Note: Descriptions are shown in the official language in which they were submitted.
r~ 1*
13 40 A~ 8
DETERMINATION METHOD, USE AND COMPCN~N.S
This invention relates to a method of determining
haptens, to the usc of that method and to component~
including kit~ useful in that ~ethod.
At present there are a number of co-mercially
availablc ~ethods of determining hapten~. However 6uch
method~ frcquently 6uffer from the dlsadvantage of
producing an increasing signal a~ the concentration of
the hapten decreases. This is often inconvenient and
can be a source of v~riablility, noise or error.
-2- 134048~
Another difficultly with many commercially available
method~ of determining haptens is that they can be
sensitive to the amounts of certain reagcnt~ employed.
I believe that it would be desirable to provide a
method that enable~ hapten~ to be deter~ned in a
manner that allow~ the determination to produce ~
response which increa~es a~ the concentration of the
haptens increase (a~ opposed to the inver~e ratio which
often applies). It would be an added ad~antage if ~uch
a method could employ excess reaqents ~och as certain
antibodie8, especially labelled antibodie~, ~o that
preci~ion would not be adversely effected by ~mall
variations in the amount of such reagentr employed.
Many of the preceding advantages are offered by the
systems described in published European Patent
Application No. 85901495.3, Publication No. EP 0177531
published on April 16, 1986; Europe~n Patent Application
No. 85903019.9, Publication No. EP 0185722 published on
July 2, 1986; and European Patent Application
No. 87308829.8, Publication No. EP 0264219 published on
April 20, 1988, in which antibodies are described which
bind a complex of a small molecule and an antibody
thereto but do not bind the small molecule or the
antibody thereto alone. Unfortunately preparing the
antibodies disclosed in those Patent Applications is
believed to be frequently tedious with considerable
repetitive procedures to be followed if antibodies having
the desired level of specifivity are to be achieved. It
is believed that high backgrounds can result if
specicivity is sacrificed to ease of preparation. Patent
~ 1340488
Application No. pcT/Gs87/oo46l~ Publication No.
Wo 8800240 published on January 14, 1988, discloses a
further class of antibodies which can bind a complex of
small molecule and an antibody thereto and can bind that
antibody against the small molecule but cannot bind the
small mol~cule al~one. T~hese could ~e used in determination
methods and can be readily produced. Unfortunately they
were not said to allow the determination of haptens other
than by limited methods such as, for example, by using
labelled haptens in competitive assays,
I believe that it would be desirable to provide a
method of determining haptens which enploys antibodies
which can be produced relatively easily but still
offers advantages as set forth hereinbefore and can be
used in a variety of manner~. Such a ne~od has now
been discovered.
The pre~ent invention provides a ~thod of
deternining a hapten which method conpr~res (i)
contacting the hapten with a binding partner of the
hapten, whereby the hapten becomes bount to some of the
binding partner, (ii) contactng the unbcund binding
partner with a secondary binding partner therefore,
(iii) contacting the binding partner with an antibody
which binds the binding partner which ha~ bound thereto
the hapten but which does not bind the bindlng partner
which has bound thereto lts secondary bindlng partner;
,
,. - 4 -
1340~88
and (iv) determining the amount of antibody bound to
the binding partner.
The antibody referrd to in (iii) und (iv) above
will often be referred to as the ~elective antibody~
hereinafter. The selective antibody nay be polyclonal
although I prefer to employ a monoclonal relective
antibody.
When the term ~antibody" i~ u~ed it ~hould be
reali~cd that the antibody can be the ~hole
immunoglobulin or fragments thereof which containing
the binding site (such a~ Fab, F~abl)2 ~ rV) . The
antibody may al~o be an aggregate or hybrid but thi~ i6
u~ually le~ preferable. using a fragmeDt a~ thc
binding partner of the hapten (such a~ ~ rab fragment)
can be particularly u~eful.
When used herein the term ~hapten- ha~ the normal
meaning of a ~mall molecule which i~ not it~-lf
immonogenic. The ~illed art worker will appreciat-
that low molecular weight material6 ~uch a~ ~mall
molecule~ for determination by thi~ invention ar-
normally non-immunogenic but that antibodie~ agaln~t
these haptens can be obtained by im~uni~ing an animal
with a con~ugate of the non-immunogenic nolecule (or
sometine~ a very clo~e analogue) and an i-nunogenic
. . .
. ~ .
1310488
material (such as bovine serum ablumin or an equivalent
agent).
It will be understood that haptens are small
molecules. Such haptens aptly have a molecular weight
of for example 100 to 1500, more suitably from 120 to
1200 and favourably from 200 to 1000. ~aptens with
molecular weights of less than 1000 are often of
particular interest for this invention. ~uropean Patent
Application No. 85901495.3, Publication No. EP 0177531
published on April 16, 1986; European Patent Applicatlon
No. 85903019.9, Publication No. EP 0185722 published on
July 2, 1986; and PCT/GB87/00461, Publication No. wo
8800240 published on January 14, 1988, may be consulted
for the types of haptens which are most suitable for
determination by the method of this invention. European
Patent Application No. 87308829.8, Publication No. EP
0264219 published on April 20, 1988, may similarly be
consulted.
~ aptens of particular interest for deter-ination
may be selected form amongst groups such as
medicaments, drugs of abuse, metabolite~, industrial
chemicals (~uch as pollutants and agrochenical~),
toxins and the li~e.
$n order to facilitate the determination of the
amount of selective antibody bound to the binding
partner it is apt that either the selective antlbody or
the binding partner i~ immobilized Precipitation of
the complex including the binding partner and the
selective antibody i~ al80 envisaged but this i~
-" ~ ' - 6 ~ 0 4 8 ~
thought to be le~ apt than employing i-~obolization of
the selective antibody or the binding partner. The
immobilization employed may be any ~uitable method but
in general attachment to a ~olid ~urface, for example
to the ~urface of a plate, tube, dip-stic~, capilliary,
paper, gel or the li~e i~ favoured.
The ~econdary binding partner for the hapten~
binding partner may be any which will bind to that
binding partner and which once bound will prcvent
~ignificant binding of the antibody thereto. Generally
the ~econdary binding partner will be an analogue or
derivative of the hapten. Mo~t ~uitably the ~econdary
binding partner will be a moiety which ir ~ufficiently
large to prevent binding of the antibod~ by ~teric
effect~. I believe that a particularly favoured cla~
of ~econdary binding partner~ are large binding
partners and that the~e are preferably large
derivative~ of the hapten, for exa-ple con~ugate~ of
the hapten with a large molecule or of a clo~e analogue
of the hapten with a large molecule. Such large
molecule~ will mo~t ~uitably have a molecule weight of
greater than 5000 and will preferably be
macromolecule~, for example proteins. Typically the~e
macromolecule~ will have molecular weight~ greater than
10000, more aptly greater than 20000 and preferably
greater than 40000. I believe conventio~al
1340 i8~
macromoleculee ~uch a~ albumin ~for exa-ple boven 6erum
albumin) are ~uitable for conjucation to the hapten.
If a conjugate of a hapten (or a cloee analogue)
and large ~olecule is u~ed to rai~e the binding
partner, then that con~ugate i~ often a prticularly
~uitable secondary binding partner. It i~ often
advantageou~ that the con~ugate or ~econdary binding
partner ~o employed are highly water ~oluble (although
thoge conjugate~ of lower ~olubility can be employed).
~he ~killed art worker will be aware that ~uch
con~ugate~ are readily available and ~ay be obtained by
many conventional method~ of con~ugatlon.
The ~econdary binding partner can advantageou~ly
be an antibody again~t the binding partner of the
hapten, for example again~t the hapten bind$ng ~ite,
for example an anti-idiotypic antibody. It can be
particularly advantageou6 to u~e a fragxent of the
anti-idiotypic antibody a~ previou61y lndicated.
The ~illed art worker will appreciate that the
binding partner~ will u~ually be ~elected to bind with
a~ high an affinity a~ conveniently obtainable but that
the affinity of the eelective antibody for the
~econdary binding partner binding partner complex
~hould be a~ low a~ conventiently obtainable.
i340~8~
The determination may employ a label if de~ired.
If this is so it will not be on an i~ obilized member
of the ~elective antibody binding partner pair.
In ~ome a~pect~ of the invention a label will not
be required; for example, where as~ociation of the
hapten, primary partner and ~elective antibody i~
measured directly, for example by electrical, optical
or other mean~ for example by modification of a ~urface
~uch a~ a diffraction granting or the li~e in which
reflectance propertie~ change of deposition.
The binding partner for the hapten nay be any
~uitable binding material ~uch a~ a ~pecific binding
protein or antibody but I believe be6t reeult~ are
obtained when a monoclonal antibody i~ e-ployed a~ the
binding partner for the hapten.
rrom the foregoing it will be clear that in a
favoured agpect the pre~ent invention provide~ a method
of deter-ining a hapten which method coepri~e~ (i)
contacting the hapten with a labelled nonoclonal
antibody therefore whereby the hapten become~ bound to
~ome of the labelled aonoclonal antibody, (il)
contacting the unbound labelled monoclonal antibody
with a large binding ~artner thereforeS (iii)
. .
- 9 - 13 40 4~
contacting the labelled monoclonal antibody with an
immobilized antibody which bind~ the labelled
monoclonal antibody which ha~ bound thereto the hapten
but which doe~ not bind the labelled monoclonal
antibody which ha~ bound thereto it~ large binding
partner; and ~iv) determining the amount of antibody
bound to the labelled ~onoclonal antibody. The
determination will be effected u~ing the label on the
labelled monoclonal antibody. Normally tbe thu~
immobilized label will be a~sayed after ~-paration of
the liquid pha~e and wa~hing the,~olid.
From the foregoing it will al60 be clear that in
a particularly favoured aspect the pre~ent invention
provide~ a method of determining a hapt-n which method
compri~e~ (i) contacting the hapten with an immob~lized
monoclonal antibody therefore whereby the hapten
become~ bound to some of the monoclonal ~ntibody,(ii)
contacting the unbound immobilized monoclonal antibody
with a large binding partner therefore, (iii)
contacting the im~obilized monoclonal antibody with a
labelled antibody which bind~ the i-mobilized
monoclonal antibody which ha~ bound thereto the hapten
but which doe~ not bind the i~mobilized ~onoclonal
antibody which ha~ bound thereto it~ large binding
partnerS and (iv) deter-ining the amount of labelled
antibody which has beco-e bound to the i- obilized
~ i ~ lO- 134048~
monoclonal antibody. The detetmination vill be effected
using the label on the labelled monoclonal antibody and
normally that which has become bound to the solid.
Normally the thus immobilized label will be assayed
after ~eparation of the liquid phase and washing thc
801 id.
The amount of binding partner for the hapten
(generally a monoclonal antibody) employed will
normally be such that it i~ not saturated on expo~ure
to the hapten; an excess i~ advantageou-ly present with
the resulting benefit that the as~ay ir not unduly
effected by the size of that excess wit~in reasonable
limit~. Thi~ is ea~ily achieveable by u~ing an excess
of b~nding partner over the likely amouDt of ~ample to
be te~ted. In practice this will provid- the skilled
wor~er with little difficulty as the li}ely
concentration of hapten in a ~ample i~ generally known
(or e~t~mateable) within broad limit~ aDd ranging
experiments can be carried out if necer~ry. Similarly
an exce~ of secondary binding partner ~11 also
normally be employed and this can be e~tiaatcd by the
skilled worker without difficulty although ranging
experiments can be carried out if nece~ry.
The ~ource of the hapten for diagnortic tests may
be blood, serum, saliva, urine or other ~ource
', ,' t ' - 11 - 134048~
,.
~uspected of containing the haptens. Oth~r ~ource~ such
a~ food ~ample~, indu~trial ~ample~, laboratory ~amplc~
are also envi~aged. ~he ~ource may be purifled or
concentrated before u~e if de~lred. Tbu~ the hapten
being introduced into the te~t method nay be in it~
original medium or in a sub~equent ~ediun. Such ~ample
handling i~ conventional and doe~ not for- part of the
pre~ent invention.
The antibody which bind~ thereto t~e binding
partner of the hapten which actually har bound thereto
hapten (ie the ~elective antibody) can ~ve varying
degree~ of affinity for the binding par~er of the
hapten which ha~ not bound thereto hapt~n (eg. the
primary antibody). In the ca~e where tb~ binding of the
antibody to the binding partner of the ~pten which has
bound thereto hapten i~ high compared to the bindinq
partner of the hapten which has not bou~ hapten, then
the ~econdary binding partner and the ~elective
antibody uay be added ~i~ultaneou~ly. ~ n the ratio ir
lower, as i~ generally the ca~e, it will be
advantageou~ to add the ~econdary bindi~ partner firrt
and then ~ubsequently add the ~elective antibody.
Since I believe that it i8 easier to produce
antibodie~ with ~ome re~idual binding of the free
binding partner for the hapten I con~e ~ ntly prefer to
- 12 - 1 3 I q ~ 8 8
u~e a method of determination in which tbe ~elective
antibody i~ introduced after the ~econdaq binding
partner has been introduced.
Generally the incubation period u~ed ln this
invention are fro~ about 1 ~inute to 2 hoor~, more
usually from 2 minutes to 100 minutes, for example 10
to 60 minute~.
The method of determining the amount of antibody
bound to the hapten'~ binding partner ~ay be any method
chosen to ~uit the user~' convenience. Ge~erally one of
the antibody and binding partner will be labelled with
a signal generating mean~. Thi~ mean~ ~ay be pre~ent ln
~itu throughout the method or may be added
~ub~equently, for example by a ~Sandwich- technique
~uch a~ u~ng another antibody which i8 it~-lf labelled
to bind to antibody or binding partner. ~o~ever, I
prefer to u~e a label which ha~ been prc~ent on the
antibody or binding partner throughout the method.
The method of determining the amouQt of antibody
bound to the hapten'r binding partner ~ay be indirect
(for example by determining the amount Dot bound and
determining the amount bound by difference) although it
i~ preferred to determine the amount of antibody bound
to the hapten' 6 binding partner directly.
1340488
., ;~
The determination will aptly employ measurement
of a label present on the selective antibody or on the
hapten' 6 binding partner. The label employed may be any
convenient label such as a radiolabel (for example C
or H~) or a luminescent label (for example a
bioluminescent, fluorescent or che~ilumine8cent label)
or enzyme label8 (such as a phosphatase, peroxidase,
~-galactosidase or the like or coenzyme label. The
label ~ay be used to produce an amperometric change ~f
desired. European Patent Application No. 85901495.3,
Publication No. EP 0177531 published on April 16, 1986;
European Patent Application No. 85903019.9, Publication
No. EP 0185722 published on July 2, 1986; and Patent
Application No. PCT/GB87/00461, Publication No. WO
8800240 published on January 14, 1988 may be consulted
for suitable labels and their measurement. The solid
surface I prefer to employ is a microtitre well. The
label I believed most apt is an enzyme label of which I
prefer an alkaline phosphatase. This may be determined
in convenient manner, for example by dephosphorylation of
p-nitrophenylphosphate or by dephosphorylation of NADP or
NADPH which then starts an amplification cycle. The
choice of such assay method is wide and the skilled art
workers may choose such a method for their own
convenience.
The various 8taqes of the method of this
invention will take place under conditions
conventionally used in immunoassays. Thus for example
the temperature will be non-extreme, for example 4-~0~C
- 14 - 1340488
, ~ .
and most suitably ambient, the aqueou~ ~olutions
employed may contain conventional bufferr and
isotonicity adjusting agents as required so that the
solutions are at non extreme pH's and ~ icitles.
Particularly suitable ~elective antibodie~ for
use in this invention can be rai~ed as ~cribed in
Patent Application No. PCT/GB87/00461, Publication No.
WO 8800240 published on January 14, 1988 because of their
easier availability.
Any convenient method of labelling of the
selective antibody may be employed but I prefer to use
the method of Voller or those of Ishikava et al (J.
Immunoa~say 1983, ~, 209-327) especiall~ those
employing the use of hetero bifunctional cros~ linking
agents.
It can be advantageou~ to use as ~econdary
binding partner the material which has ~en used to
raise the antibody (normally the monocl~l antibody)
against the hapten (for example hapten li~ed to BSA
can be used to raise antihapten monoclo~l antibody and
can be the secondary binding partner).
The selective antibodies may be rai~ed by the
nethods of patent applications refered to hereinbefore
and also Patent Application No. PCT/GB87/00461,
Publication No. wo 8800240 published on January 14, 1988.
In some
. ,= . . .
" " , - 15 - 1340 i88
case~, e~pecially where the hapten i~ ~turally pre~ent
in the ani~al or cell, it may be sufficient to immuni~e
with hapten binding partner although I prefer to u~e
hapten and hapten binding partner. Thc ~apten and
binding partner may fir~t be formed into a co~plex if
desired which complex may be covalently bound if
required.
The method of thi~ invention offe~ the advantage
of not needing a ~tep to remove hapten ~und binding
partner from hapten unbound binding par~er. Similarly
thi~ invention doe~ not require removal of the binding
partner bound by hapten prior to the deter~ination of
the amount of ~elective antibody bound to the binding
partner. Si~ilarly thi~ invention offer~ the advantage
of not requiring special dedicated cqui~ent if thir i~
not convenient.
In a preferred a~pect thi~ invention provide~ a
method of determining a hapten whihc ue~od co~pri~e
(a) ab~orbing a monoclnal antibody to th~ hapten onto a
~olid ~urface, ~b) contacting the thu~ obilized
monoclonal antibody with a ~olution of ~e hapten
whereby hapten become~ bound to ~o~e of ~onoclonal
antibody and then with a ~olution of a l~rge derivative
of the hapten whereby the remaining mon~lonal becomes
boundS (c) contacting the thu~ bound i~ obilized
.. .
.
~ - 16 - 1340488
monoclonal antibody with a labelled antibody which
binds to the hapten bound monoclonal antibody but not
the large derivative of the hapten bound ~onoelonal
antibody; ~d) ~eparate the immobilized labelled
antibody from the non-immobilized labelled antibodys
and (e) determine the amount of label whieh hae beeome
immobilized.
The present invention aleo provide~ a ~it for
determination of a hapten which co~prire~ a binding
partner for the hapten, a ~econdary binding partner for
the hapten, and a ~elective antibody.
Moet aptly the binding partner ie an i~mobilized
monoclonal antibody. moet aptly the eecondary binding
i~ a large derivative of the hapten. Moet aptly the
eelective antibody ic a labelled monoelonal antibody.
Favourably the ~it may aleo compriee reagente for
the binding of the ~elective antlbody.
Selective ant$bodie~ can be ehoeen by obtaining
antibodiee by the method~ hereinbefore deecribed and
ccreenlng the cohort of antibodiee produeed for the
desired aetivity.
A method of obtaining eelective antibody ie ba~ed
' 1340488
on a method for determining whether the combination of
the antibody under te~t with the pri~ary binding
partner ha~ taken place. Selective antibody i6 that
antibody which i- found to combine with the pri-ary
binding partner in the presence of hapten but not if
the pri~ary binding partner ha~ bound ~econdary binding
partner. Any convenient method may be e-ployed but I
prefer to u~e one of the following ~ethod~. ~n such
method~ the secondary binding partner e-ployed i~ often
the con~ugate u~ed to rai~e the primary binding
partner.
The antibody under te~t i~ bound to a ~olid
~urface (for example by absorption onto the curface or
onto an antibody already on the ~urface). To thi~ is
added a primary binding partner which ha~ been expo6ed
to either hapten or ~econdary binding partner. The
degree of a~ociation between the labelled antibody and
the ~urface i~ deternined. Tho6e te~t antibodie~ which
give ri6e to a higher level of binding in the pre~enc-
of hapten than in the pre~ence of eecondary binding
partner are cho~en a~ selective antibodie~.
The higher the level of thi~ binding the better
the ~elective antibody i~. Normally a ten fold increa~e
i~ very readily obtainable and a 100 fold increa~e
readily obtainable and even higher ratio~ obtainabl-
~ . ,
" ~ - 18 - 1340~88
.
with relative ease.
Alternatively a primary binding partner i~ bound
to a solid ~urface. ~ither hapten or ~econdary binding
partner is added. The test antibody i~ added and the
amount of test antibody bound to the solid surface i~
then determined. Those te~t antibodies vhich have a
higher level of binding in the presence of hapten than
in the presenceof secondary binding partner are cho~en
as selective antibotie~. The binding ~ar be determined
by employing a further labelled antibodr.
Those selective antibodies which e~hibit a high
level of binding to the primary binding partner in the
absence of hapten are generally employed ~equentially
in the method of this invention. If the binding i~ les~
high a simultaneous system may be u~ed but thi~ i~ not
generally so easily achieveable.
When a ~elective antibody has beeD obtained the
above methods may be adapted to ~creen for suitable
alternative secondary binding partners.
Selection of an antibody for u~e a~ an
anti-idotypic antibody secondary binding partner in the
method of this invention may use one of the many
methods available for the demonstration of
- 19- 1310488
-
anti-idiotypic antibodie~. The ma~ority of these
involve the demonstration of the competitive binding of
the antibody with hapten for a primary receptor such as
the primary bind$ng partner described herein and such
as an antibody. An example of demon~tration that an
antibody is as anti-idotypic antibody for a monoclonal
antibody again~t a hapten i as follows:
i. Surface bound monoclonal antibody is exposed to
the antibody being tested.
ii. Labelled hapten to which the monoclonal antibody
is specific i~ added to the expo~ed antibody as
well as a duplicate preparation of the monoclonal
which ha~ not been exposed to the tcst antibody.
iii. Unbound labelled hapten is washed a~ay.
iv. The amount of label associated with and without
the test antibody is mea~ured.
. When there i~ a significant reduction in the
amount of label as~ociated after the addition of
te~t antibody (such as a 50% reduction or ~uch
greater percentage as may be achieved) then the
antibody is considered a~ anti-idiotypic
antibody.
., , ~ . ? -- 20 -- 13 4 ~ 4 8 ~
ThiC antibody may then be tested for its
usefulne~s as a secondary binding partner. thi6
involves determining whether on binding the pri~ary
binding partner the anti-idiotypic antibody inhibit~
the further binding of the ~elective antibody. Again
there are many ways of carrying out thi~ determination
~uch as:
i. Surface bound monoclonal antibody (primary
binding partner) i~ exposed to the anti-idiotypic
antibody.
$i. ~abelled selective antibody is then added to this
and a duplicate preparation of the primary
binding partner which has not been exposed to thc
anti-idiotypic antibody.
iii. Unbound labelled antibody is washed away.
iv. The amount of label associated witb and without
the anti-idiotypic antibody is then ~easured.
v. Where the anti-idiotypic antibody rcsult~ in a
signicant reduction of labelled as~ociated with
the primary binding partnerthen it i~ considercd
to be u~eful a~ a ~econdary binding partner in
134048~
, . - 21 -
, .
the method of this invention. A 50% reduction in
the label measured ~ay indicate a ureful
~econdary bindinq partner but in general thi~
figure ~hould be a8 great as can be achieved and
reductions of greater than 90% ~bould be aimed
for, and reduction of greater than 98% would be
particularly useful. Other secondary binding
partners may be selected in an analoqou6 manner.
Example 1
A murine monoclonal antibody i~ rai~ed against
oestradiol by conventional procedure e~ploying ar
immunogen beta-oestradiol 6-(0-carboxy--ethyl)
omime:~SA (Sigma Chemical Company Ltd cat number
E5630).
Employing ~tandard procedures monoclonal
antibodie~ are then raised against lotr of 100~ of the
anit-oestradiol monoclonal antibody ~ixed with 200
oestradiol. The hybrido-a clones are screene)d for
production of the required (Sel~b) monoclonal antibody
as follows:
.. . .
13~0~8~
Microtltre plates (Nunc Immunoplate 1 code 4-39454) are
taken and the wells coated eac~ with 100 pl of SO ~M
bicabonate buffer p~ 9.6 containing l~g of antl-mouse
~gG antibody (Slg~a cat no. M 8642) by leaving the~
overnight at room temperatur-. The solut~on~ are
removed and the wells filled with 0.2% cate~n ln the
~ame buffer and left for one hour at room te~perature.
They are then washed four times with SO DM ~rls pH 7.4
containing 0.02% Tween 20 (TT). Quadrupllcate wells
then receive 100 ~1 each of the same hybrldo~a culture
fluid to be tested. Shey are incubatet for 2 hours at
room temperature 10 ~1 of a solution of 2 ug ~ouse IgG
is then mixed into each well and a furt~er 30 nlnute
lncubation carried out. The wells are then ~ashed four
times with 5T.
A con~ugate of the anti-oestradiol monoclonal ant~body
~s then made ~ith al~aline phosphatase e-ploylnq l.Smg
of the 00noclonal antibody and Smg o f al~al~ne
phosphatase accordinq to the method of ~oller A, .~.
~idwell and ann Barlett, Bull. World ~e~lth Orqan., 53,
55 (1976). A dilution of 1:200 is ~ad- of thi~. To
40~1 of thl~ ls added lOOug of beta-oestradiol
6-(0-carboxy-~ethyl)oxime:85A (solution I) and to
another 40~1 the ~ame amount of BSA (Sol~tion II).
~ach quadrupllcate group of wells are t~en and
1 3 4 0 4 8 8
duplicates receive 100~1 of ~olutlon ~. The re~aining
duplicate pair rece~ve 100~1 of solution r~. She wells
are then incubated for a further hour at room
temperature. The ~olutions are then di~card~d ~nd the
wells washed four times with TT. Each ~ell then
receives 100 ~1 of lOmM para-nitrophen~l phosphate in
50 mM bicarbonate buffer pH 10.3 and containing 3.3m~
MgC12. The alkaline phosphatase acti~ity is then
recorded at 405nm.
The hybridoma clones are selected for ~ose which
produce culture fluid providing the following result in
the above screen: duplicated wells con~ining solution
I - low alkallne phosphatase activity; duplicate wells
containing solution r~ - high alkaline phosphatase
activity.
The clones are purified free of conta~inating elones
and used to raise ascites by convention~l technigues.
Tbe monoclonal antibody thu~ obtained 1~ purified by
neans of Protein A fractionation. ~t 1~ then
con~ugated to alkaline pho~phata~e as abovo (giving
'SelAb-Con~'). This con~ugate is used i~ an a~say for
oestradiol a~ follow~:
~icrotitre plate well~ are coated by the addition into
each of 100~1 of 50m~ bicarbonate buffer pH 9.6
- 2~ - 1 3 ~ ~ 4 8 8
., f
contalning lpg of thc ant~-oestradlol ~onoclonal
antibody. They are left overnight at room temperature.
The wells are then glazed with 100 ~1 of 0.2% casein in
tbe same buff-r and left for a further hour at roos
tempe~ature. Shey are then washed four ti~e~ w~th TT.
A serial 1:2 dilutlon of the oestradiol:~SA con~ugate
is then made ln phosphate buffered ~aline startinq with
a concentration of l~g/~l. 100~1 of cach of these are
added to individual wells in dupl~cate and lncubated
for one hour at room temperature and a duplicate pair
of wells recei~e buffer alone. The solutions are
discarded and the wells are washed four ti~es with TT.
100~1 of a 1:400 d~lution of the SelAb-Con~ preparation
is mixed into each well and the wells ~Qcubated at roon
temperature for a further one hour. The solutions are
then discarded and the wells washed four times with TT.
100~1 of lOmM para-nitrophenyl phosphate $n SO~M
bicarbonate p~ 10.3 and containing 3.3n~ ~gCl~ i5 then
added to each ~ell and the al~aline phospbatase
activity followed at 402n~. A graph of oestradiol
concentraton against phosphata~e activit~ is then drawn
which ~hows the power of added oe~tradiol-con~uqate to
lnhibit binding of the prinary and seco~dary
antibodie~. Th- lowest concentration of th- con~ugat-
which gives nore than 90% inhibition i~ identified and
used in the following deter~ination.
- 2S - 134048~
.
,~
The microtitre plates are coated with ~~ti-oe$trad~ol
monoclonal antibody, glazed and washed as ln the
precedinq paragraph. 1:2 serial dilutions of
oestradiol are ~ade from l~g/ml in 50a~ ~ris p~ 7.~.
100~1 of each of these arc added to dupl~cate wells and
the wells incubated for one hour at roo- temperature.
10~1 of a solutlon of the oestrogen-conjugate at ten
fold higher concentration than identified abov- is then
mixed into each well. The wells are incubated for a
further hour at roo~ temperature. The ~olutions are
discarded and the wells washed four ti-es ~ith TT.
100~1 of the SelAb-Con; is then added to each well and
icubated for a further hour at room tea~rature. The
solution is then discarded and the wells Yashed four
times with TT. Each ~ell then receive 100~1 of lOmM
para-nitrophenol phosphate in 50mM bicarbonat~ p~ 10.3
and containing 3.3~M ~gCll and the alkali~e phosphate
activity followed at 402n~. Thus a standard curve of
oestradiol concentration against phcsphatase activity
is obtained.
Uknown samples are deterained by adding the~ to the
assay in place of the oestradiol serial d~lutions in
the above protocol. The degree of phosphatase actl~ty
being finally found in the well~ which had received the
unknowns ls then related to the standard cu~ve to
concentration of oestradlol in these un}nonn ta-ples.
.
! - 26 - 1 3 4 0 4 8 8
Example 2
Example 1 is repeated but the i~munisation regine for
producing the secondary (SelAb) ~onocloo~l antibody i-
via intrasplenic i~munisation with lOO~g of the
anti-oestradiol monoclonal antibody ~i~ed with 200~g of
oestradiol. The first antibody coated to the
microtitre plate is anti-mouse ~g antibody instead of
anti-mouse IqG and also the 2 hour in~tion 2~g of
mouse IgM is added in place of ~gG.
Examplc 3
Example 1 is repeated but the secondar~ antibody is
polyclonal antibody produced by conventional
immunisation of a rabbit. The antibody i5 sinilarly
purified ~ith protein A prior to con~ug~tion ~ith
alkaline phosphatase.
Example ~
Example 1 i~ repeated witb hydrocortisoDe in place of
oestradiol. ~ydrocortisone 3-(0-carbosr-nethyl)
oxime:BSA con~ugate i8 e~ployed to raise tbe
anti-hydrocortisone antibody and also ~b~e~uently in
the inhibition ~tep~.
~ ~ I L -- 27 -- 1 3 ~ O ~ 8 8
Example S
Example 2 is repeated with hydrocortisone.
Example 6
Example 3 is repeated with hydrocortlsone.
Example 7
Example 1 is repeated with progesterone in place of
oestradiol. Progesterone ~-(O-Carboxy ethyl)-Oxime:85A
conjugate is employed to raise tbe anti-progesterone
monoclonal antibody and also subsequently ln the
lnhibition steps.
Example 8
Example 2 ls repeated ~ith progesterone.
~xample 9
A uurlne monoclonal antlbody ls obtained by
conventlonal neans agalnst tbeophylline.
Mlce are then l~munised wlth the lntact aonoclonal
1340488
- 28 -
antibody by multiple intra-peritoneal ~n~ection~, their
spleens are taken and IgG class monoclonal antibodies
against the monoclonal antibody are raised. These are
~creened for anti-idiotypic activity as follow~ :
Nunc nicrotitre plates are coated per ~ell with 100~1
of SOmM bicarbonate buffer pH 9.6 containing l~g
ant~-mouse IgG antibody by overnight incubation at room
temperature. The ~olutions are removed and the wells
glazed with 200~1 per well of 0.2% casein ~n the same
buffer for one hour at room temperature. The solutions
are removed and the wells washed four tines with 50mM
Tris pH 7.4. Quadruplicate wells then each receive l~g
of an antibody to be tested in 100~1 Tris pH 7.4. They
are incubated for two hours at room te~rature. She
wells are then washed four times with SO-M Tris 7.4
containing 0.02% Sween 20 (TT). A conj~gate of the
anti-theophylline monoclonal antibody is then made with
alkaline phosphatase accordlng to the ethod of Voller
A, E. Bidwell and Ann 8arlett, Bull. ~o~ld ~ealth
Organ., 53, SS (1976). A dilution of l:SOO i~ nade of
this. Two 200~1 ~lliquots are taken and SO~l of a
lmg/ml solution of theophylline in SO~ Tr~s pH 7.~
added to one wbile buffer alone is added to the other.
Two of the quadruplicate wells then receivQ 100~1 of
the theophylline containing solution while the other
duplicate pair receive the solut~on without
~ 29 -
1340488
theophylllne. The ~olutions are incubated for a
further hour at room temperature. The ~ells are then
washed four t$mes w~th TT and each well given 100~1 of
lOmM para-nitrophenol phosphate in 50~ b$carbonate
buffer pH 10.3. The$r phosphatase content ~s assessed
by the rate of change of opt$cal dens$t~ at 405n~.
Screened antibod$es in which the addition of
theophylline very significantly reduces the amount of
phophatase conjugate bound (such as a r~ading of 2.0
optical density units without theophylline w$th 0.7
optical density units with theophylline) are considered
anti-idiotypic antibodies of use in the following
syste~.
Another group of ~ice are i~unised intro-splenically
with one immunisation each consist$ng of 100~g of the
anti-theophylline nonoclonal antibody ~u~ed with an
equal quantity of theophylline in 100~1 50~M ~r$s p~
7.4. Over the next four days the ~ice rece$ve three
further $n~ections of 100g of theophylline
intrope~itoneally.
On the fourth day the$r spleens are re-o~ed and
hybridoma~ nade by convention~l procedure ~ith uyelo~a
cell llne NSO. The hybridomas are scree~ed ~or the
required (Selab) antibody as follo~s:
~ 30 _ 13iO~3
Mlcrotitre plate~ are taken and the wells coated each
with 100~1 of 50mM b$carbonate buffer p~ 9.6 containlng
l~g of anti-mouse IgM antibody by leaving the~
overnight at rooa temperature. The solutions are
removed and the wells filled with 0.2% casein ln the
~ame buffer and left for one hour ~t room temperature.
They are then washed our times with TT. Quadruplicate
wells then receive 100~1 each of the sa-e hybrldoma
culture fluid to be tested. They are ~Dcubated for 2
hours at room temperature 100~1 of a ~olution of 2 ~g
~ouse IgM 1~ then mixed into each well and a further 30
minute incubation carried out. The wells are then
washed four times with TT.
Two 200~1 allquots of a 1:500 dilut~on of the
antl-theophylline ~onoclonal antibody - ~lkaline
phosphatase con~u~ate in SO~M Sri~ p~ 7.~ buff-
~containlng 0.1% bovine serum albumin (~ssA) are takcn.
So one is added 50~1 of the buffer containing lOO~g
theophylllne and to the other buffer alone. The
~olutions are ~ixed and incubated for teD mlnutes at
roo~ tc~perature. ~o each 18 then added 50~1 of TBSA
contain~ng lO~g of a selected antl-idlot~pic antibody.
The solution~ are then ~ncubated for ~ further then
mlnutes at roo~ te~peratur-.- 100~1 of each ~s then
added ln dupllcate to the microtitre well~ which have
been ~xposed to the antlbody to be tested. A further
. . . ~ ~ . .
~ - 31 -
134048~
lncubation of half an hour 1~ made at ~oom tempe~ature.
The ~olutions are then dlscarded and tbe wells washed
four tlmes with T~. Each well then recelves 100~1 of
lOm~ para-nitrophenyl phosphate ln 50 ~ bicarbonat-
buffer pR 10.3 and containing 3.3mM ~gCl~. The
alkallne phosphatase activity ls then determined by the
optical density change at 405nm. Where the optieal
density change with those duplicates ~hich received the
add~tlon of theophylllne is ~ignificantly larger than
those which did not (l.e. such as 2.0 OD to 1.0 OD
units) the ant~body tested ls considered to be a
selective antibody and the hybridoma fro- which it
derived purified free of contaminating clones and used
to raise ascites by conventlonal t-chnigues. The
monoclonal antibody thus obtained ls purified by neans
of Prote~n A fract~onation. It is then con~ugated to
alkal~ne phosphatase as above tgiving 'SelAb-Con~').
Thi~ conjugate ls used ln an assay for theophylline a~
follow~:
Microtitre plate well~ are coat~d by t~e addition into
each of 100~1 of 50~M bicarbonate buffer pB 9.6
contalning l~g of the ant~-theophylline onoclonal
antibody. They are left overnight at roo- te~perature.
The ~ell~ are then glazed ~th 100~1 of 0.2% ca~ein ln
tbe rame buffer and left for a further hour at roo~
temperature. They are then ~a~hed four ti-e~ ~ith TT.
.. . , ., ~.~ . ... ~ . .
~ 32 - 134048~
~.
A serial 1s2 dilution of the anti-idiotypic antlbody 18
then made in phosphate buffered sallne ~tarting with a
concentration of l~g/~l. 100~1 of each of these are
added to individual wells ln duplicate and lncubated
for one hour at room temperature and a dupllcate pair
of wells receive buffer alone. The ~olotions are
discarded and the wells are washed four times with TT.
100~1 of a l.~00 dilution of the SelAb-Con~ preparation
is mixed into each well and the wells lncubated at room
temperature for a further one hour. The solutions are
then discarded and the wells washed four times with TT.
100~1 of lOmM para-nitrophenyl phosphate in 50mM
bicarbonate ph 10j3 and containing 3.3~ MgCl~ ls then
added to each well and the alkaline phosphatase
acti~ity followed at 402nm. A graph of theophylline
concentration against phosphatase activity 18 then
drawn which shows the power of added anti-ldiotypic
antibody to inhibit binding between the primary and
~econdary antibod~es. The lowest concentration of the
anti-idiotypic antibody whlch gives ~ore than 90%
lnhibition is id~ntified and used in the following
deternlnation.
Thc ~icrotltre plate~ are coated with aati-theophylline
~onoclonal antlbody, glazcd and ~ashcd ~ ln th-
preceding paragraph. 1:2 s~rial dilutlo~ of
theophylllne are ~ade fro- l~g/ml ln 50-~ Tri~ p~ 7.~.
.. . .
- 33 - 1~404~
100~1 of each of these are added to duplicate wells and
the wells incubated for one hour at roo- temperature.
10~1 of a solution of the anti-id$otypic antibody at
ten fold higher concentration than identified abov- i~
then mixed into each well. The wells are incubated for
a further hour at room temperatur-. T~e solutions are
discarded and the wells washed four tines with TT.
100~1 of the SelAb-Con; is then added to each well and
incubated for a further hour at room te-perature. She
solution is then discarded and the wells washed four
times with ST. Each well then receive 100~1 of lOmM
para-nitrophenol phosphate in SOmM bicarbonate pH 10.3
and containing 3.3mM MgCl2 and the alkaline phosphatase
activity followed at 405nm. Thus a ttandard curve of
theophylline concentration against phosphatas- activity
is obtained.
Unknown samples are determined by adding the~ to the
assay in place of the theophylllne serial dilutions in
the above protocol. ~hc degrce of phosphatase activity
belng finally found ~n the ~ells which had recei~ed the
un~nowns i~ then relat-d to the standard curve to
concentratlon of theophylline ~n these un~nown sa~ples.
, .
", . ...
~ . - 3~ -
134U~8
Example 10
A monoclonal antibody is obtained against theophylline
and a portion labelled ~ith alkalinc phosphatase. A
monoclonal anti-idiotypic antibody ~s raised against
tbe anti theophylline monoclonal antibody. A selective
monoclonal antibody is raised against the anti
theophyll~ne monoclonal antibody theophylline complex.
An assay for theophylline is then conducted by neans of
binding selective monoclonal antibody onto solid
surfaces and exposing these to labelled
anti-theophylline antibody exposed to a ranqe of
theophylline standard preparat~ons or sa-ple to be
determined and the anti idiotypic antibody. The amount
of alkaline phosphatase bound specifically to the
surfaces is than det-r~ined and to conccntration of
theophylline in the sample calculated.
Example 11
The procedure of ~xanple 10 ~as employed ~ith
gentamycin in place of theophylline.
- 35 - 1 3 ~ O 4 8 ~
Example 12
The procedure of Example 10 wa~ repeated except
that the monoclonal antibody again~t theophylline was
fragmented and Fab fragment then labelled wth alkaline
pho~phatase and used in the determination.
Example 13
Preparat$on and use of a ~elective antibody
again~t tetrahydrocannibal.
~ y method~ analogou~ to tho~e de~cribed in
European Patent No. 0264219: (1) a ~onoclonal antibody
is obtained again~t tetrahydrocannibal (~C); (2) it i6
affinity labelled with THC derivative ar de-cribed to
form a delta-8-THC conjugate; ~3) a ~onoclonal antibody
i~ prepared again~t thi~ con~ugate. Although thi~
antibody will bind a complex of the anti-T~C monoclonal
antibody and THC it al~o bind~ the anti-T~C antibody on
its own to a not in~ignificant extent ~o that high
background~ re~ult if it i~ u~ed in the a~ray~
de~cribed in European Patent No. 0264219; however it
may be employed effectively for the deter-ination of
THC by the ~ethod of thi~ pre~ent invention a~ a
- -
r 1 3 ~ O ~ 8 ~
- - 36 -
. . .
~elective antibody. It i~ employed a~ iD Example 1 of
the present specificat$on with the folloving change~:
the hapten to be determined i~ THC; the primary binding
partner i~ the anti-THC monoclonal antibodys the
~elective antibody i~ a~ made a6 de~cribed aboves the
~econdary binding partner i~ a con~ugate of 11
carboxymethyloxime derivative of of delta-9-THC and
bovine ~erum albumin prepared in conventional manner.
