GB2029011A - The use of a synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes - Google Patents

Abstract

The specification describes an immunochemical method for detecting the fertile period of the menstrual cycle, by determining the ratio, in urine of the two hormonal metabolites oestrone-3-glucuronide (E13G) and pregnanediol-3- glucuronide (PD3G). The method depends on determining how an immunocomplex dissociates in the presence of the two metabolites. The immunocomplex comprises a solid phase with antibodies to PD3G attached, and bifunctional ligand (E13G and PD3G both covalently bound to bovine serum albumin) bound to these antibodies, finally E13G antibodies are bound to the E13G moieties of the bifunctional ligand. The immunocomplex is incubated with urine. The metabolites in the urine compete for the antibodies so the immunocomplex partially dissociates. The solid phase is washed and the number of exposed E13G moieties remaining depends on the ratio of E13G to PD3G in the urine. These are determined with enzyme labelled E13G antibodies.

Description

SPECIFICATION The use of a synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two so lutes Introduction The specification describes a simple technique (a kit) for determining reliabiy the fertile period of the human menstrual cycle. Such a kit will be suitable for home use, to enable a woman to determine when she is fertile and so avoid conception by periodic abstinence from intercourse. Conversely the ability to predict ovulation will be valuable in cases where conception is desired.

It is proposed to use a technique in which the ratio of two steroid metabolites, in urine, is measured directly; a signal being generated in direct proportion to the value of this ratio. The two metabolites are: 3-hydroxy-oestra-1 .3.5(10)- triene-1 7-one 3-P-D-glucupyranosiduronic acid (trivial name: oestrone-3-glucuronide or El 3G) and 5}ss-pregnane-3a,20-diol 3t-P-D- glucupyranosiduronic acid (trivial name: pregnanediol-3-glucuronide or PD3G). The signal will be a colour produced by an enzyme-labelled antibody. The novelty of the approach lies in the use of a mixed steroid glucuronide-protein immunocomplex to measure directly a steroid/steroid ratio. This principle can be applied to the measurement of any solute/soiute ratio.

Materials I Mixed steroid glucuronide-protein complex The scheme is based on the preparation of a macro-molecule containing both oestrone glucuronide and pregnanediol-glucuronide covalently linked to a protein, such as bovine serum albumin (BSA): (oestrone-3-glucu ronide),-BSA-( pregnaned ioi-3- glucuronide)y.

The method used will be the mixed anhydride reaction as described1 with the difference that two steroids glucuronides instead of one would be involved in the reaction. The advantage of using a protein, such as bovine serum albumin is that the number of molecules of each steroid glucuronide per molecule of BSA can be independently varied by adjusting the stoichiometry of reagents. It is intended to synthesise a range of mixed steroid glucuronide-BSA complexes with varying ratios of oestrone-glucuronide to pregnanediol g!ucuronide. Such mixed steroid glucuronide-BSA complexes will be capable of simultaneously binding both oestrone-g lucuronide antibodies and pregnanediol-glucuronide antibodies. The complex with dn optimum ratio will finally be selected after testing in the reaction scheme set out below.

Antisera Immunoglobulin enriched fractions of both oestrone-3-glucuronide and pregnanediol3- glucuronide antisera will be used.

Oestrone glucuronide antiserum labelled with the enzyme "horse radish peroxidase" prepared by a modification of the method using glutaraldehyde as the cross linking reagent2. An alternative enzyme label, alkaline phosphatase will also be investigated.

Solid Phase Polystyrene test tubes (50 x 6 mm) obtained clean and sterile from Sterilin Ltd will be used as a solid phase. The antibody coated tubes are robust and can be stored empty, in a sealed container, at -200C for many months, without loss of antibody activity. Alternative solid phase supports will be investigated such as filter paper strips, to which antibody is covalently bound.

Procedure The -method proposed is in two parts, (1) preparation of a solid phase immunocomplex (Fig.

1); (2) the testing of a sample for a El 3G/PD3G ratio (Fig. 2).

(1) Preparation of the solid phase immunocomplex 1. Coating of tubes with pregnanediolglucuronide antiserum.

2. Buffer wash.

3. Incubation of antibody coated tubes with the mixed steroid glucuronide-BSA complex (i.e.

El 3G-BSA-PD3G).

4. Buffer wash.

5. Incubation of coated tubes with oestrone glucuronide antiserum.

6. Buffer wash.

7. Store empty, but sealed at -200C until required.

(2) Use of above preparation to test for the El 3G/PD3G 1. Add (diluted) urine to immunocomplex coated tubes. Incubate.

2. Buffer wash.

3. Add excess enzyme labelled oestrnne- glucuronide antisera.

4. Buffer wash.

5. Measure amount of label, by addition of substrates followed by colorimetry.

N.B. Excess reagent in step 3 will enable this to be a very short step. Also any non-specific interfering factors from the urine sample will be washed away therefore enzyme blanks should be minimal.

Rationale The reaction end point will be determined by the amount of residual labelled oestrone glucuronide antibody bound to the solid phase, and this will be proportional to the number of exposed oestrone glucuronide residues on the solid phase after incubation with diluted urine. This in turn is References 1 Samarajeewa, P. a Kellie, A. E. (1975) Biochem. J., 151,369-376.

2 van Weemen, B. a Schuurs, A. (1974) FEBS Letters, 43, 21 5.

dependent on two factors. (1) the number of exposed El 3G residues will be proportional to the concentration of oestrogen glucuronide in the urine sample, since free oestrone glucuronide will competitively displace non labelled oestrone glucuronide antibodies from the immunocomplex.

(2) the amount of immunocomplex bound to the solid phase will be inversely proportional to the concentration of pregnanediol glucuronide in the urine sample, as free pregnanediol glucuronide will competitively displace BSA linked pregnanediol glucuronide from the solid phase pregnanediol glucuronide antibodies.

It follows that, for samples containing high concentrations of oestrone glucuronide and low concentrations of pregnanediol glucuronide (periovulatory phase), maximum binding of labelled antibody will be achieved i.e. high El 3G/PD3G ratio will give a high signal.

Three other extreme conditions are possible: (1) low oestrone glucuronide + low pregnanediol glucuronide (early follicular phase); (2) low oestrone glucuronide + high pregnanediol glucuronide; (3) high oestrone glucuronide + high pregnanediol glucuronide (mid luteal phase).

Under condition (1), unlabelled oestrone glucuronide antibodies will not be displaced from the immunocomplex, no labelled oestrone glucuronide antibodies can bind to the solid phase and a low signal will result Under conditions (2) and (3) the immunocomplex will be.cleaved from the solid phase altogether, no oestrogen residues will remain on the solid phase and no signal will be possible.

The above scheme has a number of advantages.

1. Colour development occurs when the test is positive.

2. Interfering factors from the urine sample are eliminated by washing after absorption onto solid phase.

3. The test is independent from urine volume.

4. The use of a solid phase technique enables the use of a simple "dipstick test." Optimisation and evaluation of the kit The kit will be optimised in terms of 'tuning' the colour signal response to correspond with the range of oestrone-3-glucuronide/pregnanediol-3glucuronide ratios that occur in a normal menstrual cycle. The following variables will be investigated in order to achieve this end: a) Variation of the number of residues of oestrone-3-glucuronide and/or pregnanediol-3glucuronide per molecule of mixed steroid-protein complex.

b) Variation in the dilution of pregnanediol-3- glucuronide antiserum used to coat the solid phase.

c) Variation in the characteristics of the unlabelled antisera, e.g. high versus low affinity antiserum.

d) Variation in the characteristics of the enzyme-labelled antiserum.

e) Variation in the nature of the enzyme label and/or substrates.

It is intended to examine urines from a large number of menstrual cycles using the kit. Using a simple colour chart, significant measures in colour intensity will be recorded and correlated with the fertile period.

Preliminary data on the El PG/PD3G ratio from nine normal menstrual cycles by conventional analyses have shown that the ratio increases 2 to 5 fold from the mean follicular phase baseline value (days --12 to -6) to the pre-ovulatory peak value. Differences in chromophore concentrations of this magnitude are readily detected by the human eye therefore in theory the kit is feasible.

However it has been observed that the absolute value of the ratio can differ from woman to woman. For the nine subjects examined the follicular phase baseline ratio (mean of days -12 to -6) ranges from 0.01 to 0.03 and the pre-ovulatory peak value (days -2 to 0) ranges from 0.028 to 0. 56. In terms of a kit this indicates that an absolute colour endpoint may not be suitable, rather a relative colour change would mark the end point.

It is envisaged that the kit would contain a colour chart, graduated in increasing intensities of colour. For each woman a follicular phase baseline of colour intensity would be recorded on the chart.

As mid cycle approaches an increase in colour intensity by a certain number of graduations on the chart would be taken as a positive result.

Claims (2)

1. CLAIM

   The claim covers a technique for determining the ratio by mass of two different solutes. The technique generates a measurable signal, the value of which is directly related to the value of the above ratio. The technique is applicable to determining the ratio of any two solutes, providing that each solute can be antigenic, that is capable of being the antigen component in an antibodyantigen reaction. (It is not a requirment that either solute in unmodified form be immunogenic.) The novelty of the technique on which the claim is based is the preparation and use of a "complete reagent system" (comprising a solid phase attached to an antibody, attached to a bifunctional ligand, attached to a second antibody) together with a labelled antibody, to produce a measurable signal which can then be related to the solute ratio to be determined.

   No novelty is claimed for the separate components of the above, nor for the methods of labelling antibodies to produce measurable signals; these can be prepared and carried out by published methods.

   The features of the invention are specified as follows:- Ligand 1 and ligand 2 are the two solutes and the ratio ligand 1: ligand 2 is the value which the invention determines. Antibody 1 is an immunoglobulin which will reversibly bind ligand 1, with high specificity, by means of an antibody - antigen reaction. Antibody 2 is an immunoglobulin which will reversibly bind ligand 2 with high specificity by means of an antibody antigen reaction. The bifunctional ligand consists of one or more molecules of ligand 1 and one or more molecules of ligand 2 bound to a supporting molecule. The ligand 1 functions of the bifunctional ligand are reversibly bound by antibody 1. The ligand 2 functions of the bifunctional ligand are reversibly bound by antibody
2. 2.Labelled antibody 1 has the same properties as antibody 1 except that it has been chemically modified by the covalent attachment of a marker molecule or label. This label, e.g. an enzyme, is capable of generating a measurable signal.

   In the "complete reagent system" antibody 2 is irreversibly bound to a solid phase. This attachment is made in such a way as to enable antibody 2 (plus components bound to antibody 2) to be in intimate contact with the solution in which the ligand 1: ligand 2 ratio is to be determined thus facilitating the separation of antibody 2 (plus components bound to antibody 2) from the solution.

   The invention is characterised by the following: The complete reagent system consists of antibody 2 attached to a solid phase, bifunctional ligand bound to antibody 2 by its ligand 2 function(s) and finally antibody 1 binding to the ligand 1 function(s) of the bifunctional ligand. The complete reagent is used by incubating it in contact with a solution on which it is desired to measure the ratio ligand 1: ligand 2. Competitive dissociation of the immunocomplex takes place to an extent dependant on the amounts of ligand 1 and ligand 2 present in the solution. After removal of the solution, the number of ligand 1 functions of the bifunctional ligand, not having antibody 1 bound to them, but still associated with the solid phase, via ligand 2 attachments, are determined.

   This is dong by incubating the solid phase with a solution of labelled antibody 1, followed by removal of that excess labelled antibody 1 which does not bind to the bifunctional ligand on the solid phase. The signal generated by the labelled antibody 1 finally associated with the solid phase, is then measured.