Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-23
2002-02-05
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S007200, C435S007210, C435S008000, C435S005000, C435S006120, C435S007920, C435S007930, C435S007940, C435S007950, C435S968000, C435S973000, C436S056000, C436S060000, C436S164000, C436S500000, C436S536000, C436S537000, C436S800000, C436S805000, C436S172000, C250S36100C, C250S302000, C250S259000
Reexamination Certificate
active
06344331
ABSTRACT:
This invention relates to methods and kits for detecting or assaying an analyte present in a water immiscible solvent.
The terms “water immiscible solvent” and “aqueous solution” are intended to be construed broadly, to include liquids which are partially miscible on agitation but which on settling separate into two layers to form a liquid/liquid interface.
BACKGROUND TO THE INVENTION
Ligand binding assays have been applied extensively to the detection and quantisation of compounds of biological importance present in fluids such as serum. Such assays rely on the interaction between the compound to be analysed (the analyte) and a specific binding partner such as an antibody. The extent of the binding reaction is generally monitored by the use of a marker, such as a radioactive label, which can be detected at very low concentrations. These assay techniques have been used widely for the measurement of water soluble substances such as proteins and also in the quantisation of compounds such as steroids or thyroid hormones, which are only sparingly soluble in water but which are normally associated with specific or non-specific binding proteins. Assay of these latter compounds frequently involves extraction of the substance of interest into a water immiscible solvent, removal of the solvent by evaporation and then “solubilisation” of the compound in an aqueous medium to provide essentially a single phase system in which reaction with a binding partner such as an antibody can occur. Such techniques are labor intensive and can only be performed in specialised laboratories.
The progressive devolution of ligand binding assays to non-specialist laboratories and their increasing use in analytical applications outside clinical diagnostics require simplification of the basic analytical procedures to make them accessible to non-specialist staff in what might be regarded as a “field” situation.
It has generally been considered that the assay system should essentially comprise a single liquid phase, and as will be evident from the prior art, complex and expensive procedures have been implemented to render a sample of an analyte soluble in an organic solvent suitable for analysis by ligand binding assays.
However, we have discovered, surprisingly, that an effective ligand binding assay may be set up in a two-phase liquid system in which the binding partner is in an aqueous phase and the analyte is in a substantially water immiscible solvent phase and in which binding takes place at or near the interface between the liquids.
DESCRIPTION OF PRIOR ART
The concept of a physiologically derived molecule such as an antibody retaining its activity under conditions where it is exposed to an organic solvent is both unexpected and unpredictable. Russell et al (Biochem Biophys Res Comm 1989; 158: 80) have shown that immobilised anti-hapten antibodies will bind hapten in the presence of water miscible solvents such as dioxane and acetonitrile. However, their experiments showed that the binding affinity of the antibodies was significantly reduced by the presence of the solvent and also that the binding was progressively reduced as the hydrophobicity of the solvent used was increased.
Subsequently, Weetall (J Immunol Meth 1991; 136:139) found that antibodies linked to paramagnetic particles were capable of retaining binding activity in water immiscible solvents such ar hexane.
Francis and Craston (Analyst 1994; 119:1801) developed an immunoassay for parathion in hexane in which the antibody was encapsulated in reverse micelles. In this way, the antibody was “protected” by inclusion in the micelle.
Recently, Matsuura et al (J Biochem 1993; 114:273) have described the binding of a shell fish toxin to antibodies in the presence of methanol, though they did not develop a quantitive analytical procedure.
U.S. Pat. No. 4,238,472 describes a method in which samples are extracted with a water immiscible solvent which is subsequently removed by evaporation. The dried residues are taken up in a detergent which will form an emulsion with the assay reagents, essentially producing a single phase liquid system.
Published PCT Application No. WO 92/12427 describes an assay procedure where the sample is extracted into hexane and, after further purification, the hexane is removed by evaporation and the sample re-dissolved into a water soluble solvent (methanol). This is then diluted into an aqueous assay buffer, again forming a single phase.
SUMMARY OF THE INVENTION
Broadly speaking, the present invention relates to the detection and quantisation of largely water insoluble compounds dissolved in a water immiscible solvent by being brought into contact with a specific binding reagent present in an aqueous medium so that a binding reaction takes place via the water/solvent interface, the extent of the binding reaction, i.e. the degree of association between said analyte and said binding partner being proportional to the concentration of compound in the solvent phase.
Thus, in one aspect of this invention, there is provided a method of determining the presence and/or concentration of an analyte in a substantially water immiscible solvent, which method comprises the steps of:
(i) mixing a sample of the water immiscible solvent with an aqueous solution containing a specific binding partner of said analyte to allow binding between said binding partner and said analyte (if present), and
(ii) monitoring directly or indirectly the degree of association between said analyte and said binding partner thereby to determine the presence and/or concentration of said analyte.
The benefits of this method include the facts that the method provides a two phase liquid system in which binding can occur and that the binding partner is largely preserved or protected by remaining in the aqueous phase, and the natural separate of the aqueous and solvent phases may in many cases simplify separation of the fractions of the bound and unbound analyte.
In a preferred aspect, the binding reagent is an antibody labelled with a substance which allows the extent of binding to be readily assessed.
Preferred embodiments of the present invention allow the development of quantitative or qualitative analytical methods for the detection of hydrophobic compounds present in water immiscible solvents such as hexane, xylene and toluene where the appropriate binding reagent in an aqueous medium is agitated with the solvent sample such that the compound reacts with the binding reagent at or near the water/solvent interface and so becomes effectively “trapped” by the reagent in the aqueous layer. The amount that is trapped in this way is a function of the concentration of analyte in the solvent sample and can be readily determined by standard ligand binding techniques, suitable examples of which are well known to those skilled in the art.
It will be readily appreciated that this approach represents a significant simplification for detecting or assaying an analyte initially present in a solvent, when compared with methods which require prior extraction of the sample or the manufacture of reverse micelles to encapsulate the binding reagent. Such considerations are particularly important when a method is being developed for field use.
In a preferred aspect, the invention involves the use of monoclonal antibodies in solution in an aqueous buffer which are shaken with a sample of a hydrophobic hapten present in a water immiscible solvent. The antibodies, which may be labelled with a marker, are specific for the hapten which will not normally partition into the aqueous phase. After shaking for a predetermined period of time, the mixture is allowed to stand briefly to allow the aqueous and water immiscible solvent layers to separate. The aqueous layer is transferred to a reaction tube which contains an immobilised derivative of the analyte. In a further reaction, labelled antibody which has not reacted with analyte in the sample will bind to the derivative in the tube. The amount of this bound antibody, which varies inversely with the concentration of analyte in the original solv
Aston Jeremy Paul
Ball Raymond Lathan
Cryer Jennifer
Weeks Ian
Birch & Stewart Kolasch & Birch, LLP
Chin Christopher L.
Cook Lisa V.
Molecular Light Technology Research Limited
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