CA1046936A - Analysis of biological fluids - Google Patents

Abstract

ABSTRACT

Biological fluids, such as serum, are analysed for the presence, nature and/or amount of antibodies, antigens and antibody: antigen complexes therein using as a reagent, insolubilised rheumatoid factor or insolubilined Ciq.
These regents are themselves novel. The bind to antibody:
antigen complexes, but not to free antibodies or antigens, and complexes can thus be removed from mixtures thereof with other materials, such as antibodies and antigens.

Description

- 1~46936 T~is in~ent~on ~5 concerned with the analysl~ of biological fluids, such as urine or seru~, for ~he determin-ation of the presence, amount and/or nature of antibodies, antigens and antibody:antigen complexes, and with novel materials useful therefor. ~For simplicity hereinafter the symbols "Ab", "Ag" and "Ab:Ag" are used for "antibody", "antigen", and "antibody-antigen complex", respectively.) As is well known, it is important to be a~le to analyse biological fluids for Ab, Ag and Ab:Ag complexes.
For example many diseases are characterized by the presence in the circulation of Ab:Ag complexes. The Ag may be any of a wide variety of proteins including those due to the presence of bacteria or viruses or those released from human tissues or cancer cells. The Ab are, of course, specific to the particular Ag and are predominantly immunoglobulins of the IgG class synthesised by the subject's lymphoid system. The detection of Ab:A~ complexes in blood, and their separation and characterisation, provide information of value and can be usedj ~or example, in the diagnosis of disease.
There are a number of techniques known for detectin~
and quantifying Ag, Ab and Ab:Ag complexes and particularly ~-for determining the nature and amount of Ag present. These quantiflcation techniques are called "immunoassay" procedures.
It has been known for some time that two naturally occurring substances, namely rheumatoid factor tRF) and a particular co~ponent of complement, namely CIq, have Phe property of combining with Ab:Ag complexes but not with either ` Eree Ag or free Ab~ Whilst there has been ~ prior proposal ;` tAgnello et al., J; ~xp. Med., I34, 228,1~71~ to use this , , . ~ , . . . . ..

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property In one ~a~ticular w~y ~or the detec~ion (but not the ~-quant~tative a~say or absolute det'ermination~ o~ Ab:Ag com-plexes, it has ne~er previously been realised th~t RP and Clq are potentially extremely useful reagents in the analysis of Ab~ Ag and Ab:Ag complexes.
We have now found that RF and Clq in insolubilised form are in fact ver~ widely applicable reagents in analytica procedures involving Ab, Ag and/or Ab:Ag complexes and their use can, for example, simplify and render more accurate immunoassay techniques.
RF is a known material and methods for its prepar-ation and isolation are known. It ls present, or can be made to appear, in the blood of a number of animal'species includi~g ' man. It is normally obtained from goats or rabbits by intra- ' ; dermal injections of their own purified immunoglobulins previous'ly aggregated by heating at about 63C for about 10 minutes. RF is then isolated from the serum obtained from th animals, by passing the serum through a column of aggregated ' immunoglobullns on which it is retained. The RF can then be eluted from the column using as eluant a solutlon of the appr~priate pH or salt concentration.
Clq is a natural circulating protein and methods for its separation and purification are known. It i9 usually obtàined from human, rabbit or bovine serum by a technique known as "euglobu}in precipitation" which is described in, ;~ for~ example~ J. Immunol., 106, 304-413 (1971~. ' In one aspect, the invention provides, as analyti- "
.
cal reagents, R~ and Clq in insolubilised form. By ~'ln~olubilised" RF and Clq~ we mean that the RF or Clq is

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~9L6~36 - coyalently bonded to a solid ph~se substrate which is insolu~le in a~ueous fluids, or Is adsorbed on a synthetic solid phase su~strate which is insolu~le in a~eous fluids, one exampie of such a synthetic solid phase substrate being latex.
Insolubilisation of RF and Clq may be effected by covalently linking the RF or Cl~ directly or indirectly to a solid phase. The general procedures known and used for covalently coupling proteins to insoluble subst~ates can be used for insolubilising RF and Clq. The solid phase substrate must-contain one or more reactive groups, such as amino or carboxylic groups, for the coupling reaction. Suitable solid phase substrates include naturally occurring materials such as aggregated i~munoglobulins, and synthetic materials, such as aminated agarose.
In some cases, the RF or Clq can be directly covalently linked to or absored on the solid phase, e.g.
where the solid phase is nylon, agarose, cellulcse, acrylamide or acrylic polymers, polystyrene or various glass preparations. It is generally preferred, however, to link ~he RF or Clq to the solid phase using a bridging substance such as glutaraldehyde. A preferred reagent of the invention is RF or Clq covalently linked by a glutaraldehyde bridge to aminated agarose.
Where RF or Clq are insolubilised by adsorbtion on a 3ynthetic solid phase substrate, this ~ay be effected by contacting a solution of RF or Clq with the solid phase, for exa~ple.
It is generally preferred that the reagents of the : .
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invention be in particulate or granular form, such as beads o~ amina~ed agarose havlng t~ereon a coating of ~F o~ Clq.
However, for some purposes, e.g. continuous flow analysis, it is advantageous to form the reagents as a coating over at least part of the inner surface o a tu~e, so that reactants passing throu~h the tube come into contact with the reagent coating.
It is known to isolate RF from serum by passing the serum through a column of aggregated immunoglobulins, on ~hich the RF is retained. In such a column, the ~F is temporarily adsorbed on the aggregated immunoglobulins and is not covalently linked thereto. It will be appreciated that aggregated immunoglobulins are not synthetic materials.
The insolubilised RF and Clq reagents of the invention have a very large number of uses in biological analyses. Basically, all these uses depend on the ability of the reagents to combine with Ab:Ag complexes and not with free Ab and free Ag, thereby making it possible to use the reagents to separate Ab:Ag complexes from mixtures thereof with free Ab and free Ag.
The reagents of the invention can be used to parti-cular advantage in continuous flow analysis of blological ~luid samples, and the lnvention includes such use.
In order t-hat the invention may be more fully understood, various test procedures utilising a reagent of the iuvention will now be described by way o~ illustration only.
Separstion The procèdure for separating Ab:Ag complexea from .. ~
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. : , ~ ~916~316 biological (or oth.erl ~lu-~ds is to contact the ~luid wLth.
insolubili.~ed R~F or Clq. The complexes ~ the fluid will become bound to t~e insolu~ilised RF or Clq, w~ereas other materials present, such.as Ab or Ag, will not ~or will no~ to any significant degree~. Hence, by separating the insolu-bilised RF or Clq from the mixture~ the Ab:Ag are separated out bound on th.e RF or Clq. They can then, if desired, be separated from the insolubilised RF or Clq by ~ashing with a suitable buffer of appropriate salt concentration and pH. In ~ -this way, a relatively concentrated solution of th.e complex(es) can be obtained.
This procedure can conveniently be carried out using a colum~ contai~ing the insolubilised RF or Clq. The s~rum or other sample containing an Ab:Ag complex is passed throug~ the column and the complex is retained therein bound to the ~F or ~ Clq. The complex can then be eluted. In one typical pro-: cedure, a micro-column comprising beads of aminated agarose to which RF or Clq has been coupled with glutaraldehyde, is used.
The serum is passed through. th.e column and the retained Ab:Ag complexes subsequently eluted using, for example, increasing concentrations (lM to 3M) of ammonium thiocyanate.
As an alternative to using a column, insolubilised RF or Clq can simply be mixed, for example in a flask, with - the test liquid.and then subsequently separated by centrifuging ~ or filtering.
: The separative processes of the invention are useful not only for Ab:Ag complexes but also) indirectly, for ~eparating psrticular Ab or Ag ~rom solutions. For example, if it 19 desired to separate an antigen Ag~ from a solution, .:

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4~36 ~he specific an~ibody A~' ~a~ ~e adde.d in e~cess to ~orm the .. ~
co~plex Ab~:Ag~ which ma~ then ~e sepn~ated ~si.ng a reagent ..
of t~e invention. I~ the initial solutlon contains an Ab:Ag complex, t~is must usually first be separated out by a prelimi.nar~ treatment, ~efore t~.e specific antibody Ab~ is added.
. A par~icular feature of th.ese separative procedures of the invention is that t~ey are very efficient and so allow the collection, and concentration, of Ab:Ag complexes from very dilute solutions thereof. This is a very impor~ant advantage since not in~requently the complexes are present in very low concentrations in sera or oth.er biological flui.ds, and it is extremely difficult by prior kno~n procedures~ to- -separate these complexes.
2. Separative detection and quantitation .
ta) This test involves selective remoyal of Ab:Ag com-plexes from a biological fluid ~y contacting the fluid ~ith ; insolubilised RF or Clq ~as described above) followed by , detection of th.e complexes. For example, a sample of serum : 20 is treated to separate out any Ab:Ag com~lexes therein by binding them to insolubilised R~ or Clq. Any complexes so bound are then freed from the RF.or Clq and detected, for . example by using soluble RF or Clq. Thus, ~hen red blood cells (or particles coated with immunoglobulins~ contact ~ soluble RF or Clq, agglutination begins to occur, but if th.ere ~1 is also p~esent in solution an Ab:Ag complex, this will resct ; with the RF or Clq relatively quicker and the RF and Clq ~ill ` become bound to the Ab :Ag complex in ~olution and, as a -result, no agglutination of the~coated particles ~or red blood 6~ :
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39~6~36 cella) ~ill occur. Th.u~, the ~re~ence o~ g complexes in a li~uid, ~o~ example, can ~e detected B~ con~actlng the serum wit~ (solu~le~ ~ and Clq and wit~ particles coated with immunoglobulins. If agglutl.nation is .o8served, t~e serum does not contain an~ Ab:Ag complexes. This.novel technique is more fully described in our copending application no.

tb~ This test can also be useful ~or detecting the presence of a particular Ab or ~g, in a sample. For example, ~hen testing for a particular antigen, Ag', th.ere is first added to the serum the appropriate specific anti~ody Ab', and then the mixture is contacted with a reagent of the invention to separate out any complexes therein. The presence of any such complexes may then be detected as in (a~ above. If the ; serum sample originally contains other Ab:Ag complexes, these can be removed before adding t~e Ab'.
~ ~c) Another detection procedure involves competition betwe2n : two Ab:Ag complexes for a limited amount of RF or Clq. Thus, for example, i an excess of a labelled Ab:Ag complex is added to a llmited amount of insolubilised RF or Clq, then all~the RF
, 20 . or Clq will become bound to tho complex. If, then, in additio~
.~ to the labelled complex, a serum sample is added containing unlabell~ed Ab:Ag complex, the labelled and unlabelled complex .
: will compete on molsr terms for the limited amount o~ RF or Clq.
If, after equilibrium is reached, the RF or Clq i9 removed, toget~er with the complexes bound thereto, the prese~ce (or the presence of a particular minimum amount) of labelled complex in the remaining solution indicates that the serum sample con- .
.
` tai~ed an Ab:Ag complex. This method can be operated quanti-.
tatively to measure the smQunt o~ complex in the serum 3ample.
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This techn~ue ~y al50 ~e used generally as described above, for detecting the presence o~ a pareieular Ag or ~b.
~d~ A ~urther detection procedure (whic~ may be operated quantitatively), especially for a particular Ag or Ab, is as follows. If it is wis~ed to establis~ whether a sample of serum, for example, contains par~icular antigen Ag1, the specific antibody A~' is prepared and labelled with, for example, an enzyme. The labelled Ab' is then mixed with insolu-bili-sed RF or Clq, and the serum sample added thereto. The insolubilised R~ or Clq (with the adhering Ab':Ag' complexes) is then separated cut and the remaining liquid tested for the presence ~or amount) of la~elled Ab' therein. If this is less than the amount originally added, the specific antigen Ag' must have been present in the serum sample. As an exa~ple, one can detect the presence (or absence~ of a specific Ag, for example IgE, in serum as follows. Anti-IgE antibodies, labelled with catalase, are added to insolubilised RF or Clq ; (eg. agarose beads), in an amount in excess of that required to complex with any IgE in the sample to be tested. The serum sample i9 then added and thé mixture incubated. After separ-atlon of the insolubllised RF or Clq ~carrying with it any IgE: snti IgE complexes formed), the residual enzymatic activity o~ the remaining liquid can be measured. If IgE is p~esent in tha serum sample, this residual enzymatic activity ~ill be less than the original activity of the added anti-IgE
antibodies since some of the latter will have complexed with the IgE in the serum and been removèd with the insolubilised ~ . . . . .
- RF. This method can be carried out quantitatively to determine the amount of IgE in the serum.
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6~36 Anothe~ exa~ple o~ th.is. gene~al procedure concerns the detec~i.on of, for e~ample, t~.e antigen morp~ine. I.n this procedure, morph.ine is labelled ~ith an enzyme, eg. am~lase.
Specific anti-morphine antibodies, Ab" are prepared. T~.e - sample of serum or urine, for example, to ~e tested for morphine, is mixed with t~e Ab". Any morphine present will form a complex with the Ab". Th.ere is then added the enzyme- .
labelled morphine. This ~ill only be able to complex with the Ab" in pro~ortion ~o the concentration of morphine in the serum or urine.
Insolubilised RF or Cl~ is added to absorb the Ab:Ag complexes formed between the Ab" and the morphine in the serum, and between the Ab" and the:labelled morphine. The insolubi- ~\
lised RF or Clq ~carrying with it these complexes~ is then removed from the solution. There remains in solution labelled morphine and by measuring its enzymatic activity, it is possible to determine whether or not the original serum sample contained morphine and, ii i.t did, how much morphine was presen.t.
It will be appreciated that whilst the above example refers o.nly to morphine, the same procedure can be used for the detection of other antigens, and mutatis mutandis antibodies.
The labelling need no~ be enzymatic.
(e) The reagents of the invention are also useful in ~ nephelometric immunoassays, in particular to improve the sensi-: tlvity of such assays. In particular, the use of insolubilised RF and Clq has greatIy facilitated the procedure of nephelometric inhibition immunoassays in which the residual activity of Ab is measured after a~sorption with the Ag to be ~ .
determined. In this procedure or measuring al-foetoprotein for example`, the serum is miXed with anti-alfoetoprotein _9_:
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sntiserum. ~h.e A~:A~ comRlexes ~ormed a~e in too low a concentratI.on ~or them to be separated out by~ centri~uging, and insolu~ilised RF or Clq is used to bind wit~ t~ese com-plexes to remove them from solution. The Ab remaining ~ree in solution are then mixed with ~l~foetoprotein and the . particle density of the solution, which. depends on the resid-ual concentration of Ab, is measured ~y nephelometry.

3._Characterisation It will he understood that many of the procedures outlined above.involvi.ng th.e use of insolubilised RF or Clq, are useful preliminaries in the ch.aracterisation of Ab, Ag or Ab:Ag complexes. Some of the procedures do directly result in identificat'ion of a particular Ab or Ag, for example, those procedures where the presence of a particular Ab is suspected and subsequently confirmed by adding the specific Ag and detecting t'ne presence of the Ab:Ag complex. The reagents of the invention are .very useful reagents in the characterisation of Ab, Ag and Ab:Ag complexes, as ~ill be clear from th.e fore-going description.
Identification (i.e. c~aracterisation) of an antigen is generally effected by various proce~ures, eg, spectro-pho'tometry to tetect'the presence of nucleic acids, electron microscopy to identlfy viruses, immunofluorescence with :'' specific antisera directed against virus or tissue antigens. .
The last mentioned procedure can be used with insolubillsed RF
or Clq (eg. agarose beads) hsving Ab:Ag complexes bound thereto, i.e. the complex need not first be removed.
For certain purposes 3 it ma~ be convenient to label~
insolubillsed RF or Clq and thi5 can be effected with, ~or 10, "

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~L~46936 example, I125 or ~luo~eacent or co-enz~e la~lling ~NA~H~.
Insolu~ilised R~ and Clq com~in~ not only w~th A~:Ag complexes but also wit~ aggregated immunoglo~ulins. This fact should, o~ course, be ~orne in mind when carrying out the procedures described above, as- will ~e clear to t~ose skilled in the art. Aggregated immunoglobulins can be la~elled, such as with-radioactive iodine or a fluoroc~rome, and as such can be used in place of labelled A~:Ag complexes in the analytical procedures described a~ove.
In order that the invention may be more fully under-stood, the following Examples are given which illustrate various aspects of the invention.
Example 1 Coupling of RF or Clq to a solid phase 1 ml of a 25% aqueou~ solution of glutaraldehyde was added to 5 ml of aminated agarose (A~-Sepharose~) which had pre~iously been swollen with a saline solution, in 5 ml of carbonate buffer at pH 8.5. The mixture was stirred for 15 minutes at room temperature and ~hen washed with 100 ml of the carbonate buffer. The mixture was centrifuged, the supernatan~ ;
was decanted off, and a further 5 ml of the carbonate buffer was added to the solids. While continuously shaking, 1.25 mg of purified RF (or Clq) and sufficient glycine to make the final ~olution 0.2 molar in glycine, were added O Shaking was con- ~;
tlnued for 10-12 hours, the solid phase was then separated by centrifuging and washed with physiological saline.
Example 2 - .
Preparation of labelled RF or Clq.

200 ml of 50 mM phosphate buffer at pH 7.4 was added .
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to 100 ~g of RF (or Clq~.
The mixture was diYided into 20 ~1 al~quots and 10 ~C
Il25 ~llo~ed ~y 50 ~g of C~loramine-T were added to each aliquot. Oxidation was allowed to proceed ~or 30 seconds and the reaction was t~en terminated by the addition of 50 ~1 of aqueous sodium metabisulphite solution, containing 50 ~g of the sait.
Labelled RF (or Clq) was then separated from the I125 by passing the above mixture do~n a column of Sephadex~ G25 and eluting the desired labelled material with the above-mentioned phosphate buffer. Eluate fractions were assayed for ~-activity and the fractions having peak activity ~ere pooled~ stoppered and frozen until required.
Example 3 Separation of Ab:Ag complex 100 ~1 of insolubilised RF prepared as described in Example 1, were. added to lO0 ~1 of a sample containing a naturally occurring Ab:Ag complex in serum. The mixture was shaken for 1/2 hour at room temperature and was then centri-fuged at 3000 g for 5 minutes. It was found that the Ab:Ag - complex had been removed from the liquid phase and was attached ; to the solid phase.
Example 4 Determination of tetanus anotoxin + IgG
- Rabbit antibodies to tetanus anotoxin were labelled with peroxidase using the technique described by Miles and Hales, Nature, 21g9 186 (1968).
To ~1 of serum suspected of containing tetanus ano-toxin A~:Ag complexes, enough of the labeIled Ab solutlon to -.~ ., : , , ' ' ' ~ . ' , ' :: .

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contain 10 mg of IgG was added. The mi.xture was shaken at 4C overnigh~. 100 ~1 of insolubilised RF prepared as described in Example 1, were added to the resulting ~olution, the mixture was shaken for 1/2 hour at room temperature and was then centrifuged at 3000 g for 5 minutes. The supernatant was removed with a Pasteur pipette and introduced into a separate tube containing 1 ml of an aqueous solution containing 100 mg of phenol, 30 mg o~ 4-amino-phanzone and 300 ~1 of 30~ H2)2 per 100 ml.
The mixed solutions were incubated at 37C for 10 minutes. The colour developed at 520 m~ was then colori-metrlcally estimated. The concentration of tetanus anotoxin was found to be,inversely proportional to the peroxidase activity and could be calculat~d from a calibration cur~e previously established with solutions of labelled antibody.
Example 5 Use of insolubilised RF in automatic analyser ' _of the continuous flow type.

4.0 mg of RF were adsorbed on to 200 mg of 0.8 micron latex particles in a carbonate buffer solution having p~ 8.4.
The particles were then washed with 0.1~ bovine albumin solutiou to block further absorption.
' In an automatic analyser of the continuous flow ,type, a sample to be tested was asplrated at 0.1 ml/minute and ~oined a stream flowing at 0.4 ml/minute rabbit antiserum ,~
against HPL (dilution 1 in 2000) and this stream was ~oined by '~ a stream containing 1125 HPL flowing at 0.4 ml/minute.
', The mixed streams were then pas~5ed through a mixing ' .. ..
` coil maintained at 37~C with a 10 minute time delay and the -' . ~ -13- , .
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~916~36 ml,xture ~as then ~oined by a flo~ of Q.3 ml/minute o~ the RF/late~ material prepared ab,oYe containing 100,000 particles'/-ml.
The mixed stream was again heated for 10 minutes at 37C. On emerging from the heating bath, the latex partlcles were separated from the stream and aspirated to waste using any convenient technique, for example the technique used in automated blood typing apparatuses.
The stream was then passed through a gamma counter and the residual counts are recorded. The counts are inverse-ly prop~r.ion to the conc~n~ation ot EIPL.

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Claims (23)

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

1. A reagent useful in analysis which comprises RF or Clq characterized in that the RF or Clq is covalently bound to a solid phase substrate which is insoluble in aqueous fluids or in that the RF or Clq is adsorbed on unagglutinated particles of a synthetic solid phase substrate, said particles being insoluble in aqueous fluids and being suspended in a liquid in the form of a latex.

2. A reagent useful in analysis which comprises RF or Clq, characterized in that the RF or Clq is covalently bound to a solid phase substrate which is insoluble in aqueous fluids.

3. A reagent according to claim 1, characterized in that the solid phase substrate is a naturally occurring material.

4. A reagent according to claim 2, characterized in that the solid phase substrate is aggregated immunoglobulins.

5. A reagent according to claim 1, characterized in that the solid phase substrate is a synthetic material.

6. A reagent according to claim 4, characterized in that the solid phase substrate is aminated agarose.

7. A reagent according to claim 5, which comprises beads of aminated agarose having a coating of RF or Clq covalently bonded thereto.

8. A reagent useful in analysis which comprises RF or Clq, characterized by comprising unagglutinated particles of a synthetic solid phase substrate, having RF or Clq adsorbed thereon, the particles being insoluble in aqueous fluids, and being suspended in a liquid in the form of a latex.

9. A reagent according to claim 2 or 8 characterized in that the RF or Clq is chemically labelled with an identifying atom or molecule.

10. A reagent according to 1 or 7 characterized in that the RF or Clq is chemically labelled with a radioactive atom or an enzyme or coenzyme.

11. A reagent according to claim 2, 3 or 4, characterized in that it is in particulate form.

12. A reagent according to claim 5, 6 or 7, characterized in that it is in particulate form.

13. A method of separating an antibody: antigen complex from a liquid, characterized by contacting the liquid with a reagent as claimed in claim 2 to bind the said complex to the reagent, and then separating from the liquid the reagent with the complex bound thereto.

14. A method according to claim 13, characterized in that the reagent is confined within a tube and the liquid is passed through the tube.

15. A method according to claim 14, characterized in that the reagent is in the form of a coating on the inner wall of the tube.

16. A method according to claim 14, characterized in that the reagent forms a column within the tube.

17. A method according to claim 13, 14 or 15, characterized in that after separating the reagent from the liquid, the reagent is treated to liberate the complex therefrom.

18. A method of competitive binding assay by which the presence, in the sample, of a particular Ab, Ag or Ab:Ag complex may be detected, and the amount thereof determined, characterized by the steps of:
a) adding to the sample a quantity of the specific Ag or Ab for the Ab or Ag, respectively, to be determined, or a quantity of the Ab:Ag complex to be determined, which said quantity is chemically labelled with an identifying atom or molecule;
b) contacting the mixture with a reagent as claimed in claim 2, 3 or 4 to bind the complex to the reagent;
c) separating from the mixture the reagent with the complex bound thereto; and d) measuring by the label the amount of labelled Ab, Ag or Ab:Ag complex remaining in the mixture or separated bound to the reagent, and thereby determining the presence and amount of Ab, Ag or Ab:Ab complex in the original sample.

19. A method of analysing a fluid sample for the presence, amount and/or nature of an Ab, Ag or Ab:Ag complex therein, characterized by the step pf contacting the sample with a reagent consisting of insolubilized Rf or Clq, the said reagent comprising RF or Clq covalently bonded to a solid phase substrate which is insoluble in aqueous fluids, or adsorbed on a synthetic solid phase substrate which is insoluble in aqueous fluids.

20. A method of continuous flow analysis of fluid samples, characterized by the step of contacting the samples with a reagent as claimed in claim 2, 3 or 8.

21. A method of assaying a liquid sample for the presence therein of antibody: antigen complexes which includes the step of adding to the sample a reagent as defined in claim 2 whereby the RF or Clq reacts with the antibody: antigen complexes present in the mixture.

22. A method of assaying a liquid sample for the presence therein of antibody: antigen complexes which includes the step of adding to the sample a reagent as defined in claim 8 whereby the RF or Clq reacts with the antibody: antigen complexes present in the mixture.

23. A method according to claim 21 or 22 wherein the antibody:
antigen complexes are formed by adding to a liquid containing an antibody or antigen the corresponding antigen or antibody, respectively.