Note: Descriptions are shown in the official language in which they were submitted.
WO95/16026 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.
1 0
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
3
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.
1 0
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
5
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
7
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 ~
WO 95/16026 2 1 7 ~ ~ 9 ~ PCT/US9~1/13558
9
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
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
1 1
~.~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
12
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
1 3
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
14
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
1 5
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~
17
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
1 5
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
1 9
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
21
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
