CA2178590A1 - Reagents and methods for the detection of methotrexate - Google Patents
Reagents and methods for the detection of methotrexateInfo
- Publication number
- CA2178590A1 CA2178590A1 CA 2178590 CA2178590A CA2178590A1 CA 2178590 A1 CA2178590 A1 CA 2178590A1 CA 2178590 CA2178590 CA 2178590 CA 2178590 A CA2178590 A CA 2178590A CA 2178590 A1 CA2178590 A1 CA 2178590A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- methotrexate
- cell line
- test sample
- hydroxymethotrexate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
A murine monoclonal IgG antibody is provided which is useful in the detection of methotrexate in body fluids. This monoclonal antibody, designated 4-351-178, may be employed in an assay for methotrexate, preferably a fluorescence polarization immunoassay (FPIA) of urine. Monoclonal antibody 4-351-178 is characterized by having no detectable cross-reactivity with 7-hydroxymethotrexate. Also disclosed is a hybridoma cell line, ATCC HB 11467, which produces the monoclonal antibody of the invention.
Description
2 ~ 7 8 ~ 9 0 PCTIUS94113558 REAGENTS AND MErElODS FOR T~E DETECTION OF
METHOTRl~ATh Field sf th.~ Invrntinn The present invention relates to the ;mmllnnln~ir~l detection of methotrexate. In particular, the invention relates to a cell line which secretes a mnnnrlnnAl antibody specific for methotrexate and imm~lnnlngi~Al methods for detecting the presence or amount of methotrexate in a test sample.
R~3rk~rollnrl of thr Tnv~ntinn Methotrexate is an antineoplastic agent which is commonly used for treating patients having solid tumors and/or h.omAtnlngirAl mAlignAn~ The dosages of methotrexate received by these patients 1 ~ can vary widely because the therapeutic effect of methotrexate varies with the dosage. Unfortunately, however, patients on high methotrexate regimens may experience toxic side effects.
In order to avoid the toxic side effects Acqor;At.od with high doses of methotrexate, immllnnA.~sAys have been developed to monitor patients' methotrexate levels. These immunoassays commonly use Ant.;hn~
which are specific for methotrexate, to assist in the detection of the presence or amount of methotrexate in a patient's test sample. Results from these assays can be used to modify, if necessary, a patients methotrexate regimen to prevent the toxic side effects that may result 2 5 from high doses of methotrexate or to optimize a patients methotrexate regimen.
TmmllnnA~cAys designed to ~ trrminp the presence or amount of methotrexate in a test sample require highly specific antibodies to avoid erroneous assay results which may lead to inJ~lu,ul u~ Le dosage changes 3 0 which, can in turn, lead to toxic side effects or the administration of less than optimal methotrexate doses. Erroneous assay results can occur when the antibody used to detect methotrexate cross reacts with other rnmrollntlq For example, 7-hydroxymethotrexate is a m~t~holit.f~ of methotrexate that is structurally similar to methotrexate and cross 3 5 reacts with anti-m~llu~ d antibody.
Because 7-~1yLu~ylllt ~llotrexate is a m~t~hnl;t~ of methotrexate, nearly every test sample rnntAininF m~Ulu~ will also contain 7-wosSll6o26 21 785qa 2 PCTIUS9~/13~8 hydroxymethotrexate, and, in some inct~nr~5c, the concentration of the metabolite can exceed the concentration of the par~nt compound. Thus, an assay designed to determine the presence or amount of methotrexate in a test 6ample should employ Antiho~ A that have very little cross 5 reactivity with 7-hydroxymethotrexate. Most desirably, such antibodies should have no lP+~t~hl~ cross reactivity with 7-hydroxymethotrexate.
European Patent Application No. 0044441 generally describes the production of mnnnrlnnAl antibodies specific for drugs, but it does not describe the production of a mnnnrlnn~1 antibody specific for 10 methotrexate. Moreover, the disclosure does not describe an anti-methotrexate antibody with any particular specificity.
Assays which detect the presence or amount of mG~llo~G~te in a test sample and which are currently available, employ polyclonal nntiho~ .c to assist in the detection of methotrexate. However, according 15 to the methods of drt~rmininE~ cross reactivity used herein, such polyclonal antibodies can display cross reactivity with 7-hydl u~ylllethotrexate.
IIence there is a need for an antibody that is more ~liqrriminzltin~
than those currently available which can be used in an ;mmllnn~c.c~y to 2 0 accurately ~PtPrmin~' the presence or amount of methotrexate in a test sample .
Sllmm~ry of thP Inv~ntinn The present invention provides a mnnnrlnnz~l IgG antibody that is 2 5 specific for methotrexate and has no tl~tect 3hlP cross reactivity with 7-yd~u~y~ethotrexate. A-lrlit;~ lly, the invention provides a hybridoma cn~ ; eell line which secretes the above mnnnrlnn~l antibody and has been 11PCi~tPd cell line number A.T.C.C. HB 11467.
The mnnnrl~ l antibody of present invention ean be used in 30 various immllnnACc~y formats. Aeeording to one Pmho~limPnt, a nuulesCGllce polarization immllno~Cc~y (FPIA) for ~iPtPrminin~ the presence or amount of methotrexate in a test sample is provided. The FPIA rnmrriqPC the steps of (a) cnnt~r+in~ the test sample with (i) the mnrl~rlnnAl IgG antibody provided herein and (ii) a nuul~S..~u~ tracer 35 moleeule, to form a solution; (b) cnnt~t~tin~ the solution with plane polarized light; and (c) detecting a fluu-GscG..ce pnl~ri7~tinn response.
Aeeording to another PmhûrlimPnt, an improyed immunoassay for WO 9~116026 2 1 ~ ~ 5 ~ ~ PC~J~vS941~3SS8 determining the presence or amount of methotrexate in a test samp~e is provided. The improved immunoassay is of the type having a step of rnntArtin~ the test sample with Antiho~ q specific for methotrexate, wherein the hl.l.lv~l.lcnt comprises cnntActin~ the test sample with the monoclonal IgG antibody herein proYided.
The present invention also provides a test kit for ~ terminin~ the presence or amount of methotrexate in a test sample. The test kit comprises (a) the mnnnrlnnAl IgG antibody of the invention; and (b) a fluorescent tracer molecule.
Detail~rl Descr~tion of thl~ Jny~ntinn The following ~ finitinn.q are ArrlirAhl~ to the invention.
The term "analyte analog", as used herein, refers to a gllhctqnr~
which cross-reacts with a methotrexate-specific antibody, although it may do so to a greater or lesser extent than does the analyte (methotrexate) itsel The analyte analog can include a modified analyte as well as a frA~m-~nt.~(l or synthetic portion of the analyte molecule, 90 long as the analyte analog has at least one epitope in common with the analyte of interest.
The term "ill. uuuv~ ", as used herein, refers to a substance which, when illLIùvlu~t:d into an animal having an immune system, is capable of s~imlllatin~ an immune response. ~ertain low molecular weight sllhctanrPq which are not capable of stimulating an immune response are referred to as haptens. However, haptens can be made immunogenic by conjugating a hapten to a carrier molecule, which may itself be immunogenic. Thus, while generally not immunogenic, haptens conjugated to carrier mnl~clll.o.q are immunogenic and can be considered immunogens.
The term "signal generating group" as used herein refers to a nuvl~ ,e ,t cnmrolln-l (snm~tim~q referred to as a fluorophore) which is capable of absorbing energy and emitting light or fluorescing. F~amrl(~q of signal ~..( ., Liug groups include, but are not intended to be limited to nuul~s~ ;u, cascade blue, TEXAS REDTM, p-phthallocyanines, cyanine dyes, thiazoles, dansyl, narthAl~nl~, p-toluidinyl napthalene sulfonic 35 acid, rollmarin~ pllyuu~l~U~ , allop~l~.v~.~d~iue and the like.
The term "test sample", as used herein, refers to a material sllqpected of cnnt.Ainin~ m~LlluLlt.,dLe. The test sample can be used WO 95/16026 2 1 7 ~ 5 9 0 PCT/IJS94/13~8 directly as obtained from the source or following a pretreatment to modify the character of the sample. The test sample can be obtained from any biological source, such as a physiological fluid, including, but not limited to blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like; fermentation broths; cell cultures; chemical reaction mixture3 and the like. The test sample can be lu~elledLed prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, rlictill~t;r~n, concentration, inactivation of interfering ~nmrrnPntq~ and the addition of reagents. In addition to biological or physiological fluids, other liquid samples can be u6ed such as water, food products and the like for the performance of ellviluu~ell~dl or food production assays. In addition, a solid material suspected of rnnt:~inin~ the analyte can be used as the test sample. In some jn.ctqnrPc~ it may be beneficial to treat a solid test sample to form a liquid medium or extract the analyte.
It has been lmP~rPctP~1ly di;~ uveled that according to the present invention, a cell line can be produced which secretes a mr~n~lrlr n~l antibûdy which is specific for methotrexate and displays, surprisingly, no rlPtPrt~h1~ cross-reactivity with 7-llydlu~ylllethotrexate. Moreover, the antibody m~int~in.C its specificity over a range of ~el:l~eld~ules.
Cuus~luèll~ly~ the antibody herein provided can be employed in an immllntl~Cc~y which is more rlicrrimin~tin~ than the methotrexate immunoassays currently available.
2 5 The mt~nf.rl~nAI antibody of the instant invention can be produced following the tPrhniql1Pc described generally in Kohler and Milstein, I~Cuu~iuluuus Culture of Fused Cells Secreting Antibody of Predefined Specificity", ~, 256, 495 (1975) and iu~ Uu~ a~illg the mollifir~ti~n.~
described herein. Briefly, an i~ luuo~eu is created via the conjugation 3 0 of methotrexate to an immumogenic carrier molecule to produce an immunogen. The immunogen can be used to ;.. ".. ;,.- animals such as guinea pigs, sheep, rats and the like, preferably mice. Although virtually any animal capable of producing an immune response can be ;.,....II.~;,.-c1 with the above immunogen to form antibody producing cells, 3 5 mice are preferred and will be used llel ehlclr~ for illustrative purposes.
The imml1ni7~ti~n is pPrio-lirsll1y boosted to increase the titer of the mice antisera, and mice producing antisera specific for methotrexate are WO 95116026 ~ 5 9 0 PC'r/US94113558 i~pnt.ifi~ Splenocytes from the mice which produce antisera to methotrexate can then be removed and fused with a mouse myeloma cell line. The resulting hybrids are then screened for secretion of methotrexate specific antibody and positive hybrids are selected and 5 P~rr7~nrlP~ or cloned, to produce a sufficient quantity of antibody for purification. Expansion of a positive clone can be ~c( nmrlichPd using mPthnflnlogieq well known in the art such as, for example, in tissue culture or by injecting mice with the clone and collecting the ascites fluid from the mice after sufficient time has elapsed to allow the clone to 10 replicate and produce antibody.
Immunogens which can be used to assist in the production of the antibody herein provided can generally be described as carrier-molecule-fimrtinn~li7Pd methotrexate. Structurally, the hapten methotrexate is shown below as the compound of the formula X.
N ~N` N ~N ~OH
H2NlN N'rCH3 H
1 5 (X) Carrier mnlPclllPq that can be used to synthesize an immunogenic form of methotrexate can be selected from any of those conventionally used in the art, and in most instances will be a protein or polypeptide, although other materials such as carbohydrates, polysaccharides, 2 0 lipopolysaccharides, poly(amino) acids, nucleic acids, and the like of sufficient size and immunogenicity can also be employed. ~ ml)lp.q of carrier mnlPclllPA include, but are not intended to be limited to thyroglobulin, keyhole limpet h~lllo.yc~ (KLH), and the like, preferably bovine serum albumin (BSA). As it will be understood, of course, some 25 carrier mnlPclllPc are not i,lherently reactive with methotrexate, however, carrier mnlPclllPq can be made reactive through the introduction of an ~ 7,~7LU~ 7~ reactive moiety using methodologies well known in the art.
Methotrexate can be made illll.~UllOo~lliC by filnrti~m7l1i7in~ or 3 0 cuujuc7~iug it with at least one carrier molecule to synthesize a compound of the formula Y, shown below, wherein A and Z are inllP~Pn~lPntly -OH or a carrier molecule. Methods of sYnthPqi7inF
immunogens of the formula Y are well known in the art. For example, rnmhininF methotrexate and a carrier molecule in the presence of a WO 95/16026 2 1 7 ~ 5 9 0 PCT/US9.1/13558 dehydrating agent will yield an immunogen~of the formula Y. Suutable dehydrating agents include, but are not intended to be limited to dicyclohexyl ~a~o liilllide (DCC), 3 ethyl 1-(3-dimethylaminopropyl) call,o~ii..lide (EDAC) and the like. Heternhifi~nrti~n~l or 5 hnmnhifi-nrtinnA1 linking agents, which are well known in the art, can also be used to synthesize an immunogenic methotrexate molecule.
Such coupling agents include but are not intended to be limited to m-m~leimitlnbPn7oyl-N-llydlul~yb.. ~ ester (MBS), sulruauc~.;llilllidyl 4-(p-m~lPimirlnphenyl) butyrate (S-SMPB), m-10 m~l~imirlobenzoy1AIllfnqllrrinimi~1~ ester (S-MBS), N-g-m~ imi~nhutyrylu~y~ .."";~lP ester (GMBS) and the like. It will be umderstood, of course, that the instant invention is not limited by the methods employed to synthesize the immunogens taught herein.
A
1 5 N ~ ~rN ~NI ~
The present invention also provides improved immllnns~A~slys for rl~t~rminine the presence or amount of methotrexate in a test sample.
An improved immllno~AA~y includes or comprises a step of cnnt~Art;n~ a 2 0 test sample, which may contain methotrexate, with the antibody herein provided. It is cnnt.~mrl~t~d that any immllnn~AA~y for methotrexate utilizing the antibody herein provided is within the scope of the present invention. F,Y~mrl-~ of immllnns~A~ys include, but are not intended to be limited to, r~linimmllno~AcS.ys (RLAs), enzyme immunoassays 25 (EIAs), enzyme linked immllnosnrbent assays (ELISAs), particle concentration lluulcs~.lce immumoassays (PCFIAs) and preferahly nuu.~sc~uL polarization immumoassays (FPIAs).
In general, the antibody herein provided can be utilized in competitive binding immunoassays to (~t~rmjn~ the presence and/or 3 0 amount of an analyte in a test sample. Cu.. ,I.~LiLivt: binding immlln~ AA~ys are typically used for mP?~Ilrin~ analytes in a test sample, and for purposes of the present invention, the analyte is methotrexate. The analyte and an analyte analog compete fûr the binding site on the antibody specific for the analyte and analyte analog.
WO 95/16026 2 ~ 7 ~ ~ 9 ;~ PCIIUS9~113559 The concentration of analyte in the test sample ~l~tsrmin~q the amount of analyte analog which binds to the antibody, and the amount of analyte analog that will bind to the antibody is inversely proportional to the concentration of analyte in the test sample because the analyte and the 5 analyte analog each bind to the antibody in proportion to their respective concentrations .
In one ~mhorlim~nt of the invention, FPIA techniques are utilized for rlf~t~rminin~ the amount of tracer-antibody complex produced in a CUlUpt~iiiV~ binding immunoas6ay. Such procedures are based on the 10 principle that a fluol~ic.du~1y labeled compound or tracer, when excited by plane polarized light, will emit fluorescence having a degree of po1Ari7Atirn inver6ely related to its rate of rotation. Accordingly, when a tracer is bound by an antibody and the signal generating group associated with the tracer is excited with plane polarized light, the 15 emitted light remains highly polarized because the nuOld~..dllt label is constrained from rotating between the time that light is absorbed and emitted. In contrast, when a signal generating group associated with an unbound tracer i8 excited by plane polarized light, its rotation is much faster than the ~ ulld"~uulldillg tracer-antibody complex and the mol~c11l 2 0 become more randomly oriented. As a result, the light emitted from the unbound tracer mol~oc~ is l~pOlAri7~-1 A preferred FPIA method of the present invention for llst~rminin~
the presence or Amount of methotrexate in a test sample comprises the steps of: (i) forming a solution by c~ a test sample with (a) the 2 5 monoclonal antibody of the present invention and (b) a fluorescent tracer wherein the tracer is capable of producing a ~ tectshl~ fluorescence pr~lAri7Ati~m in response to the presence of the antibody; (ii) t-nntAl tin~ thesolution formed in step (i) with polarized light to obtain a lluul~cdllce pr~lAri7Ation response; and (ui) detecting the LlUul~.. ~ce p/~1Ari7Atinn 3 0 response of the solution of step (ii) as a measure of the presence or amount of methotrexate in the test sample.
By adding a constant cuu~ Ll~lion of the antibody and tracer to a test sample, the ratio of methotrexate-antibody cnmrl-y~ to tracer-antibody rnmrl-Y~ that are formed is directly ~ululuul~iu~al to the amount 3 5 of methotrexate in the test sample. Upon exciting the mixture with linearly polarized light and meAcl1ring the polarization (in units of polarization) of the nUUl~L~dLlC~ emitted by the unbound tracer and WO 95116026 2 ~ 7 ~ 5 ~ a PCTIUS94/135~8 tracer-antibody rt~mrlrYf~c, one is able to quantitatively determine the amount or qualitatively determine the presence of methotrexate in a test sample. The results can be quantified in terms of net millipolarization units (mP) and span (in millirrlRri7Rtion units) The measurement of 5 net millipolarization units indicates the maximum polarization when a mR~imllm amount of the tracer is bound to the antibody (in the absence of any methotrexate) and the higher the net millirolAri7Rt;~n units, the better the binding of the tracer to the antibody. Assay span is the difference between the net millipolarization values obtained when the 1 0 ..,.Y;~ 1., amount of tracer is bound in the absence of any methotrexate and the net millipolarization obtained when a specific amount of methotrexate is present in the test sample. A larger span allows for more millipolarization units to be placed between each of the calibrators of the standard curve generated for the assay, thereby providing better 15 assay precision which, in turn, results in a better numerical analysis of the data obtained. It is illlpUl ~:llli to note that the span varies ~l~rPntlin~on the sample size and the amounts of antibody and tracer can be altered accordingly.
FluolGscall~ tracer reagents (tracers) that can be used, in 2 0 conjunction with the antibody of the instant invention, to perform FPIAs, sllhetqntiRlly correspond to the ~rul~ .ionl~d i~ lUllo~GIls. However, instead of a carrier molecule, mGLIlu~lG~tG mnl~clll~c or analogs thereof are filnrt;~mRli7ed with signal generating groups. MPthnrlolr,~iec well known in the art can be used to synthesize tracers using any of the signal 2 5 generating groups previously defined. While any of the previously m~nt;~ l signal generating groups can be used to produce a tracer, nuOl~ ~cdhl and dGliV~iVGs thereof are preferred. Preferably, the tracer cu~ uu.ld:, to a culll~uuulld having the formula (Q) shown below wherein F is a signal generating group. ~ost preferably, F is 5-lluula~GIu. It 3 0 will be understood, of course, that the invention herein provided is not limited by the signal generating group used to synthesize a tracer.
H N 1`~N ~rCH ~
METHOTRl~ATh Field sf th.~ Invrntinn The present invention relates to the ;mmllnnln~ir~l detection of methotrexate. In particular, the invention relates to a cell line which secretes a mnnnrlnnAl antibody specific for methotrexate and imm~lnnlngi~Al methods for detecting the presence or amount of methotrexate in a test sample.
R~3rk~rollnrl of thr Tnv~ntinn Methotrexate is an antineoplastic agent which is commonly used for treating patients having solid tumors and/or h.omAtnlngirAl mAlignAn~ The dosages of methotrexate received by these patients 1 ~ can vary widely because the therapeutic effect of methotrexate varies with the dosage. Unfortunately, however, patients on high methotrexate regimens may experience toxic side effects.
In order to avoid the toxic side effects Acqor;At.od with high doses of methotrexate, immllnnA.~sAys have been developed to monitor patients' methotrexate levels. These immunoassays commonly use Ant.;hn~
which are specific for methotrexate, to assist in the detection of the presence or amount of methotrexate in a patient's test sample. Results from these assays can be used to modify, if necessary, a patients methotrexate regimen to prevent the toxic side effects that may result 2 5 from high doses of methotrexate or to optimize a patients methotrexate regimen.
TmmllnnA~cAys designed to ~ trrminp the presence or amount of methotrexate in a test sample require highly specific antibodies to avoid erroneous assay results which may lead to inJ~lu,ul u~ Le dosage changes 3 0 which, can in turn, lead to toxic side effects or the administration of less than optimal methotrexate doses. Erroneous assay results can occur when the antibody used to detect methotrexate cross reacts with other rnmrollntlq For example, 7-hydroxymethotrexate is a m~t~holit.f~ of methotrexate that is structurally similar to methotrexate and cross 3 5 reacts with anti-m~llu~ d antibody.
Because 7-~1yLu~ylllt ~llotrexate is a m~t~hnl;t~ of methotrexate, nearly every test sample rnntAininF m~Ulu~ will also contain 7-wosSll6o26 21 785qa 2 PCTIUS9~/13~8 hydroxymethotrexate, and, in some inct~nr~5c, the concentration of the metabolite can exceed the concentration of the par~nt compound. Thus, an assay designed to determine the presence or amount of methotrexate in a test 6ample should employ Antiho~ A that have very little cross 5 reactivity with 7-hydroxymethotrexate. Most desirably, such antibodies should have no lP+~t~hl~ cross reactivity with 7-hydroxymethotrexate.
European Patent Application No. 0044441 generally describes the production of mnnnrlnnAl antibodies specific for drugs, but it does not describe the production of a mnnnrlnn~1 antibody specific for 10 methotrexate. Moreover, the disclosure does not describe an anti-methotrexate antibody with any particular specificity.
Assays which detect the presence or amount of mG~llo~G~te in a test sample and which are currently available, employ polyclonal nntiho~ .c to assist in the detection of methotrexate. However, according 15 to the methods of drt~rmininE~ cross reactivity used herein, such polyclonal antibodies can display cross reactivity with 7-hydl u~ylllethotrexate.
IIence there is a need for an antibody that is more ~liqrriminzltin~
than those currently available which can be used in an ;mmllnn~c.c~y to 2 0 accurately ~PtPrmin~' the presence or amount of methotrexate in a test sample .
Sllmm~ry of thP Inv~ntinn The present invention provides a mnnnrlnnz~l IgG antibody that is 2 5 specific for methotrexate and has no tl~tect 3hlP cross reactivity with 7-yd~u~y~ethotrexate. A-lrlit;~ lly, the invention provides a hybridoma cn~ ; eell line which secretes the above mnnnrlnn~l antibody and has been 11PCi~tPd cell line number A.T.C.C. HB 11467.
The mnnnrl~ l antibody of present invention ean be used in 30 various immllnnACc~y formats. Aeeording to one Pmho~limPnt, a nuulesCGllce polarization immllno~Cc~y (FPIA) for ~iPtPrminin~ the presence or amount of methotrexate in a test sample is provided. The FPIA rnmrriqPC the steps of (a) cnnt~r+in~ the test sample with (i) the mnrl~rlnnAl IgG antibody provided herein and (ii) a nuul~S..~u~ tracer 35 moleeule, to form a solution; (b) cnnt~t~tin~ the solution with plane polarized light; and (c) detecting a fluu-GscG..ce pnl~ri7~tinn response.
Aeeording to another PmhûrlimPnt, an improyed immunoassay for WO 9~116026 2 1 ~ ~ 5 ~ ~ PC~J~vS941~3SS8 determining the presence or amount of methotrexate in a test samp~e is provided. The improved immunoassay is of the type having a step of rnntArtin~ the test sample with Antiho~ q specific for methotrexate, wherein the hl.l.lv~l.lcnt comprises cnntActin~ the test sample with the monoclonal IgG antibody herein proYided.
The present invention also provides a test kit for ~ terminin~ the presence or amount of methotrexate in a test sample. The test kit comprises (a) the mnnnrlnnAl IgG antibody of the invention; and (b) a fluorescent tracer molecule.
Detail~rl Descr~tion of thl~ Jny~ntinn The following ~ finitinn.q are ArrlirAhl~ to the invention.
The term "analyte analog", as used herein, refers to a gllhctqnr~
which cross-reacts with a methotrexate-specific antibody, although it may do so to a greater or lesser extent than does the analyte (methotrexate) itsel The analyte analog can include a modified analyte as well as a frA~m-~nt.~(l or synthetic portion of the analyte molecule, 90 long as the analyte analog has at least one epitope in common with the analyte of interest.
The term "ill. uuuv~ ", as used herein, refers to a substance which, when illLIùvlu~t:d into an animal having an immune system, is capable of s~imlllatin~ an immune response. ~ertain low molecular weight sllhctanrPq which are not capable of stimulating an immune response are referred to as haptens. However, haptens can be made immunogenic by conjugating a hapten to a carrier molecule, which may itself be immunogenic. Thus, while generally not immunogenic, haptens conjugated to carrier mnl~clll.o.q are immunogenic and can be considered immunogens.
The term "signal generating group" as used herein refers to a nuvl~ ,e ,t cnmrolln-l (snm~tim~q referred to as a fluorophore) which is capable of absorbing energy and emitting light or fluorescing. F~amrl(~q of signal ~..( ., Liug groups include, but are not intended to be limited to nuul~s~ ;u, cascade blue, TEXAS REDTM, p-phthallocyanines, cyanine dyes, thiazoles, dansyl, narthAl~nl~, p-toluidinyl napthalene sulfonic 35 acid, rollmarin~ pllyuu~l~U~ , allop~l~.v~.~d~iue and the like.
The term "test sample", as used herein, refers to a material sllqpected of cnnt.Ainin~ m~LlluLlt.,dLe. The test sample can be used WO 95/16026 2 1 7 ~ 5 9 0 PCT/IJS94/13~8 directly as obtained from the source or following a pretreatment to modify the character of the sample. The test sample can be obtained from any biological source, such as a physiological fluid, including, but not limited to blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like; fermentation broths; cell cultures; chemical reaction mixture3 and the like. The test sample can be lu~elledLed prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, rlictill~t;r~n, concentration, inactivation of interfering ~nmrrnPntq~ and the addition of reagents. In addition to biological or physiological fluids, other liquid samples can be u6ed such as water, food products and the like for the performance of ellviluu~ell~dl or food production assays. In addition, a solid material suspected of rnnt:~inin~ the analyte can be used as the test sample. In some jn.ctqnrPc~ it may be beneficial to treat a solid test sample to form a liquid medium or extract the analyte.
It has been lmP~rPctP~1ly di;~ uveled that according to the present invention, a cell line can be produced which secretes a mr~n~lrlr n~l antibûdy which is specific for methotrexate and displays, surprisingly, no rlPtPrt~h1~ cross-reactivity with 7-llydlu~ylllethotrexate. Moreover, the antibody m~int~in.C its specificity over a range of ~el:l~eld~ules.
Cuus~luèll~ly~ the antibody herein provided can be employed in an immllntl~Cc~y which is more rlicrrimin~tin~ than the methotrexate immunoassays currently available.
2 5 The mt~nf.rl~nAI antibody of the instant invention can be produced following the tPrhniql1Pc described generally in Kohler and Milstein, I~Cuu~iuluuus Culture of Fused Cells Secreting Antibody of Predefined Specificity", ~, 256, 495 (1975) and iu~ Uu~ a~illg the mollifir~ti~n.~
described herein. Briefly, an i~ luuo~eu is created via the conjugation 3 0 of methotrexate to an immumogenic carrier molecule to produce an immunogen. The immunogen can be used to ;.. ".. ;,.- animals such as guinea pigs, sheep, rats and the like, preferably mice. Although virtually any animal capable of producing an immune response can be ;.,....II.~;,.-c1 with the above immunogen to form antibody producing cells, 3 5 mice are preferred and will be used llel ehlclr~ for illustrative purposes.
The imml1ni7~ti~n is pPrio-lirsll1y boosted to increase the titer of the mice antisera, and mice producing antisera specific for methotrexate are WO 95116026 ~ 5 9 0 PC'r/US94113558 i~pnt.ifi~ Splenocytes from the mice which produce antisera to methotrexate can then be removed and fused with a mouse myeloma cell line. The resulting hybrids are then screened for secretion of methotrexate specific antibody and positive hybrids are selected and 5 P~rr7~nrlP~ or cloned, to produce a sufficient quantity of antibody for purification. Expansion of a positive clone can be ~c( nmrlichPd using mPthnflnlogieq well known in the art such as, for example, in tissue culture or by injecting mice with the clone and collecting the ascites fluid from the mice after sufficient time has elapsed to allow the clone to 10 replicate and produce antibody.
Immunogens which can be used to assist in the production of the antibody herein provided can generally be described as carrier-molecule-fimrtinn~li7Pd methotrexate. Structurally, the hapten methotrexate is shown below as the compound of the formula X.
N ~N` N ~N ~OH
H2NlN N'rCH3 H
1 5 (X) Carrier mnlPclllPq that can be used to synthesize an immunogenic form of methotrexate can be selected from any of those conventionally used in the art, and in most instances will be a protein or polypeptide, although other materials such as carbohydrates, polysaccharides, 2 0 lipopolysaccharides, poly(amino) acids, nucleic acids, and the like of sufficient size and immunogenicity can also be employed. ~ ml)lp.q of carrier mnlPclllPA include, but are not intended to be limited to thyroglobulin, keyhole limpet h~lllo.yc~ (KLH), and the like, preferably bovine serum albumin (BSA). As it will be understood, of course, some 25 carrier mnlPclllPc are not i,lherently reactive with methotrexate, however, carrier mnlPclllPq can be made reactive through the introduction of an ~ 7,~7LU~ 7~ reactive moiety using methodologies well known in the art.
Methotrexate can be made illll.~UllOo~lliC by filnrti~m7l1i7in~ or 3 0 cuujuc7~iug it with at least one carrier molecule to synthesize a compound of the formula Y, shown below, wherein A and Z are inllP~Pn~lPntly -OH or a carrier molecule. Methods of sYnthPqi7inF
immunogens of the formula Y are well known in the art. For example, rnmhininF methotrexate and a carrier molecule in the presence of a WO 95/16026 2 1 7 ~ 5 9 0 PCT/US9.1/13558 dehydrating agent will yield an immunogen~of the formula Y. Suutable dehydrating agents include, but are not intended to be limited to dicyclohexyl ~a~o liilllide (DCC), 3 ethyl 1-(3-dimethylaminopropyl) call,o~ii..lide (EDAC) and the like. Heternhifi~nrti~n~l or 5 hnmnhifi-nrtinnA1 linking agents, which are well known in the art, can also be used to synthesize an immunogenic methotrexate molecule.
Such coupling agents include but are not intended to be limited to m-m~leimitlnbPn7oyl-N-llydlul~yb.. ~ ester (MBS), sulruauc~.;llilllidyl 4-(p-m~lPimirlnphenyl) butyrate (S-SMPB), m-10 m~l~imirlobenzoy1AIllfnqllrrinimi~1~ ester (S-MBS), N-g-m~ imi~nhutyrylu~y~ .."";~lP ester (GMBS) and the like. It will be umderstood, of course, that the instant invention is not limited by the methods employed to synthesize the immunogens taught herein.
A
1 5 N ~ ~rN ~NI ~
The present invention also provides improved immllnns~A~slys for rl~t~rminine the presence or amount of methotrexate in a test sample.
An improved immllno~AA~y includes or comprises a step of cnnt~Art;n~ a 2 0 test sample, which may contain methotrexate, with the antibody herein provided. It is cnnt.~mrl~t~d that any immllnn~AA~y for methotrexate utilizing the antibody herein provided is within the scope of the present invention. F,Y~mrl-~ of immllnns~A~ys include, but are not intended to be limited to, r~linimmllno~AcS.ys (RLAs), enzyme immunoassays 25 (EIAs), enzyme linked immllnosnrbent assays (ELISAs), particle concentration lluulcs~.lce immumoassays (PCFIAs) and preferahly nuu.~sc~uL polarization immumoassays (FPIAs).
In general, the antibody herein provided can be utilized in competitive binding immunoassays to (~t~rmjn~ the presence and/or 3 0 amount of an analyte in a test sample. Cu.. ,I.~LiLivt: binding immlln~ AA~ys are typically used for mP?~Ilrin~ analytes in a test sample, and for purposes of the present invention, the analyte is methotrexate. The analyte and an analyte analog compete fûr the binding site on the antibody specific for the analyte and analyte analog.
WO 95/16026 2 ~ 7 ~ ~ 9 ;~ PCIIUS9~113559 The concentration of analyte in the test sample ~l~tsrmin~q the amount of analyte analog which binds to the antibody, and the amount of analyte analog that will bind to the antibody is inversely proportional to the concentration of analyte in the test sample because the analyte and the 5 analyte analog each bind to the antibody in proportion to their respective concentrations .
In one ~mhorlim~nt of the invention, FPIA techniques are utilized for rlf~t~rminin~ the amount of tracer-antibody complex produced in a CUlUpt~iiiV~ binding immunoas6ay. Such procedures are based on the 10 principle that a fluol~ic.du~1y labeled compound or tracer, when excited by plane polarized light, will emit fluorescence having a degree of po1Ari7Atirn inver6ely related to its rate of rotation. Accordingly, when a tracer is bound by an antibody and the signal generating group associated with the tracer is excited with plane polarized light, the 15 emitted light remains highly polarized because the nuOld~..dllt label is constrained from rotating between the time that light is absorbed and emitted. In contrast, when a signal generating group associated with an unbound tracer i8 excited by plane polarized light, its rotation is much faster than the ~ ulld"~uulldillg tracer-antibody complex and the mol~c11l 2 0 become more randomly oriented. As a result, the light emitted from the unbound tracer mol~oc~ is l~pOlAri7~-1 A preferred FPIA method of the present invention for llst~rminin~
the presence or Amount of methotrexate in a test sample comprises the steps of: (i) forming a solution by c~ a test sample with (a) the 2 5 monoclonal antibody of the present invention and (b) a fluorescent tracer wherein the tracer is capable of producing a ~ tectshl~ fluorescence pr~lAri7Ati~m in response to the presence of the antibody; (ii) t-nntAl tin~ thesolution formed in step (i) with polarized light to obtain a lluul~cdllce pr~lAri7Ation response; and (ui) detecting the LlUul~.. ~ce p/~1Ari7Atinn 3 0 response of the solution of step (ii) as a measure of the presence or amount of methotrexate in the test sample.
By adding a constant cuu~ Ll~lion of the antibody and tracer to a test sample, the ratio of methotrexate-antibody cnmrl-y~ to tracer-antibody rnmrl-Y~ that are formed is directly ~ululuul~iu~al to the amount 3 5 of methotrexate in the test sample. Upon exciting the mixture with linearly polarized light and meAcl1ring the polarization (in units of polarization) of the nUUl~L~dLlC~ emitted by the unbound tracer and WO 95116026 2 ~ 7 ~ 5 ~ a PCTIUS94/135~8 tracer-antibody rt~mrlrYf~c, one is able to quantitatively determine the amount or qualitatively determine the presence of methotrexate in a test sample. The results can be quantified in terms of net millipolarization units (mP) and span (in millirrlRri7Rtion units) The measurement of 5 net millipolarization units indicates the maximum polarization when a mR~imllm amount of the tracer is bound to the antibody (in the absence of any methotrexate) and the higher the net millirolAri7Rt;~n units, the better the binding of the tracer to the antibody. Assay span is the difference between the net millipolarization values obtained when the 1 0 ..,.Y;~ 1., amount of tracer is bound in the absence of any methotrexate and the net millipolarization obtained when a specific amount of methotrexate is present in the test sample. A larger span allows for more millipolarization units to be placed between each of the calibrators of the standard curve generated for the assay, thereby providing better 15 assay precision which, in turn, results in a better numerical analysis of the data obtained. It is illlpUl ~:llli to note that the span varies ~l~rPntlin~on the sample size and the amounts of antibody and tracer can be altered accordingly.
FluolGscall~ tracer reagents (tracers) that can be used, in 2 0 conjunction with the antibody of the instant invention, to perform FPIAs, sllhetqntiRlly correspond to the ~rul~ .ionl~d i~ lUllo~GIls. However, instead of a carrier molecule, mGLIlu~lG~tG mnl~clll~c or analogs thereof are filnrt;~mRli7ed with signal generating groups. MPthnrlolr,~iec well known in the art can be used to synthesize tracers using any of the signal 2 5 generating groups previously defined. While any of the previously m~nt;~ l signal generating groups can be used to produce a tracer, nuOl~ ~cdhl and dGliV~iVGs thereof are preferred. Preferably, the tracer cu~ uu.ld:, to a culll~uuulld having the formula (Q) shown below wherein F is a signal generating group. ~ost preferably, F is 5-lluula~GIu. It 3 0 will be understood, of course, that the invention herein provided is not limited by the signal generating group used to synthesize a tracer.
H N 1`~N ~rCH ~
6 2 1 7 ~ ~ 9 ~ PCT/US9~1/13558 As previously mentioned, an FPIA is the preferred method for using the antibody herein provided. Reagents that can be used for such an assay comprise: 1) a monnrlnnAl antibody specific for methotrexate and 2) a fluorescent tracer reagent. A~-litinn~lly~ conventional reagents - 5 including a ~ al~lllent solution, a dilution buffer, methotrexate calibrators and controls are desirably prepared. These reagents are commercially available in assay "kits" from Abbott Laboratories, Abbott Park, IL.
A test kit according to the present invention comprises all of the essential reagents required to perform a desired immllnns~ y for the qll~nt.ifir~tinn or qualitative ,1..~...., .; ., .tinn of methotrexate in a testsample. The test kit is presented in a commercially packaged form as a r,nmhins~tion of one or more containers holding the necessary reagents, as a rnmrnAit.jnn or admixture where the rnmrAtihility of the reagents 1 5 will allow. Padrticularly preferred is a test kit for a FPL~ qlls~ntifin~tinn of methotrexate in a test sample, rnmrricin~ the antibody herein provided and a tracer having the formula Q shown above. It will be understood, of course, that the test kit can include ot_er materials well known in the art and which may be desirable from a users s~n~rnint, such as buffers, diluents, standards and the like.
The preferred ~,- U~ dUI t: iS particularly designed to be used in conjunction with the Abbott TDXe3 Clinical Analyzer, the Abbott TDXFLXlM or the Abbott ADX~) Drugs of Abuse Systems, all of which are available from Abbott Labuldtul;es. The calibrators, controls, or unknown samples, which do not require any priûr preparation, are pipetted directly into the sample well of the TDX~19 sample cartridge and the remaining assay procedure is fully ~lltnmslt~rl All references to patents or pllhlir~tinnq in this ~pf,ri~r~tinn are hl~ol~uld~ed herein by reference.
3 0 The following examples are provided to further illustrate ~mhollim~nt~ of the invention and should not be construed as a limit~tinn on the scope of the invention. All animals, media, reagents, and equipment used in the production of the cell line and mnnnclnn~l antibody of the invention are publicly available. Those skilled in the art will readily recognize that ".n.l;l;. ~ and Vdl;a~iUI~S of the disclosed protocols and methods exist and are within the scope of the invention.
wo 95/16026 ~ t 7 ~ ~ ~ Q 1 o PCTIUS94/13558 E~;ample 1 IMMUNONOGEN SYNTEIESIS
~At.f~r;~l.q 5 1. (+)-Amethopterin (methotrexate) - available from Sigma Chemical Co. (Sigma); St. Louis, Missouri 2. 1-(3-Dimethylaminopropyl) carbodiimide HCl (EDAC) - available from Sigma 3. Bovine serum albumin (BSA) - available from Intergen; Purchase, New York 4. SEPHADEX(~) G50-300 - available from Pharmacia-LKB;
Piscataway, New Jersey 5. Phosphate buffered saline (PBS) - 0.01 M NaPO4, 0.15 M NaCl, pH
A test kit according to the present invention comprises all of the essential reagents required to perform a desired immllnns~ y for the qll~nt.ifir~tinn or qualitative ,1..~...., .; ., .tinn of methotrexate in a testsample. The test kit is presented in a commercially packaged form as a r,nmhins~tion of one or more containers holding the necessary reagents, as a rnmrnAit.jnn or admixture where the rnmrAtihility of the reagents 1 5 will allow. Padrticularly preferred is a test kit for a FPL~ qlls~ntifin~tinn of methotrexate in a test sample, rnmrricin~ the antibody herein provided and a tracer having the formula Q shown above. It will be understood, of course, that the test kit can include ot_er materials well known in the art and which may be desirable from a users s~n~rnint, such as buffers, diluents, standards and the like.
The preferred ~,- U~ dUI t: iS particularly designed to be used in conjunction with the Abbott TDXe3 Clinical Analyzer, the Abbott TDXFLXlM or the Abbott ADX~) Drugs of Abuse Systems, all of which are available from Abbott Labuldtul;es. The calibrators, controls, or unknown samples, which do not require any priûr preparation, are pipetted directly into the sample well of the TDX~19 sample cartridge and the remaining assay procedure is fully ~lltnmslt~rl All references to patents or pllhlir~tinnq in this ~pf,ri~r~tinn are hl~ol~uld~ed herein by reference.
3 0 The following examples are provided to further illustrate ~mhollim~nt~ of the invention and should not be construed as a limit~tinn on the scope of the invention. All animals, media, reagents, and equipment used in the production of the cell line and mnnnclnn~l antibody of the invention are publicly available. Those skilled in the art will readily recognize that ".n.l;l;. ~ and Vdl;a~iUI~S of the disclosed protocols and methods exist and are within the scope of the invention.
wo 95/16026 ~ t 7 ~ ~ ~ Q 1 o PCTIUS94/13558 E~;ample 1 IMMUNONOGEN SYNTEIESIS
~At.f~r;~l.q 5 1. (+)-Amethopterin (methotrexate) - available from Sigma Chemical Co. (Sigma); St. Louis, Missouri 2. 1-(3-Dimethylaminopropyl) carbodiimide HCl (EDAC) - available from Sigma 3. Bovine serum albumin (BSA) - available from Intergen; Purchase, New York 4. SEPHADEX(~) G50-300 - available from Pharmacia-LKB;
Piscataway, New Jersey 5. Phosphate buffered saline (PBS) - 0.01 M NaPO4, 0.15 M NaCl, pH
7.2to7.5 1 5 Procedllre.
Methotrexate (251.2 mg) is dissolved in 4.0 ml of distilled water and 1.0 ml of 2.0 N sodium hydroxide (NaOH) to form a solution. The pH of the resulting solution is adjusted to 7.5 with 0.1 N hydrochloric acid (HCI). The total volume of the solution is d,U~JI U~ilUdLt:ly 10.0 ml (approximately 25 mg/ml methotrexate). BSA (77.7 mg) is dissolved in 2.0 ml of distilled water to form a second solution. A third solution is prepared and ,IJIll,Ul ;~es 250 mg of EDAC in 5.0 ml of distilled water.
1.5 ml of the m~LlluLl~d~ solution is added to the BSA solution and dissolved at room L~ aLule. Over a three hour period, 0.7 ml of 2 5 the EDAC solution is added to the methotrexateMSA solution in 100 ,ul aliquots, and thereafter, 0.8 ml of the EDAC solution is added all at once for a total EDAC solution addition of 1.5 ml. This reaction mixture is allowed to mix overnight and after the mixing period the solution turns to a clear bright-yellow-solution.
A SEPHADEX(~ G50-300 column (2.5 mm ~ 400 mm) is equilibrated with PBS before the reaction mixture is applied to it and eluted with PBS. The eluant from the column is ul~ ulqd for absorbance (A2go) and the il-'l"UI10~ (BSA-methotrexate complex) is contained in the first peak eluted from the column. The volume of the 3 5 collected portion of the first peak is 44.5 ml with a protein concentration of 2.0 mg/ml which yields 89.0 mg of immunogen.
WO 95116026 2 ~ 7 ~ ~ 9 ~ PCTrUS94113558 ~.~s~mple 2 Tra~cer ~ 'h~
Mater;~l c 1. Methotrexate - available from Sigma 5 2. 5-Amint)flllorescein - available from Aldrich Chemical Co.
(Aldrich); Milwaukee, Wiscon3in 3. SiO2 Thin Layer Chromatography (TLC) plates - available from EM Reagents; Gibbstown, New Jersey 4. Clg TLC plates - available from Whatman Inc.; Clifton, New 1 0 Jersey Proced-lre:
The following solutions are prepared: 0.2305 g of methotrexate in enough dimethylsulfoxide (DMSO) to dissolve the methotrexate; 0.09 g of p-nitrophenol in enough DMSO to dissolve the p-nitrophenol; 0.11 g of 15 dicyclohexyldi~lbor~ihLIide (DCC) in enough DMSO to dissolve the DCC;
and 0.1747 g of 5-~minr~flllllrescein in enough DMSO to dissolve the 5-Amin~ s~ir~ The methotrexate (approximately 1.0 ml), DCC
(approximately 1.0 ml), and p-nitrophenol solutions (approximately l.0 ml) are combined to form a reaction mixture that is allowed to stir for 4 20 hours at room t~ ,UI~. Then, the 5-~min~.nll..,t:~r ~ solution ly 1.0 ml) is added to the reaction mixture while it is stirring. The resulting reaction mixture is protected from light and allowed to stir overnight. After the mixing period, the reaction mixture is applied to eight, 2.0 mm SiO2 TLC plates. The full length of the plates 2 5 are developed with (2~ 1.1roform and allowed to dry prior to developing the plates half-way with (l:l) chloroform mPth~n~ll and allowing them to dry. The bands from one developed plate are separately collected and eluted from the SiO2 plates with methanol before the bands from the plate are applied to a Clg TLC plate. The Clg plate is developed 3 0 in 50% methanol with 0.05% sodium dodecyl sulfate (SDS) and 'iO% 0.2%
KH2PO4 in distilled water and show that the product is in the region corrP.~rnnrlin~ to Rf 0.05 to 0.38 of the SiO2 plates. The remaining SiO2 plates are scraped from R~ 0.05 to 0.38 and the product is eluted with mPtll~nn1, conc".~ d to ~ u~hllately 4.0 ml, and applied to ten l.0 3 5 mm Clg TLC plates. The plates are developed as above and the brightest band, as determined by an ultraviolet lamp, is at Rf 0.69 to 0.76. The band is collected, eluted with ethanol and concentrated as before. The WO 95/16026 2 ~ 7 ~ 5 q ~ PCT/US94/13SS8 concentrated product is applied to eight C1g TLC plates, developed, and the final product is collected as above and is showrl below as the compound of the formula (P).
HO _~0 O CO H
H2N ~N CH~ N ~NH
Ex~nple 3 An~: '' ' G ' ~r ...., ~ ~yl~u~iiv -Tmmiln;7:~tir)n strateev A 2.0 mg/ml slurry, rrmt~inin~ the methotrexate conjugate of 10 Example 1 ~mlllRifi~d in Freund's Complete Adjuvant (Difco Inc, Detroit, Mirhii~An) and 0.9% saline, is used to Rllhcllt~nr~ously immunize ten RFBlDnJ mice (available from Jackson Laboratories; Bar Harbor, Maine) such that each mouse received a total of 100 llg of conjugate.
Four weeks later each mouse is ;.,.,...~.I;,.~d a second time using the 5 same ~IU~,~,dUl~ specified above except that the conjugate is emulsified in Tnrnmrlrte Freund's Adjuvant (Difco Inc., Detroit, MirhiE~n) and each mouse is i~..~..~",;,._rl with 20 ilg of corljugate. A third 20 ilg/mouse j""",..,;,.~ n is pr.~fr~rmr~d after 8 weeks from the date of the original ;.".,.u.,i~ ion with the TnrrmrlPt~ Freund's Adjuvant protocol and a 20 final 20 ilg/mouse ;l~ rln is rrmrlrt~d 12 weeks after the original ;"",...";,.,.1 r)n The mice are bled 6, 10 and 15 weeks after the initial immumization and sera from the bleeds is assayed for an immune response to methotrexate.
25 Serllm T~',v5~ ti~1n Utilizing a co..,~ binding assay format, the mouse sera (from above) is assayed for anti-ll-eulu~ e activity. The samples are tested on a TDX(!~) Clinical Analyzer (Abbûtt Laboratories) using a reagent configuration which includes reagents labeled S pot, T pot, and P pût 30 which contain the following:
WO 95/16026 2 1 7 8 5 9 ~ PCIIUS94/13558 S pot: 0.1 M rhncrhslte buffer, 2% ethylene glycol, 5% BSA, 0.1% sodium azide, 0.01% bovine gamma globulin (BGG) T pot: 62 nM nuult~SC~iu tracer in 0.1 M tris, 0.7% sodium dodecyl sulfate (SDS), 0.5% lithium dodecyl sulfate (LDS), 0.01% (BGG), 0.1% sodium azide P pot: 0.1 M tris, 0.7% SDS, 0.5% LDS, 0.01% BGG, 0.1%
sodium azide Before running the assay, the mouse sera iB serially diluted in the S pot diluent which is dispensed in the sample cup of the sample cartridge.
The dilutions range firom a neat sample to a 1:32 dilution. Antibody dic~ ( Prnf~nt is tested by spiking 8.0 ,ul of the A and F calibrators in the predilute cup of the sample cartridge. The sample cartridges are placed on the carousel and the samples are tested using mode 11 pipetting 6equence which has a 10 ,ul sample size. Dicrl~r~m~nt of the A-F (0-1.0 llmol/L) calibrators ranged from 28-65 mP. Because the serum sample firom mouse ~1PC; ns~tl~rl 1-lA gave the best ~i.c~l~n~nn~nt., a curve is generated for the serum from this mouse and the serum is assayed for 7-Il~u;~y~ethotrexate~ 4-deoxy-4-amino-N10-m~Lllyl,uL~lu:c acid (DAMPA) 2 0 and aminopterin cross reactivity. The results firom the assay show low cross reactivities for all three of the cross reactants and a decision is made to use cpl~- , Lt:~ firom mouse 1-lA for fiusion.
Pre-Fllcinn Tmmllni7~tinn 2 5 Three days prior to cell filsion and 12 weeks after the original immllni7~tinn mouse 1-lA is given a 20 ~g injection of the methotrexate conjugate (firom Example 1) diluted in 0.1 ml rhncrhslt~ buffered saline (PBS - pH 7.2) via the tail vein.
3 0 Ela~
On the day of the fusion, mouse 1-lA is ellth~ni7f~d by cervical orsltinn and the spleen is removed. The splenocytes are washed one time in Iscove's Modified Dulbecco's Medium (IMDM - GIBCO, Grand Island, New York) ana u ~:uLliru~d at 1000 RPM for 7 minutes. The 3 5 supernatant is removed and the pelletted splenocytes are combined with SP2/0 myeloma cells (firom the laboratory of Dr. Milstein, Cambridge, United Kingdom) at a 1:1 ratio, washed in IMDM, and r~nt.rifil~fl The WO 9~/16026 2 1 7 8 5 q ~ PCT/llS94113558 supernatant is removed from the pelletted cells and l.0 ml of 50% PEG
(polyethylene glycol available from American Type Culture Collection, Rockville, Maryland) is added to the pellet and the pellet is gently dispersed for approYimately 1 minute by tapping and swirling. 30.0 mls 5 of IMDM is added to the miYture and centrifuged as previously described. The supernatant is decanted and the pellet is resuspended in IMDM with HAT (hypnYAnt.hinf', aminopterin, thymidine available from GIBCO), 10% Fetal Bovine Serum (FBS - Hyclone Laboratories, Logan, Utah) and 0.5% STM v/v (RIBI Tmmllnorh~nn Research, Inc., T-TAmiltnn, 10 Montana). STM denotes ,sAlmnnf?llA tv~ .l mitogen, a B-cell specific mitogen, and is used to enhance fusion frequency. The fusion cell suspension is plated into 96-well tissue culture plates.
F~lcinn Scr~ninF
The primary screening of the 96-well fusion sll4r~qncinn supernatant occurred on day 10 of confluent cultures. A SCREEN
MACHINElM, (IDEXX Labul~lL~/LI~A7, Westbrook, Maine) is used according to mAnllfA~tllrerls instructions for the primary fusion screen.
Microparticles coated with goat anti-mouse IgG ~ M (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Maryland) and the mnnnrlnnAl antibody fusion supernatant sample are added to the 96-well IDEXX
FLUORICON(g~ assay plates. Following a 15 minute in~-llhAtinn, the plates are washed and a methotrexate-fluorescein coruugate is added to each sample well. After a 10 minute inrnhAtinn the plates are washed a final time and the relative nuul~ s~ t intensity is read. Values with elevated signals indicate the pre6ence of anti-methotrexate antibody in the culture. Cultures with a signal greater than 3 times the negative control are e~panded to 24-well tissue culture plates for further evaluation.
3 0 Positive Hybrids from the IDEXX screen are then tested on the Abbott TDX~E9 Clinical Analyzer~ according to a modified screeni~g format which allows the monoclonal antibody supernatant to be tested in the sample wells. According to the modified assay format, the methotreYate reagent pack ~....I.~i..i..e the S pot, T pot and P pot reagents 35 (described above) and pipetting mode 11 are used. All-litinnAlly, the 6ample size is 10.0 ,ul and the sera from the hybrids is used as the sample. Polarization of the test samples is evaluated in the absence and WO 95116026 2 ~ 7 8 5 ~ ~ PCTIUS9 1113558 presence of free methotrexate and the cross reactivity of the antibody with 7-llydlu~ylllethotrexate is also evaluated. Antibody from hybrids that had a milli~ ri7Ati-)n (mP) value for 7-hydroxymethotrexate and the A
calibrator within 20 mP of each other are selected for cloning.
~vbrid clnnin,~r Seventy hybrids are cloned by limiting dilutions starting at a 1-100 dilution, 10-fold down to 10-6. The cloning media is IMDM with 10% v/v FBS and 1% v/v HT supplement (hylJ~7 A ~ 1 i I IP, thymidine available from 1 0 GIBCO). A 100 111 cell s~lqpPnCilm is added to each of the 96 wells in the tissue culture plate and on day 7 the plates are fed with 200 Ill/well of cloning media. The antibody from the resulting clones is assayed for anti-methotrexate activity and 7-hydroxymethotrexate cross reactivity using the same method used to screen the hybrids. Based on the clone 5 screening, 23 clones are selected for antibody production and further screening.
Antihf~dv Pro~llrtifm The selected clones are scaled up in ascites fiuid by injecting 2 0 approximately 106 cells into the pPrit.onPAl cavity of pristaned primed female BALB/c mice (Charles River Labs; Charles River, New York).
Two weeks later the animals are ellt.h7~1ni7Pd and the ascites fluid is harvested. The antibody cr~ntAinin~ fluid is ut~ulliru~;t~d at 1000 RPM for 10 minutes to remove the cells. The ascites fluid is then screened for 2 5 specificity and temperature sensitivity.
Specificitv ~n(l T~nlpPrature Spncitivitv ScrPRnin,~
A modified screening format allows selection of a methotrexate clone based on temperature sensitivity (airset studies) and specificity 3 0 (cross-reactivity studies). The antibody from the methotrexate clones is tested on the TDX(E9 Clincal Analyzer (Abbott LabuL~Lul;t7b) using pipetting mode 11, a sample size of 17.5 ~1, 13.~ ~Ll of altering A & F
calibrator in the pre-dilute cup, and antibody straight or titered in the sample cup. The reagent pack configuration comprises the the S pot, T
35 pot and P pot diluent and reagents ~viuu~,ly described.
All Antih~rliPc from the anti-methotrexate producing clones are initially screened using the above procedure and the titer is selected to WO 9S/160Z6 2 1 7 P~ S q ~ 1 6 PCT/US94/135S8 have a Po value of between 170 and 190. When an antibody dilution gave a Po value between 170 and 190, the antibody is 6creened at that dilution for temperature st,ll,,iLiviiy. The L~ ld~ulr~ sensitivity protocol requires three runs. All three runs follow the modified screening format 5 previously described with the e2ception that, the A and F calibrators are run in duplicate on an instrument with a correct airset setting (34.0C), a -2 airset setting (32.0C) to lower reaction t~ ra~Ult, and +2 airset setting (36.0C) to rai6e the reaction temperature. Actual temperatures of the liquid in the cuvette are measured with an external temperature 1 0 probe in the last cuvette on the carousel at the connpl~ti~m of the run. The mPs are compared, looking for the least amount of change seen in the A
and F calibrators for the 32.0C and 36.0C runs. For ~ ix.", an unpurified antibody having undesirable ~ laluu~ and cross reactivity charA~ t~riPt;~ x (UP935) and the commercial polyclonal antibody (S pot) 15 are used as positive and rlegative controls respectively. The data for the antibody from 23 clones assayed for the t~lu~ Ul~ sensitivity is shown in Table 1. A~1~1iti~nAlly, the values shown for the controls are an average taken from multiple runs.
WO 95/16026 2 1 7 ~ S 9 ~ PCT/US9411355~
IY, 0 CD O O C~ --~ O ~ O ~ O O O O ~ ~ D O ~ O ~ O 0 I~ ~ ' ' ~O ' 10 ' C~ O C~ CD O O~ q CD C~ O o o~
O O O C~ Cl a N a ~ a o ~ a a c~ o o o o o~ c~ ~ a o~
o ~ N O ~P ~P CD ~ ~r C~ N 0 N 0. O, 1 CD t` N ~C) X ~0 CD 0 0~ 0~ 0 N eq ~ 0 a ~ ~ o r~ o - 1: 0 c~:i ~i o 1~ ~ r ~i t; N CD ~ ~ 15~ i O ~ N ~5 ~ ~ ~ ~ C- ~ 0 0 t~ 0 0 1` 0 1~ ) 0 0, 0 t` 1-- C` I` ~ I` 1- 1--N N ~ N N g~ N ~ N N N ~ N N ~ g~ N ~ a N N g~ N N g~ N N a~ c~
' P~ 0 N N ~i 0 od N 0 CD N ~ 0 0 ~` t; ~ ~.d U ) ' ~i t; ~ CC ~ N CO N ~:P
~ El ~ 0 1-- --I 0 g~ N ~g 0 ~ æ N ~a g c~ ~ 0 ~ ~ x ~ o ~ ~
~ _I N ~1 ~ ~1 ~ X ~ X ~ 0 O ~g 3 ~ ~ r 0N t o ~ c~ CD a N ~ --~ ~
~ ~ N C17 0 co ~ 1~ 0 0 10 O t` t~ 0 0 N ~ ~P
0 [~ ~ o tl~ o 0 I X ~ ~-- Lt ' C ~ I ~N ~ e' N ~ 10 ~D ~ t ~ ~1 . e ~ CD ~ e ~ . ~I N ~
WO9S/16026 ~ ~ 7~ 8 PCT/US94/13558 Antibody from clones ~ci~n~t.~rl 4-757-121, 4-1034-293, 4-3~1-178, 4-704-151, 4-768-178, 4-957-150, 4-175-150, and 4-363-113 are selected as possible r~n~ t.l~R from the airset e~periment. The antibody from the eight clones is further screened for cross-reactivity.
Initial cross-reactivity testing is performed on the antibody from the eight clones using the TDX~) Clinical Analyzer (Abbott Laboratories) and the cross-reactants are made in recalcified human plasma without the presence of methotre~ate. The cross reactants (shown in Table 2) are added to the antibody solutions such that their conc~ul~a~iull in the sample is as shown in Table 2. The respective cross-reactivities ~cAnris~ted with the antibody form the eight clones are also shown in Table 2.
Cro~s-Reactant 4-175-150 4-363-113 4 351-17B 4-704151 Aminfq~b~rin @ 39 5 1.4% 2.2% ~2.5% l~o ol/L
DAMPA @ 6 llmol/L 5.5% 16.û% 4.8% 7.8%
7-l~y~llu y - rexate @ 0.04% 0.06% 0.05% 0.06%
100 llmol/L
Cro~-Reactant 4-757-121 4-768-178 ~957-150 4-1034-293 Am;nrll~t~rin @ 39 5 19% 1.9% ~2.5% 2.1%
ILmo3JL
DAMPA @ 6 umol/L 3.5% 3.8% 5.0% 4.2%
7-l-y~l / y - rexate @ 0.04% 0.04% 0.05% 0.05%
100 umol/L
A-1~1itil-n~1 airset studies and cross-reactivity testing are performed on the remaining seven antibodies usirlg the S pot, T pot, and P pot reagents as previously ,l"c~hf,~ New calibration curves are 2 0 generated using pipetting mode 11 and sample volumes between 3.5 and 10.0 ,ul in order to optimize the calibration curves. The calibrators are run in duplicate at the optimal airset (34C). The airset studies are then repeated by running the calibrators as samples (in duplicate) at :: 2 airset units to change the reaction ~tulllu~laiul~ in the cuvettes to 2 5 approximately 32C and 36C. The mP values of the calibrators from the two airset runs are evaluated for their degree of change. Cross-reactivity is then repeated by running the aforementioned cross-reactants a6 samples (in duplicate) using the above reagents on the new calibration curve at the optimal airset setting of 34C. The commercial methotrexate 21 7~9~
WO 9S/16026 PCTIUS941135Sg assay (Abbott) is also repeated as a control. Results from the second set of testing un the seven antibodies, are shown in Table 3. As shown in Table 3, the AmP, FmP and C describe the total changes between 32C and 36C reaction temperatures. The A-F, A-B, and E-F columns represent 5 the spans seen with éach antibody reagent configuration.
.
WO 9S/16026 : PCT/llS94/13SS8 ~ 1 7~q~ 20 1~
C Amin- 7-hydrox Altb~ody AmP FmP E~fc-.ct optenn DAMPA metho- A-F A-B E-F
-ec Commer~ial . .~ . -. % 5.2% . . ~ : 20 .
,~ ; . , ~ Oto . - _30 Ot O~
~ ~'- ~ 7 o ~
~ ,~ ., . . %
- 4- -3 . . . ._~C ~`
Clone Selprtinn Based on the the results shown in Table 3, Clone 4-351-178 i6 6elected for antibody production because the antibody that it secretes has a potential for a larger curve and ha6 low cross reactivity values.
I6Otvre 1 0 The isotype of the mnnnrlnn~l antibody secreted from the cell line 4-361-178 is 11PtPrminPd to be an rgGI with a kappa light chain. The isotype wa6 identified with a Mou6e Mnnnrlnnr~l Antibody I60typing Kit (Amer6ham Searle Corporation; Arlington Height6, Illinoi6) according to m~nllfr~rt~lrer'6 in6truction6.
F,IPrtrophoretic ev~ tinn The iqoPlPrtrir point (pl) of the mnnr~rlnn~l antibody 6ecreted from the cell line identified a6 4-351-178 and purified u6ing a protein A column i6 llPtPrminPd on an i.qoPlPctrir focu6ing a~ alal,u:i (Bio Rad, Rirhmnn~l 2 0 California). The gel i6 ca6t and run according to vendor recommPn~r~ti~mq and the re6ult6 indicate a pI of 6.7+0.2.
According to the m~ntlfArt~lrer'6 in6truction, SDS-PAGE
evaluation i6 p~lrolllled with the S&S PROFILETM Mini-Electrophor~tic Sy6tem (Schleicher & Schuell, Keene, New Frr~mrRhine)~ The antibody is run under 2-mc.~ Lu~ anol reduced rnn~itinnc Cooma66ie 6taining of the SDS-PAGE profile identifie6 the typical antibody banding pattern of a 6ingle light chain band at 24 kD and a 6ingle heavy chain band at 56kD.
wo gs/16026 2 ~ 7 8 ~ ~ ~ PCT~Sg41l35S8 Cell T.ine De~ qit In accordance with the Budapest Treaty, the hybridoma cell line, designated as hybrid 4-351-178, is deposited with the American Type Culture Col~ection (ATCC), 12301 Parklawn Drive, Rockville MD, 20852, United States of America. The deposit date is October 19, 1993, and the ATCC number assigned to the cell line is HB 11467.
Example 4 '~IJ- - :fi- :~y and C~ss R- vlLy This example is intended to compare a prior art commercial assay (TDXg) methotrexate assay - Abbott Laboratories), which uses a polyclonal antibody, to an assay uding the antibody from the cell line tlr~qi~n~te~ 4-361-178 and uu~ l reagents. The cross-reactants were made in recalcified human plasma, as above, without the presence 1 5 of methotrexate. The commercial assay is run according to the m~ l;.. L-~rr-d instructions and the assay using the antibody from clone 4-351-178 is run according to the commercial assay except that the pipetting mode is mode 43, and the antibody from cell line 4-361-178 (diluted in 0.001 M rhrqrh~t~ buffer, 0.15 M NaCl, 10% glycerol, 5% BSA, 2 0 0.01% BGG, and 0.1% sodium azide) is cllh=~itllt,,d for the antibody reagent (S pot). The T pot ctlmrricpA 250 nM methotrexate fluorescein tracer in 0.1 M tris, 0.7% sodium dodecyl sulfate (SDS), 0.5% lithium dodecyl sulfate (LDS) and 0.1%sodium azide; and the P pot comprises 0.1 M tris, 0.7% SDS, 0.5% LDS, 0.35% lithium ~ 1rl--c~lirylate (LIS), and 2 5 0.1% sodium azide.
The results from the two assays are shown in Table 4 which shows the increased specificity of the monoclonal antibody secreted by the cell line ~ cign~trtl 4-351-178 and the possibility of the v, ~ ial assay antibody cross reacting with 7-l-y~llu~y-ll~Lllotrexate.
I`~.ble 4 Cross-~` actant Commercial 4-35:.-178 DAMP. . @ .. llmoVL 47% 3 %
DAMP @ ..... IlmoVL >16.7% 1 %
7-llyd~ w~yllle, lotrexate <1% None ~etected @ 1000 llmo /L
Methotrexate (251.2 mg) is dissolved in 4.0 ml of distilled water and 1.0 ml of 2.0 N sodium hydroxide (NaOH) to form a solution. The pH of the resulting solution is adjusted to 7.5 with 0.1 N hydrochloric acid (HCI). The total volume of the solution is d,U~JI U~ilUdLt:ly 10.0 ml (approximately 25 mg/ml methotrexate). BSA (77.7 mg) is dissolved in 2.0 ml of distilled water to form a second solution. A third solution is prepared and ,IJIll,Ul ;~es 250 mg of EDAC in 5.0 ml of distilled water.
1.5 ml of the m~LlluLl~d~ solution is added to the BSA solution and dissolved at room L~ aLule. Over a three hour period, 0.7 ml of 2 5 the EDAC solution is added to the methotrexateMSA solution in 100 ,ul aliquots, and thereafter, 0.8 ml of the EDAC solution is added all at once for a total EDAC solution addition of 1.5 ml. This reaction mixture is allowed to mix overnight and after the mixing period the solution turns to a clear bright-yellow-solution.
A SEPHADEX(~ G50-300 column (2.5 mm ~ 400 mm) is equilibrated with PBS before the reaction mixture is applied to it and eluted with PBS. The eluant from the column is ul~ ulqd for absorbance (A2go) and the il-'l"UI10~ (BSA-methotrexate complex) is contained in the first peak eluted from the column. The volume of the 3 5 collected portion of the first peak is 44.5 ml with a protein concentration of 2.0 mg/ml which yields 89.0 mg of immunogen.
WO 95116026 2 ~ 7 ~ ~ 9 ~ PCTrUS94113558 ~.~s~mple 2 Tra~cer ~ 'h~
Mater;~l c 1. Methotrexate - available from Sigma 5 2. 5-Amint)flllorescein - available from Aldrich Chemical Co.
(Aldrich); Milwaukee, Wiscon3in 3. SiO2 Thin Layer Chromatography (TLC) plates - available from EM Reagents; Gibbstown, New Jersey 4. Clg TLC plates - available from Whatman Inc.; Clifton, New 1 0 Jersey Proced-lre:
The following solutions are prepared: 0.2305 g of methotrexate in enough dimethylsulfoxide (DMSO) to dissolve the methotrexate; 0.09 g of p-nitrophenol in enough DMSO to dissolve the p-nitrophenol; 0.11 g of 15 dicyclohexyldi~lbor~ihLIide (DCC) in enough DMSO to dissolve the DCC;
and 0.1747 g of 5-~minr~flllllrescein in enough DMSO to dissolve the 5-Amin~ s~ir~ The methotrexate (approximately 1.0 ml), DCC
(approximately 1.0 ml), and p-nitrophenol solutions (approximately l.0 ml) are combined to form a reaction mixture that is allowed to stir for 4 20 hours at room t~ ,UI~. Then, the 5-~min~.nll..,t:~r ~ solution ly 1.0 ml) is added to the reaction mixture while it is stirring. The resulting reaction mixture is protected from light and allowed to stir overnight. After the mixing period, the reaction mixture is applied to eight, 2.0 mm SiO2 TLC plates. The full length of the plates 2 5 are developed with (2~ 1.1roform and allowed to dry prior to developing the plates half-way with (l:l) chloroform mPth~n~ll and allowing them to dry. The bands from one developed plate are separately collected and eluted from the SiO2 plates with methanol before the bands from the plate are applied to a Clg TLC plate. The Clg plate is developed 3 0 in 50% methanol with 0.05% sodium dodecyl sulfate (SDS) and 'iO% 0.2%
KH2PO4 in distilled water and show that the product is in the region corrP.~rnnrlin~ to Rf 0.05 to 0.38 of the SiO2 plates. The remaining SiO2 plates are scraped from R~ 0.05 to 0.38 and the product is eluted with mPtll~nn1, conc".~ d to ~ u~hllately 4.0 ml, and applied to ten l.0 3 5 mm Clg TLC plates. The plates are developed as above and the brightest band, as determined by an ultraviolet lamp, is at Rf 0.69 to 0.76. The band is collected, eluted with ethanol and concentrated as before. The WO 95/16026 2 ~ 7 ~ 5 q ~ PCT/US94/13SS8 concentrated product is applied to eight C1g TLC plates, developed, and the final product is collected as above and is showrl below as the compound of the formula (P).
HO _~0 O CO H
H2N ~N CH~ N ~NH
Ex~nple 3 An~: '' ' G ' ~r ...., ~ ~yl~u~iiv -Tmmiln;7:~tir)n strateev A 2.0 mg/ml slurry, rrmt~inin~ the methotrexate conjugate of 10 Example 1 ~mlllRifi~d in Freund's Complete Adjuvant (Difco Inc, Detroit, Mirhii~An) and 0.9% saline, is used to Rllhcllt~nr~ously immunize ten RFBlDnJ mice (available from Jackson Laboratories; Bar Harbor, Maine) such that each mouse received a total of 100 llg of conjugate.
Four weeks later each mouse is ;.,.,...~.I;,.~d a second time using the 5 same ~IU~,~,dUl~ specified above except that the conjugate is emulsified in Tnrnmrlrte Freund's Adjuvant (Difco Inc., Detroit, MirhiE~n) and each mouse is i~..~..~",;,._rl with 20 ilg of corljugate. A third 20 ilg/mouse j""",..,;,.~ n is pr.~fr~rmr~d after 8 weeks from the date of the original ;.".,.u.,i~ ion with the TnrrmrlPt~ Freund's Adjuvant protocol and a 20 final 20 ilg/mouse ;l~ rln is rrmrlrt~d 12 weeks after the original ;"",...";,.,.1 r)n The mice are bled 6, 10 and 15 weeks after the initial immumization and sera from the bleeds is assayed for an immune response to methotrexate.
25 Serllm T~',v5~ ti~1n Utilizing a co..,~ binding assay format, the mouse sera (from above) is assayed for anti-ll-eulu~ e activity. The samples are tested on a TDX(!~) Clinical Analyzer (Abbûtt Laboratories) using a reagent configuration which includes reagents labeled S pot, T pot, and P pût 30 which contain the following:
WO 95/16026 2 1 7 8 5 9 ~ PCIIUS94/13558 S pot: 0.1 M rhncrhslte buffer, 2% ethylene glycol, 5% BSA, 0.1% sodium azide, 0.01% bovine gamma globulin (BGG) T pot: 62 nM nuult~SC~iu tracer in 0.1 M tris, 0.7% sodium dodecyl sulfate (SDS), 0.5% lithium dodecyl sulfate (LDS), 0.01% (BGG), 0.1% sodium azide P pot: 0.1 M tris, 0.7% SDS, 0.5% LDS, 0.01% BGG, 0.1%
sodium azide Before running the assay, the mouse sera iB serially diluted in the S pot diluent which is dispensed in the sample cup of the sample cartridge.
The dilutions range firom a neat sample to a 1:32 dilution. Antibody dic~ ( Prnf~nt is tested by spiking 8.0 ,ul of the A and F calibrators in the predilute cup of the sample cartridge. The sample cartridges are placed on the carousel and the samples are tested using mode 11 pipetting 6equence which has a 10 ,ul sample size. Dicrl~r~m~nt of the A-F (0-1.0 llmol/L) calibrators ranged from 28-65 mP. Because the serum sample firom mouse ~1PC; ns~tl~rl 1-lA gave the best ~i.c~l~n~nn~nt., a curve is generated for the serum from this mouse and the serum is assayed for 7-Il~u;~y~ethotrexate~ 4-deoxy-4-amino-N10-m~Lllyl,uL~lu:c acid (DAMPA) 2 0 and aminopterin cross reactivity. The results firom the assay show low cross reactivities for all three of the cross reactants and a decision is made to use cpl~- , Lt:~ firom mouse 1-lA for fiusion.
Pre-Fllcinn Tmmllni7~tinn 2 5 Three days prior to cell filsion and 12 weeks after the original immllni7~tinn mouse 1-lA is given a 20 ~g injection of the methotrexate conjugate (firom Example 1) diluted in 0.1 ml rhncrhslt~ buffered saline (PBS - pH 7.2) via the tail vein.
3 0 Ela~
On the day of the fusion, mouse 1-lA is ellth~ni7f~d by cervical orsltinn and the spleen is removed. The splenocytes are washed one time in Iscove's Modified Dulbecco's Medium (IMDM - GIBCO, Grand Island, New York) ana u ~:uLliru~d at 1000 RPM for 7 minutes. The 3 5 supernatant is removed and the pelletted splenocytes are combined with SP2/0 myeloma cells (firom the laboratory of Dr. Milstein, Cambridge, United Kingdom) at a 1:1 ratio, washed in IMDM, and r~nt.rifil~fl The WO 9~/16026 2 1 7 8 5 q ~ PCT/llS94113558 supernatant is removed from the pelletted cells and l.0 ml of 50% PEG
(polyethylene glycol available from American Type Culture Collection, Rockville, Maryland) is added to the pellet and the pellet is gently dispersed for approYimately 1 minute by tapping and swirling. 30.0 mls 5 of IMDM is added to the miYture and centrifuged as previously described. The supernatant is decanted and the pellet is resuspended in IMDM with HAT (hypnYAnt.hinf', aminopterin, thymidine available from GIBCO), 10% Fetal Bovine Serum (FBS - Hyclone Laboratories, Logan, Utah) and 0.5% STM v/v (RIBI Tmmllnorh~nn Research, Inc., T-TAmiltnn, 10 Montana). STM denotes ,sAlmnnf?llA tv~ .l mitogen, a B-cell specific mitogen, and is used to enhance fusion frequency. The fusion cell suspension is plated into 96-well tissue culture plates.
F~lcinn Scr~ninF
The primary screening of the 96-well fusion sll4r~qncinn supernatant occurred on day 10 of confluent cultures. A SCREEN
MACHINElM, (IDEXX Labul~lL~/LI~A7, Westbrook, Maine) is used according to mAnllfA~tllrerls instructions for the primary fusion screen.
Microparticles coated with goat anti-mouse IgG ~ M (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Maryland) and the mnnnrlnnAl antibody fusion supernatant sample are added to the 96-well IDEXX
FLUORICON(g~ assay plates. Following a 15 minute in~-llhAtinn, the plates are washed and a methotrexate-fluorescein coruugate is added to each sample well. After a 10 minute inrnhAtinn the plates are washed a final time and the relative nuul~ s~ t intensity is read. Values with elevated signals indicate the pre6ence of anti-methotrexate antibody in the culture. Cultures with a signal greater than 3 times the negative control are e~panded to 24-well tissue culture plates for further evaluation.
3 0 Positive Hybrids from the IDEXX screen are then tested on the Abbott TDX~E9 Clinical Analyzer~ according to a modified screeni~g format which allows the monoclonal antibody supernatant to be tested in the sample wells. According to the modified assay format, the methotreYate reagent pack ~....I.~i..i..e the S pot, T pot and P pot reagents 35 (described above) and pipetting mode 11 are used. All-litinnAlly, the 6ample size is 10.0 ,ul and the sera from the hybrids is used as the sample. Polarization of the test samples is evaluated in the absence and WO 95116026 2 ~ 7 8 5 ~ ~ PCTIUS9 1113558 presence of free methotrexate and the cross reactivity of the antibody with 7-llydlu~ylllethotrexate is also evaluated. Antibody from hybrids that had a milli~ ri7Ati-)n (mP) value for 7-hydroxymethotrexate and the A
calibrator within 20 mP of each other are selected for cloning.
~vbrid clnnin,~r Seventy hybrids are cloned by limiting dilutions starting at a 1-100 dilution, 10-fold down to 10-6. The cloning media is IMDM with 10% v/v FBS and 1% v/v HT supplement (hylJ~7 A ~ 1 i I IP, thymidine available from 1 0 GIBCO). A 100 111 cell s~lqpPnCilm is added to each of the 96 wells in the tissue culture plate and on day 7 the plates are fed with 200 Ill/well of cloning media. The antibody from the resulting clones is assayed for anti-methotrexate activity and 7-hydroxymethotrexate cross reactivity using the same method used to screen the hybrids. Based on the clone 5 screening, 23 clones are selected for antibody production and further screening.
Antihf~dv Pro~llrtifm The selected clones are scaled up in ascites fiuid by injecting 2 0 approximately 106 cells into the pPrit.onPAl cavity of pristaned primed female BALB/c mice (Charles River Labs; Charles River, New York).
Two weeks later the animals are ellt.h7~1ni7Pd and the ascites fluid is harvested. The antibody cr~ntAinin~ fluid is ut~ulliru~;t~d at 1000 RPM for 10 minutes to remove the cells. The ascites fluid is then screened for 2 5 specificity and temperature sensitivity.
Specificitv ~n(l T~nlpPrature Spncitivitv ScrPRnin,~
A modified screening format allows selection of a methotrexate clone based on temperature sensitivity (airset studies) and specificity 3 0 (cross-reactivity studies). The antibody from the methotrexate clones is tested on the TDX(E9 Clincal Analyzer (Abbott LabuL~Lul;t7b) using pipetting mode 11, a sample size of 17.5 ~1, 13.~ ~Ll of altering A & F
calibrator in the pre-dilute cup, and antibody straight or titered in the sample cup. The reagent pack configuration comprises the the S pot, T
35 pot and P pot diluent and reagents ~viuu~,ly described.
All Antih~rliPc from the anti-methotrexate producing clones are initially screened using the above procedure and the titer is selected to WO 9S/160Z6 2 1 7 P~ S q ~ 1 6 PCT/US94/135S8 have a Po value of between 170 and 190. When an antibody dilution gave a Po value between 170 and 190, the antibody is 6creened at that dilution for temperature st,ll,,iLiviiy. The L~ ld~ulr~ sensitivity protocol requires three runs. All three runs follow the modified screening format 5 previously described with the e2ception that, the A and F calibrators are run in duplicate on an instrument with a correct airset setting (34.0C), a -2 airset setting (32.0C) to lower reaction t~ ra~Ult, and +2 airset setting (36.0C) to rai6e the reaction temperature. Actual temperatures of the liquid in the cuvette are measured with an external temperature 1 0 probe in the last cuvette on the carousel at the connpl~ti~m of the run. The mPs are compared, looking for the least amount of change seen in the A
and F calibrators for the 32.0C and 36.0C runs. For ~ ix.", an unpurified antibody having undesirable ~ laluu~ and cross reactivity charA~ t~riPt;~ x (UP935) and the commercial polyclonal antibody (S pot) 15 are used as positive and rlegative controls respectively. The data for the antibody from 23 clones assayed for the t~lu~ Ul~ sensitivity is shown in Table 1. A~1~1iti~nAlly, the values shown for the controls are an average taken from multiple runs.
WO 95/16026 2 1 7 ~ S 9 ~ PCT/US9411355~
IY, 0 CD O O C~ --~ O ~ O ~ O O O O ~ ~ D O ~ O ~ O 0 I~ ~ ' ' ~O ' 10 ' C~ O C~ CD O O~ q CD C~ O o o~
O O O C~ Cl a N a ~ a o ~ a a c~ o o o o o~ c~ ~ a o~
o ~ N O ~P ~P CD ~ ~r C~ N 0 N 0. O, 1 CD t` N ~C) X ~0 CD 0 0~ 0~ 0 N eq ~ 0 a ~ ~ o r~ o - 1: 0 c~:i ~i o 1~ ~ r ~i t; N CD ~ ~ 15~ i O ~ N ~5 ~ ~ ~ ~ C- ~ 0 0 t~ 0 0 1` 0 1~ ) 0 0, 0 t` 1-- C` I` ~ I` 1- 1--N N ~ N N g~ N ~ N N N ~ N N ~ g~ N ~ a N N g~ N N g~ N N a~ c~
' P~ 0 N N ~i 0 od N 0 CD N ~ 0 0 ~` t; ~ ~.d U ) ' ~i t; ~ CC ~ N CO N ~:P
~ El ~ 0 1-- --I 0 g~ N ~g 0 ~ æ N ~a g c~ ~ 0 ~ ~ x ~ o ~ ~
~ _I N ~1 ~ ~1 ~ X ~ X ~ 0 O ~g 3 ~ ~ r 0N t o ~ c~ CD a N ~ --~ ~
~ ~ N C17 0 co ~ 1~ 0 0 10 O t` t~ 0 0 N ~ ~P
0 [~ ~ o tl~ o 0 I X ~ ~-- Lt ' C ~ I ~N ~ e' N ~ 10 ~D ~ t ~ ~1 . e ~ CD ~ e ~ . ~I N ~
WO9S/16026 ~ ~ 7~ 8 PCT/US94/13558 Antibody from clones ~ci~n~t.~rl 4-757-121, 4-1034-293, 4-3~1-178, 4-704-151, 4-768-178, 4-957-150, 4-175-150, and 4-363-113 are selected as possible r~n~ t.l~R from the airset e~periment. The antibody from the eight clones is further screened for cross-reactivity.
Initial cross-reactivity testing is performed on the antibody from the eight clones using the TDX~) Clinical Analyzer (Abbott Laboratories) and the cross-reactants are made in recalcified human plasma without the presence of methotre~ate. The cross reactants (shown in Table 2) are added to the antibody solutions such that their conc~ul~a~iull in the sample is as shown in Table 2. The respective cross-reactivities ~cAnris~ted with the antibody form the eight clones are also shown in Table 2.
Cro~s-Reactant 4-175-150 4-363-113 4 351-17B 4-704151 Aminfq~b~rin @ 39 5 1.4% 2.2% ~2.5% l~o ol/L
DAMPA @ 6 llmol/L 5.5% 16.û% 4.8% 7.8%
7-l~y~llu y - rexate @ 0.04% 0.06% 0.05% 0.06%
100 llmol/L
Cro~-Reactant 4-757-121 4-768-178 ~957-150 4-1034-293 Am;nrll~t~rin @ 39 5 19% 1.9% ~2.5% 2.1%
ILmo3JL
DAMPA @ 6 umol/L 3.5% 3.8% 5.0% 4.2%
7-l-y~l / y - rexate @ 0.04% 0.04% 0.05% 0.05%
100 umol/L
A-1~1itil-n~1 airset studies and cross-reactivity testing are performed on the remaining seven antibodies usirlg the S pot, T pot, and P pot reagents as previously ,l"c~hf,~ New calibration curves are 2 0 generated using pipetting mode 11 and sample volumes between 3.5 and 10.0 ,ul in order to optimize the calibration curves. The calibrators are run in duplicate at the optimal airset (34C). The airset studies are then repeated by running the calibrators as samples (in duplicate) at :: 2 airset units to change the reaction ~tulllu~laiul~ in the cuvettes to 2 5 approximately 32C and 36C. The mP values of the calibrators from the two airset runs are evaluated for their degree of change. Cross-reactivity is then repeated by running the aforementioned cross-reactants a6 samples (in duplicate) using the above reagents on the new calibration curve at the optimal airset setting of 34C. The commercial methotrexate 21 7~9~
WO 9S/16026 PCTIUS941135Sg assay (Abbott) is also repeated as a control. Results from the second set of testing un the seven antibodies, are shown in Table 3. As shown in Table 3, the AmP, FmP and C describe the total changes between 32C and 36C reaction temperatures. The A-F, A-B, and E-F columns represent 5 the spans seen with éach antibody reagent configuration.
.
WO 9S/16026 : PCT/llS94/13SS8 ~ 1 7~q~ 20 1~
C Amin- 7-hydrox Altb~ody AmP FmP E~fc-.ct optenn DAMPA metho- A-F A-B E-F
-ec Commer~ial . .~ . -. % 5.2% . . ~ : 20 .
,~ ; . , ~ Oto . - _30 Ot O~
~ ~'- ~ 7 o ~
~ ,~ ., . . %
- 4- -3 . . . ._~C ~`
Clone Selprtinn Based on the the results shown in Table 3, Clone 4-351-178 i6 6elected for antibody production because the antibody that it secretes has a potential for a larger curve and ha6 low cross reactivity values.
I6Otvre 1 0 The isotype of the mnnnrlnn~l antibody secreted from the cell line 4-361-178 is 11PtPrminPd to be an rgGI with a kappa light chain. The isotype wa6 identified with a Mou6e Mnnnrlnnr~l Antibody I60typing Kit (Amer6ham Searle Corporation; Arlington Height6, Illinoi6) according to m~nllfr~rt~lrer'6 in6truction6.
F,IPrtrophoretic ev~ tinn The iqoPlPrtrir point (pl) of the mnnr~rlnn~l antibody 6ecreted from the cell line identified a6 4-351-178 and purified u6ing a protein A column i6 llPtPrminPd on an i.qoPlPctrir focu6ing a~ alal,u:i (Bio Rad, Rirhmnn~l 2 0 California). The gel i6 ca6t and run according to vendor recommPn~r~ti~mq and the re6ult6 indicate a pI of 6.7+0.2.
According to the m~ntlfArt~lrer'6 in6truction, SDS-PAGE
evaluation i6 p~lrolllled with the S&S PROFILETM Mini-Electrophor~tic Sy6tem (Schleicher & Schuell, Keene, New Frr~mrRhine)~ The antibody is run under 2-mc.~ Lu~ anol reduced rnn~itinnc Cooma66ie 6taining of the SDS-PAGE profile identifie6 the typical antibody banding pattern of a 6ingle light chain band at 24 kD and a 6ingle heavy chain band at 56kD.
wo gs/16026 2 ~ 7 8 ~ ~ ~ PCT~Sg41l35S8 Cell T.ine De~ qit In accordance with the Budapest Treaty, the hybridoma cell line, designated as hybrid 4-351-178, is deposited with the American Type Culture Col~ection (ATCC), 12301 Parklawn Drive, Rockville MD, 20852, United States of America. The deposit date is October 19, 1993, and the ATCC number assigned to the cell line is HB 11467.
Example 4 '~IJ- - :fi- :~y and C~ss R- vlLy This example is intended to compare a prior art commercial assay (TDXg) methotrexate assay - Abbott Laboratories), which uses a polyclonal antibody, to an assay uding the antibody from the cell line tlr~qi~n~te~ 4-361-178 and uu~ l reagents. The cross-reactants were made in recalcified human plasma, as above, without the presence 1 5 of methotrexate. The commercial assay is run according to the m~ l;.. L-~rr-d instructions and the assay using the antibody from clone 4-351-178 is run according to the commercial assay except that the pipetting mode is mode 43, and the antibody from cell line 4-361-178 (diluted in 0.001 M rhrqrh~t~ buffer, 0.15 M NaCl, 10% glycerol, 5% BSA, 2 0 0.01% BGG, and 0.1% sodium azide) is cllh=~itllt,,d for the antibody reagent (S pot). The T pot ctlmrricpA 250 nM methotrexate fluorescein tracer in 0.1 M tris, 0.7% sodium dodecyl sulfate (SDS), 0.5% lithium dodecyl sulfate (LDS) and 0.1%sodium azide; and the P pot comprises 0.1 M tris, 0.7% SDS, 0.5% LDS, 0.35% lithium ~ 1rl--c~lirylate (LIS), and 2 5 0.1% sodium azide.
The results from the two assays are shown in Table 4 which shows the increased specificity of the monoclonal antibody secreted by the cell line ~ cign~trtl 4-351-178 and the possibility of the v, ~ ial assay antibody cross reacting with 7-l-y~llu~y-ll~Lllotrexate.
I`~.ble 4 Cross-~` actant Commercial 4-35:.-178 DAMP. . @ .. llmoVL 47% 3 %
DAMP @ ..... IlmoVL >16.7% 1 %
7-llyd~ w~yllle, lotrexate <1% None ~etected @ 1000 llmo /L
Claims (12)
1. A monoclonal IgG antibody which specifically binds to methotrexate having less than 0.01% cross reactivity with 7-hydroxymethotrexate.
2. The antibody of Claim 1, wherein the antibody is raised in response to an immunogen of the formula (Y) (Y) wherein one of A and Z is -OH and the other is a carrier molecule selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin.
3. The antibody of Claim 2, wherein the carrier molecule is bovine serum albumin.
4. The antibody of Claim 1, wherein the antibody is secreted by a hybridoma cell line designated A.T.C.C. HB 11467.
5. A continuous hybridoma cell line which secretes IgG antibody which specifically binds to methotrexate wherein the antibody has less than 0.01% cross reactivity with 7-hydroxymethotrexate.
6. The cell line of Claim 5, wherein the antibody is raised in response to an immunogen of the formula (Y) (Y) wherein one of A and Z is -OH and the other is a carrier molecule selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin.
7. The cell line of Claim 6, wherein the carrier molecule is bovine serum albumin.
8. The cell line of Claim 5 which is designated A.T.C.C. HB 11467.
9. A monoclonal IgG antibody secreted by the hybridoma cell line designated as A.T.C.C. HB 11467 which specifically binds to methotrexate.
10. The hybridoma cell line designated as A.T.C.C. HB 11467.
11. A fluorescence polarization immunoassay for determining the presence or amount of methotrexate in a test sample comprising the steps of:
a) contacting the test sample with i. a monoclonal IgG antibody specific for methotrexate wherein the antibody has no detectable cross reactivity with 7-hydroxymethotrexate, and ii. a fluorescent tracer molecule, to form a solution;
b) contacting the solution with plane polarized light;
and c) detecting a fluorescence polarization response.
a) contacting the test sample with i. a monoclonal IgG antibody specific for methotrexate wherein the antibody has no detectable cross reactivity with 7-hydroxymethotrexate, and ii. a fluorescent tracer molecule, to form a solution;
b) contacting the solution with plane polarized light;
and c) detecting a fluorescence polarization response.
12. A test kit for determining the presence or amount of methotrexate in a test sample comprising:
a) a monoclonal IgG antibody specific for methotrexate having no detectable cross reactivity with 7-hydroxymethotrexate; and b) a fluorescent tracer molecule.
a) a monoclonal IgG antibody specific for methotrexate having no detectable cross reactivity with 7-hydroxymethotrexate; and b) a fluorescent tracer molecule.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US16512193A | 1993-12-10 | 1993-12-10 | |
| US08/165,121 | 1993-12-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2178590A1 true CA2178590A1 (en) | 1995-06-15 |
Family
ID=22597511
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2178590 Abandoned CA2178590A1 (en) | 1993-12-10 | 1994-11-22 | Reagents and methods for the detection of methotrexate |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0733102A1 (en) |
| JP (1) | JPH09506260A (en) |
| AU (1) | AU1186695A (en) |
| CA (1) | CA2178590A1 (en) |
| WO (1) | WO1995016026A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107219357A (en) * | 2017-04-21 | 2017-09-29 | 中国人民解放军第三军医大学第附属医院 | A kind of ELISA kit for detecting methotrexate (MTX) and its application |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5710009A (en) * | 1994-02-17 | 1998-01-20 | Serex, Inc. | Receptor:release ligand (reland) complexes and release assays using said reland and methods and kits based thereon |
| AU2678997A (en) * | 1996-04-19 | 1997-11-12 | Lexigen Pharmaceuticals Corp. | Inhibition of the binding of human ige to its receptor by tetracyclic compounds for the alleviation of ige-mediated immune response |
| JP2011523853A (en) | 2008-06-03 | 2011-08-25 | アボット・ラボラトリーズ | Dual variable domain immunoglobulins and uses thereof |
| SG178602A1 (en) * | 2009-09-01 | 2012-04-27 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
| MX341579B (en) | 2010-08-03 | 2016-08-25 | Abbvie Inc * | Dual variable domain immunoglobulins and uses thereof. |
| AU2011293253B2 (en) | 2010-08-26 | 2014-12-11 | Abbvie Inc. | Dual variable domain immunoglobulins and uses thereof |
| CA2861610A1 (en) | 2011-12-30 | 2013-07-04 | Abbvie Inc. | Dual specific binding proteins directed against il-13 and/or il-17 |
| TW202210507A (en) | 2012-11-01 | 2022-03-16 | 美商艾伯維有限公司 | Anti-vegf/dll4 dual variable domain immunoglobulins and uses thereof |
| US9062108B2 (en) | 2013-03-15 | 2015-06-23 | Abbvie Inc. | Dual specific binding proteins directed against IL-1 and/or IL-17 |
| US10093733B2 (en) | 2014-12-11 | 2018-10-09 | Abbvie Inc. | LRP-8 binding dual variable domain immunoglobulin proteins |
| CN104569373B (en) * | 2015-01-27 | 2016-08-17 | 苏州博源医疗科技有限公司 | A kind of methotrexate homogeneous enzyme immunoassay detectable and preparation thereof and detection method |
| TW201710286A (en) | 2015-06-15 | 2017-03-16 | 艾伯維有限公司 | Binding proteins against VEGF, PDGF, and/or their receptors |
| WO2016210054A1 (en) * | 2015-06-23 | 2016-12-29 | The Board Of Trustees Of The Leland Stanford Junior University | Novel rhodol fluorophores for near-infrared imaging |
| WO2017007889A1 (en) * | 2015-07-08 | 2017-01-12 | Defined Diagnostics, Llc | Compounds and methods for the detection of methotrexate |
| CN108614107A (en) * | 2016-12-12 | 2018-10-02 | 上海复星长征医学科学有限公司 | A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof |
| CN110357886B (en) * | 2018-04-09 | 2022-06-24 | 浙江准策生物技术有限公司 | Methotrexate hapten and complete antigen as well as preparation method and application thereof |
-
1994
- 1994-11-22 JP JP7516209A patent/JPH09506260A/en not_active Ceased
- 1994-11-22 WO PCT/US1994/013558 patent/WO1995016026A1/en not_active Application Discontinuation
- 1994-11-22 CA CA 2178590 patent/CA2178590A1/en not_active Abandoned
- 1994-11-22 AU AU11866/95A patent/AU1186695A/en not_active Abandoned
- 1994-11-22 EP EP95902688A patent/EP0733102A1/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107219357A (en) * | 2017-04-21 | 2017-09-29 | 中国人民解放军第三军医大学第附属医院 | A kind of ELISA kit for detecting methotrexate (MTX) and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1995016026A1 (en) | 1995-06-15 |
| AU1186695A (en) | 1995-06-27 |
| EP0733102A1 (en) | 1996-09-25 |
| JPH09506260A (en) | 1997-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2178590A1 (en) | Reagents and methods for the detection of methotrexate | |
| JP6068520B2 (en) | Risperidone immunoassay | |
| EP1875237B1 (en) | Docetaxel immunoassay | |
| US5688921A (en) | Reagents and methods for the detection and qualification of thyroxine in fluid samples | |
| US5593896A (en) | Reagents and methods for the detection and quantification of thyroxine in fluid samples | |
| US4900662A (en) | CK-MM myocardial infarction immunoassay | |
| JP4435305B2 (en) | Topiramate immunoassay and analogs and antibodies | |
| JP4896959B2 (en) | Doxorubicin immunoassay | |
| EP2467139B1 (en) | Imatinib immunoassay | |
| EP2349335B1 (en) | Levetiracetam immunoassays | |
| EP1771732B1 (en) | Taxol immunoassay | |
| CN110938082A (en) | Olanzapine artificial antigen and preparation method thereof | |
| CA2028849A1 (en) | Monoclonal antibodies, assay method, reagent kit, sarching method and drug missiles using them | |
| CN104066749B (en) | For the monoclonal antibody of specific recognition people liver Procaine esterase 1, the hybridoma cell strain generating monoclonal antibody and uses thereof | |
| CN110357886B (en) | Methotrexate hapten and complete antigen as well as preparation method and application thereof | |
| CN101995460A (en) | Ractopamine residual time resolution immunoassay kit and detection method thereof | |
| CN101988923A (en) | Time resolution immunoassay detection kit of diethylstilbestrol residuals and detection method thereof | |
| CN111154000A (en) | Anti-cimaterol monoclonal antibody and application thereof | |
| JPS63209596A (en) | Monoclonal antibody and use thereof | |
| JPH11510591A (en) | Haptens, tracers, immunogens and antibodies for immunoassays of propoxyphene | |
| CN117603340A (en) | Gentamicin complete antigen and preparation method and application thereof | |
| CN110950808A (en) | Clozapine artificial antigen and preparation method thereof | |
| JPH09154596A (en) | Antibody specific to para-nitrophenoxy organic phosphorous compound, its production, and measurement of para-nitrophenoxy organic phosphorous compound | |
| JPH0346562A (en) | Immunization and immunoassay | |
| EP2535333A1 (en) | Generically binding antibodies with multiple applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |