making the sample contact have the immune binding partners of specificity binding affinity, and detect generation or its amount of this immunity binding partners specificity combination the aggrecan G2 domain that has the keratan sulfate chain.
- 2. method as claimed in claim 2, wherein when described G2 domain has the keratan sulfate chain, and when described G2 domain was expressed with recombinant forms, described immune binding partners all had the specificity binding affinity to the G2 domain of aggrecan.
- 3. method as claimed in claim 1 or 2, it carries out according to the sandwich immunoassay method, adopts described immune binding partners as trapping agent or detection agent.
- 4. method as claimed in claim 3, it carries out according to the sandwich immunoassay method, adopts described immune binding partners as trapping agent, and adopts described immune binding partners as detection agent.
- 5. method as claimed in claim 4, wherein said trapping agent is provided by the described immune binding partners that is fixed on the solid surface, and described detection agent is provided by the described specific binding partner that has detectable label.
- 6. method as claimed in claim 1 or 2, it carries out according to the competitive immunometric assay method, wherein (a) is fixed in solid surface with described immune binding partners, and hatch with described sample and the competitive reagent of mark that comprises described G2 domain or its antibody-binding fraction, perhaps (b) will comprise described G2 domain or its antibody-binding fraction competitive immobilization of reagents in solid surface, and with described sample and the described immune binding partners of mark hatch.
- 7. method as claimed in claim 1 or 2, it carries out according to the sandwich immunoassay method, use (a) described immune binding partners and (b) the N terminal amino acid sequence that comprises FFGVG.. or comprise ARGS.. is had the immune binding partners of specificity binding affinity, wherein (a) and (b) in any in described determination method, be used as trapping agent, (a) and (b) in another is as detection agent.
- 8. method as claimed in claim 6, wherein said immune binding partners (b) are the antibody A F-28 that is produced by hybridoma cell line ATCC HB 11671.
- 9. each described method in the claim as described above, wherein said sample are synovia, serum or cultivate or the sample of the conditioned medium of cultured chondrocytes or contain their sample from the cartilage explant.
- 10. one kind is detected or quantitative in-vitro method the cartilage updating mark thing in the sample, comprise making the sample contact when the G2 domain has the keratan sulfate chain and when the G2 domain is expressed with recombinant forms, all have the immune binding partners of specificity binding affinity to described aggrecan G2 domain, and generation or its amount of measuring this immunity binding partners specificity combination.
- 11. method as claimed in claim 9, wherein said sample are the sample that derives from the patient, this method also comprise with measure in conjunction with level with corresponding to existing and/or not existing the calibration value of cartilage degradation disease illness to compare.
- 12. an immune binding partners, it has the specificity binding affinity to the aggrecan G2 domain that has the keratan sulfate chain.
- 13. immune binding partners as claimed in claim 11, it all has the specificity binding affinity to aggrecan G2 domain when described G2 domain has the keratan sulfate chain and when described G2 domain is expressed with recombinant forms.
- 14. as claim 11 or 12 described immune binding partners, its form is monoclonal antibody or its fragment with specificity binding characteristic.
- 15. hybridoma cell line of expressing monoclonal antibody as claimed in claim 13.
- 16. as claim 11 or 12 described immune binding partners, it is recombinant expressed.
- 17. an immunoassay kit, it comprises as claim 11 or 12 described immune binding partners, and one or more:Anti-aggrecan antibody in addition; WithPeptide competition reagent with anti-aggrecan antibodies; With optional one or more:Washing reagent, damping fluid, termination reagent, enzyme labeling thing, enzyme base number of a tender thing, anti-mouse antibodies, calibration criterion product and instructions.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0521392.1 | 2005-10-20 | ||
| GB0521392A GB0521392D0 (en) | 2005-10-20 | 2005-10-20 | Detection or quantification of aggrecan and its fragments |
| GB0608947.8 | 2006-05-05 |
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| CN101317089A true CN101317089A (en) | 2008-12-03 |
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| CNA200680044851XA Pending CN101317089A (en) | 2005-10-20 | 2006-10-17 | Detection or quantification of aggrecan and its fragments |
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| GB (1) | GB0521392D0 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108828224A (en) * | 2018-04-24 | 2018-11-16 | 上海市第十人民医院 | Liver cancer serum tumor markers ACAN |
| CN110944684A (en) * | 2017-06-25 | 2020-03-31 | 科.东股份公司 | Method for preparing transplantable cartilage tissue |
| CN110997716A (en) * | 2017-06-02 | 2020-04-10 | 埃博灵克斯股份有限公司 | aggrecan-binding immunoglobulin |
| CN114217078A (en) * | 2021-12-23 | 2022-03-22 | 西安依朵生物科技有限公司 | Test strip, kit and detection method for detecting aggrecan |
-
2005
- 2005-10-20 GB GB0521392A patent/GB0521392D0/en not_active Ceased
-
2006
- 2006-10-17 CN CNA200680044851XA patent/CN101317089A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110997716A (en) * | 2017-06-02 | 2020-04-10 | 埃博灵克斯股份有限公司 | aggrecan-binding immunoglobulin |
| CN110997716B (en) * | 2017-06-02 | 2024-03-15 | 埃博灵克斯股份有限公司 | aggrecan-binding immunoglobulins |
| CN110944684A (en) * | 2017-06-25 | 2020-03-31 | 科.东股份公司 | Method for preparing transplantable cartilage tissue |
| CN110944684B (en) * | 2017-06-25 | 2022-03-18 | 科.东股份公司 | Method for preparing transplantable cartilage tissue |
| CN108828224A (en) * | 2018-04-24 | 2018-11-16 | 上海市第十人民医院 | Liver cancer serum tumor markers ACAN |
| CN114217078A (en) * | 2021-12-23 | 2022-03-22 | 西安依朵生物科技有限公司 | Test strip, kit and detection method for detecting aggrecan |
Also Published As
| Publication number | Publication date |
|---|---|
| GB0521392D0 (en) | 2005-11-30 |
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