Note: Descriptions are shown in the official language in which they were submitted.
-- 2 --
RAN 4093/24
The present invention relates to novel reagents for the
determination of immunologically active materials and to
immunological methods using said reagents.
In the last years many analytical systems based on
competitive protein binding analysis (also called saturation
analysis) have been developed.
The terms 'saturation analysis' and 'competitive protein
binding analysis', which are synonymous, refer to analytical
systems used for the determination of immunologically
active materials (ligands). The results of these determinations, `
in biological fluids, are emplo~ed in medical and veterinary
diagnosis. The dia~nosis is dependent upon the level of the
determined substance being normal or pathological. The
analytical principle is e.g. based upon the competition
between a ligand and a labelled ligand for a common specific
binding agent (receptor), as illustrated in the following
equation:-
. , , _
, .
Label Label Lalel
Ligand ~ Ligand + Receptor _ Ligand + Ligand + Ligand + Ligand Equation 1
Receptor Receptor
(1) (2) (3) (4)
.
Hen/ 7.2.1978
~ .
-- 3
The primary reaction is a combination of one molecule of ligand with
one molecule of receptor to form a bimolecular ligand/receptor
complex (1). A secondary reaction is caused by addition of labelled
ligand which similarly combines with receptor to form a labelled
ligand/receptor complex (2). In saturation analysis the concentrations
of the receptor and of the labelled ligand are constant. The concentration
of receptor is limited so that the labelled ligand is in excess relative
to the receptor. Under these conditions the addition of ligand causes
competition between ligand and labelled ligand for binding with receptor.
Hence an increase in ligand concentration lowers the amount of labelled
ligand/receptor complex. The assay principle is based on the determination
of the percentage of the total labelled ligand bound to the receptor.
This percentage is inversely proportional to the amount of ligand added
to the reaction mixture from the specimen to be measured or from the
standard used in the assay. The decrease in the concentration of
labelled ligand/receptor complex or the increase in the concentration
of labelled ligand in the reaction mixture can be used to determine
the ligand concentration.
The sensitivity of saturation analysis depends upon the use of a
receptor which has a very high affinity for the ligand and labelled
ligand. Secondly, the sensitivity also depends upon the use of a
label which can be detected at very low concentration.
The specificity of saturation analysis is dependent upon the
capacity of the receptor to bind exclusively the ligand and labelled
ligand in a complex mixture of different molecules.
Saturation analysis has been employed using many different techniques,
the differences primarily being related to the type of label used.
Generally these techniques are classified by using the broad titles
'radioassay' or 'non-radioassay', depending upon whether or not
a radioactive tracer is used as the label.
Radioassay has been utilised to a greater extent than non-radioassays.
Radioassay can be further classified as radioimmunoassay or radio-receptor
.
.
. . , .. , :
_ 4 _ ~L~l~3 2 7 ~
assay, depending upon the type of receptor employed in the assay. In
radioimmunoassay an antibody is used which specifically binds the ligand
and labelled ligand. Alternatively, radio-receptor analysis refers to the
use of any other type of biological receptor which will similarly
specifically bind the ligand and labelled ligand.
In all radioassay techniques it is essential to physically separate
the bound fraction (1 and 2 of Equation 1) from the unbound fraction
(3 and 4 of Equation 1) of the reaction mixture. An index of the
ligand concentration can subsequently be obtained by counting these
fractions in a radioactivity counter and comparing the counts obtained
for the unknowns specimens with those obtained for appropriate
standard ligand samples subjected to the same assay. Many different
and diverse methods have been described for the separation of the
bound and free fractions of the radioassay reaction mixture utilising
techniques including gel fitration, absorption and ion exchange
chrom~tography, fractional precipitation, solid phase or electrophoresis.
Subsequent to the development of radioassay techniques in saturation
analysis, methods employiny non-radioactive labels have been developed.
Methods utilising enzymes as the label have been
demonstrated. These methods can have the advantage that physical
separation of the bound (1 + 2) and free (3 + 4) fractions of labelled
- ligand is not necessary ;n the assay procedure.
When antibody binds ligand labelled with an enzyme, the activity of
the enzyme is modified. The degree of modification of the enzyme
activity is indicative of the concentration of the labelled ligand
in the bound fraction and hence gives an index of the concentration
of ligand in the reaction mixture.
The chemical structure of the ligand-enzyme complex is extremely
difficult to assess and this is a major disadvantage of enzyme-
~0 immunoassay. This is undoubtedly due to the great multiplicity of
amino acid side chains which are available on the enzyme surface for
` - 5 ~ 7~
complexing with the ligand. This causes great difficulty in
reproducing the ligand-enzyme in different preparations of the complex.
The general lack of control of the complexing reaction results in the
attachment of many ligand molecules to one enzyme molecule, although
it is likely that the binding of only a few of these ligand molecules
by antibody is involved in the inhibition of enzyme activity. Hence
not all antibody/labelled antigen interactions result in a
modification of the enzyme activity, thereby lowering the
sensitivity of the technique.
Protein-protein interaction between different antibody molecules
and antibody and enzyme molecules is another consequence of the
multiplicity of ligand molecules on the enzyme surface. Protein-protein
interaction is further increased if the ligand is also a polypeptide.
Hence the enzyme molecule induces a localised micro-environment of
high protein concentration. In such situations it has been
demonstrated that protein precipitation occurs, thereby causing a loss
of one of the main advantages of enzymeimmunoassay by necessitating
separation of antibody bound and free fractions.
A modification of enzymeimmunoassay has been described which
partially overcomes these problems in that the ligand is labelled
with a detector molecule which is ~f low molecular weight. In
~ this assay antibody binding of labelled ligand sterically hinders
binding of detector molecule by another antibody specific for the
detector molecule. The degree of hindrance is determined by
competition for binding of the free ligand-detector molecule and
detector molecule labelled enzyme with detector-molecule antibody.
The degree of modification of enzyme activity, as determined in normal
enzymeimmunoassay, is indicative of the concentration of free
ligand-detector molecule which is likewise indicative of the
concentration of ligand in the reaction mixture. The advantage
of this technique is that a small molecule rather than an enzyme is
attached to the ligand, thereby allowing the chemical structure of
the labelled ligand to be determined and thus overcoming many of the
disadvantages of enzymeimmunoassay described previously. However,
-, . . , - ~ .
.. . .
.
.
.
- 6 - ~ ~'Q2~
this system only overcomes the disadvantages of the primary blnding
reaction involving the ligand and labelled ligand and transfers them
to the detector system for determining the degree of antibody bound
and antibody free fractions of the labelled ligand.
The disadvantages of the prior art methods are overcome by the
present invention according to which an enzyme modifier is
used as the label.
More particularlv the present invention relates to a reaaent for
the determination of an immunologically active material,
comprising the immunologically active material or a receptor
which can specifically bind said immunologically active
material,labelled with a substance capable of modifying the
extent or the mode of activitv of an enzvme.
Furthermore the present invention relates to a method for the
determination of an immunologically active material in a
sample wherein the sample is contacted with a receptor which
can specifically bind the immunologically active material, the
immunologically active material labelled with a substance
capable of modifyina the activity of an enzyme, an enz~me and
an enzyme substrate, and wherein the extent or the mode of the resultina en-
- zy~e activit~v is measured and ccmpared with that obtained with a standard.
Furthermore the present invention relates to a method for the
determination of an immunologically active material in a
sample wherein the sample is contacted with a receptor which
can specifically bind the immunologically active material and
which is labelled with a substance capable of modifyins the
activity of an enzyme, with the immunologically active material
in insolubilized form, and with an enzyme and an enzyme sllhstrate,
and wherein the extent or the mode of the enzyme activitv, after
the separation of the solid phase, is measured and compared with
that obtained with a standard.
.
.
', . : - :
. . : - - .
- 7 ~
The terms 'immunologically active material' or 'ligand'
within this specification refer to any immunologically
active substance or part thereof capable of
being immunologically determined e.g. by using
saturation analysis techniques. The essential requirement
is that there be a receptor which will specifically bind the
ligand. When the receptor is an antibody the ligand will be
haptenic or ahtigenic such that specific antibody can be
formed. A ligand is referred to as a hapten when it will only
elicit antibody formation when attached to a compound having
antigenic properties. Alternatively, a ligand is referred
to as an antigen when it will elicit antibody formation
without chemical modification. The ligand may vary widely
in molecular weight ranging from approximately 100 - 1,000,000.
This range of molecular weight is not limiting in the assay
providing receptor is available which will specifically bind
the-ligand.
The ligand may be either polymeric or non-polymeric in
structure. When polymeric, the ligand will usually be
biological in origin and be classified as a nucleic acid
polysaccharide and/or polypeptide. Alternatively, when the
ligand is non-polymeric, it will generally have a molecular
weight of less than 2,000 and may have a wide variety of
structures, functionalities and physiological properties.
Ligands of ~articular i~portance which mav be used in the ~resent invention
are amines, aminoacids, peptides, proteins, lipoproteins, gl~copr~teins,
~ sterols, steroids, lipoids, nucleic acids, mono- and polvsaccharides, alka-
loids, vitamins, druas, narcotics, antibiotics, metabolités, pesticides,
toxins industrial pollutants, flavourinq agents, hormDnes, enzymes, coenzymes,
cellul~r or extracellular ccmponents of tissues and isolated antibodies from
humans or animals. The assay is,however,not limited to only these li~ands.
- 8 -
1 Components of immediate potential for analysis with the system
are hepatitis B-surface antigen; ferritin;tumour antigens like
CEA; a-feto protein; rheumatoid factor; C-reactive proteins;
imnunoglobulin classes IgC-, IgM or IgP.;myoglobin, th~roi~ hormones includina
T3 and T4, insulin; steroid horniones including testosterone or estra-
diol; drugs of abuse including narcotic analgesics, like morphine;
barbiturates; stimulants like amphetamine; drugs for the treat-
ment of epilepsy including diphenyl hydantoin and phenobarbjtal;
cardiac glycocytes like digoxin; vitamines like vitamine Bl?~nd
folic acid. Furthermore, antibodies which have immediate po-
tential for analysis with the system are those associated with
infection in syphilis, gonorrhoea, brucellosis, rubella and
rheumatism.
The term 'labelled immunologically active material' or
'labelled receptor' in this specification refers to a
ligand, analog of a ligand or part ther~of or to a receptor which is
labelled with an enzyme modifier. One, more than one, and
generally less than lOO molecules of label, can be attached
to a ligand or receptor molecule. Similarly, one, more than one
and usually less than 5 ligand or receptor molecules can be
attached to a label molecule. The attachment of extra label
molecules to a ligand or receptor molecule generally
increases the sensitivity of the assay provided the extra
labels do not affect binding.
Attachment of a modifier molecule to a ligand or receptor
molecule involves the formation of intermolecular bonds
which in most cases, but not necessarily, are covalent in
nature. Attachment may be accomplished in some cases in
the presence of a coupling agent by the insertion of a
linking group between the label and the ligand or receptor.
The mcdifier molecule can be attached directly to the liqand or
receptor molecule. However it may be desirable to insert chemical
bridges of various length between the mcdifier rolecule and the
ligand or receptor molecules depending on the speciic assay
- 9 - ~
1 en~isa~ed. In some cases it may even be advanta~eous to attach
themodifier molecule and the ligand or receptor molecules sepa-
rately to the same carrier molecule e.~. a macromolecule like a
polypeptide or a polysacch~ride.
The term'receptor'in this specification refers to any substance
which can specifically bind ligand and labelled ligand or part
thereof. Generally the receptor used in the assay is a specific
antibody to the ligand formed in the blood of vertebrates
following injection of appropriate hapten or antigen.
Alternatively, receptors which occur naturally can also be used
in the assay. This latter group includes,but is not limited to,
proteins, nucleic acids and cellular membranes. Such receptors
have been used in radioassay techniques for thyroxine, insulin,
angiotensin and various steroid hormones.
In case the ligand is an antibody the receptor may be the
antigen utilized for inducing said antibody in a host animal.
In an another embodiment the receptor can be an antibodv against
the antibody to be determined.
It is not possible to ascertain with certainty the mode of receptor -
action which, on binding of the labelled ligand, reduces interaction
between enzyme and modifier. The most likely explanation is that
the affinity of the enzyme for the modifier is reduced as a result
of a change in size and net charge of the receptor/ligand-modifier
complex compared to that of the ligand-modifier alone.
The tërm'modifier'within this specification refers to any
substance which is capable of interacting with an enzyme such
that the extent or the mode of enzyme activity is modified.
This modification may result in an inhibition, activation or
a change in specificity or any other property of the enzyme
which is detectable either directly or indirectly by a change
in enzyme activitv or in the mode of the activity e.g. a chanae
in reaction conditions like co-factor requirements or pH-opti-
mum, in kinetic properties, or in activation energy. The
:
-- 10 --
1 modifier can range in size from a small molecule to a
macromolecule, and its interaction w;th an enzyme molecule can
be either reversible or irreversible depending whether t~e
inter-molecular association is ionic or covalent in nature.
Sensitivity of the assay is dependent amon~ others upon
the affinity of the receptor for the ligand and the
ability of the modifier to cause a change in the extent or the
mode of enzyme activity. Preferably, modification of the extent
or the mode of enzyme activity is achieved with a minimal con-
centration of modifier. The closer this concentration is to
that of the enzyme on a molecular basis, the ~reater will be the
sensitivity of the assay.
Preferably, the modifier will be an enzyme inhibitor which, on
interaction with an enzyme, will inhibit its activitv. The mode
of action of the inhibitor may be competitive, non-competitive,
uncompetitive,allosteroic or a combination of two or more of
these modes. Preferably the inhibitor should ha~e an inhibition
z~ constant (concentration of inhibitor necessary for 50% inhibition
of the enzyme system) below lO ~ moles/l, dependin~ on the assay
performed. ~ost preferably the inhibition constant is between
lO 5 and lO 5.
Any enzyme ~an be used in the assay provided there exists a
modifier which will specifically modify the enzyme activity
in the manner described above Enzymes of choice are stable,
easily obtained at low cost and have a high turnover number and
a simply performed assay system. Preferably the turnover
number (molecules of product formed per molecule of enzyme
in one minute) is superior to lO0 dependinq on the specific test
performed. Most preferably, the turnover number is as
hi~h as possible and at least 200.
Enzyme modifier systems which are particularly suitable
for the present invention are dihydrofolate reductase/
methotrexate, dihydrofolate reductase/4-aminopterin,
.
.
,
.
.
8~3
1 dihydrofolate reductase/other specific inhibitors of this enzyme;
~-glucoronidase/4-deoxy-5-amino glucarlc acid and its derivatives;
biotin containing enzymes/avidin like carboxylase/avidin;
chymotrypsin/TPCK CH2-Ph
~M3 ~ H 0
0
~-cystathionase/propargylalycine; alanine racemase/tri-
fluoroalanine; trvptophanase/trifluoroalanine; tryptophan
svnthetase/trifluoroalanine; ~-cystathionase/trifluoro-
alanine; pyruvate-glutamate transaminase/trifluoro-
alanine; lactic acid oxidase/2-hydroxy-3-butynoic acid;
monoamine oxidase/N,N-dimethyl nropargyl amine; and
diamine oxidase/~2N-CH2-C_CCH2-NH2;
Determination of enzyme activity may be performed by moni-
toring directly or indirectly the consumption of substrate or
production of product at suitable pH and temperature using de-
tection systems including colorimetry, spectrophotometry, fluoro-
spectrophotometry gaseometry, thermometry (heat production),
scintillation counting.
In order to increase the sensitivity of the system it is possible
to use bioluminescence and enzymic cycling techni~ues, e.a. the
techniques described by J. Lee et al in Liquid Scintillation
~ Counting:~ecent nevelopments, Stanley P.E. and Scoqqins, B.A.,
Academic Press, New York p. 403 and Lowry O.H. et al in J. Biol.
Chem 2_6, p. 2746-2755.
'
- 12 - ~ t7~
1 In one embodiment of the present invention a labelled
ligand can be used for the determination of the presence
of a ligand in an unknown specimen by the simultaneous or
sequential addition of the labelled ligand and the un~nown
specimen to an aqueous medium, at appropriate pH, containing
a receptor specific for the ligand and labelled ligand.
After a suitable incubation period the distribution of
receptor bound to ligand and labelled ligand is
determined by the addition of enzyme and substrates. The
receptor which is specific for the ligand also binds the labelled
ligand and, in so doing, reduces the interaction between the modifier
and the enzyme thus decreasing the modification of enzyme activity.
Addition of ligand to the assay results in competition with the
labelled ligand for binding with receptor and thereby increases
the concentration of free labelled ligand in the assay. Interaction
between ligand-modifier and enzyme is increased and the enzyme
activity is once again affected. The modification of enzyme activity
is therefore a function of l;gand concentration in the assay and is
caused by unbound labelled ligand. Consequently the difference
between the resulting enzyme activity and that obtained in the
absence of ligand ;s indicative of the concentration Gf ligand in
the unknown specimen.
One of the major advantages of this method is that separation
of bound and free fractions in the procedure is unnecessary.
This does not preclude,however,the use of such a separation
step in the assay after incubation of ligand and labelled ligand
with receptor and prior to the estimation of enzyme activity.
Separation of fractions may be desirable in some instances for
removal of substances in the specimen which may interfere with
the enzyme assay. Separation may be achieved using any of the
many techniques described for radioimmunoassay including gel
filtration, adsorption and ion exchange chromatography, fractional
precipitation, solid phase and electrophoresis.
.
- 13 - ~ ~Z7~
1 This assay is of course not limited to the determination of
haptens and antigens as the ligand but can also be adapted
to the identification and measurement of antibodies as the
ligand.
This can e.g. be performed by labelling the antibody with
an enzyme modifier and measuring the extent of enzy~e acti-
vity modification following incubation of labelled antibody
and specimen antibod,v with a limiting concentration of antiaen
or hapten. The modification of enzyme activity is related to
the specimen antibody concentration. If necessary the bound and
free fractions may be separated before addition of the enzyme
and the substrate.
The invention permits also for the determination of
a ligand the use of a labelled receptor rather than
that of a labelled ligand. Ligand in the unknown
specimen reacts with excess receptor labelled with an enzyme
modifier and, after incubation, excess solid phase insolubilized
ligand is added and reacts with the free labelled receptor
remaining. After separation of the solid phase, the modification
of enz~me activity associated with the soluble ligand is
measured and related to the concentration of ligand.
Z5 The reagents of the present invention can also be used in a
'sandwich' techni~ue provided the ligand has at least two
binding sites. Ligand reacts with excess solid phase receptor
and after incubation followed by washing, the solid phase
receptor bound ligand is reacted with excess receptor
labelled with an enzyme modifier. Free labelled receptor is
removed by washing and the extent of enzyme modification
in the separated fractions is determined. Tkis then gives an
index of liqand concentration.
-.
~7~
The followin~ exam~les illustrate the invention.
.
Example 1
Preparation of "Amino-digoxin"
To a suspension of 156 mg (0.2 mmol) digoxin in 5 ml absolute ethanol
was added 10 ml 0.2 M sodium metaperiodate with stirring. The mixture
became homogeneous after 10 m;nutes and then a precipitate slowly formed.
After 2 hours, 5 ml water plus 5 ml ethanol were added. 122 ~l (2.2 mmol)
of ethylene glycol was added after a further 30 minutes and a dense
white precipitate started to form immediately. After 80 minutes'
stirring, 133 yl (2.0 mmol) ethylene diamine was added and the
resultant pH of ,11.0 was adjusted to 9.5 with 0.1 M HCl and the
~ reaction mixture was allowed to stand at room temperature for
18 hou~s. The pH did not change during this time.
151.4 mg (4 0 mmol) sodium borohydride was then added and the
mixture stirred for 3~ hours. The pH was then adjusted from
10.5 to 6.5 with IM formic acid (about 3 ml) and TLC of this
mixture showed a single major spot with an Rf 0.15 (silica on aluminium
developed in butanol:acetic acid:water/4:1:1). Digoxin had an Rf 0.7
when developed in the same system.
The solvents were evaporated to near dryness on a rotary evaporator
under Yacuum using a water bath at 60. The last few ml of water
- were removed by adding 95% ethanol (3 x 20 ml) and repeating the
evaporation as above.
The resultant pale yellow solid was extracted three times with
absolute ethanol and these combined extracts concentrated to
about 4 ml and centrifuged to separate a small amount of salt
which was discarded. The pale yellow supernatent solution was
evaporated to dryness under a stream of dry nitrogen to give a
yellow oil which showed the same Rf on TLC as the reaction mixture
as described above. The product was shown to contain a free amino
group b~ reacting it with "Fluram" (4-phenylspiro [fluran-2(3H),
1-phthalan]-3,~-dione) to form an intensely fluorescent compound.
The product exhibited a spectrum in concentrated sulphuric acid
. ..
27~3~
- 15 -
similar to that of digoxin with absorption peaks at 385 and 495 m~.
The product also exhibited a strong affinity for antisera (rabbit)
specific for digoxin. The most likely structure of the isolated
product is as follows:
O~
c~C~
H2~_c"2cR2~
.. _ .
.
Example 2
Preparat;on of methotrexate -aminod;qoxin conjugate
11 mg methotrexate was dissolved in 5 ml H20 and adjusted to pH 6.5.
10 mg "aminodigoxin" was dissolved in this solution and the volume
adjusted to 20 ml with H20. The pH was readjusted to 6.5 and 484 mg
N-ethyl-N"-(3-dimethyl am;no) propyl-carbodiimide hydrochloride,
dissolved in 5 ml H20, was added to the reaction mixture. The pH
was maintained at 6.2 for 24 hours at room temperature. The conjugate
- was purified on a silica gel column, using 3X ammonium citrate as a
solvent. Fractions containing the desired product were combined and
shown to have the dual capacity to bind strongly antisera (rabbit)
specific for digoxin and also to inhibit strongly the enzyme
dihydrofolate reductase (chicken liver). The most likely structure
of the methotrexate-aminodigoxin conjugate is as follows:
`
:
7~
- - 16 -
.'
~j-CIl~c~ ,~o~
~F
o~
Ht~'--C112C112--~ ~\
Example 3
Enzyme Inhib;tor Immunoassay for Digoxin
100 ~l serum was incubated at 30C for 15 minutes with 100 ~l antidigoxin
antibody solution, 100 ~l NADPH solution, 100 ~l 2-mercaptoethanol
solution and 550 ~l sodium phosphate buffer pH 7.5. 100 ~l Methotrexate-
digoxin conjugate of example 2 (70 ~g/ml) was added and the
mixture incubated for 15 minutes, after which, 100 ~l dihydro-
folate reductase solution was added. The dihydrofolate reductase
preparation employed was isolated from chicken liver by the
method of Kaufman, B.T., & Gardiner, R.C., Journal of Biological
Chemistry, Vol. 211, P 1319 (1966). The mixture was incubated
for a further 3 minutes and the enzyme activity determined by
the addition of 100 ~l dihydrofolate solution and monitored at
340 nm with a varian recording spectrophotometer. The results
are shown in Table.
- . ~. , . , . ~
,
,, , . - . : .. , - ,, : .
,: . , . ,:: : . . -
. .
-
- 17 -
TABLE I
Reactants Enzyme Activity
Digoxin Antidigoxin Methotrexate- Enzyme assayOD/min I Inhibition
(sample conc.) antibody digoxin conjugate components
(in assay)
O absent O present 0.150 O
O absent 7 ng present 0.100 33
O present 7 ng present 0.145 3
_ 5 ng/ml present 7 ng present 0.125 16
10 ng/ml present 7 ng present 0.1l5 23
The reactants were added in the sequence described above.
OD = optical densitv
It is evident from the above results that a few ng digoxin in serum
can be de$ermined in the system described.
_ .
' . ' ' ' : :
.
- . - . ,:
, , .. - - ~
:. ,, - .. ,. . . . ~ . -
- 18 - i
Example 4
Preparation of Methotrexate-Human Serum Albumin Conjugate
45 mg methotrexate was dissolved ;n 1.0 ml N,N-dimethyl formamide
and 25 mg N-hydroxysuccinimide was added.
41 mg N,N-dicyclohexyl carbodiimide was dissolved and the mixture
kept at room temperature for 13 hours. The insoluble urea
byproduct was removed by filtration and 100 ~1 of the filtrate
was added to a solution containing 5 mg human serum albumin in
800 ~1 O.lM sodium phosphate buffer pH 7.5 plus 100 ~1 dioxane.
,lO This reaction mixture was kept at room temperature for 30 minutes
and then loaded onto a Sephadex G-25 column equilibrated with
O.lM sodium phosphate buffer pH 7.5. The column was developed
with this buffer and two elution peaks containing methotrexate
were obtained, the first of which contained the methotrexate-human
serum albumin conjugate.
Example 5
Enzyme Inhibitor Immunoassay for Human Serum Albumin.
1OOJU1 diluted serum was incubated at 30C for 15 minutes with
1OOJU1 antihuman serum albumin antibody (rabbit) solution,
100 ~1 NADPH solution, 100 ~1 2-mercaptoethanol solution and
550 yl sodium phosphate buffer pH 7.5. 100 ul methotrexate-
human serum albumin conjugate (8Jug/ml) was added and the
mixture incubated for 15 minutes, after which, 100Jul dihydrofolate
reductase solution was added. The mixture was incubated for a
further 3 minutes and the enzyme activity determined by the
addition of lOO~ul dihydrofolate solution and monitored at
340 nm with a varian recording spectrophotometer.
The results are shown in Table II.
*Trade Mark
~.~,. . .
~`1
:.: . '.--: -, ' .'': ' - ' : , : ~ - . . ' ' :
. .:, . : '. : ~: ' ' ~ . .
::. : '- ' '
,' :" ' '. ' ' " '' ~ ' ~ '
. . .
- 19 - ~ 7~
TABLE I I
- ' . . . _ _ _
Reactants Enzyme Activity
Human Serum Antihuman Methotrexate- Enzyme OD/min % Inhibition
Albumin Serum albumin human serum Assay
(Sample antibody albumin conjugate Component
Conc.) (in assay) _ _
O absent O present 0.123 O
O absent 0.8 ~g present 0.068 45
O present 0.8 ~9 present 0.105 16
5~ug/ml present 0.8Jug present 0.092 25
10 ~g/ml present 0.8 ~9 present 0.085 31
.
.
It is evident from the above results that a few ~9 human serum albumin can
be determined in the system described.
- . . - ~ -
- . -. : ,: :
.. , ., ~ . .
- 20 -
1 EXAMPLE 6
Synthesis of methotrexate-aminoethylmorphine conjugate.
a) Synthesis of 03-aminoethylmorphine
In 10 ml of tetrahydrofuran (THF) freshly distilled from
lithium aluminium hydride (LA~) was suspended 400 mg of LA~ un-
der nitrogen. A solution of 400 mg of morphine and 400 mg of
chloroacetonitrile in 4 ml of freshly distilled THF was added
over 5 minutes, followed by refluxing for 1 hour. The mixture
was allowed to cool and 0.6 ml of water was added followed by
0.6 ml of 10 weight % sodium hydroxide and 2 ml of water. After
filtering the mixture, the salts were washed with THF, the THF
fractions combin~d, dried with magnesium sulphate under nitrogen,
filte~-ed and the filtrate evaporated to yield 380 mg of 03-
aminoethylmorphine.
b) Synthesis of N-t-Butoxycarboxyl-~-(03-aminoethylmorphine)
glutamic aci*~ -benzylester.
A mixture of 03-aminoethylmorphine (50 mg prepared as ;,
above), N-t-BOC-glutamic acid-~-benzyl ester(34 mg)and
dicyclohexylcarbodiimide (25 mg) in dichloromethane (5 ml) was
stirred for 4 hours at room temperature. The mixture was diluted
with ethyl acetate and washed with dilute sodium carbonate so-
lution. The ethyl acetate solution was then extracted twice with
0.1 N hydrochloric acid and the combined!acidic extracts were
.
: . .
~ .
- ' ' , , ,: .
.:
. ~
- 21 - ~Z7~
1 treated with sufficient 10 weight % sodium hydroxide solution to
adjust the pH to 8Ø The solution was extracted twice with ethyl
acetate and the combined ethyl acetate extracted were washed
with brine, dried (anhydrous sodium sulphate) and evaporate~ to
give a colourless oil (36 mg).
c) Synthesis of ~lutamic acid-~-(03-amidoethylmorphine) -~-
benzyl ester-trifluoroacetic acid salt.
A solution of N-t-BOC-~lutamic acid-morphine derivative
(36 mg, prepared as described above) in dichloromethane (3 ml)
was stirred at room temperature and trifluoroacetic acid (1 ml)
was added. The mixture was stirred for 15 minutes and then eva-
porated to dryness. The residue was a colourless glass (40 mg).
d) Synthesis of methotrexate ~- (03- amidoethylmorphine)
-~- benzyl ester.
A solution of 4-amino-4-deoxy~N10-methylpteroic acid
(37 mg) in 3 ml di~ethylsulfoxide (DMSO) stirring at room tem-
perature was treated with triethylamine (28 ~1) and i-butyl-
chloroformate (25 ~1) and the mixture was stirred for 30 minu-
tes. It was then added to a mixture of the morphine derivative
(75 mg prepared as descrl~ed in ~) and triethylamine (28~1)
in DMSO (2 ml) and the mixture was stirred at 60 for one hour.
The cooled mixture was diluted with water and extracted twice
with ethylacetate. The combined ethyl acetate extracts were was-
hed withwater and then extracted twice with O.IN hydrochloric
acid. The combined acidic extracts were treated with sufficient
10 weight % sodium hydroxide to raise the pH to 8Ø The mix-
ture was extracted three times with ethyl acetate and the com-
. .. : . .. . : . - . - :
.
'
- : '
- 22 -
1 bined extracts washed with brine, dried (anhydrous sodium sul-
phate) and evaporated to give a yellow oil (15 mg). This was
purified using prepæative thin layer chromatography (TLC) on silica gel
developing with chloroform~methanol, 4:1. The desired o~ound was
obtained as a yellow solid (4 mg).
e) Synthesis of MethotreXate ~-(03-amidoethylmorphine).
The product prepared as described in d (~ mg) was
mixed with O.INsodium hydroxide solution (5 ml) and the mix-
ture was stirred at room temperature for 8 hours resulting in a
clear yellow solution.
To this was added O.IN hydrochloric acid (5 ml) and the
mixture was made upto 25 ml with sodium orthophosphate buffer
(0.05 M, pH 7.4) to give a solution of the desired compound
suitable for use in the enzyme assay.
EXAMPLE 7
Enzyme inhibitor immunoassay for morphine
A mixture of 10~1 of morphine solution of suitable con-
centration, 50~1 of methotrexat~-(03-amidoethylmorphine)
solution (4 ng/25 ml), 50~1 of antimorphine antibody solution,
10~1 NADPH solution, 10~1 2-mercaptoethanol solution, 50~1 po-
tassium chloride solution, 150~1 Tris-HCl,buffer solution (p~
7.5)containing EDTA and 10~1 of dihydrofolate reductase (E.~
casei) solution was incubated for 20 minutes at 37. The enzyme
activity in the mixture was determined after the addition of
10~1 dihydrofolate solution by monitoring the change in extin-
z~
-- 23 --
ction of the solution at 340 nm using a Centrifichem
centrifugal analyser. The results are shown in Table III
TABLE III
__
~CIA~S ENZYME ACTIVITY
~)~PHINE ANTI~ PHINE ~PHINE- ENZY~!IE ASSAY . __
ME~OT~IE DD/ITin ~61NHIBITION
pg/assay ANTIBODY CONJUG~TE ~llENr3 __ _
O absent absent present O.54 O
O ~sent 3xlO M present 0.14 76
O ~resent 3xlO 8 M present 0.41 29
0.04 present 3xlO 8 M present 0.37 36
0.40 present 3xlO 8 M present 0.35 40
4.0 present 3xlO 8 M present 0.31 46
present 3xlO 8 M present 0.27 54
¦ presen~ ¦ 3xlO a M ¦ presen~ 10.23 ¦ 60
'
,~
g
- 24 -
1 The above table shows that with increasing concentration of
morphine, there is decreasing activity of dihydrofolate reduc-
tase. In this manner solutions of morkhine containing from 4~g
per ml to 4 mg per ml of morphine could be assayed where only
10~1 of the solution was available. This is not intended to in-
dicate however, that this is the range required, but rather only
that it can be successfully emploved.
EXAMPLE 8
.
Synthesis of ferritin-methotrexate conjugate.
A solution of huma~ liver ferritin (1.3 mg) in sodium
phospate buffer (0.05 M, pH 8.0, 5 ml) and 1 ml dimethyl formamide
(DMF) was stirred at room temperature and 100~1 of a solution
obtained by treating methotrexate (23 mg) in DMF (2 ml) with
triethylamine (21~1) and i-butylchloroformate (15~1) at room
temperature for 30 minutes, was added. The mixture was stirred
at room temperature for 2 hours and then dialysed against 2x2
litres of sodium orthophosphate buffer (0.05 M, pH 7.4 con-
taining sodium chloride O.IM and sod;'~m azide 0.05 weight %)
for 24 hours. The solution was then passed through a column of
Sephadex G-25 equilibrated with the same buffer and the ferritin
containlng fractions combined and made upto 10 ml with the
buffer.
The resulting solution is suitable for use in an enzyme
immunoassay for the determination of ferritin
.
.. . .~ ~ . : :
