MEASUREMENT OF ANALYTE CONCENTRATION

Abstract: The concentration of an analyte, such as a hormone or other biologically active material, in a fluid, especially a body fluid, is measured by contacting the fluid with a trace amount of a binding agent, such as an antibody, specific for the analyte, determining a figure representative of the proportional occupancy of binding sites on the binding agent and estimating from that figure the analyte concentration. Provided that the amount of binding agent is sufficiently small that its introduction has no significant effect on the total free analyte concentration, the proportional occupancy of binding sites is independent of the volume of the fluid and hence it is not necessary to measure accurately beforehand the volume of the fluid or fluid sample being tested. It is therefore possible to design a concentration-measuring device for insertion into a body fluid of a living creature for in situ measurement of concentration.

Description: Measurement of analyte concentration Technical Field The present invention relates to the measurement of am¬ bient analyte concentrations in fluids, primarily the con¬ centrations of hormones and other biologically active subst- ances in body fluids such as saliva, serum, blood and urine. Background Art It is known to measure the concentration of hormones in body fluids by contacting a fluid sample of accurately determ¬ ined volume with a binding agent having binding sites specific for the hormone, usually an antibody, and radioactively lab¬ elled hormone. The binding agent binds a proportion of the unlabelled hormone and a proportion of the labelled hormone, the relative amount of labelled hormone bound being a function of the amount of unlabelled hormone present in the sample. The results obtained are calibrated by comparison with the results obtained with standard solutions containing known concentra¬ tions of unlabelled hormone, and thus the actual amount of un¬ labelled hormone in the unknown sample is determined. Important disadvantages of the known technique are that a sample of the body fluid in question has to be removed from the body and placed in a test tube or the like and its absol¬ ute volume needs to be known. It would be an advantage to avoid one or both of these difficulties. Disclosure of Invention According to the invention means are provided whereby the ambient concentration of an analyte, such as a hormone, in a fluid can be measured without the need to know the volume of the fluid being measured and hence, in the case of body fluids, without the need to remove the body fluid from the body. This invention is based on the fact, not hitherto appre¬ ciated, that when a fluid containing an analyte such as a hormone is contacted with an antibody or other binding agent having binding sites specific for the analyte, the occupancy of the binding sites by the analyte (proportion of binding sites occupied) is independent of the absolute volume of the fluid and the absolute number of binding sites, and hence independent of the absolute amount of binding agent, provided only that the relative amounts of analyte and bind¬ ing agent and the affinity between them are such that the introduction of the binding agent into the fluid has no sig¬ nificant effect on the concentration of the analyte. Thus, for example, if only a trace amount of binding agent is used, such that only an insignificant fraction of the ana¬ lyte becomes bound to the binding agent, then the overall analyte concentration in the fluid will not change notice¬ ably. Provided that the above condition holds, the concentra¬ tion [ϊ of analyte in the fluid is related to the fraction of binding sites occupied (Ab/Ab ) by the equation where K afe is the equilibrium constant for the binding of the analyte to the binding sites and is a constant for the given analyte and binding agent at a given temperature. A close analogy to this basic idea is provided by the use °f a simple thermometer for the measurement of ambient temperatures. The introduction of a thermometer into - for example - " a room generally implies uptake of heat by the thermometer and hence a (usually insignificant) disturbance to the pre-existing temperature of the room. Provided the thermal capacity of the room and its contents are large as compared with that of the thermometer, the temperature ulti¬ mately recorded by the thermometer essentially reflects the original room temperature. Likewise the binding-site occu¬ pancy of an antibody or other binding agent probe introduced into a biological or other fluid will, assuming the condi¬ tions mentioned above are adhered to, reflect the analyte concentration originally present in the fluid. Accordingly it is possible to design a probe which con¬ tains immobilised binding agent in low concentrations, to insert this into the fluid whose ambient analyte concentra¬ tion is to be measured and, once equilibrium has been reached, to determine the proportion of antibody sites occupied by the analyte. Such a determination will frequently be per¬ formed on the probe after withdrawal from the fluid, al- OMPI though this is not an essential feature of the invention, and it would be advantageous in some cases to make the de¬ termination in situ arri perhaps to couple it with feed-back measures to correct any imbalance of analyte concentration 5 detected, for example to maintain a particular hormone lev¬ el in a body fluid. Therefore, the present invention provides in broad terms in one aspect a method of measuring ambient analyte concentration in a fluid, the method comprising contacting an unmeasured volume of the fluid with a trace amount of binding agent having binding sites specific for the analyte and estimating from the proportional occupancy of the bind¬ ing sites the concentration of analyte in the fluid. In this context the term "trace" denotes an amount which has only an insignificant effect on the total concentration of free analyte in the fluid. The method may be used for the estimation of analytes of all types provided that a specific binding agent is avai¬ lable. However, it is likely to be of greatest value in the estimation of biologically active materials such as drugs, viruses and particularly hormones, where other esti¬ mation methods are more complex. The analytes may be pre¬ sent in any fluid from simple aqueous solutions up to bio¬ logical fluids of all types but estimation of concentration? in body fluids provides a particularly important area. The presence or absence of other ingredients is immaterial pro¬ vided that they do not interfere with the binding of the analyte. The hormones may be present in fluids also con¬ taining endogenously bound hormone but this need not be the case. A wide variety of binding agents may also be used pro¬ vided that they have binding sites which are specific for the analyte in question, as compared with any other ingredient in the fluid in question. When estimating concentrations of hormones or other naturally occurring body chemicals it may be advantageous to use antibodies for the chemical in question where these are readily obtainable. However, other binding agents such as binding proteins or receptor prepara ¬ tions (preparations containing receptor sites and derived - ή - from areas of the body where the chemical in question nor¬ mally becomes bound) may also be used. Conveniently, the binding agent used will be immobil¬ ised on a solid support (although soluble binding agents could be used and later precipitated or otherwise separa¬ ted to enable the binding site occupancy to be estimated). The solid supports used may be those which are conventional for this purpose, incluidng cellulose, polysaccharide such as Sephadex (Registered Trade Mark) and the like. When, according to a preferred embodiment of the invention, the concentration of an analyte in a body fluid is to be esti¬ mated without removing the body fluid from the body, the support may be in any form convenient for insertion into an appropriate part of the body, for example a probe made of polystyrene or other rigid non-harmful plastics material. Preferably the binding agent chosen will be one whose equilibrium constant K . in the above equation is such that the proportion of binding sites occupied by the analyte at its expected concentration in the fluid will be considerably less than 10θ£, more preferably less than 7555. This gives greater sensitivity to variations in concentration. The binding agent chosen will therefore normally be different in its thermodynamic characteristics from the antibody chosen for use in known radioimmunoassay determinations of hormone concentration, where it is desirable to have as high an occupancy of binding sites on the antibody, and hence as high an equilibrium constant, as possible. When the occupancy of binding sites on the binding agent is to be determined by methods involving binding the unoccu- pied sites with a reagent whose presence is discernible, for example a radioactively labelled form of the analyte, the equilibrium constant K-. is preferably such that a sub¬ stantial proportion of the binding sites are occupied, ad¬ vantageously at least 2555, because this gives greater sensi- tivity in the final measurement. Under such circumstances, the equilibrium constant of the binding agent is preferably close to the reciprocal of the expected analyte concentra¬ tion because this will lead to a binding site occupancy close to 5055. The nature of the method of estimating the occupancy of binding sites on the binding protein is not an essential part of the present invention in its broadest form and a variety of methods can be used. The simplest of these is 5 a back-titration of unoccupied sites by the use of a la¬ belled reagent which binds with unoccupied sites but it is also possible to use a sandwich-type or two-site approach. Alternatively, the extent of occupancy can be measured by biochemical or other means in situ. Where back-titration assay methods are being used, it is preferred to use a binding agent whose dissociation con¬ stant for the uncoupling of analyte is low in order to avoid measurement errors as a result of premature dissociation of the analyte from the binding agent. The rate at which equilibrium is reached may also be slow, although if suffi¬ ciently small amounts of binding agent are used relative to analyte the equilibrium should be reached relatively quickly. It is also possible to make measurements before equilibrium is reached and to deduce from them the concen- trations involved, but this adds to the complexity of the operation -and reduces the accuracy and consequently it is not recommended. With the use of small amounts of binding agent for the test methods it becomes of greater importance to have a labelled reagent of high specific activity for the back- titration to determine the proportion of unoccupied bind¬ ing sites because, in general, only small absolute numbers of occupied and unoccupied binding sites will be present. Accordingly, instead of conventional radioisotopic labels it may be desirable to employ labels of other types such as fluorescent labels. An added advantage of the use of fluorescent labels or others of very high specific activity for analyte-labelling is that they make possible the development of very high sen- sitivity, ultiple-analyte, assays relying on the scanning of the distribution of fluorescent labels (comprising la¬ belled antibodies and/or labelled analytes) deposited on the surface of - for example - a suitable plastics material. Such a surface - "printed" with a mixture of different antibodies and subsequently exposed to the biological fluid under test - can potentially be used to reveal the concentrations of many different analytes in the same sample - a requirement which is likely to become increas¬ ingly pressing in the monitoring of blood for the presence of complex mixtures of viral antigens and/or anitbodies, tumour antigens, hormones etc. The concept of the "immunometer" - i.e. the analyte concentration sensing device discussed in the preceding paragraph - is likely to bring about significant changes in -research and of routine clinical diagnosis. For ex¬ ample, steroid and thyroid hormone levels may ultimately be monitored - not by analysis of blood samples as is cur- rent normal practice - but by examination of the antibody- binding site occupancy of a plastic probes following their insertion, for a few minutes, into the subject's mouth, and their exposure to ambient hormone levels present in the saliva. The following Examples illustrate the basis for the invention * EXAMPLE 1 Antibody directed against a hormone occurring in a body fluid is diluted in a 0.05M barbital buffer at pH 8.7 and the resulting fluid is exposed to a probe in the form of a plastics support made of polystyrene for 2 to 16 hours at ambient temperature. The plastics support is then re¬ moved and thoroughly washed and can then be used as a probe to estimate the concentration of the hormone in appropriate fluids by the method of the invention after its affinity (equilibrium constant) and binding capacity have been assessed by a known method. EXAMPLE 2. Antibody directed against hydrocortisone (cortisol), obtained from the Tenovirus Institute for Cancer Research, Welsh National School of Medicine, Cardiff, Wales, was coupled to a solid support and the equilibrium constant (K at) ) and binding capacity of the resulting material were mea ¬ sured, by Scatchard analysis, and found to be 2 x 10 .^JRI " litres/mole and 100 pmoles/ml respectively. Standard cortisol solutions containing cortisol con¬ centrations of 100 pM, InN, lOnM, lOOnM and 1 ,uM were prepared by dissolving pure cortisol (H 001, from Sigma Chemical Co., Poole, England) in a buffer solution of 0.05M KH 2 PO i| and 0.15M NaCl (pH 7.4) (hereinafter re¬ ferred to as PBS) containing 0.1? by weight gelatine (No. 440 , from BDH Chemicals Ltd., Poole, England). Amounts of the antibody preparation having a binding capacity of less than 10 fmoles of cortisol were incubated to equilibrium at 20°C (16 hours, although equilibrium had for practical purposes been achieved within 20 minutes) with samples of each of the standard cortisol solutions having volumes of 0.2, O.lj and 0.8 ml. After incubation the samples were cooled, on ice, to 4°C and the solid ma- terial washed thoroughly with PBS. The extent of occupancy of the antibody binding sites by cortisol was determined in each case by a radioimmuno- assay back-titration using as the labelled material a high 125 specific activity iodinated cortisol (-^1000 Ci/m mole I ) , obtained from RIA Ltd., Cardiff, Wales. Concentrated ^I-cortisol was added and mixed with the solid material in each sample and incubation was continued for 1 hour at 4°C. The solid material was again thoroughly washed and the bound radioactivity determined. The accom- panying drawing is a graph showing the relationship between the observed radioactivity (in counts per minute) and the cortisol concentration (in nM) of the samples of the stan¬ dard solutions. Within the limits of experimental error, the level of bound radioactivity was the same for all three samples of identical cortisol concentration and was un¬ affected by their differences in volume.

Claims: CLAIMS 1. A method of measuring ambient analyte concentra¬ tion in a fluid by contacting the fluid with a binding agent having binding sites specific for the analyte as compared with the other components of the fluid, determin- ing a figure representative of the extent of binding of the analyte to the binding agent and estimating there¬ from the analyte concentration in the fluid, characterised in that the binding agent is used in a trace amount which has at most an insignificant effect on the total concentra- tion of free analyte in the fluid, and a figure represent- ive of the proportional occupany of the binding sites on the binding agent is determined and used to estimate the analyte concentration in the fluid, whereby it is not necessary to measure accurately the volume of the fluid contacted with the binding agent. 2. A method as claimed in claim 1, characterised in that the analyte is a biologically active material and the binding agent is an antibody for the analyte. 3. A method as claimed in claim 2, characterised in that the biologically active material is a hormone, the fluid contains endogenously bound hormone as well as free hormone and the. concentration of free hormone alone is estimated. 4. A method as claimed in claim 1, characterised in that the volume of the fluid contacted with the binding agent is not measured accurately. 5. A method as claimed in claim 1, characterised in that the binding agent is contacted with the fluid whilst immobilised on a solid support, and the binding agent and solid support are separated from the fluid before the figure representative of the proportional occupancy of the binding sites is determined. OMPI 6. A method as claimed in claim 1, characterised in that the fluid is a body fluid and the binding agent is immobilised on a probe capable of being inserted into and withdrawn from the body fluid. 7. A method as claimed in claim 1, characterised in that a binding agent is chosen whose equilibrium const¬ ant for the binding of the analyte to the binding sites is such that less than 75% of the binding sites of the binding agent will be occupied by the analyte at its expected concentration in the fluid. 8. A method as claimed in claim 7, characterised in that a binding agent is chosen whose equilibrium const¬ ant for the binding of the analyte to the binding sites is such that more than 25% of the binding sites of the binding agent will be occupied by the analyte at its expected concentration in the fluid. 9. A method as claimed in claim 5, characterised in that the figure representative of the proportional occupancy of the binding sites on the binding agent is determined by back-titration using a fluorescent labelled reagen . 10. A device for measuring the concentration of an analyte in a body fluid of a living creature without removing a sample of the body fluid from the living crea- ture, characterised by a solid probe designed to be introd¬ uced into and withdrawn from the body fluid and having immobilised thereon a binding agent having binding sites specific for the analyte as compared to the other compon¬ ents of the body fluid, the binding agent being present in a trace amount such that the insertion of the probe into the body fluid has at most an insignificant effect on the total concentration of the free analyte in the body fluid.

Inventors: EKINS ROGER PHILIP, EKINS, ROGER, PHILIP
Applicants: EKINS ROGER PHILIP, EKINS, ROGER, PHILIP
Application year: 1983
Main classification: G01N33
IPC codes: G01N33