Immunoassay and immunometric assay of free ligand concentrations in biological fluids

A method of measuring the concentration of a free ligand in a biological fluid containing the free ligand and ligand bound to endogenous binding agent, by the steps of PA0 (a) mixing a sample of the fluid with an analogue of the ligand, a specific binder with which the free ligand and the ligand analogue bind, and an exogenous binding agent which binds the ligand analogue but not the ligand, either the ligand analogue or the specific binder being labelled, PA0 (b) incubating the resulting mixture, PA0 (c) determining either the amount of the labelled analogue bound or the amount of labelled specific binder bound, or not bound, to the ligand analogue, and PA0 (d) correlating the determined amount to the amount of free ligand present in the sample. The method is useful to measure concentration of free thyroid hormones and other hormones in body fluids, employing antibodies specific to the ligand analogue as the exogenous binding agents.

TECHNICAL FIELD 
The present invention relates to a method for measuring the concentration 
of free ligand in a biological fluid containing free ligand and ligand 
bound to endogenous binding agent. 
BACKGROUND ART 
Immunoassay techniques have been developed in recent vears to measure 
concentrations of free hormones and other ligands in sera and other 
biological fluids which contain free ligand in equilibrium with ligand 
bound to endogenous binding agents such as binding proteins. They are 
based on the principle that if a specific binder for the ligand, usually 
an antibody, is brought into contact with the sample to be tested the 
extent of occupancy of the binding sites on the specific binder by the 
ligand is a measure of the concentration of free ligand, provided that the 
amount of specific binder is sufficiently low that the equilibrium between 
free and endogenously bound ligand is not significantly affected. By 
measuring the extent of occupancy for the unknown sample and calibrating 
such a measurement using standard samples containing known free ligand 
concentrations it is possible to determine the free ligand concentration 
in the unknown sample. 
Initially, the extent of occupancy of binding sites was measured by 
removing the specific binder containing bound ligand from the sample and 
determing the proportion of unoccupied sites by back-titration using an 
appropriately labelled material (e.g. radioactively labelled material) 
which binds at the unoccupied sites. The process was thus effectively a 
two-step process. 
Subsequently it has been proposed to carry out the `back-titration` without 
removing the specific binder from the sample, thus converting the two-step 
process into a one-step process. This can be done either by using as the 
labelled material a labelled analogue of the ligand or by using as the 
labelled material a specific binding agent. 
Thus, it has been proposed in published European Patent Application No. 
0,026,103 which is equivalent to U.S. Pat. No. 4,366,143 of Midgley et al 
to measure the concentration of free ligand in such a biological fluid by 
a radioimmunoassay technique comprising (a) admixing a sample of the fluid 
with a labelled derivative of the ligand and with a specific binder for 
the ligand, (b) effecting reaction between the free ligand, the labelled 
derivative and the specific binder, (c) if necessary, separating that 
portion of the ligand and labelled derivative that has become bound to the 
specific binder from that portion not so bound, (d) measuring the amount 
of the labelled derivative that is, or is not, bound to the specific 
binder, and (e) using that measurement to determine the concentration of 
free ligand in the biological fluid. According to the process disclosed 
there, the labelled derivative of the ligand is chosen to bind strongly to 
the added specific binder but to bind not at all, or much more weakly than 
does the ligand, to the endogenous binding agent. 
In an alternative procedure a method of determining the free ligand 
concentration involves an immunoradiometric assay comprising admixing a 
sample of the fluid with a labelled specific binder and an unlabelled 
analogue of the ligand, incubating the resulting mixture to permit the 
free ligand and the unlabelled analogue to compete for the labelled 
specific binder, determining the amount of labelled specific binder bound 
either to the ligand or to the unlabelled ligand analogue, and correlating 
the amount of bound labelled specific binder to the amount of free ligand 
present in the sample. 
However when practical assay kits embodying the principles of EPA No. 
0,026,103 have been employed to assay free thyroid hormone in samples 
taken from patients suffering, for example, from certain non-thyroidal 
illnesses or having serum protein abnormalities unrelated to free thyroid 
hormone concentration, the assay results appear to show an anomalous free 
thyroid hormone concentration, contrary to the correct position. It has 
also been found that the concentration of antibody (acting as specific 
binder) in those kits can be up to 100 times greater than would have been 
expected on the simplified theoretical explanation of this technique 
hitherto proposed. 
Further investigation into the operation of those kits has revealed that, 
far from the ligand analogue being totally unbound to endogenous binding 
agents or being bound to only a small extent, it is bound to a very 
substantial extent, at least 90% and probably as much as 99%, not only to 
the albumin present in the sample but also to the other binding proteins 
TBG and TBPA. 
It is therefore an object of the present invention to devise an alternative 
and improved technique for assaying free ligand concentrations which is 
not subject to the disadvantages inherent in the previous technique.

DISCLOSURE OF INVENTION 
It has now been found that, for the immunoassay technique using labelled 
ligand analogue, when any fraction of the ligand analogue becomes bound to 
other binding agents in the sample as well as to the specific binder the 
fraction, b, of the ligand analogue bound to the specific binder is 
represented by the following equation (provided that the concentration of 
specific binder is sufficiently low that the equilibrium between free and 
bound ligand in the sample is not significantly disturbed): 
##EQU1## 
where, K.sub.H is the equilibrium constant for the ligand/specific binder 
reaction, 
K.sub.An is the equilibrium constant for the analogue/specific binder 
reaction, 
[fH] is the free ligand concentration, 
[An] is the analogue concentration, 
[Ab] is the specific binder concentration, 
EQU .SIGMA.K.sub.p [p] is K.sub.p1 [P.sub.1 ]+K.sub.p.sbsb.2 [P.sub.2 ] . . . 
+K.sub.p.sbsb.n [P.sub.n ], 
[P.sub.1 ], [P.sub.2 ] . . . [P.sub.n ] are the concentrations of the 
various (endogenous or added) binding agents other than Ab in the sample, 
and 
K.sub.p.sbsb.1, K.sub.p.sbsb.2 . . . K.sub.p.sbsb.n are the corresponding 
equilibrium constants for the reactions between the ligand analogue and 
the various (endogenous or added) binding agents. 
A similar equation, in which the term 1+.SIGMA.K.sub.p [P]--hereafter 
referred to as S--also appears, can be formulated for the immunometric 
assay technique in which labelled binding agent is used. 
On the basis of this theoretical equation it is possible to design an 
improved assay technique. 
Firstly, in situations where the ligand analogue binds with endogenous 
binding agent and the extent of binding varies from sample to sample of 
the biological fluid because of variations in the concentration of 
endogenous binding agent and/or the equilibrium constant for the 
analogue/endogenous binding agent reaction, it is possible to reduce the 
significance of those variations by adding a further binding agent (X) 
having a concentration [P.sub.x ] and an equilibrium constant 
K.sub.p.sbsb.x for reaction with the analogue such that K.sub.p.sbsb.x 
[P.sub.x ] contributes significantly to the term S and the contribution to 
that term from the products K.sub.p.sbsb.1 [p.sub.1 ] . . . K.sub.p.sbsb.n 
[p.sub.n ] for the endogenous binding agent(s) is proportionately reduced. 
Secondly, even in situations where the ligand analogue does not bind at all 
or to any significant extent with endogenous binding agent present in the 
sample or where the concentrations and equilibrium constants for 
endogenous binding agent do not vary significantly from sample to sample, 
the addition of a further binding agent for the ligand analogue enables 
the term S to be increased. The constraints imposed by the equation then 
allow [Ab] to be increased without sacrificing the accuracy or 
responsiveness of the technique, which means that the assay can be 
completed more quickly or that it is possible to use a specific binder of 
lower specific activity. 
According to the invention therefore there is provided an improved method 
of measuring the concentration of a free ligand in a biological fluid 
containing the free ligand and ligand bound to endogenous binding agent, 
comprising 
(a) mixing a sample of the fluid with an analogue of the ligand, a specific 
binder with which the free ligand and the ligand analogue bind, and an 
exogenous binding agent which binds the ligand analogue but not the 
ligand, either the ligand analogue or the specific binder being labelled, 
(b) incubating the resulting mixture so that the ligand and ligand analogue 
compete for the specific binder, 
(c) determining either the amount of the labelled analogue bound to the 
specific binder or the exogenous binding agent or the amount of labelled 
specific binder bound, or not bound, to the ligand analogue, and 
(d) correlating the determined amount to the amount of free ligand present 
in the sample. 
The exogenous binding agent functions in the method of the present 
invention as a buffering system for the ligand analogue, having the effect 
of reducing or eliminating irrelevant fluctuations in the composition of 
the fluids being tested. Essentially the ligand analogue is the subject of 
competition between two binding agents, namely the specific 
binder--hereafter referred to as A--which is also a binder for the ligand, 
and the exogenous binding agent--hereinafter referred to as X--which is 
not a binder for the ligand. 
Where the contribution to S from the endogenous binding agents is zero or 
is substantially constant for all samples the exogenous binding agent X 
and its concentration are advantageously chosen so that K.sub.p.sbsb.x 
[P.sub.x ] and S are both at least 10, S preferably being 50-500. When the 
contribution to S from the endogenous binding agents is liable to vary 
significantly from sample to sample the exogenous binding agent X and its 
concentration are advantageously chosen so that K.sub.p.sbsb.x [P.sub.x ] 
is comparable with or larger than the expected variation in S and 
preferably constitutes at least half of S, for example two thirds to nine 
tenths of S. 
It will be appreciated however that the ranges of optimum utility for the 
product K.sub.p.sbsb.x [p.sub.x ] as a fraction of S will vary from case 
to case depending on the clinical acceptability of inaccuracies in the 
measurement of free ligand and the extent to which the contribution to S 
from the endogenous binding agents alone is likely to vary. 
A practical upper limit on the amount of exogenous binding agent X may 
often be imposed by the fact that increases in the amount of the exogenous 
binding agent X will in general be accompanied by increases in the amount 
of the specific binder A and that too great an increase in the amount of 
the specific binder A will lead to a significant disturbance of the 
equilibrium between free and bound ligand in the biological fluid. 
Expressed in terms of competing equilibrium reactions, the method of the 
present invention can be depicted as shown in FIG. 1 of the accompanying 
drawings. An equilibrium is set up between free ligand, endogenously bound 
ligand, ligand bound to specific binder A, free ligand analogue, ligand 
analogue bound to specific binder, ligand analogue bound to exogenous 
binding agent X and, in the usual case, endogenously bound ligand 
analogue. Thus the invention differs from the system described in EPA No. 
0,026,103 by the provision of a ligand analogue which can be extensively 
bound to endogenous binding agents and is buffered by the presence of the 
additional exogenous binding agent X so that the effects of fluctuations 
in the equilibrium reactions with endogenous binding agents can be 
proportionately reduced. 
The choice of the exogenous binding agent X is dependent upon the nature of 
the ligand and the ligand analogue because it is essential that it should 
bind with the ligand analogue and not with the ligand. It is also an 
essential requirement for this binding agent, as for the specific binder 
A, that it must not through its inherent nature or its concentration 
disturb the equilibrium between the free ligand and the endogenously bound 
ligand, nor must it in turn be influenced by the endogenous binding agent 
or by drugs or any other ingredients likely to be present in the fluids 
being tested. The exogenous binding agent X may for example be a physical 
encapsulation of the analogue. Preferably, however, it will be a reagent, 
especially an antibody, which is tallored according to the chemical 
differentiation between the ligand and analogue so as to bind the latter 
but not the former. Advantageously, the antibody acting as binding agent X 
does not have a very high affinity for the analogue (provided that its 
affinity for the ligand is lower by at least about 2 orders of magnitude). 
Such antibodies may then be used at fairly high concentrations to provide 
the required value for the product K.sub.p.sbsb.x [p.sub.x ]. Those 
familiar with immunoassay techniques will be able to design an appropriate 
exogenous binding agent X without difficulty. 
The method of the present invention is applicable not only to immunoassay 
techniques (eg. radioimmunoassay) in which the ligand analogue is labelled 
(eg. radioactively) but also to immunometric assay techniques (e.g. 
immunoradiometric assays) in which the specific binder is labelled (e.g. 
radioactively). 
The method may be used to measure concentrations of free hormones in 
biological fluids, especially free thyroid hormones T.sub.3 and T.sub.4 
but also other hormones such as cortisol, progesterone, oestradiol and 
testosterone. The specific binders A used may be those known to be useful 
for this purpose in previous immunoassay techniques or may be formulated 
according to known principles. The ligand analogues described in EPA No. 
0,026,103 and No. 0,073,865 may be used in the method of the present 
invention, as may other ligand analogues and it will be appreciated that 
it is no longer necessary to attempt to design a ligand analogue which 
will not be bound to endogenous binding agents but merely one which can be 
bound to a binding agent X which does not bind the ligand itself. The 
ligand analogues can be labelled in any appropriate manner, for example as 
described in EPA No. 0,026,103 when immunoassay techniques are to be used. 
Alternatively, the analogues may be used in an unlabelled state together 
with a labelled specific binder as described in greater detail in 
International patent application WO 83/03306. The other operational 
conditions appropriate for the method of the present invention may be the 
same as those known or conventional in previous immunoassay techniques. 
The invention and the improvement achievable by its use, are illustrated by 
the following example. 
EXAMPLE 
An analogue of thyroxine (T.sub.4) suitable for the immunoassay of free 
T.sub.4 (fT.sub.4) as described in EPA No. 0,026,103 was prepared by 
chemical modification of the amino acid structure of T.sub.4. An antibody 
(X) against this analogue was produced by well known immunological 
techniques and shown to have a relative affinity for analogue as compared 
to its affinity for T.sub.4 of 10.sup.3. 
The analogue was radiolabelled with .sup.125 I by the well known "exchange" 
method and shown to have much lower affinity constants than T.sub.4 for 
the normal T.sub.4 binding proteins thus satisfying the requirements of 
EPA No. 0,026,103. 
A specific antibody against T.sub.4 (A), with an equal affinity for the 
modified T.sub.4 analogue was coupled to solid particles. 
A mixture was prepared of 0.5 ml of a suspension of the solid-phase 
antibody reagent (2 nm) and 0.5 ml of the .sup.125 I T.sub.4 analogue (2 
nM), both diluted in 4% BSA, PBS pH 7.4 and a 100 ul aliquot of normal 
human serum containing various concentrations of fT.sub.4 (prepared by 
well known techniques). The extent of binding of the .sup.125 I analogue 
to the specific binding reagent was correlated with fT.sub.4 concentration 
as shown in FIG. 2(a). 
A similar mixture was prepared containing identical concentrations of 
specific antibody and analogue but with 100 ul aliquots of samples 
containing 3 nM oleic acid and varying concentrations of fT.sub.4. (Oleic 
acid is one of a class of compounds known as non-esterified-fatty acids 
which are known to be increased in serum samples following the 
administration of some drugs and during non-thyroidal illness.) 
The extent of binding was found to correlate with fT.sub.4 as shown in FIG. 
2(b). Thus a sample containing, e.g. 20 pM fT.sub.4 and 3 mM oleic acid 
would, because of the increase in the extent of analogue binding, be 
interpreted as containing 10.6 pM fT.sub.4, a bias of 47%. 
The K.sub.p.sbsb.n [P.sub.n ] in the incubation conditions of this assay is 
estimated to be 70. 
According to the method of this invention, the additional binding agent 
prepared as previously described, was added to identical mixtures of 
antibody and antigen at a concentration of 0.2% such that .SIGMA.K.sub.x 
[P.sub.x ]=150. Again the extent of binding of the labelled analogue with 
the specific antibody was correlated with the fT.sub.4 concentration 
before and after the addition of 3 mM oleic acid as shown in FIG. 2(c and 
d) respectively. 
In this example of the invention a sample containing 20 pg/ml fT.sub.4 and 
1 mM oleic acid would be interpreted as containing 17 pg/ml fT.sub.4, a 
negative bias of only 15%. Further increases in the addition of the 
binding agent cause additional decreases in the observed bias of the 
method and the required concentration of binding agent will depend on the 
bias permissible in the estimation of fT.sub.4 for clinical reasons.