Patent Application: US-62349700-A

Abstract:
a predictive test for rheumatoid arthritis comprises the detection of antibodies to collagen in a biological sample from a patient by contacting the biological sample with an antigen comprising the cb10 peptide of mammalian type ii collagen , or an antibody - binding fragment or variant thereof , for a time and under conditions for an antibody - antigen complex to form , and detecting the antibody - antigen complex , for example by immunoassay . a diagnostic test kit is also disclosed .

Description:
the diagnosis of rheumatoid arthritis in its early stages is difficult . until characteristic signs of cartilage and bone destruction appear on x - rays , rheumatoid arthritis cannot be reliably distinguished from certain limited forms of arthritis which are of different pathogenesis . traditionally aspirin and / or other non - steroidal anti - flammatory drugs are given as a first - line of treatment for rheumatoid arthritis , and more potent agents are added if and when the disease deteriorates . however this sequence of treatment does not optimally halt progressive bone and joint destruction . because the greatest loss of function occurs early in the disease , progressively within the first four years , rheumatologists now believe that giving the most powerful drugs early in the course of treatment is more likely to prevent the destruction that leads to crippling disability . however , since these potent disease - modifying drugs can have significant adverse effects , a marker to distinguish those patients who will develop crippling disease is urgently needed . as noted previously , there have now been several studies that suggest that antibodies to collagen are present very early in rheumatoid arthritis , before the diagnosis is clearly established , and before irreversible joint destruction will have occurred . accordingly , a method of measuring collagen antibodies that can be reliably performed in routine diagnostic laboratories would have considerable utility . most current immunoassays to detect to the entire type ii collagen molecule antibodies do not show sufficient specificity and sensitivity for the diagnosis of rheumatoid arthritis , noting that these antibodies may be demonstrable in other diseases , or even in healthy individuals , or may be lacking in cases of established rheumatoid arthritis particularly cases of long duration . in accordance with the present invention , it has now been shown that the use of the particular cb peptide , cb10 of mammalian type ii collagen , or an antibody - binding fragment or variant thereof , instead of the entire collagen molecule , provides for an immunoassay to detect antibodies to collagen that greatly increases the specificity and sensitivity of the immunoassay as a diagnostic test for rheumatoid arthritis . an “ antibody - binding fragment ” or variant thereof contemplated by the present invention includes a fragment of the cb10 peptide of a mammalian type ii collagen comprising at least 50 % of the sequence of said cb10 peptide . preferably , the percentage of the cb10 sequence is at least about 60 %, more preferably at least about 70 %, even more preferably at least about 80 % and still even more preferably at least about 90 % or greater . such a fragment could be prepared from collagen purified from cartilage , or expressed as a recombinant protein in an appropriate expression system , or derived by any other means . most preferably said fragment exhibits the helical structure of the cb10 peptide and retains the ability to bind to collagen antibodies . suitable antibody - binding fragments may be identified by simple trials to ascertain their ability to bind to collagen antibodies . the present invention also extends to the use of variants of the cb10 peptide or antibody - binding fragment thereof as described above , particularly substitution variants which are conservative in nature and result from replacing one amino acid in the naturally - occurring cb10 amino acid sequence with another having similar structural and / or chemical properties , such as the replacement of a leucine with an isoleucine or valine , an aspartate with a glutamate , or a threonine with a serine . other suitable variants of the naturally - occurring sequence include insertion or deletion variants , typically of from about one to five amino acids . suitable variants will preferably exhibit the helical structure of the cb10 peptide and retain the ability to bind to collagen antibodies , and may be identified by simple antibody - binding trials . the detection of the presence of antibodies to collagen in accordance with the present invention may be accomplished in a number of immunoassay procedures , such as by elisa procedures . a wide range of immunoassay techniques is available as can be seen by reference to standard immunoassay textbooks . these , of course , include both singlesite and two - site or “ sandwich ” assays of the non - competitive types , as well as the traditional competitive binding assays . sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention . a number of variations of the sandwich assay technique exist , and all are intended to be encompassed by the present invention . briefly , in a typical assay to detect antibodies in a sample , an unlabelled antigen is immobilized on a solid substrate and the sample to be tested brought into contact with the bound antigen molecule . after a suitable period of incubation , for a period of time sufficient to allow formation of an antibody - antigen complex , a second antibody such as anti - human igg , labelled with a reporter molecule capable of producing a detectable signal , is then added and incubated , allowing time sufficient for the formation of another complex of antibody : antigen : labelled antibody . any unreacted material is washed away , and the presence of the antibody to be detected in the sample is determined by observation of a signal produced by the reporter molecule . the results may either be qualitative , by simple observation of the visible signal , or may be quantitated by comparing with a control sample containing known amounts of the antibody to be detected . variations on this assay include a simultaneous assay , in which both sample and labelled antibody are added simultaneously to the bound antigen . these techniques are well known to those skilled in the art , including any minor variations as will be readily apparent . in the typical sandwich assay , antigen is immobilised , for example by being either covalently or passively bound to a solid surface . the solid surface is typically glass or a polymer , the most commonly used polymers being cellulose , polyacrylamide , nylon , polystyrene , polyvinyl chloride or polypropylene . the solid supports may be in the form of tubes , beads , discs , or microplates , or any other surface suitable for conducting an immunoassay . the binding processes are well - known in the art and generally consist of crosslinking , covalent binding or physical adsorption . the immobilised antigen is then washed in preparation for the test sample . an aliquot of the sample to be tested is then contacted with the immobilised antigen and incubated for a period of time sufficient ( e . g . 2 - 40 minutes ) and under suitable conditions ( e . g . 25 ° c .) to allow binding of any antibody to collagen present in the sample . following the incubation period , the immobilised antigen with any bound antibody is washed and dried , and incubated with a second antibody specific for the bound antibody , for example anti - human igg . the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antibody : immobilised antigen complex . by “ reporter molecule ” as used in the present specification , is meant a molecule which , by its chemical nature , provides an analytically identifiable signal which allows the detection of antigen - bound antibody . detection may be either qualitative or quantitative . the most commonly used reporter molecules in this type of assay are either enzymes , fluorophores or radionuclide - containing molecules ( i . e . radioisotopes ), or chemiluminescent molecules . in the case of an enzyme immunoassay ( eia ), an enzyme is conjugated to the second antibody , generally by means of glutaraldehyde or periodate . as will be readily recognized , however , a wide variety of different conjugation techniques exist , which are readily available to the skilled artisan . commonly used reporter enzymes include horseradish peroxidase , glucose oxidase , beta - galactosidase and alkaline phosphatase , amongst others . the substrates to be used with the specific enzymes are generally chosen for the production , upon hydrolysis by the corresponding enzyme , of a detectable colour change . it is also possible to employ fluorogenic substrates , which yield a fluorescent product rather than the chromogenic substrates noted above . in all cases , the enzyme - labelled antibody is added to the first antibody - antigen complex , allowed to bind , and then the excess reagent is washed away . a solution containing the appropriate substrate is then added to the complex of antibody : antigen : labelled antibody . the substrate will react with the enzyme linked to the second antibody , giving a qualitative visual signal , which may be further quantitated , usually using a spectrophotometric instrument , to give an indication of the amount of antibody which was present in the sample . “ reporter molecule ” also extends to use of cell agglutination or inhibition of agglutination , such as red blood cells on latex beads , and the like . alternately , fluorescent compounds , such as fluorescein and rhodamine , may be chemically coupled to antibodies without altering their binding capacity . when activated by illumination with light of a particular wavelength , the fluorochromelabelled antibody adsorbs the light energy , inducing a state to excitability in the molecule , followed by emission of the light at a characteristic colour visually detectable with a light microscope . as in the eia , the fluorescent labelled antibody is allowed to bind to the first antibody - antigen complex . after washing off the unbound reagent , the remaining tertiary complex is then exposed to light of the appropriate wavelength and the fluorescence observed indicates the presence of the antibody of interest . immunofluorescence and eia techniques are both very well established in the art and are particularly preferred for the present method . however , other reporter molecules , such as radioisotope , chemiluminescent or bioluminescent molecules , may also be employed . further features of the present invention are more fully described in the following examples . it is to be understood , however , that this detailed description is included solely for the purposes of exemplifying the present invention , and should not be understood in any way as a restriction on the broad description of the invention as set out above . human and bovine type ii collagen was prepared from cartilage by pepsin digestion and differential salt precipitation ( rowley , et al ., 1988 ). bovine type ii collagen was digested with cyanogen bromide ( cnbr ) and the resultant fragments of type ii collagen , known as cb peptides , were purified according to the method of miller and lunde ( 1973 ) with slight modifications ; 1 . 5 g of bovine type ii collagen was dissolved in a minimum volume of 70 % ( v / v ) formic acid , containing 1 . 5 g of cnbr ( sigma , st louis mo ., usa ) and cleavage was achieved by the method of scott and veis ( 1976 ). the cb peptides were initially resolved into 2 major fractions by chromatography on a 2 . 4 × 82 cm column of bio - gel p - 2 ( 100 - 200 mesh , exclusion limit 2600 daltons , globular proteins , biorad , usa ) equilibrated with 0 . 1 m acetic acid . the peptides obtained after cyanogen bromide digestion were dissolved in 12 ml of 0 . 1 m acetic acid and 3 ml ( 500 mg ) was applied to the column at a time . the larger peptides , eluted in the excluded volume of the p - 2 column , were pooled , lyophilized , and rechromatographed on a 2 . 5 × 12 cm cation exchange column of carboxymethylcellulose ( cm - cellulose , whatman cm - 52 ). the column was equilibrated with 0 . 02 m sodium citrate , ph 3 . 6 containing 0 . 01 m nacl , designated as the start buffer , and the peptides were dissolved in this start buffer for application to the column . chromatography was performed at 42 ° c . over 10 hours using a linear salt gradient obtained by the addition of 1 % 0 . 16 m nacl in 0 . 02 m sodium citrate , ph 3 . 6 , to the start buffer every 6 minutes , with a flow rate of 1 . 75 ml / mm , using the biorad econo system ( biorad , usa ). fractions of 17 ml were collected at 10 minute intervals , and were run on 15 % tricine gels and stained with 0 . 2 % ( w / v ) coomassie blue ( biorad , usa ). fractions containing cb10 identified by comparison with published type ii cb peptide maps ( bornstein et al , 1980 ; miller et al , 1982 ) were pooled , as were fractions containing cb11 , desalted on the bio - gel p - 2 column and lyophilized . cb10 was purified further , and in particular separated from cb8 and cb9 , 7 , and cb11 was separated from cb12 , by applying each fraction to a 1 . 0 × 75 cm column of p - 30 ( 100 - 200 mesh , exclusion limit 40 kd , globular proteins , biorad , usa ) equilibrated in 0 . 1 m acetic acid . the purity of the peptides after each column run was determined by separation on 15 % tricine gels and staining with 0 . 2 % ( w / v ) coomassie blue ( fig2 a , 2 b ). purified cb10 and cb11 were renatured by step - wise cooling from 20 ° c . to 0 . 2 ° c ., as described by terato et al , 1985 . antibodies to native human type ii collagen , and native bovine type ii collagen , were measured by an enzyme linked immunosorbent assay ( elisa ). microtitre plates ( dynatech , germany ) were coated with 100 μl / well of 10 μg / ml human or bovine type ii collagen overnight at 4 ° c . plates were washed 3 times in 1 % ( w / v ) skim - milk powder in pbs , ph 7 . 4 ( sm - pbs ) containing 0 . 05 % tween 20 ( sigma , usa ) and 3 times in distilled water ( dh 2 o ), blocked with 1 % sm - pbs , 0 . 05 % tween 20 for 2 hours at room temperature , then washed again as above . sera were tested in duplicate at a dilution of 1 : 50 in the presence or absence of antigen . antibodies were detected using horseradish peroxidase conjugate ( hrp ) anti - human igg ( silenus , hawthorn , australia ), with 0 . 5 mg / ml 2 , 2 - azino - di -[ 3 -: ethyl - benzthiazoline sulfonate ( 6 )] ( abts ) ( boehringer , germany ) in 0 . 03 m citric acid , 0 . 04 m na 2 hpo 4 , 0 . 003 % h 2 o 2 , ph 4 as substrate . plates were read after 30 minutes at 41 5 nm on a biorad platereader ( biorad , usa ). non - specific binding of immunoglobulins to the plates was allowed for by subtracting the optical density ( od ) obtained in the absence of collagen from that obtained in wells coated with collagen . sera from two patients with high levels of antibodies to native and denatured type ii collagen , were used throughout as positive controls . measurements by elisa of antibodies to purified cb peptides of type ii collagen antibodies to the purified cb 10 peptide of bovine type ii collagen were measured by elisa as described above for intact type ii collagen with the following modifications . plates were coated with 100 μl of 10 μg / ml of purified cb10 peptide and sera were tested at a dilution of 1 : 50 . the peptide antigen was coated onto the plates overnight at 4 ° c ., blocked with 1 % sm - pbs , 0 . 05 % tween 20 for 90 minutes at room temperature with shaking , and sera were incubated for 2 hours at room temperature with shaking . plates were washed 3 times in tris buffered saline ( tbs ) containing 0 . 05 % tween 20 using an automatic plate washer ( wallac , finland ) in all steps prior to the addition of the secondary antibody and in distilled water ) immediately before and in all steps subsequent to the addition of the secondary antibody . bound antibodies were detected using hrp - conjugated anti - human igg ( silenus , hawthorn , australia ), and the appropriate substrate . results are expressed in terms of the number of standard deviations above the mean for controls as described above . antibodies to intact human type ii collagen measured by elisa were present in sera of 25 % of 96 patients with rheumatoid arthritis . by contrast , antibodies to collagen were present in 15 % of 34 patients with osteoarthritis and in 15 % of 33 patients with inflammatory arthritis associated with psoriasis ( psoriatic arthritis ), representing other non - autoimmune articular diseases . these figures are comparable with those reported by others for rheumatoid arthritis and other articular disease using an elisa in a similar format ( choi et al ., 1988 ; fujii et al ., 1992 ). the levels as well as frequencies of antibodies to type ii collagen were generally higher in rheumatoid arthritis than in other diseases ( fig3 ). fig4 compares the frequency of antibodies tested in a similar elisa in which cb10 peptide prepared from bovine type ii collagen was used as antigen . antibodies to the cb10 peptide of bovine type ii collagen were present in the sera of 88 % of the same 96 patients with rheumatoid arthritis , but in 6 % of patients with osteoarthritis and in 12 % of patients with psoriatic arthritis . in other words , the replacement of the intact type ii by the cb10 peptide greatly increased the sensitivity of the assay for the diagnosis of rheumatoid arthritis , from 25 % to 88 %, without any decrease in specificity , since the frequency of positive reactions to the cb10 peptide in the sera from patients with diseases of the joint other than rheumatoid arthritis was no greater than the frequency of positive reactions to intact type ii collagen , i . e . less than 15 %. this increased sensitivity was shown not to be due to the use of bovine ( versus human ) cb10 peptide of collagen in the assay , since the frequency of reactivity by elisa with intact collagen is very similar whether bovine collagen or human collagen is used as antigen in the elisa ( see table i below ). in addition , there was substantial specificity to the reactivity against cb10 since reactivity observed in rheumatoid arthritis with the cb11 peptide , a peptide of collagen of very similar size , was only 50 %. gioud , m ., meghliaoui , a ., costa , o ., monier , j . c . 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