Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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~ his invention rela-tes to methods and equipment
designed for use in the analysis, assay, or location o~
proteins and other substances of biological interest by
linking ("labelling") them to another molecule or
molecules which can take par-t in a reaction resulting in
-the emission of light. ~he labelled substance, to be
termed "luminescent reagent", may be used in various
ty~es of biological investigations; such as:-
(a) Immunoassay and ~rotein binding assays.
(b) ~urnover of substances in vivo and in
vltro .
(c) ~ooalisation of substances histol~ogically.
(d) Tracing of substances undergoing re-
distribution in biological systems,
or in vitro separation procedures
such as centrifugation, chromatography -
and electrophoresis~
In the context of this Specification the term
"luminescent reagent" is used to describe a complex
betwean a molecule not normally capable of -taking part
in a luminescent reaction and another molecule which is
capable of participat m g in a luminescent reaction~ A
luminescent reaction is one which invol~es a chemical
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reaction that results in the emission of light. The
amount of the "luminescent reagent" is measured b~
recording the light e~itted after producing the appropriate
conditions required for the luminescent reaction to taXe
place. The light may be intense or quite weak~ bu-t
of sufficient duration, to enable the
light to be detected and measured. ~his luminescent is
to be distinguished clearly from fluorescence and
phosphorescence.
A luminescent reaction is normally one between
at least t~o molecules (S and L) with or without other
rea~ents, cofactors, or a catalyst (D) or under the
influence of a physical trigger. L is the substance which
generates light, such as luminol. S is the substance
which reacts with ~ to cause excitation, for example oxygen
or hydrogen peroxide. D (if present) is a cofactor~ and/or
catalyst or trigger such as an enzyme, a luciferase, or
pottassium ferricyanide. The reaction between ~ and S
results ~n the conversion of L to an excited molecule ~*
and the return of this excited molecule to a non-excited
state results in the emission of a photonO The reaction
be-tween L and S and the decay of L* to the non-excited
state may take place spontaneously or may require the
presence o~ the cofactor or catalys-t D~ or a physical
trigger such as temperature or radiation. An example of
such a reaction is the oxidation by H202 of luminolO
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The catalysts and cofactors are often inorganic compounds
as here, but may also be extracted from biological material
such as the enzyme peroxidase which catalyses the
luminesce~t reaction involving luminol~ I
~he formation and rneasurernent of the "lu~inescent .
reagent" can be described by the fo].lowing ~reneral
reactions:
~he synthesis of the "luminescent reagent" may
be written thus:
A ~ B + other reagents and/or catalysts ~ .
AB ~ other products.
Where A = substance not noImally capable of
participating in a lumi~escent reaction,
B = substance capable of taking part in a
luminescent reaction, and
AB = "Luminescent reagent".
A is normally ~but not always) the substance
of interest, requiring anal~sis or de-tection, and may be
of biological or chemical nature. For example, it may
be a protein, antibody, antigen, hapten, hormone, a drug
or other substance of pharmacological interest, a
metabolite, nucleic acid~ or in general any macromolecule
or non-pol~eric molecule. . .
B is normally (but no~ always) the label, and
is essentially stable in terms of luminescence, a~d any
other possibla reactions which might affect the ;
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procedures. B may consist of L and/or S and/or D7 and
in general may comprlse any one or more s-table cornponents
of a multiple-component luminescent rea~ent~ Usually the
..... .
synthetic reac-tion for AB will omit one or more of the
essential components or ingredients, such that the lumin-
escent reaction can be triggered by adding -the missin~
component(s).
In some cases the substance A alone requires to
be analysed, measured or detected. The stable label B will
then normally be added to h at an earlier stage in -the
procedure, and the remaining component(s) of the
luminescent reagent will be added at the moment when the
emit-ted ]ight is to be sensed.
In other cases the substance A is to be reacted
with another substance C, and either A or C may be of
interest, and require analysis or detection~ ~he
reaction to be analysed may be written:
n~B ~ C ~ C(AB)n
- Where C is a substance, not normally
luminescent, which reacts with ~.
n _ number of molecules of AB which
combine with C~
The analysis involves the measurement of C(AB)n
or the loss of nAB from the initial pool of AB molecules~
In the final deterirnination of the "luminescent
reagent" the reaction may be written:
AB ~ omitted component (~, S or D~ or
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physical trigger ~ product + lighto
The amount of light emi-t-ted (lu~inesce~ce) CQn
be related directly to the concentration
of AB added to the reaction mixture.
Currently the most popular substance ~or
labelling proteins is a radioactive isotope such as 125I.
With this method the amount of AB reactio~ with C, as
shown above, is deter~ined by measurement o~ radioactivit~
Radioactive reagents have three major disadvanta~es~
~ir~tly, the method of labelling invol~es the use of
hi~hly radioactive and hence potentially hazardous
reagentsO Secondly, the shelf life of the radioactively
labelled substance is often relatively shoxt not only
because by its very nature the radioactive isotope is
continuously decaying but also radioactively labelled
proteins are of-ten unstable. Thirdly, it is often
difficult to label proteins suf~iciently to provide a
sensitively and rapidly detectable reagent. The measure-
ment of luminescence is both highly sensitive and very
rapid, the time of measurement being of the order of
seconds rather than the several minutes normally required
for measurement of radioactivity. The attachment~ either
covalently or non-covalently, to substances not normally
capable of taking part in a luminescent reac-tion of a
substance which lS capable of taking part in a luminescent
reaction provides a reagent which can be rapidly
~JR~l~MD _ 6-- -
measured in ve~y small quantities.
A substance to be used as a "label" in the
preparation of a "luminescent reagent" shGuld preferably
satisfy four reojuirements:
~a) It should be capable of taking p~rt
in a luminescent reaction.
(b) It should be possible -to attach i-t to
the substance not normally luminescent
to form a re~gent which is relatively
stable.
(c) It should still be able to participate
in a luminescent reaction after being
coupled to form the reagent~
(d) It should not sig~nificantl~ alter -the
properties of the molecule to which it
is attached~
Broadly stated the invention consists of a
method of detecting, analysing, quantifying or locating a
substance, in which the substance, or an associated
substance, is labelled by lin~-.ing one or more components
which can participate in a luminescen-t reaction, and a
luminescent reaction is subseauently ~rig~ered, the emitted
li~ht being sensed, observed, measured and/or recorded, to
provide information concerning the substance of interest~
Preferably, the labelling of the substance produces a
luminescent reagent.
The reaction may be triggered chemically by
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means of a chemical reagent or catalyst, or by a change
in pH, or it may be triggered physically, for exa~ple by
....
heat or radiation. In some preferred ernbodiments the
luminescent reagent comprises at least two chemical
components, with or without a coYactor or catalyst, and
the label comprises an incomplete number of the components
or elements, the lurninescent reaction being triggered by
subsequent addition oi the essential missing component(s)
or element(s).
In any case the labelling component(s) Or the
reaction are preferably stable, and do not si~nifican-tl~
alter or effect the substance to which they are linkeda
Preferably, the substance of interest or the
associated substance is a protein, antibody, antigen,
hapten, hormone, metabolite, nucleic acid or steroid. In
a particularly preferred aspect of the invention, the
luminescent reagent comprises antibodies labelled with
a luminescent material. ~he labelled antibodies are
preferably used to detect, anal~se, quantify or locate
correspo~di~g antigens.
In a further aspect of the invention, there is
provided a luminescent reagent comprising antibodies
labelled with a luminescent label. Preferably, at
least 6~,b, more preferably at least 8~/5 of the anti-
bodies in the luminescent reagent are biologically active,
i.e. they are capable of binding to corresponding antigens.BJR/MMD - 8 -
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The labelled antibodies ma~-be prepared from
a medium containing the antibodies, such as serum, by
contacting the medium with the corresponding antigens,
preferably in a solid phase, to cause antibody-anti~en
association, a :luminescent label being added beIore or
after association occurs, and subsequently dissociating
the labelled antibodies from the anti~ens. ~he anti~ens
only bind the antibodies specific to them, ancl -the
subse~uent dissociation produces luminescent labelled
antibodies ~ith a high degree of biological activity.
The dissociation may take place by adjusting the pH of
the system or by a reduction reaction. I'his preparation
effectively produces "~urified" labelled antibodies due
to the selectivity of the antigen-antibody reaction.
The antigens are preferably linked to a solid phase
immuno-adsorbent, and the luminescent label may elther
be added to the antibody-containing medium before or
after the medium contacts the antigens~ The result is
the same in tha-t the remaining components of the medium
which do not bind with the antigens may be washed away,
leaving a labelled anti~en~antibody com~lex from which
labelled antibodies can be dissociated. This type of
purification is known as immunological purification and,
where the antigen is itself an immunoglobulin~ it car be
used to produce labelled anti-immuno~:lobulins which can
be used as universal reagents in i~munological assays.
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A labelled an-tibody is by nature an
i~munoglobulin. In most instances these can be directed
at antigens such as protein ho~nones, h~ptens or other
molecules. In special cases immuno~lobulins may
themselves be used as antigens, e.g. rabbit IgG (an
immunoglobulin) w~ll behave as an antigen when injected
into sheep. The antibody produced will bind rabbit IgG
~hich w;ll include specific ant-ibodies raised in rab~1-ts.
Thus for example alphafoetoprotein (~P) may be quantified
by the binding of labelled rabbit antibody -to AFP.
Alternatively~ A~P can be quantified by first reacting
with rabbit anti-A~P antibody (unlabelled) and then
detecting the complex by the uptake of labelled sheep
(anti rabbit IgG) antibody. ~his indirect procedure has
the advantage that many immunological assay systems
utilize rabbit antibodies. Instead of labelling these
individually, the labelled sheep~ ~anti rabbit IgG)
antibody can be used as a '~universal" reagent~ Other
preferred universal reagents ~re antibodies -to ~uinea pig
IgG, sheep IgG, goat IgG and donkey IgG, since antisera
to certain molecules may be raised in any of these species.
~lost commonly used universal reagents would however be
sheep- ~anti rabbit IgG) and sheep anti- ~guinea pig Ig~
~he "purified" labelled antibodJ preparation is
extremely stable, especiallv if stored at a pH of from
i
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about 7 to 8 and a-t a temperature below 0C, preferably
about -20C, and has a shelf life of many months. This
is a great advantage over radio-isotopes which have a
limited shelf life due to their conti.nual decay~ A
further advantage is that radio-isotopes can dama~e
protei.ns, whereas the luminescent labels do not.
~abelled antibodies may be obtained for -the lollowing
hoI~nones and proteins: In.sulin, Growth hormone,
Parathyroid hormone, ~ollicle s-timulatin~ ho~mone,
Lu-teinizing hormone, ~hyroid stimulating hoxmone,
Adrenocorticotrophic hor.~one, Gluca~on, Prolactin,
Galcitonin, ~erritin, Alphafetoprotein, Human immuno-
globulin G ~IgG), Rabbit IgG, Sheep IgG, Guinea pig IgG,
Donkey IgG, Human Immunoglobulins ~ and M, Cell surface
antigens. ~abelled antibodies to haptens can also be
produced which bind to drugs or cyclic nucleotides. ~he
- labelled antibodies derived from these hormones~ proteins
and haptens are particularly useful for carrying out
rapid immunological assays. Such assays are con-
veniently carried out by using so-called immuno.radiometric
or "two-site assay".
~ ccording to a further aspect of the invention~
therefore~ there is provided a method of carrying out an
assay in ~Jhich a medium containing the substance of
.25 interest, such as antigens, is contacted with antlbodies
so that the antibodies selectively bind the substance,
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the product of the binding reaction is subsequently
contacted with luminescent labelled antibodies and a ,
luminescent reaction is triggered, -the a~ount of the
substance of in-terest which is present bein~ indicated
by the amount of luminescence.
Preferably the substance of interest is bound
to antibodies on a solid r~)hase which ~ay be cellulose
powder or the wall of a reaction vessel, such a~ a glass
or plastics tube. Other substances in the medium are
not bound by the antibodies and so can be washed away.
This "two-site assay" technique is especiall~
effective usin~ luminol or a derivative thereof as the
luminescent label, coupled to sheep (anti-rabbit IgG)
antibody. The performance of this luminol-labelled
antibody is unaffected following 9 months storage at
-20C.
The variety of luminescent labels which may
be used in the present invention is extremely wide.
In the present invention lumlnescent labels
can be conveniently divided into -two classes:
1. Chemi-Luminescent labels
2. Bio-Luminescent labels.
Chemi-Luminescent labels include inorganic
or organic molecules which can react with other
molecules to produce light. These substances may be
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extracted from biological sources but in this case are
not normally involved in the emission of light in their
natural environmentO The luminescent reaction involving
the chemi-luminescen-t label is triggered by addition o~
the appro~riate reagent or reagents.
~ he trigger may.be an o~idising agent
preferably in an alkaline medium, or a catalyst. A
preferred cheJDiluminescent label is a compound of the
general ~ormula (I)(a) or (I)(b).
0 R3 0 R~ c~
R2~ Rs~
~ J ~ (I)~l)
in ~hich each f R1, R2 and R3 is H, C1-C10 optionally
substituted alkyl or alkenyl, amino, substituted amino~
carbox~l or hydro~yl, each of R4 and R5 is the same as R1'
R? or R~, or is a diazo-linXage, a hemi-dicarboxylate
linkage, an amide linkage,an organic acid halide, or an
isothiocyanate group~ with the proviso that at least one
of R1 to R5 is amino or substituted ami.no. Preferably,
the compound is luminol (R1 to R3 are each hydro~en).
Preferably~ each of R4 and R5 is amino sub~ :
stituted by hemisuccinate, amino ethyl-, or chloroacetyl~ -
An important advantage of the.compounds of
formula (X) is that they can readily form bridges with
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antibodies by, for example7 peptide linka~es. Peptide
bond formation may be carried out by usin~ mixed
anhydride, carbodiimide, suberimidate or hemisuccinabe
coupling. Other preferred.chemiluminescent labels are
(i) lophine and deriva-t~esthereof, i.e. co-ilpounds of
the general formula (II)
h '\
~8 \ / --R (II)
in which each of R6, ~ and R8 is an optionally
substituted phenyl radical
(ii) luci~enin and derivatives thereof, i.e. compounds
of the general formula (III) -
R~---~, (III)
R~
,
in which each of R9,R10,R11 and R1? is the same as R1,
R2, R~, R~ or R5 as defined in formula (I)
(iii) compounds of the ~eneral formula (IV3
R
~13 ~- Rl~ (IV)
25 P~
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- in which each of R13 and R1~ is the same as R1, R2, ~3,
and R5, as defined in formula (I)
(iv) trans-azodicarbo~ylates of the gene~al formula (~)
~- N~CG2, ~l~ (V)
in whlch each of R15 and R16 is H or lower alkyl
(v) Qxalàte-esters of the general forrnula (~I)
C00~217
COOR18
in which each of R17 and R18 is lower al~;yl.
(vi) pyrogallol
Preferred -triggers are h~drogen peroxide, potassium
permanganate, potassium ferricyanide, or oxygen in the
presence of dimethyl sulphoxide. Catalysts from biological
sources, such as enzymes, may be used as trigp;ers. A
preferred catalyst is peroxidase~
Examples of reactions involving l~minOl which result is
the emission of light are:
+ H202 ~ K3~e(CN)6 + OH
7 + N2 + hv(light)
NU~
2. luminol ~ H202 ~ KMnO4 ~ oxidised luminol + hv(light)
, .
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3- luminol ~ H22 ~~ peFXidase -~oxidised luminol ~ hv
, The luminescent reac-tion may also ~e triggered
by a change in p~ or in the physical conditions such as
te~nperature, polarity of the so:Lvent and emissions such
as ~-rays,.~.rays a~c~ particles from radioac-tive isotopes~
Bio-luminescent labels are components which take
part in a bio-luminescent reac-tion, which is defined as
a reaction resulting in the production of ligh-t in which
at least one of the components has been extracted from a
biological source and which in its na-tural environment
is involved in the production of light. Under this
heading are included reactions involving synthetic compound
of identical or ~imilar structures which have been
s~nthesised in order to mimic the naturally occurring
compound.
Preferred bi.o-luminescent labels are
(i) firefly luciferin, and derivatives thereof, of the
general formula (~II)
Xl ~ :
~ ~ ~ G42X~ (~II)
y~o~~f S S Xl
in which each of X1, X2, X3 and X4 has the same meaning
as R1, R2, R3~ R4 and R5 in formula (I),
and,
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(ii) Coelenterate chromophores of the general forrnula (VIII)
o
~ Xs
X6~, N ~ N
~l ~ (VIIL)
X 6 N ~\X 7
in which each of Xs~ X6 and X7 has the same meaning as Rt, Rz,
R3l R4 and R5, in formula (I), or is
I
CH2 CH2
Rl `~, Rl R3 `~, R3 R2`'~ : .
Rl~ Rl R3~ R3 R2 R2
ORl R3 ORl
In general, any stable luciferin or coelenterate derivativeswill be suitable.
A large number of bio-luminescent reactions require oxygen
as part of the reaction and an example is as follows:
oxyluciferin +
Luciferin + other cofactors + 02 ~ o-ther products
(L) (D) (S) -~ light
The reaction is catalysed by an en~y~e known as a luciferase.
Other oxidising agents such as those used for the chemi-luminescent
labels, may be used in the reaction.
The luminescent reactions isolated from some luminous
organisms however do not conform to this scheme. In some
instances of the present invention these reactions are triggered
by addition of the
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appropriate cofactors. An example is the addition of
calcium ions to the photo-proteins isolated fro~ a
.....
number of coelenterates;
~ ot~
in which each of X5, X6 and X7 is as defined iD formula (VIT)
In this invention any one of ths components
involved in a luminescent reaction may be used as a
label to form a "luminescent reagent". Thus a chemi-
lumînescent label may be an organic compound such as
1~ luminol, an inorganic ion or molecule, or a catalyst
such as the enz~me peroxidase. Similarly a bio-
luminescent label ma~ be any luciferin or luciferase,
it may be a structural analogue of a luciferin, or it
~ay be one of the following co-factors: ;-
1. ~Irefl~ A~P
2. ~acteria NADH
~MNH
R.CH0 where R = aliphatic
group
3. Fungi - NADH
NADPH
In performing the invention the amount of
luminescent rea~ent (AB) is noxmally determin2d b~ -
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placi~g a sample in a tube, ornitting one or more
components, or the trigger needed ~or the luminesce~t
....
reaction. The reaction is then triggered, physically,
or chemically by the addition of the remaining component(s~
or catalyst. The light emitted ma-y be loca-ted or
quantified by a standard measuring device such as a
photo multiplier t-ube the signal froM which is fed to
and displayed or recorded on a recoxder, oscilloscope,
or scalar. The light may also in some cases be observed
by the naked eye~ or recorded on a photographic plate,
or by means of an ima~e intensifier especially when -
positional information is required, Eor example the
position o~ rnaximum light intensity for histological
purposes, or the colour, or the in-tensity relative to
a standard.
~ he present invention involves the
application of "luminesce~t reagents" to a variety of
procedures, including in particular the following four
major analytical problems.
1. Immunoassay and protein binding assays.
This analytical procedure is in common use
and normall~ involves the reaction of the molecule
requiring analysis (A) with the binding protein (C),
which in the case Or immunoassay is an antibody~
~he essential feature of this method of
analysis is that it is necessary to separate the A-bound-
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to~antibody from the fxee A or to ~eparate bound antibod~
from free antibod~ The reaction is quankified by
label'ling eikher the antibody~ A, or another molecule
which can react with the free or bound moieties af-ter
separation The label will be either a chemi- or bio-
luminescent label as defined above. An example of this
type of assay is the "-two-site assay" described abovev
2. ~urno~er.
There are many situations where it is
~0 necessary to quantitativel~ follow the turnover of a
molecule in vivo or in vitro. The molecule may be of
biolo~ical origin or it may be a pharmacological compound.
In order to measure -the turnover of such substances a
small, standard quantity of the substance labelled with
a chemi- or bio- iuminescent label (as defined above)
is added to medium containing the unlabelled substance.
The turnover of the substance can then be measured by
measuring the loss of the labelled substance.
3. ~ocalisation of substances histologically.
In order to localise a substance inside or
outside a cell using a "luminescent rea~ent" the
appropriate reagent (e.g. a labelled antibody) is added
to a tissue preparation and after washing is examined
under a microscopeO The "Luminescent reagent" is then
located by eye or by using an image intensifier; the
image can then be photographed.
BJR/MMD _ 23
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4. ~racing of substances undergoin~ re-
distribution.
In a number of biological systems and in vitro
separation procedures, such as centrifugation, chromato-
graphy, and electrophoresis it is necessa~7 to follow asubstance undergoing redistribu-tion. This is done by
adding to the initial preparation a small standard quantity
of the substance labelled with a chemi- or bio-luminescent
label. The distribution of the substance being studied
can then be monitored by ~ollowing the distribution of
the "luminescent reagent".
~ he invention also has considerable value in
carryin~ out homo~eneous assays, particularly for small
molecules. Such assays have the advantages of rapid
analysis and no reguirement for a separation s-tage. ;
In a conventional radioimmunoassay technique ;;
an antigen is reacted with a limited ouantity of binding
reagent (i.e~ antibod~ he proportion of antigen
bound varies inversely with the concentration added. ~he
reaction is monitored by the incorporation of a trace
amount of labelled antigen. An essential feature of the
technique is the ability to separate bound antigen from
unbound antigen.
A homogeneous assay is designed so that a
property of the labelled antigen is altered upon reaction
with antibody. ~hus the reaction is monitored without
BJX/I'~ 21 _
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the necessity of separation of bound and unbound
fractions. Radioisotope labels can not be used in this
way~ In a homogeneous assag the lipht emitted from a
luminescent labelled antigen (or antibody) causes the
emission of light of different wavelength from antibody
(or antigen) labelled with a fluorescent ~nolecule. Thus
the use of an appropria-te filter i.n the detection
apparatus would result in the measurement of li~ht deri~ed
from the fluorescence resulting from antigen-~tibody
interaction. ~he system is homogeneous because it is
unnecessary to remove unreacted anti.gen from the assay
since its emission is of shorter wavelength and not
therefore registered.
Thus, the invention also provides a method of
carrying out a homogeneous assay in which a luminescent
labelled substance is reacted with an an-tibody or antigen
labelled with a fluorescent label and a luminescent
reaction is trig~ered, the energy from the lu~inescent
reaction exciting -the fluorescent label on the antibody
or anti~en to pro2uce a waveleDgth shift in light emission
or a change in qu~ntum yield. -
Preferably -the antibody or an-tigen is .-
labelled with fluoroscein. ~he luminescent reaction is
conveniently triggered by addin~ peroxidase.
. .
BJR~MMD _ 22 ~-
: :
Preferably, the substance of~ interest is a
hapten, and includes the following substances: Cyclic
nucleo't'ides (cyclic AMP, c~clic GMP and cyclic CMP),
25-hydroxycholecalciferol 9 .1 ,25--dihydroxycholecalciferol
~hroxine, Triiodothyronine, Pro~resterone, Oestradiol,
Oestriol, ~estosterone, Aldosterone, Cortisol,
Glycocholic acid, Taurocholic acid, Barbiturates,
Salicylates, Phenitoin, Morphine, Heroin, Methotrexate~
Digoxin.
The preferred chemiluminescent label for the
haptens, especiall~ thyroxine~ is an oxalate ester.
E~ LES '
1. ~xcitation of a luminol-Sheep (anti-rabit I~G~
A luminescent reagent is prepared by coupling ~he
diazonium salt of lu~inol to sheep ~anti-rabbit IgG)
antibody. The'luminescent reaction is triggered by '
excitinE the reagent with ox~gen in the presence of
alkali, light being emitted at a wavelength of 400 - 500
rm. and detected with a photon counting meter.
~he light e~iscion from the reagent following
excitation is extremely rapid, less than 2 seconds -
being required to quantify accurately the luminol
present. ' - -
' -'' ' ' '
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More detailed examples of the invention are
as follows: -
2. ,, Immunoassay - Assay of ~arathyroid hormone
using luminol Iabelled antibodies.
a. General method
A l~ethod for measu~ing the concentration of
parathyroid hormone in hu~an serum is known (~Joodhead et
al; Brit. Med. Bull. ~0, 44-49, 1974), using antibodies
labelled with 125I~
~0 b. Preparation of antibodie~, labelled wi-th lurninol.
The principle chemical reaction described here
is a diazotisa-tion between luminol and antibodies to
human parathyroid hormone. 2 mg of luminol are suspended
in 1 ml of NHC1 at 0C. 10 m~r of NaN02 are added and
the'solution gently mixed for approximately 3 min. The
pH is the~ adjusted to 8.0-9.0 with NaOH~ 2 mls of a
solution of antibodies to human parath-~roid hormone bound
to human parathyroid hormo~e on aminocellulose
~immu~oadsorbant) in 0.2 M sodium borate, pH 8.2 are
then added. After incubatin~ this mixture for approximltely
16 h at 4C the immunoadsorbant is washed four ti~e5with
borate buffer ~he
i~munoadsorbant is then washed three,times with 0.9
(w/v) sodium chloride. The antibodies labelled with
luminol are eluted with buffer at pH 2.
c. Assay of parathyroid hormone (P~H)
This is done by a method similar to a immuno-
BJR/MMD _ 2~ _
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radiometric assay except that the antibodies are
labelled with luminol rather than 125I. P'l'H stanaards
(0.08 - 50 ng/ml) are made up in NIGP ~20.6 g sodium
barbitone, 10 g NaCl, l g sodium azide~ 2 g human serum
albumin, 40 mg non-immune ~uinea pig y-globulin~ made up
to 2 l, pH aajusted to 8.0 wlthl~O N ~ ). lOO ~l of each
standard is put into Bec~aa ~polyethylene tube (~E'~' 23)
in quadruplicate. 50JIl of sanple to be assayed is put;
into similar -tubes and 50 ~ll NIGP buffer added. 10 ul of
"luminescent reagent" (P'rH antibody labelled with luminol)
are added. After mixing, the tubes are incubated for 3
days at 4C. 50 ~l of PTH ImAd (P~H covalently
linked to cellulose) are added. After 20 min -the tubes
are centrifuged for 1 min and 90 ~l of the supernatant
assayed for "luminescent reagent" by assaying for luminol.
d~ Assay of "lumineccent reagent"
90 ul of the supernatant described above is
added to 900 ul of N/lO NaOH, 10 ul of H202 are added and
the tube placed in front of a photomultiplier tube. ~he
total counts recorded in the first 10s after the addition
of 1 ml of 1 r~l K3 ~e(CN)6 are plotted against P~H
concentration. The values in the samples can then be
read off this standard curve.
3. '~urnover - Turnover of albumin in a liver
omo~enate
Bovine serum albumin (BSA) labelled with luminol
is prepared as described in ~xamples 2 for antibodies to
BJR~ ~D - 25 -
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P~H. An homogenate of rat liver (2:1 w/~) is prepared
in 15~ mM KCl~ 2 mM MgC12, 1 ~M mercaptoethanol,
..... .
20 mM tris pH 7.4 'I ml samples are placed in ~uckam
Iæ3 plastic tubes, 10 ul sa~ples of ~SA labelled with
luminol + 10 ul of unlabelled BSA (0.01 - 100 mg/ml)
~qre added and the tubes incubated at 37C for up t~ 60
min. After a defined interval -the albumin is
precipitated by addition oI an equal volume of saturated
(NH4)2$04. ,The ~ubes are then centrifuged, the pellet
washed and redissolved in 0.5 ml N/50 naOH. 100 ul
samples are taken for assay of the 'lluminescent reagent"
as described in Example 2 for luminol labelled antibodies.
1'he decrease in luminescent activity from this pellet is
a measure of the rate of destruction of the albumin. ,
4. Histochemical localisation - Localisation
of anti~;ens on eryt ocytes
Antibodies to human erythrocy~es are labelled
with luminol as described in Example 2 for antibodies
to PTH~ 10 ul of this "luminescent reagent" are added to
1 ml 0.~ (w/v) NaCl containing 10~ human erythrocytes.
After a 30 min incubation at 37C the cells are centrifuged
and washed three times with 0,~,~ (w/v) NaCl. ~he cells
are then smeared on to a microscope slide and observed
under a light microscope with an image intensifier
attachment. 1 mM KMnO4 is added to stimulate the
luminescence of the luminol. Cells with antibodies
attached to them (and hence anti~ens on their cell
~JR~1D _ 26 -
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surface) emit li~ht which is visualised by the image
mtensifier and then photographed. '~he cells con-taining
anti~ens can then be localised by comparing the image
intensified photograph with a photograph taken unde
phase con-trast.
5. Distribu-ti.on of cell membrane anti~ens during
fractionation of xat adipocytes~
Antibodies to rat adipoc~tes are labelled with
luminol as described in ~;.xanple 2 for an-tibodies to PT~.
10 ul salples of -the "luminescent reagent" are added to
1 ml of a solution containing 144 mM Na ~, 5.9 mM K~,
1.2 mM Mg2~, 2.6 T~M Ca 2~, 128.9 mM Cl , 1.2 mM So4? ,
1.2 mM pi, 25 mM HC03 , 45~ BSA 2/v, pH 7.4 and 106-107
rat adipocytes. After a 30 min incubation at 37C the
suspension is centrifuged and the cells washed three times.
The cells are then homogenised in 5 ml 150 mM KC1, 2 mM -;
MgC12, 1mM mercaptoetha~ol, 20 mM tris, pH 7~4. A
convential differential centrifugation procedure is
carried out, the 500 g, 10,000 g, 100,00 g pellets and
100,000 g supernatant being collected. 100 ul samples
of each fraction are then assayed for luminol as
described in Example 2~ ~ comparison of the quantity of
luminol in each fraction compared l~ith that on the whole
cells enables a quantitative estimate of the amount of
cell membrane antigens in each fraction to be obtaine~.
B.~R/MM~ - 27 _
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6. 2-Site Qssay of AlPhafoetoprotein Usin~
~uminol Labelled Antlbodies
An immunoglobulin fraction of an antiserum to
.....
alphafoetoprotein (AFP) is prepared by sodiwn sul-phate
precipitation, ~he precipitate containing antibodies
to AFP is coupled covalen-tly using glutaraldehyde to a
silane derivative of borosilica-te ~lase ln the form of
reaction tubes. Sa~ples containing AFP are incubated ~or
a period of 3 hours in the tubes durin~ which time the
h~P binds to the antibodies. Antibodies to A~P, labelled
with luminol prepared as described for the Pl'H assay are
then added. After an overnigh-t incubation period, the
tubes are emptied and ~:ashed twice in phosphate (0.05 M
pH 7.4) buffered saline containing 0~1% bovine serum
albumin. ~he tubes are transferred to the photon counter
and the llght emitting reaction is triggered as in the
PTH assay. The amount of light emitted is a function of
the amount of luminol present and hence of the concen-tra-
tion o~ AFP in the samples~
7. Homo~eneous Assa~ of C~clic AMP
An antibody to cyclic AMP (or GMP) is labelled
with luminol as de5cribed in Examples 2 and 3. Succinyl
cyclic hMP (or Gr~P) is labelled with fluorescein. The
labelled antibody is reacted with the labelled cyclic AMP
(or G~P) in reaction tube at pH 7.4~ Peroxidase (10 mU)
~5 and hydrogen peroxide (1 mM) are added and the light
e~ission at 540 nm measured. Emission at this wavelength
BJR/MMD - 2~ _
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.: . : , . -
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can only derive from the fluorescein labelled component
which is bound to antibody. Unbound antibody will ernit
at a different wavelength (i.e. 4~D nrn).
~ he li~ht emitted by lu7ninol is absorbed by
fluorescein most efficiently when -the two molecules are
in close pro~imity io eO ~ 100 ~ Thus the wavelength
shift occurs only when labelled cy~lic A~ and antibody
are bound together. The sarne reaction may be carried
out when the cyclic hl`~ labelled with lurninol and the
antibody wi-th fluorescein.
- 29 _
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