Fluorescent immunoassay employing total reflection for activation

United States Patent im

Kronick et al.

[ii] 3,939,350 [45] Feb. 17, 1976

bound to the epitopic sites on the surface and light of predetermined wave length is directed toward the surface, the fluorescing molecules are activated and fluoresce.

In carrying out an assay, receptor is combined with an unknown suspected of containing molecules having the same epitopic sites bound to the surface. The receptor will bind to these molecules reducing the number of receptor sites available for binding to the epitopic sites on the surface. When the assay medium is contacted with the surface, the amount of receptor which binds to the surface, will be a function of available binding sites and, therefore, to the number of the molecules present in the unknown. Upon irradiation of the surface, substantially only the fluoroescent molecules bound to the surface will fluoresce. By monitoring the fluorescence, one can determine the presence and number of molecules of interest present in the unknown.

The apparatus consists of a transparent solid sheet, conveniently as part of or optically connected to a prism, a light source set at an angle to provide total internal reflection at the sheet, a cell which includes the reflecting" surface as a wall, and a fluorescence detector. Various optics and filters may be employed to modify the light source beam and the fluorescence beam.

This work was carried out under a grant of the National Science Foundation.

15 Claims, 4 Drawing Figures

3,939

1

FLUORESCENT IMMUNOASSAY EMPLOYING
TOTAL REFLECTION FOR ACTIVATION

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BACKGROUND OF THE INVENTION 1. Field of the Invention

There is a continuing and expanding interest in the ability to measure small quantities of naturally occurring and synthetic compounds or compositions. A 1° broad category of methods fall in the classification of immunoassays. These methods depend on the ability of a receptor, usually an antibody, to recognize a particular spatial and polar configuration and bind to such configuration. As a result of this binding, the resulting 15 complex can be differentiated from molecules which are present, which are not bound to the receptor.

For purposes of convenience, the compound to be determined will be referred to a "ligand." In immunoassays, a ligand analog is provided which is capable of 20 competing with the ligand for the receptor. That is, the ligand analog has a spatial and polar configuration analogous to the ligand and is also tagged, so as to allow for its detection. In the immunoassay, any ligand present in an unknown and ligand analog compete for the 25 receptor. The amount of ligand analog bound to receptor will be related to the amount of ligand present in the unknown. Ligand analog bound to receptor can be distinguished from ligand analog which is unbound.

In one technique, referred to as radioimmunoassay, 30 the ligand analog has a radioactive atom. Where the receptor is antibody, which is the conventional receptor, ligand analog bound to antibody can be separated from ligand analog which is unbound. By determining the distribution of radioactive labeled ligand, between 35 bound and unbound, one can determine the amount of ligand present in the unknown.

An alternative method employs a stable free radical tag, such as a small nitroxide molecule. With small ligands, molecular weights below about 50,000, the 40 rate of tumbling of the ligand analog in solution is sufficiently fast, so as to provide a relatively sharp peak in the EPR spectrum of the free radical. When the ligand analog is bound to receptor, which is normally of high molecular weight, the peak is broadened to a much 45 greater half-width. Therefore, by measuring a point near the maximum of the peak, the height at that point can be related to the distribution of bound and unbound ligand analog. This in turn can be related to the concentration of ligand in an unknown. 50

A third technique employs an enzyme as the detector. The technique can be carried but homogeneously or heterogeneously. In U.S. Pat. Nos. 3,654,090 and 3,791,932, heterogeneous systems are described. The heterogeneous system requires binding one of the rea- 55 gents involved in the determination to a solid support, for example, the receptor. By allowing competition for the receptor bound to solid support between the ligand and the ligand analog, and separating the solid support, one can then determine the enzyme activity in the 60 supernatant. The amount of ligand analog remaining in the supernatant, as determined by the enzyme activity in the solution, is related to the amount of ligand present.

An alternative system is homogeneous. This is based 65 on a reduction in enzyme activity, when ligand analog is bound to receptor. The reduction in enzyme activity is related to the amount of ligand analog bound to

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receptor. By using standards, which is conventional with the other immunoassay techniques, one can relate change in enzyme activity to the amount of ligand present in the unknown.

Each of the above systems have advantages and disadvantages as applied to specific situations. In one or more of the systems, expensive equipment is required. Working with radioactive materials is undesirable. Furthermore, the radioactive materials have only a limited shelf life. The free radical technique is limited as to the molecular weight of the ligand. The enzyme technique is subject to interfering substances present in the unknown. There is, therefore, a continued interest in finding new systems, which may avoid the deficiencies of the earlier systems, and have substantial advantages in particular applications.

2. Description of the Prior Art

Radioimmunoassays are described in Murphy, J. Clin. Endocr., 27, 973 (1967); ibid, 28, 343 (1968). Free radical immunoassays are described in U.S. Pat. No. 3,690,834. Enzyme immunoassays are described in U.S. Pat. Nos. 3,654,090 and 3,791,932 and U.S. Pat. Application Ser. No. 143,609, filed May 14, 1971, now abandoned. Techniques employing total internal reflection are described in Herrick, et al., Anal. Chem., 45, 687 (1973) and Amer. Laboratories, 5, 63 (1973). See also, Kronick, et al., Bull, of the Amer. Physical Society, 18, 782 (1973).

SUMMARY OF THE INVENTION

Method and apparatus are provided for carrying out immunoassays, using the amount of fluorescence as an indication of the presence of a compound or composition ("ligand") to be detected. A ligand, having one or more epitopic sites, is bound to the flat surface of an optically transparent sheet. The surface is contacted with an aqueous solution—assay medium—containing the unknown and antibody to the ligand. The antibody is tagged with fluorescing molecules. Depending on the amount of ligand present in the solution, the available sites for binding to the surface will vary, and the amount of antibody bound to ligand on the surface will proportionately vary, when the surface is contacted with the assay medium.

The sheet is irradiated with light at the wave length of absorption of the fluorescing molecule bonded to the antibody. The angle of irradiation provides total internal reflection, so that fluorescence can occur within only a few hundred Angstroms of the surface. By measuring the amount of fluorescence, for example, with a photomultiplier tube, the amount of ligand present in the solution can be determined.

The apparatus is comprised of a light source, an optically transparent sheet in appropriate juxtaposition to provide total internal reflection, a cell having the transparent sheet as one wall, and a light detector situated so as to receive fluorescent light from the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elevational cross-sectional view of a cell and prism;

FIG. 2 is a diagrammatic view of an immunoassay apparatus;

FIG. 3 is a stylized illustrative view of the reaction occurring in the cell;

FIG. 4 is a plot of three curves obtained following the method of this invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

A method and apparatus are provided for carrying out immunoassays, whereby the amount of fluorescence obtained from a sample cell is related to the amount of material being determined. In discussing the method, the following terminology will be employed. A compound or composition recognizable by a single receptor, haptens or antigens, will be referred to as ligand. "Receptors" are high molecular weight molecules, usually proteins, which are capable of binding to a particular spatial and polar organization. For the most part, receptors are antibodies and antibodies will be illustrative of receptors.

The immunoassay is carried out by providing a competition for receptor to which fluorescer molecules are bound, between epitopic or determinant sites of molecules in an aqueous buffered assay medium and the same epitopic sites bound directly or indirectly to a transparent surface. The transparent surface serves as one wall of a cell containing the assay medium. The assay medium is constituted of an aqueous solvent, normally buffered, fluorescer-bound-receptor, unknown to be assayed, and other additives which may be appropriate in particular situations.

By appropriate choice of the material for the transparent surface, a material having a refractive index greater than the assay medium, the surface can be irradiated with light at an angle which provides total internal reflection. Under conditions of total internal reflection, only fluorescent molecules within a few hundred Angstroms of the surface will be activated and fluoresce. The number of receptor molecules bound to the surface, and, therefore, the number of fluorescent molecules sufficiently close to the surface to be activated will be proportional to the number of molecules to which receptor is bound in the assay medium.

By measuring the amount of fluorescence upon irradiation, one can obtain a determination of the presence of molecules in the unknown having the same epitopic sites as the molecules bound to the surface. By using standards having known amounts of such molecules, one can prepare a curve relating the amount of fluorescence to the amount of such molecules present in the assay medium.

The method employed in the immunoassay depends upon total internal reflection. At an interface between two materials of different refractive indicies, the angle of incidence is related to the angle of reflection by the following formula:

n,sine, = n2sin82

where «i and n2 are the refractive indicies of the two materials and 01 and 62 are the angles from the norm which the incident radiation makes at the interface. When the sine of the angle of incidence is equal to or greater than the ratio of the refractive indicies, n2/nu total internal reflection occurs, and the light does not penetrate the second medium. Some light energy does in fact penetrate the second medium over relatively short distances, usually not exceeding about 1,000A. Depending upon the variables involved, the distance of penetration of light energy can be diminished to as little as 500A. If a fluorescing molecule is positioned at the interface in the second medium, so as to be within the range of light energy which penetrates the second medium, when the wave length of the light is within the adsorption peak of the fluorescing molecule, the fluorescing molecule will

fluoresce. Fluorescing molecules that are outside this narrow band will not fluoresce.

The subject immunoassay can be used for detecting a wide variety of compounds, both haptenic and antigenic. The significant factor is that a receptor can be provided which recognizes a specific spatial and polar organization—an eptiopic or determinant site—and is capable of having one or more fluorescing molecules bound to it.

In the subject invention, for the most part, the receptors will be macromolecules, which have sites which recognize specific structures. The recognition of the specific structures will be based on Van der Waals forces, where the receptor provides a specific spatial environment which maximizes the Van der Waals forces; dipolar interactions, either by permanent or induced dipoles; hydrogen and ionic bonding; coordinate covalent bonding; and hydrophobic bonding.

The macromolecules of greatest interest are proteins and nucleic acids which are found in cell membranes, blood, and other biological fluids. These compounds include enzymes, antibodies, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and natural receptors. Of particular interest are the antibodies, particularly the -y-globulins, which have two binding sites or can be split in two so as to have a single binding site.

The receptors are modified with fluorescing molecules. Methods for linking fluorescing molecules to proteins are well-known in the art. Various linking groups include activated carboxylic acid groups, for example, by employing the mixed anhydride or with carbodiimide, isothiocyanate, nitrophenyl or nitrobenzyl esters, Or the like. Many of the commercially available fluorescing compounds have groups for linking to proteins. The preparations are normally carried out under mild conditions in aqueous media.

In choosing the fluorescing compound to be linked to the antibody, a number of considerations come into play . If the ligand to be determined fluoresces, then the choice of fluorescer will be such as to have a longer wave length higher absorption maximum than the ligand. Also, the fluorescer should not enhance nonspecific binding to glass surfaces or other proteins. In addition, since proteins adsorb at a wave length of about 280nm, the fluorescer should have an adsorption maximum above 300nm, usually above 35 Onm and preferably above 400nm. The extinction coefficient should be greatly in excess of 10, preferably in excess of 103, and particularly preferred, in excess of 104. The number of fluorescing molecules per receptor will be from about 1 to 40, usually from about 1 to 30, and preferably about 5 to 25.

A number of different fluorescers are described in Brand, et al., Annual Review of Biochemistry, 41, 843-868 (1972) and Stryer, Science, 162, 526 (1968).

One group of fluorescers of particular interest are xanthene dyes, which include the fluoresceins, derived from 3,6-dihydroxy-9-phenylxanthydrol and rosamines and rhodamines, derived from 3,6-diamino-9-phenylxanthydrol. The rhodamines and fluoresceins have a 9-o-carboxyphenyl group and are derivatives of 9-ocarboxyphenylxanthydrol.

These compounds are commercially available with substituents on the phenyl group which can be used as the site for bonding or as the bonding functionality. For example, amino and isothiocyanate substituted fluorescein compounds are available.