Note: Descriptions are shown in the official language in which they were submitted.
lOS'7~94
This invention is concerned with the analysis
of fluids, such as urine or serum, for the determination
of the presence, amount and/or nature of antibodie~,
antigens and antibody:antigen complexes. tFor ~implicity
hereinafter the symbols "Ab", "Ag" and "Ab:Ag" are used
for "antibody", "antigen", and "antibody-antigen complex",
respectively.)
As iq well known, it is important to be able to
analyse biological fluids for Ab, Ag and Ab:Ag complexesO
For example many diseases are characterised 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 cour~e~
specific to the particular Ag and are predominantly immuno-
globulins of the IgG class synthe~ised by the ~ub~ect's
lymphoid system. The detection of Ab:Ag complexe~ in blood,
and their ~eparation and characterisation, provide infor-
mation of value and can be used, for example, in the
diagnosis of disease.
There nre a number of techniques known for
detecting and quantifying Ag, Ab and Ab:Ag complexe~ and
particularly for determining the nature and amount of Ag
present. These quantification techniques are called
"immunoassay" procedures.
It has been known for some time that the naturally
occurring substance Clq ha~ the property of combining with
Ab:Ag complexes but not with either free Ag or free Ab.
Whilst there has been a prior propo~al (Agnello et al,
J. Exp. Med., 134, 228, 1971) to use this property in one
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particular way for the detection (but not the quantitative
assay or absolute determination) of Ab:Ag complexe~, it has
never previously been realised that Clq is potentially an
extremely useful reagent in the analysis of Ab, Ag and
Ab:Ag complexes.
We have now devised a method of immunoassRy using
Clq.
According to the invention, there is provided a
method of assaying a fluid sample for Ab:Ag complex, which
comprises adding to the sample a solution of Clq, charac-
terised in that there is also added a material which is
caused to agglutinate on contact with Clq, and wherein the
extent of the agglutination or non-agglutination of the
material is detected.
]5 Clq is a natural circulating protein and method
for its separation and purification are known. It is
usually obtained from human, rabbit or bovine serum by a
technique known as "euglobulin precipitation" which is des-
cribed in, for example, J. Immunol., 106, 304-413 (1~71).
The Ab:Ag complex to be assayed may be formed by
adding to a fluid containing an Ab or Ag, the corresponding
Ag or Ab, respectivelyO In this way it is posslble to
determine the amount of an Ab or Ag in a fluid.
In the method of the invention, the sald m~terial
which is caused to agglutinate can vary widely. Thu~, Clq
causes agglutination of red blood cells. Also, it wlll
cause agglutination of materialq which, for example, com-
prise immunoglobulins, e.g. have a coating or outer ~urface
of immunoglobulins, such as polystyrene particles coated
with immunoglobulins. Such coatings can be formed by
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immunological reactions between Ab and membrane antigens,
or by physical adsorption or chemical reaction. Poly-
styrene particles coated with immunoglobulini are commer-
cially available but can, in any event, easily be prepared.
When such coated particles or red blood cells
contact Clq, agglutination begins to occur, but if there
is also present in solution an Ab:Ag complex, thi~ will
react with the Clq relatively quicker and the Clq will
become bound to the Ab:Ag complex in solution and~ a~ a
result, no agglutination of the coated particles (or red
blood cells) will occur. Thus, the presence of Ab:Ag
complexes in serum, for example, can be detected by con-
tacting the ~erum with Clq and with particles coated with
immunoglobulins. If agglutination is observed, the ~erum
does not contain any Ab:Ag complexes.
This is a very simple and accurate te~t, and is
applioable to the detection of all Ab:Ag complexes. An
example of the procedure is as follows:
50 ~1 of serum are added to 50 ~1 of a ~olution
of soluble Clq, and the mixture i~ combined with
50 ~1 oP a ~u~pen~ion oP poly~tyrene particle~
coated with immunoglobulin~. Any re~ulting
agglutination of the particles can readil~ be
observed.
The above test can also be used for detectlng the
presence of a particular Ag or Ab in serum, For example,
when testing for a particular antigen, Ag', there i~ added
to the serum the appropriate specific antibody Ab', and
then the test is made for the presence of an Ab:Ag complex,
3o
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namely Ab':Ag'. If the fferum originally contain~ other
Ab:Ag con~plexes, tllcse must first be removed (for example
using insolubili~ed RF or Clq as described in our co-
C~
pending~application no. 227,481, filed May 21, 1975).
The method of the invention may also be effected
quantitatively. Eor examplc, the sample under te~t 18
mixed with a solution of Clq and with a known amount of
latex particles coated with IgG, whereby Clq becomes bound
to any Ab:Ag complex present and the remainder of the Clq
causes agglutination of the latex particles. The amount of
the said Clq remainder can be assayed by counting the
agglutinated or non-agglutinated latex particles and com-
paring the results with standard results. From the result,
the amount of Ab:Ag complex can be determined.
In~order that the invention may be more fully
understood, the following Examples are given by way of
illustration only~
EXA~LE 1
Determination of IgE
0.1 ml of the sample containing IgE wa~ pip~tted
int,o a 5 ml tube and 1.0 ml of rabbit anti-human IgE anti-
serum (diluted 1:128) was added. The mixture was incubated
for 1 hour at 37C and 1 ml of a solution containing 60000
latex particles of o.8 micron diameter which had previously
been incubated in a solution of physiological saline con-
taining 1 mg of IgG/ml, wa~ added. The mixture,was shaken
and 0.1 ml of a solution containing 600 nanograms of Clq was
added.
'The-mixture was incubated overnight and then
-counted in a particle counter capable of rejecting partlcles
`~ 1057194
groater than 1 micron in diameter. The concentrRtion o~
IgE was inver~ely proportional to the particle count and
could be determined from a pr~viouYly prepared cAlibrAtion
curve obtained with -qolutionq containing known ~oncentration~
of IgE.
E3CAMPLE 2
Automatic analysis for C~1 foetal proteln
~ _ .
A flow of 0.1 ml/minute of the sample to be te~ted
wa~ pumped into an automatic analyser of the con~lnuou~ flow
type, together with the same flow Gf-a ~olution contAlning
- 60000 particles of latex per ml. The latex particle~ were
.o.8 micron in diameter and the solu~ion had previou~ly been
incubated with an antiserum to ~ foetal protei~ diluted
64 with physiological saline~ The mixed streamn ~ere
qegmented with air pumped into the system at 0.32 ml/~inute.
Following the introduction of the air, a flow of 0.4 ml~
minute of A solution of Clq was introduced.
The segmented stream wa~ heated to 37C for 10
-minutes and then further heated to 63C for 2 minutec to
~0 Adestroy the Clq and hence release nll latex par~icle~ bound
by Clq, but not tho~e bound by Ag:Ab coupling.
The stream was then passed through a pArticle
counter which is capable of rejecting particles $reator than
1 micron in diameter. The concentration ofa~1 ~oetnl protein
~was inversely proportional to the particle count and c4uld be
-~determined from a previously prepared calibration cur~e
obt~ined with solutions containing known concentrntionJ of
~oetal protein~
_This-application is a division of copending Canadian
_application Serial No. 227,493, filed May 21, 1975.
