Note: Descriptions are shown in the official language in which they were submitted.
161 ~:
T~ inV(~lti.OII iS COnCOrll~ Wi.t;l; ':illlmllnOas~;ays
alld Wit~l .1 rl{~v~l J~CaSCnt fOI' llSC t~l~r~
It i~ wt~ll lcnown to assay ant:i~t~n~ nnc1 ant:i.-
bodics (and other bindins prote:ins) by a com~)ctitive b:ind-
S ing roaction USiJI~ a l.abclle(i rtasor1t, tllo assny ~)c.ingr
effccted by nleasurin~ ti~e amo~mt of lctbcl e:i.tllor bourld in
a complax or remain:ing llnboun-l i.n so1uti.on. An cxam~llo of
SUCtl a procedllre i.s the we].l knowrl rnd:io~ lno~...say
techniqllo in which a rad-ioactivo ].ab(31 is usod. Simi].nr
techniques etro known us:ing otller lul)cls sucll a8 enzym~s
ancl fluoro1-~horos.
In the~o p1occ!clllros, :i.t: i..s nocos.~.lry e:it;llor to
~eparate tllo com~)i.oxcs formr3d fr-oln the relllLI:irldor of tllc
reaction m:i.xture, or to ~e~arnto tho freo unboulld 1LIbO13 od
reactant from the remainder of tlle roaci:ion tll:i.xt:~lre, i.n
order to measure the amollnt of la~ol iin o:i.thc:l part. 1`h:is
separation step is se.1.-10lll ea~y to per:rorm alld :is a source
Or error in such n.ssay.q.
W.-i.th a vic3w to a~o:i(l:i.ng or ~l: ^i.OLI,~t; 1"0 dllC` 'i.~l~s
~(~ this problom, W(3 hnv~ pJ~0l)0~(3~ i.n O~lr CO])Ol'ld.i.n~; ~tllla~litlll patellt nppli.cL~tioll no. 273,631 - tto wh1c~l
reParence silould bo made Por furtllor dot;.~ ) t;llo II.YC i.ll
inllllllrloa~say~ Or m.lgnot:i.cnlly ~Ittracta~lo pnrticlas h.~v:i.ng
on tllei.r ~ori~he:ral ~lrface n ro~!crlt. co~alolltly bol11ld
thor~to. The particles (togatllcr W:i.tll th(. procil1ct of
reactioll botween the rea~ont Ind n l..-llel:Lod com~onent of
the rcaction mi.xture) can bc clenrly an~l(3~sily sepuratccl
fr~lll tll(3 r~mail~der o~ t:lle rcaction nl;.xl~ rt, ancl tl1e l.lbal
:i.n thc saicl ronl.~i.nder or on tllo part:ic1.os sul)scquelltly
3CI rllens~lrod.
;
A limitation of this procedure à~ hitherto
proposed is that the only reagents whicll can be coupled
to the magnetic particles have been substances which
- could be covalently bound to the pol~neric matrix of the
particles. Thus, the particles have consisted of
magnetically attractable material embedded in a matrix
of a polymer such as cellulose, and the reagent is co-
valently bound to the cellulose. It has thus not been
possible to use reagents which could not be so bound to
the magnetic particles. Among the reagents which could
not be 80 used are inert solid absorbent~, such as charcoal
and the like, and theso material~ are useful in immuno-
assays in that they can be used to absorb free unbound
label from the reaction mixture and can then be separated
as solids from the reaction mixture.
We have now found a way of combining the ~se of
magnetically attractable particles (with their inherent
advantages in ~eparation) with the use of absorbents such
as charcoal and the like (with their inherent advantages
in ~elective absorbtion of re~gent~). In particular, we
~have ~ound that these ab~orbent~ can be incorporated, as
discrete solid particles, in a polymeric matrix which al~o
contains magnetically attractable particles, so forming a
magnetically attractable particulate reagent for use in
immunoassays. Hereinafter, the magnetically attractable
particulate materials of the invention, comprisin~ discrete
particulate magnetically attractable material, and discrete
particulate absorbent distributed in a polymeric matrix,
are referred to ~imply as ~the particles of the invention".
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161
In one aspect the invention provides a
particulate reagent for use in an immunoassay of a
liquid samp~e to selectively absorb from the liquid
one or more components therein, wherein each particle
comprises a continuous phase of a water-insoluble
polymeric matrix having incorporated therein a
magnetically attractable material and discrete solid
particle~ of an absorbent material selected from
charcoal, talc, ion-exchange resins, T~`uller's earth,
silicon dioxide, oxides of zirconium or aluminium or
titanium, porous glass, zeolites, natural or synthetic
polymers, polymerised first or Aecond antibodies or
enzymes, cell surface antigens or receptors, sub-
cellular particles, viruses and bacterial cells.
In another aspect, the invention provides a
method of carrying out an immunoassay for a constituent
of interest in a liquid sample, which comprises forming
a reaction mixture of the sample with a substance which
~will react with the said constituent of inte:rest thereln;
~ adding a particulate rea~ent o~ the lrlverltlorl to
selectively ab~orb a component of the reaction mixture;
magnetically separating the said particulate reagent from
the reaction mixture; and determining the constituent of
interest by analysis of the separated particulatc reagent
or the separated reaction mixture.
The invention also includes a method o~ carrying
out the immunoassay of two or more liquid samples for
different constituents of interest in each, usi-ng
continuous-flow techniques, comprising the steps of
forming a reaction mixture of each of` said liquid samples
161
: with a substance which will react with the constituent
of interest thereill, flowing said reaction mixt~es of
each of said liquid samples successively and on a
discrete basis along a conduit, introducing into each
of said reaction mixtures while flowing along said
conduit a particulate reagent of the invention in which
the absorbent is capable of dissolving the bound or free
fraction in each of said reaction mixtures, magnetically
separating said reagent from each of said reaction
mixtures and analysirlg the sepnrated reag~erl-t or reaction
mixture to determine the constituent of interest in each
of said liquid samples~
- In the particles of the invention the
absorbent material is embedded in the polymeric matrix,
in contrast to prior art reagents in which a material is
covalently bonded to a polymeric matrix~
.
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61
Tlle l)articles Or tl~c :inverlt:ion coinpt:isc a con-
t.inllOUs p)laSO of a water-:insolubJ~ polylllcr:ic m.l-tr:i.x
Ilavin~ the masnetically attractal)le part:i.cles ~nd tbc
absorbent particles embadd~d therein. The matrix may be
of a wide variety of matcrials, incllld:in~ bot;l~ natllral and
syntllctic polymels, such as polyacryl.~ ido, cellulose,
cellulose estors, polyamidc, poJ.ycarborlr1to, polyvinyl
resins, methyJ.methacrylates, polystyrenc, polystyreno-
acryloni.trile, epoxy rcsills, nle].allli.no-:forlllaJ.dollydo, phcnol
formaldellyde, ~lrea forlllald-3tly(le polyest(3r, polyacryloni.trile,
polyacroloirl, po].yacet:nl, cro~3s-l:;nko-.l prote:ins, arld t}le
l.ik~.
The matrlx serves to hold to~etller t}-e ma~notic
and absorbent particles :in .- un;.tary palticulate rorm
w.ithout either componont bei.n~ cholll:i.cal.ly bollnd to tllc
matrix. Si.nce some, :i.f not IllOSt, Or th~ absorbent yarticles
will bo wholly wit}lin the matri.x (as opposed to b~i.ns
partially exposed at the nlatr:i.x pcripllcry), tho mntri.x
sllould ~rofor~lbly b~ poro~l~ to ~In ~XtOIIt~ Cr.lcl~Jrl1: to
allow in~ro-3t3 Or tho roclct:l.on nllxt:uro. 'rll~ po:ro~i.t;y o:f
the matrix can wi.th ad~antaso be controlll~(l to allow
in$res~ only of seJ.cct:i.vc ~ol.ut~s (C . ~'. to allolr:i.nsross
o~ smaJ.l urltl~on~ l~ut not larso antibodi.o~), and tlli.3 so-
callod "gol filtrat:ion~' offoct ;.g :in itself woll krlown.
Polyacrylam-Lde is a parti.cuJ.arly su:itablc polym~r.i.c
material for makins matrice~ o.f se].ecte~l pore si~e.
Suital~o ma$n(-t.ical.1y al;tractablo matc~r:inls are
describ~d in our ~aid copendills a}~l~li.cal:ioll no. 273,631
' to wll.icll rercrellco Yhoul.d bc Illadc L'or furt}l(~r d~tnil~.
L;Z161
The nature of the absorbent matèrial can vary
widely, depending on the ultimate u~e of the particle~
of the invention. Charcoal is highly preferred, but
other uaeful materials include talc, ion-exchange resins,
; 5 Fuller's earth, ~ilicon dioxide, oxides of zirconium,
aluminium and titanium, porou~ gla~s, zeolites, finely
divided natural and synthetic pol~llers, polymerised first
or second antibodies or enzymes, cell ~urface antigens
and receptors, ~ub-cellular particles, and viru~es and
bacterial cells.
The function of the absorbant is to selectively
ab~orb, or bind by reaction to, a part of the immunoa~say
reaction mixture 80 that that part of the mixture is
removed with the particles of the invention. For example,
in the a~ay of a particular antigen, a reaction mixture
is formed compri~ing the sample, a quantity of labelled
antigen, and an antibody. There i9 formed in the mixture
complexes between the antibody and tho anti~en~ and between
the antibody and the labellod antL~en. ParticleH of the
invention are added to the mixture and the~e may, for
example~ ~electively ab~orb the free unbound antigen (both
labelled and unlabelled). Separation of the particles by
a magnetic trap leaves only the complexes in ~olution,
and either the solution or the separated particle~ can be
a~sayed for labelled antigen content.
It is a sub~tantial advantage of the present
invention that the particle~ of the invention are non-~pecific
rather than immunoapecific and therefore have a wide range of
applicability. For example, a particle of the invention
3o containing charcoal
161
as the absorbent, will absorb many different small
antigens. Tl~us, these particles of tho invention can be
used in immunoas~ays of many different antigens. Thi~ is
highly advantageous, ~ince it avoids the necessity in
prior art procedures of providing a different reagent on
the magnetic particles for each different immunoassay
undertaken. The applicability range of the particles of the
invention will of course vary with the absorbent involved,
but with materials such as charcoal and other similar
solid ab~orbents, it can be highly significant.
The partlcles Or the invontion are particularly
useful in radioimmunoas~ay~. The particular absorbent
appropriate in any particular immunoassay (or other
analy~is) will either be clear to those skilled in the
art or can be determined by routine trial and experiment.
Some examples of particular uses are as follows: charcoal
in assays of steroids, th~roid hormones T~ and T3, ACTH,
angioten~in II, insulin and glucagon, human growth hormone,
vitamin B12, folate, and other~; t~lo or ~lica in A~Uy~
o~ ACTH, growth hormone, purathroLd ~lo~lone, in~ulin and
calcitonin, and vitamin B12; anion exchange resins in
assays of insulin and growth hormone, digoxin, cyclic AMP,
cyclic GMP, insulin, angiotensin I and thyroxine (T~
polymerised first or second antibodies for absorbing
antigens and first antibodies, respectively, or for pre-
paring sera free from thyroxine and cortisol etc.; cell
surface antigens and receptor~ and sub-cellular particles
for separation purposes; and bacterial cell~ and viruses
for detecting antibacterial and antiviral antibodies in a
sample~
~L;2iGl
Tho particlcs of thc invontion may be usod.in
discrete (manual~ ns~ays or in automated continuous flow ''
systems (such as doscribod, for example, in our copcnding
, C~nadian application no. 273,631 corresponding to U.S. Patent
No~ 4,141,687 issued Feb..27~79). In continuous flow
~ystemq particularly, the R~ecific gravity of the particles
;o~ the invention should be reasonably cloge to that of the
reaction mixture so that no undue floating or ~ettling
occurs and the particleq can be maintained in suspension
in the reaction mixture. Specific gravities of about 1.4
to ~.2 are generally satisfactory. The preqent invention
includes a method of i,mmunoassay as de~cribed in our said
copending application in which there is used particles of
the present invention. Thu~, the present invention
includes a method of analysing a fluid for a constituent
Or interest, which comprise~ the qteps of
(a) fonming a mixture of a sample of the fluid and a
reagent which w,ill react with the constituent of
interest in the fluid;
~b) flowing the ~ixtura along a conduit;
(c) including in the mixture particles of the inven.tion
including as particulato absorbent a material which
will selectively absorb a component of the reaction
~ixture;
(d) magnetically trapping the p~rticles Or the invention
in the conduit to hold them against flow and thereby
separate them from the reaction mixture; and . .
(e) determin.ing the constituent of intere~t by analysis
on the separated particles and/or separated reaction
mixture.
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1~2~L`Çi1 -
In one proforrod ~c1~ met11od, t1~e ~id r~a~ont
i~ a predeterminod amount Or a sub~tance which reacts with
the con~tituent of interest to form therewith a complex,
and wherein the particles of the inv~ntion bind with exce~s
unreacted said rea$ent in the mixture, and either the
~eparated liquid or separated particles are analysed to
determine the con~tituent of intere~t in the ~ample.
The particles of the in~ention may be made by
disper~ing the particulate absorbent and ~articulate
0ngnetically attractable material in a polymer or polymer-
forming mix. In the ca~e of a polyacrylamide matrix, for
oxample~ the two particulate matërial~ may be mixed with
liquid acrylamide monomer(s) and a selected amount of
cro~s-linking agent (e.g. methylene bis-acrylamide).
Polymerisation is initiated (using ammonium persulphate)
and the resulting solid mas~ is dried, ground and sieved
to the de~ired particlc ~ize. This may vary widely, but
for most a~say purposes a particle size in the range l to
20 ~ ia generally ~ati~fActory.
The wel~ht ratio o~ particul~te matorial~ in
the ~atrix ~ay vary widely a~ desired.
In order that the lnvention may be more fully
under~tood, the following Example~ are ~iven by way of
illu~tration only.
In the Drawings:
Figures 1 and 2 compare the dilution curves obtained in
Examples 2 and 3 respectively, with that obtained using the standard
free charcoal procedure.
Figure 3 is a graph which confirms the utility of the
present invention as exemplified in Example 4.
_ 10-- .
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~L~1;Z161
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EXAMPLE 1
Prcparation of ~rticlo~ o~ tho inv~n~ion contalning
charcoal
*
A stock su~pen~ion of charcoal NORIT OL (~DI-I, 5g)
in assay diluent (0.05M phosphate buffer, pl1 7.4, 200 ml)
i .
wa~ prepared. To 152 ml of stock ~uspension wa~ added
ucrylamide (3.ô g), N~N-methylenebisacrylamide (0.2 g),
rerric oxide (Fe304, Fi~on~, 3.8 g) and N,N,N',N'-tetra-
methylethylenediamine (1 ml).
Ammonium per~ulphate (1.2 g) in as~ay diluent
~2 ml) was added with vigorou~ agitation. An exothermic
reaction ensucd, leading to the formation of a homogeneouq
gel ~dry yield 13.2 g).
The gel was cut into small pieces (about 0.25 cm3)
and dried in a vacuum desiccator for 2~ hour~ and the
resultant hard dry granules were crushed in a coffee grinder
~Braun) and dispersed in water (2.0 g in 30 ml). The qus-
pen~ion was then milled in a McCrone microniser mill for
20 minutes snd after washing (assay diluent, x4), the gel
was re~uspended (2 g in 30 ml). 0.11 ml of thi~ suspen~ion
waJ used per assay tube for the separation stepO
,: .
EXAMPLE 2
ntlbody dilution curves (Figure 1)
Doubling dilutions of anti~erum were prepared
and 50 ~1 aliquots added to tubes ln duplicate at dilutions
. .,
ranging from 1 : 400 to 1 : 25,600. A ~olution of 125I-
labelled digoxin was prepared by dilution of ~tock solution
1 : ô i~ a~say diluent. 100 ~1 o~ the 125I-labelled dioeoxin
,
~ Trademark
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~.. ~ )
,~" .. . ~ - : ' ' ' : '
~$~2i61
solution was dispensed into each tubc. 200 ~1 o~ digoxin
free plasma was aliquoted into each tube and the tubes were
' then incubated overnight.
. .
: 400 pl of the particle suspension prepared in
Example 1 were then added to each tube. After a 10 minute
incubation, assay diluent (2 ml) was added and the particle-~
~i were rapidly sedimented by placing the tubes on a multi-
" polar ferrite plate magnet (Magnet Applications Ltd.). The
supernatant was aspirated and the particles washed once with
assay diluent ~2 ml) and then the 125I activity measured in
a gamma counter. The dilution curve obtained is shown in
Figure 1, together with that obtained using the ~tandard,
free charcoal procedure. It is apparent that the new
''~ material of the invention ha~ similar absorbent properties
' 15 to free charcoal.
EXAMPLE 3
Standard curve~
,
Antiaerum was dilute,d wlth n~s~y diluen t
6,400). Digoxin Atand~rd~ (0.5~ 1.0~ 2.0~ 3.0~ 4.0
and 6.o ng~ml pooled serum)were dispensed (200 ~1) into
aH~ay tubes followed by addition of 125I-labelled digoxin
(104 ~1) into each tube. To this was added 50 ~1 of anti-
~erum solution. The tubes were incubated for 30 minute~
at room temperature~ followed by addition of 400 ~l,of
the particulate suspen~ion prepared in Example 1. After
10 minutes, the particulate material was separated in a
magnetic field (as Example 2). The standard curve obtained
is shown in Figure 2, together with that obtained using
the conventional free charcoal separation technique, and
.:
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it is apparent that the new material has similar absorptive
properties to the old.
EXAMPLÆ 4
The utility of the particulate material of the
invention was confirmed by the following experiment.
Sera from 25 patients were assayed in duplicate
by both the conventional method and the new method of the
invention. An acceptable correlation was obtained between
both methods (r = 00959) (Figure 3). Statistically the
~lope of the li,ne was not significantly different from 1oO
nor the intercept ~igni~icantly different from 0Ø
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In a modification of the invention, the absorbent
material of the particles may itself be used as the matrix
for the magnetic particles, and no separate polymeric matrix
ia required. Thio may be effected using, for example, AniOn
exchange re~in~ as the ab~orbont.
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