CA1116079A - Immunoassay with fluorescent label excited by luminescent reaction - Google Patents

Abstract

ABSTRACT
The invention relates to a method of detecting, analysing, quantifying or locating a protein, antibody, antigen, hapten, hormone,. metabolite, nucleic acid or steroid, in which the substance of interest is linked to a chemi-luminescent or bio-luminescent label.
luminescent reaction is then triggered by the addition of an oxidising agent or a catalyst and -the emitted light is observed in order to provide information about the substance. The invention employs a luminescent reagent which consists of antibodies labelled with a luminescent material such as luminol. The luminescent reagent can be used to quantify antigens in an immunological assay.
The luminescent labelled antibodies selectively bind to the antigens and the amount of light emitted in a luminescent reaction gives an indication of the amount of antigens present. A luminescent labelled substance can also be reacted with an antibody or antigen labelled with a fluorescent material in order to carry out a homogeneous assay.

Description

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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 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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: ., e7~3 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 BJRfMMD - 5 -.~

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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 BJR/MMD _ 7 _ .

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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.
BJR/~D _ 9 _ , 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 BJR/MMD _~0 -` !

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~ ~ ~ s 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, BJR/~D _ 11 _ , .
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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 BJR/~D - 12 -.~ . .
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, ' ~ 0~9 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 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

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 ~ jl/$~ -17-, . , ~ , ' ~ ! . . . i :

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appropriate cofactors. An example is the addition of calcium ions to the photo-proteins isolated fro~ a .....
number of coelenterates;

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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-BJR~I~D - 19 -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.

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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 _ , 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.

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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. ' - -' -'' ' ' ' ~JR~MMD _ 23 _ .

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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 -.i. .

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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~ _ ~ .

.: . : , . -., . , ~ . .

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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Claims (6)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of carrying out a homogeneous assay in which a luminescent labelled substance of biological interest is reacted with an antibody or antigen labelled with a fluorescent label and a luminescent reaction is triggered such that the energy from the luminescent reaction excites the fluorescent label.

2. A method according to Claim 1, in which the substance of interest is a hapten.

3. A method according to Claim 2, in which the hapten is a cyclic nucleotide (cyclic AMP cyclic GMP, cyclic CMP), 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol, Thyroxine, Triiodothyronine, Progesterone, Oestradiol, Oestriol, Testosterone Aldosterone, Cortisol, Glyocholic acid, Taurocholic acid, a Barbiturate, a Salicylate, Phenitoin, Morphine, Heroin, Methotrexate or Digoxin.

4. A method according to Claim 1, in which the antibody is labelled with fluorescein.

5. A method according to Claim 1, in which the luminescent reaction is triggered by peroxidase or hydrogen peroxide.

6. A method according to Claim 1, in which the excitation of the fluorescent label produces a detectable wavelength shift in light emission or a change in quantum yield.