WO1995015496A1

WO1995015496A1 - Ultrasensitive competitive immunoassays using optical waveguides

Info

Publication number: WO1995015496A1
Application number: PCT/US1994/013935
Authority: WO
Inventors: Stanley M. Klainer; Hong Li
Original assignee: Fci-Fiberchem, Inc.
Priority date: 1993-12-01
Filing date: 1994-12-01
Publication date: 1995-06-08
Also published as: TW371714B; JPH09509480A; EP0731916A1

Abstract

Sensitivity of a competitive immunoassay optical sensor is increased by selecting a tag or altering the attachment of a tag to an antigen so that the binding of tagged antigen to an antibody is relative to the binding of untagged antigen to the antibody. The indicator material is selected of a suitable size, decreased antibody, or the indicator material is attached to the antigen through a long chain high molecular weight compound, or the indicator material is attached directly to the antigen with a long chain high molecular weight compound attached elsewhere to the antigen. Pretreatment of the antibody/bound tagged antigen to remove unbound antigen, and optical isolation of the substrate also increase sensitivity. If the tag cannot be attached to the antigen, it is attached to the antibody. The tag is selected or attached to increase sensitivity to antigen.

Description

ULTRASENSITIVE COMPETITIVE IMMUNOASSAYS USING OPTICAL

WAVEGUIDES

BACKGROUND OF THE INVENTION

The invention relates generally to optical, chemical, and biochemical sensors and more particularly to sensors using competitive immunoassays with monoclonal or polyclonal antibodies.

In a competitive immunoassay optical sensor, an antibody is immobilized in the sensor, and a tagged antigen is bound to the antibody. The tag is typically a fluorophore. The target (untagged) antigen competitively binds to the antibody and displaces the tagged antigen, causing a change in sensor optical properties, e.g. fluorescence of the tagged antigen, which is related to the concentration of target antigen. U.S. Patent 4,321,057 issued March 23, 1983 to Buckles describes a fiber optic sensor based on competitive immunoassay between tagged and untagged antigen.

Immunoassays have become a well-accepted method of analysis in medicine because of their unquestionable specificity to the target compound of interest. If a monoclonal antibody is used, then its reaction is specific to a particular antigen (compound of interest) . If, on the other hand, a polyclonal antibody is used, then its reaction is specific to a particular chemical structure rather than a precise chemical compound.

This invention primarily addresses new approaches to competitive binding fluoroimmunoassays. Many of the techniques presented, however, are also applicable to immunoassays in general. For example, although this application focuses on fluorescent tags, the technique is amenable to radioactive tags as well.

The use of competitive antibody-antigen reactions has been primarily limited because of: (1) Lack of sensitivity, i.e. high background noise, inadequate antibody loading on the substrate and inefficacious exchange between antibody bound to tagged antigen and untagged antigen; and (2) The inability to measure small molecules.

For the molecules which can be measured, the minimum detection limits (MDL) is in the high parts-per-billion (ppb) to low parts-per-million (ppm) range while the limit of quantification (LOQ) is a factor of 3.3 higher. These limitations exclude their use in such important areas as: (1) Environmental monitoring, especially measuring pollutants in drinking water, and in the work place or home; (2) Measuring contamination in chemical processes; (3) Examining personnel for alcohol, drug or other substances abuse; (4) Determining exposure to and presence of toxic substances and infectious diseases; and (5) Diagnosing and evaluating maladies such as cancer, heart infarctions, arthritis, gastrointestinal ailments, abnormal blood panels, and urological problems.

The production of high sensitivity competitive immunoassays for an extended list of antigens is the primary focus of this invention. This provides the ability to measure and quantify numerous antigens in the low ppb to parts per trillion (pptr) range irrespective of their molecular size. Thus the analysis of small molecules is also part of this invention.

As a result of the chemical systems developed according to the present invention for high sensitivity measurements and the analysis of small molecules, the drawback that existing prior art immunoassays must be done by trained personnel and are subject to human error, is overcome. In particular, many of the up-to-date prior art assays require mixing of chemicals, such as the addition of enzymes and dyes, and the results are, therefore, only as accurate as the technician. The use of immunological systems, where there is no human participation in the chemistry, is an additional part of this invention.

Optical waveguide chemical sensors (O CS) , optical waveguide biochemical sensors (OWBS) , fiber optic chemical sensors (FOCS) , fiber optic biochemical sensors (FOBS) , optical chip chemical sensors (OCCS) , and optical chip biochemical sensors (OCBS) are all. transducers in an information acquisition strategy which obtains real-time data about the presence and concentration of specific species, or chemical groups of compounds, in chemical and biochemical systems. Optical waveguides include flat channeled and non-channeled waveguides as well as chips with waveguides on them. Some waveguide sensor configurations are illustrated in Figs 1A-D. Figure 1A shows a typical waveguide system with sensor chemistry attached. Figure IB shows more than one sensing chemistry on a single waveguide while Figure 1C is a miniaturized waveguide which is totally covered with sensing chemistry. In order to have an internal reference, the waveguide is half coated and the uncoated section is used to obtain a reference signal. Figure ID. In order for this to work, the uncoated portion of the waveguide must face the illumination source and the reference signal must be taken before that of the coated section. The miniaturized waveguide can be « 2«2*0.1 cm, or less or can be formed on a chip.

Optical chemical and biochemical sensors are devices with indicators for pre-selected chemical and/or physical properties attached to their surfaces, so that sensitive, specific, real-time analyses can be made. These can be based on fluorescence, absorption, Raman, polarization, refraction, reflection, or radio-chemical measurements. The species or group-specific chemistry can be selected from organics, inorganics, metals, enzymes, monoclonal and polyclonal antibodies, biochemicals and polymers or combinations thereof. Interaction of an analyte with the sensing reagent (in this case a tagged antigen or tagged antibody) produces a change in one of the above- mentioned spectroscopic parameters. For sensitive measurements using antibody-antigen reactions fluorescence and polarization are the preferred measured properties, depending on the molecular size of the target molecule. A read-out device electronically converts light flux into voltage. Modulation in the voltage reading directly correlates with the analyte concentration.

Figures 2A,B show the basic reactions in a competitive immunoassay. In this Figure Y represents an antibody, *v (or v*) is the tagged antigen and v is the untagged antigen, i.e., the target compound of interest. The greater the exchange rate between v and *v the more sensitive the reaction. Ideally, most of the *v will be lost at the actual (or integrated) concentration of the compound to be measured. This is not the case under normal circumstances; however, the present invention provides a method of making this happen. Figure 3A shows how an antibody (Y) is attached to a glass substrate and saturated with a tagged antigen (*v) . Figure 4A shows the same arrangement on a membrane substrate.

SUMMARY OF THE INVENTION Accordingly, it is an objective of the invention to increase sensitivity of a competitive immunoassay.

It is also an object of the invention to provide an improved optical sensor based on competitive immunoassay.

It is another object of the invention to provide method and apparatus for competitive immunoassay which has sensitivity to low ppb and even pptr levels.

The invention is method and apparatus for competitive immunoassay having a tagged antigen bound to an antibody immobilized in a sensor, in which the binding of the tagged antigen to the antibody is altered or distorted so that the untagged target antigen more easily displaces the tagged antigen. The binding of the tagged antigen is controlled by selecting a suitable large tag, or by making the tag larger by adding a long chain to a smaller tag, or by making the tagged antigen larger and attaching a long chain elsewhere on the tagged antigen. Optical isolation is also provided. The antibody-tagged antigen is pretreated by a special washing technique. The sensor can be configured to detect a single antigen and multiple antigens, and can be placed on a chip. When the antigen cannot be tagged, the antibody can be tagged. BRIEF DESCRIPTION OF THE DRAWINGS

Figs 1A-D illustrate waveguide sensor configurations with single and multiple sensing chemistries.

Figs 2A,B illustrate the basic reactions of a competitive immunoassay .

Figs 3A-C illustrate tagged antigen bound to an antibody attached to a glass substrate.

Figs 3D-F are the systems of Figs 3A-C including an additional optical isolation layer. Figs 4A-F are similar arrangements as Figs 3A-F on a membrane substrate.

Figs 5,6 are the chemical formulas of a pair of fluorescein based fluorescent tags.

Fig 7 shows the fluorescent spectrum of a sensor using the tag of Fig 6 with no cocaine and with 160 ppb cocaine.

Fig 8 shows the fluorescent spectrum of a sensor using the tag of Fig 6, optical isolation, and special washing, with no cocaine and with 4 ppb cocaine. Fig 9 shows another fluorescent spectrum as in Fig

8, showing that saturation is reached before 80 ppb cocaine.

Fig 10 illustrates the repeatability of 4 ppb cocaine as a function of time.

Fig 11 illustrates the loading effect of a greater amount of antibody-tagged antigen than in Fig 10.

Fig 12 illustrates detection of sub-ppb concentration of cocaine.

Fig 13 is the fluorescent spectra of various concentrations of anti-mouse IgG. Fig 14 illustrates a tagged antibody.

Fig 15 illustrates the reaction of an immunoassay with tagged antibody.

Fig 16 is the hydrolysis reaction of cocaine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The basic reactions in a competitive immunoassay are illustrated in Figs 2A,B. A substrate 10 with antibodies 12 immobilized thereon is contacted with a solution containing tagged antigen 14' which is composed of antigen 14 with attached tag 16. The tagged antigen 14' binds to the antibody 12, producing sensor 15. When sensor 15 is brought into contact with a sample containing antigen 14, antigen 14 competes with tagged antigen 14' for binding sites on antibody 12. Antigen 14 displaces tagged antigen 14 producing sensor 15 which has different optical characteristics than sensor 15. The sensitivity of sensor 15 will be determined by the ease with which antigen 14 can competitively displace tagged antigen 14' . The invention alters the binding of tagged antigen 14 so that antigen 14 more easily displaces tagged antigen 14-'. Substrate 10 may be an optical fiber or other waveguide structure, or other support through which a light signal may be input and output to measure changes in sensor 15.

Since the interaction between an antibody and antigen is a "lock-and-key" fit, i.e. only one (1) antigen will react with a monoclonal and only (1) chemical structure will react with a polyclonal, it is necessary to come up with an approach whereby the tagged antigen is sufficiently distorted so that its ability to bind with the antibody is impaired but not negated. On the other hand, the binding between *v and the antibody must be strong enough so that it cannot be removed except by an antigen which makes a better fit, i.e. v, the specific analyte of interest.

There are three (3) approaches for distorting *v, i.e., making *v of suitable size to decrease binding relative to v: (1) a large is selected so that the tagged antigen has sufficient size, Figs 3A, 4A, (2) a long-chain, high molecular weight compound can placed between the antibody and the tag as shown in Figures 3B and 4B and (3) a simple tagging compound is used and a long-chain high molecular weight compound can be attached elsewhere on the antigen. Figures 3C and 4C. In either case it is important to use "distorting compound" which does not change the shape of the antigen to the point where it is not recognized by the antibody.

As shown in Figs 3A, 4A, tagged antigen 14 formed of antigen with attached tag 16, binds to antibody 12 which is immobilized a substrate. In Fig 3A, antibody 12 is attached to a glass substrate 22 by a silane 24 and glutaraldehyde 26. In Fig 4A, antibody 12 is directly attached to membrane substrate 10. The same methods are used to immobilize the antibody 12 to the substrate in Figs 3B-C, 4B-C. Since the tagged antigen differs from the untagged antigen in the presence of the tag, proper selection of a suitably sized tag, if one is available, can produce a tagged antigen with the requisite decreased binding to the antibody.

However, if a suitable tag is not available, then the invention can be implemented with available tags by changing the tagged antigen to alter the relative binding strength compared to untagged antigen. As shown in Figs 3B,4B, tagged antigen 14a is formed by attaching tag 16 to antigen 14 through a long chain high molecular weight compound 18. Alternatively, as shown in Figs 3C, 4C, tagged antigen 14b is formed by attaching tag 16 directly to antigen 14, but additionally attaching a long chain high molecular weight compound 20 elsewhere on antigen 14.

From the standpoint of simplicity and reproducibility, the use of a tagging compound which also distorts the antigen is the best approach, i.e., the use of a suitably large tag. This means that only one (1) synthetic operation has to be performed on the antigen. For example, if cocaine, morphine or heroin are the antigens, they cannot be measured below 1 ppm when the simple tag, fluorescein isothiocyanate (FITC) , Figure 5 is used. If fluoresceinthiocarbamyl ethylenediamine is used. Figure 6, then 160 ppb of cocaine can be detected. Figure 7. Note that this compound has a higher molecular weight than the FITC and also has a distortion chain. Thus the tag provides both the indicator (fluorescein) and the distorting compound. This tag was selected because it distorted the antigen to make the exchange between *v and v optimum. If only FITC had been available, it could have been attached to the antigen through a long chain compound, or it could have been attached directly to the antigen and another compound attached elsewhere on the antigen. The choice of tags which distort the antigen are made based on experimental data. Each tag must be selected based on the antigen to which it is being attached. In cases where the target antigen cannot be tagged, i.e. the target compound has no active group to which a tag can be attached, then it is necessary to tag the antibody, as further described below. The tags are based on an active indicator material, e.g., a fluorophore, which is preferably a laser dye because of its high quantum efficiency. Illustrative compounds suitable for tags for antigens include, but are not limited to: fluoresceinthiocarbamyl ethylenediamine rhodamine B isothiocyanate eosin-5-isothiocyanate malachite green isothiocyanate rhodamine X isothiocyanate

Lissamine™ rhodamine B sulfonyl chloride 6-carboxyrhodamine 6G hydrochloride 5-(and-6)-carboxy-X-rhodamine

6-(fluorescein-5-(and-6)-carboxamido)hexanoic acid succinimidyl ester Texas Red® sulfonyl chloride Other tags can be found in Handbook of Fluorescent Probes and Research Chemicals, 5th ed. , 1992-1994, Richard P. Haugland, Molecular Probes, Inc., which is herein incorporated by reference.

The tag is selected, or attached to the antigen tagged additional compounds, or additional compounds are attached to the antigen, to produce ideally the lowest binding energy of the tagged antigen to the antibody so that the tagged antigen does not come off unless the untagged antigen is present, but the tagged antigen is easily displaced by the untagged antigen. Although the ideal lowest binding energy may not be achieved, significant reduction of the binding energy of the tagged antigen will greatly increase sensitivity to untagged antigen. By following the principles of the invention, suitable tagged antigen can be produced by routine experimentation.

The background noise, which reduces the signal to noise ratio of a measurement can come from three

(3) sources: (1) Unused active sites on the substrate, (2) Reflections and light scattering from the substrate, and (3)

Loose tags on the antibody or antigen.

The easiest first step to sensitivity is to make sure that the tagging compound is present only when attached to either the antigen or antibody. In the conventional method of tagging this is no problem since no effort is made to reduce the bond strength between *v and v and traditional washing techniques suffice. When the bond between the antibody and *v is deliberately weakened, it is necessary to use milder washing techniques, i.e. neutral buffer and triply distilled water. Washing is done repetitively until there is absolutely no indication of fluorescence in the used wash solution. This washing treatment to remove loose tags forms an additional part of the invention.

Two (2) approaches are used to make sure there are no unused active sites on the substrate to which interfering compounds can attach: (1) The substrate is allowed to react with the antibody containing the tagged antigen for an extended period of time (hours to days) . This assures that the maximum number of antibodies are attached to the substrate. (2) The remaining sites are bound to an inert blocking compound such as bovine serum albumin (BSA) . This is required because geometric and stearic hindrances do not allow all the active sites to be bound to antibody.

Reflections and light scattering from the substrate is normally handled using spectroscopic techniques, i.e. using a narrow slit or narrow band filter to sort the excitation light (reflected and scattered) from the fluorescence emission signal. In order to get maximum sensitivity, however, it is necessary to use wide slits or broad band filters to collect as much light as possible so as to enhance light collection. Since spectral sorting cannot be used, two (2) ways to handle this problem have been invented for glass: (1) The glass can be made opaque or frosted both to reduce the scattering and reflections from the excitation light and (2) an inert non-reflecting compound, such as charcoal, can be added to the blocking agent to render the substrate "flat black" and non-reflective. Figures 3D-F. It is imperative that the addition of charcoal or other optical isolation follow the immobilization of the antibody (with bound tagged antigen) or the active sites will be blocked or no (or minimum) antibody will be attached to the substrate. In the case of the membrane substrate, only the use of charcoal or other optical isolation is possible since pretreatment would destroy the membrane. Figures 4D-F. For both types of substrate the non-reflecting compound can be added at any compatible step in the chemistry after immobilization. Figs 3D-F, 4D-F show the addition of an optical isolation layer 28 or 10 to the structures of Figs 3A-C, 4A-C.

The optical isolation layer can be of a variety of materials. The optical isolation can be a dispersion of a black, white, red or reflective material in an inert and antigen-permeable polymer. Examples for colored materials include carbon black, barium sulfate, titanium dioxide, red or black ferrous oxide, gold particles, or glimmer pigments. In addition to BSA, antigenpermeable polymers into which the colored materials are dispersed include silicone, polystyrene or ethyl cellulose. As shown schematically and not to scale in Figs 3D-F, 4D-F. The antibodies extend far beyond the optical isolation layer. However, the optical isolation layer which is formed on the substrate will typically cover the antibodies but allow passage of the antigen. The optical isolation layer may also form a non-reflective surface below the antibody binding sites.

Figure 8 shows the improvement that can be made when the above sensitivity improvement methodologies are applied. In this Figure, 4 ppb of cocaine is shown with a signal-to-noise ratio that indicates parts-per-trillion (pptr) sensitivities are attainable. A comparison of Figs 7 and 8 shows that in Fig 7, which used only the improved tag, 160 ppb cocaine produced an intensity change of about 0.2 units while in Fig 8, which also used the wash and optical isolation, 4 ppb produces an intensity change of about 1.0 unit. Figure 9 shows both 4 ppb and 80 ppb. Here it can be seen that the sensor is so sensitive and the exchange rate so efficient that saturation is reached before 80 ppb, i.e., there are not enough tagged sites left and there is no linear relationship between 0, 4 and 80 ppb. Figure 10 shows the repeatability at 4 ppb as a function of time. Figure 11 shows that there can be a time dependence depending on the "loading factor" on the substrate. What is important, however, is that the change in counts-per-second (CPS) is the same for both sensors at the ten minute exposure time. At ten minutes, both sensors produced an intensity change of about 2 units for 4 ppb. The difference between Figs 10 and 11 is that the substrate in Fig 11 is "loaded" with more antibody than the substrate in Fig 10. Over longer time, more tagged antigen is displaced in Fig 11 while in Fig 10 the total amount of tagged antigen is quickly removed. Fig 12 shows spectra at various cocaine concentrations which demonstrate that very low (pptr) concentrations can be detected. Figure 13 shows the quantitative aspects of this technology using mouse IgG as the antibody and tagged anti-mouse IgG as the antigen.

There are two (2) special cases which must be addressed to increase the number of antigens that can be measured: (1) Small molecules where there are active functional groups to attach a tag, but where the tag would distort the antigen to an extent that it would not be compatible with the antibody and (2) Molecules of any size where there are no functional sites to attach the tag. In these cases the tag would have to be put on the antibody. Figure 14. A suitable tag 30 is attached to antibody 12 through a long chain high molecular weight compound 31, if necessary. The reaction of the immunoassay using tagged antibody is shown in Fig 15. Antibody 12 with attached tag 16 is contacted with a sample containing antigen 14, which binds to the tagged antibody, producing sensor 17. The optical characteristics of the sensor 17 are a function of the amount of antigen which binds to the tagged antibody. The tag 16 is selected, or attached to antibody 12 in such a manner, so that the sensor 17 has increased sensitivity to antigen 14. The preferred tag is a fluorescent compound but other tags, such as radiochemical, will also work.

In the absence of an antigen, the fluorophore attached to the antibody would be free to move around, and therefore, its fluorescence will be highly depolarized. When an antigen binds with the tagged antibody, the movement of the fluorophore becomes restricted, leading to a more polarized luminescence. Polarization measurements, therefore, can be used for quantifying antigens for which a matching tagged antibody is available. The choice of fluorescent tag is still the key to sensitivity. It should be chosen to give the greatest polarization change and this may be different in each case depending on the antibody and target molecule. The use of a long chain compound for attaching the tag permits greater motion of the fluorophore in the absence of antigen, and greater sensitivity in measuring polarization change when antigen binding occurs. Thus, the molecular shape, size and charge distribution, etc. and the method of attachment of the tag can be controlled to produce greater sensitivity of the tagged antibody to the antigen and greater change in the measured optical effect.

Another measurement technique can also be used, i.e., fluorescence. The required measurement involves the modulation of fluorescence lifetimes. This is a different approach than the routine relationship of antigen concentration to light intensity that has been previously described. Lifetime modulation is very specific to the presence of the target molecule sought. Fluorescence lifetime is controlled by the manner of binding the fluorophore to the antibody. An illustrative example of the tagged antibody process is an alternative method for detecting cocaine. Although cocaine can be detected directly, as shown above using a suitable tag and antibody specific to cocaine, with great sensitivity, an indirect approach can also be used. Cocaine and its hydrochloride have very low volatility at ambient conditions. Thus, an alternate approach is to focus on a cocaine derivative having a far greater volatility which will be easier to detect. When cocaine is shipped, stored or transported, environmental conditions cause degradation. Temperature, humidity, pressure and other environmental variations lead to the production of cocaine derivatives. One of these derivatives (which is always found) is methylbenzoate, a transesterification product formed during the hydrolysis of cocaine, as shown in Fig 16. Methylbenzoate constitutes a good chemical marker for cocaine, since it is liquid at room temperature, and therefore, has a significantly higher vapor pressure. However, methylbenzoate is a small molecule, devoid of functionalities that could be used for tagging with a fluorophore. Therefore, the antibody is tagged with a fluorophore. Polarization measurements for quantifying methylbenzoate will reveal the presence of cocaine. The approaches introduced can also be applied to the use of multiple sensors on a single substrate. This is accomplished by simply changing the tag while applying all of the other enhancement parameters, Figure IB. The first step is to use antibodies which are specific to each of the antigens (target molecules) of interest. The next stage is to select different tags for each antigen or antibody. When fluorescence tags are used: (1) They must have an active group where attachment can take place and (2) They must have very high quantum efficiency. Simplicity is added to the total system if these fluorophores excite at the same wavelength and emit at well-separated different wavelengths. Fluorescein and rhodamine are a pair of the better choices because they meet these criteria. For example, a multiple sensor for morphine and cocaine could be made using the antibodies specific to each of these and tagging the antigens differently, i.e., one with a fluorescein compound and the other with a rhodamine compound. This concept could be extended to several sensors on a single substrate by choosing additional tags. The best way to accomplish this is to mask the substrate into as many sections as there are antibodies and immobilize these individually. The use of antibody mixtures, in exactly known concentration mixes, does not assure these will be attached to the substrate in these ratios or that the relationship between these will be the same from sensor to sensor. Each tag will have its own specific emission wavelength which means there will have to be a fixed spectral channel for each these or a tunable detection system.

As shown in Fig 1A, a waveguide 32 is positioned between a source 38 and a detector 40 with filters 36. A sensing region 34 is made up of an immobilized antibody with bound tagged antigen is formed on waveguide 32. The antigen is tagged in accordance with the invention. Fig IB illustrates a multi-sensor configuration where additional sensing regions 42,44 are added to waveguide 32. Each sensing region 42,44 is made up of a different immobilized antibody with associated bound tagged antigen. The tags are different so the responses can be differentiated. In Fig 1C, sensing region 34 covers the entire waveguide, which can be a miniaturized structure, e.g. a chip. The configuration of Fig 1C :an be modified to provide an internal reference as shown in Fig ID by coating only half the waveguide 32 with sensing region 34.

EXAMPLES

An example of how to make a sensor in accordance with the invention is set out hereunder. A glass or quartz slide or waveguide is cleaned with 1% nitric acid (HN0₃) and then silanated in a 20% solution of 3-amihopropyl-triethoxysilane (APTS) in acetone.

It is then washed with acetone and water. The slide/waveguide is then placed in a 50% aqueous solution of glutaraldehyde for about 5 hours. Thereafter, it is washed in a carbonate buffer at a pH of 9.6. The buffer is 0.05M sodium carbonate. The slide or waveguide is then placed into a solution of cocaine antibody (lg/L in the pH 9.6 buffer) for 15 hours. It is then washed with pH 7.4 buffer (0.05M sodium phosphate) . At this point in the sequence, bovine serum albumin (BSA, 1% in carbonate buffer, pH 9.6) is usually added as a "blocking agent". However, this step may be eliminated if the purity of the system can be controlled at satisfactory levels. The slide or waveguide is then placed into tagged antigen (drug) (300nM in phosphate buffer at pH 7.4) for about 1 hour. For greater sensitivity, an optical isolator such as active carbon powder can be used at this step.

The slide or waveguide is then washed with a pH 7.4 buffer until a stable fluorescence baseline is obtained indicating that no free tag is present.

Certain criteria are used for selecting the tag. In this present invention, fluorochrome was used as the tag. However, in other examples, iodine, biotin, enzymes and the like can be used. In selecting a tag, the following consideration should be taken into account:

1. Materials that produce a good optical signal should be selected.

2. Materials with a high fluorescence quantum efficiency should be selected.

3. Materials which have a long lifetime, namely, materials that do not easily bleach, should be used.

4. Materials which excite and emit at suitable wavelengths should be selected. In other words, where good LED's and detectors are available.

5. Materials with good binding groups, to attach to the antigen, are preferred.

6. Materials should be used having a proper chemical structure to match the antigen as well as having the optimum antibody-antigen binding site for the tag and minimum interference from steric hindrance.

The competitive immunoassay described in this application is designed to be as general as possible. It will work for any reaction whether it is an antigen with matching antibody and a site for attaching a tag. Sensitivity advantages should be available in all situations whether size, shape and other characteristics of the antigen and tag do not hinder the interaction of the target molecule with the sensing system.

Changes and modifications in the specifically described embodiments can be carried out without departing from the scope of the invention which is intended to be limited only the scope of the appended claims.

Claims

1. A method for increasing the sensitivity of a competitive immunoassay in which untagged antigen replaces tagged antigen bound to an antibody, comprising: decreasing the binding of the tagged antigen to the antibody relative to the binding of untagged antigen to the antibody to increase the exchange rate with untagged antigen.

2. The method of Claim 1 wherein the binding is decreased by forming the tagged antigen of a suitable size.

3. The method of Claim 2 wherein the tagged antigen is formed by attaching a suitably sized tag to the antigen.

4. The method of Claim 2 wherein the tagged antigen is formed by attaching a tag to the antigen through a long chain high molecular weight compound.

5. The method of Claim 2 wherein the tagged antigen is formed by attaching a long chain high molecular weight compound on the antigen molecule in addition to a tag.

6. A method for competitive immunoassay, comprising: immobilizing an antibody on a substrate; attaching an indicator tag to an antigen to produce a tagged antigen; binding tagged antigen to the immobilized antibody; contacting the immobilized antibody and bound tagged antigen with a sample containing untagged antigen so that the untagged antigen displaces tagged antigen; measuring an optical change produced by the change in amount of tagged antigen bound to the antibody; wherein the tagged antigen is formed to decrease the binding of the tagged antigen to the antibody relative to the binding of untagged antigen to the antibody.

7. The method of Claim 6 further comprising selecting the tag of a sufficient size.

8. The method of Claim 6 further comprising selecting the indicator tag from fluoresceinthiocarbamyl ethylenediamine, rhodamine B isothiocyanate, eosin-5-isothiocyanate, malachite green isothiocyanate, rhodamine X isothiocyanate, Lissamine_TM rhodamine B sulfonyl chloride, 6-carboxyrhodamine 6G hydrochloride, 5- (and-6) -carboxy-X-rhodamine, 6- (fluorescein-5- (and -6)-carboxamido)hexanoic acid, succinimidyl ester, Texas Red® sulfonyl chloride.

9. The method of Claim 6 wherein the indicator tag is attached to the antigen through a long chain high molecular weight compound.

10. The method of Claim 6 further comprising attaching a long chain high molecular weight compound to the antigen in addition to the indicator tag.

11. The method of Claim 6 further comprising pretreating the immobilized antibody and bound tagged antigen to remove unbound tagged antigen prior to contacting with the sample.

12. The method of Claim 6 further comprising forming an optical isolation layer on the substrate with immobilized antibody.

13. The method of Claim 6 further comprising binding tagged antigen to substantially all antibodies immobilized on the substrate.

14. The method of Claim 13 further comprising binding an inert blocking compound to any antibodies to which tagged antigen is not bound.

15. The method of Claim 6 further comprising selecting the antigen from cocaine, morphine or heroin.

16. A method for immunoassay, comprising: immobilizing an antibody on a substrate; attaching an indicator tag to the antibody to produce a tagged antibody; contacting the tagged antibody with a sample containing an antigen so that the antigen binds to the tagged antibody; measuring an optical change produced by the amount of antigen which binds to the tagged antibody; wherein the tagged antibody is formed to increase sensitivity to the antigen.

17. The method of Claim 16 wherein the tagged antibody is formed with a tag of sufficient size, shape and the like and the tag is attached in a manner to increase sensitivity to the antigen.

18. A competitive immunoassay sensor, comprising: a waveguide; an antibody immobilized on the waveguide; a tagged antigen bound to the antibody, the tagged antigen comprising an antigen and an active indicator material, the tagged antigen having sufficient size to lower the binding energy to provide a desired level of sensitivity to untagged antigen; means associated with the waveguide for detecting an optical change produced by a change in the amount of tagged antigen bound to the antibody when the antibody with tagged antigen contacts a sample containing untagged antigen.

19. The sensor of Claim 8 further comprising a distorting compound through which the active indicator material is attached to the antigen.

20. The sensor of Claim 9 further comprising a distorting compound attached at a separate location on the antigen from the active indicator material.

21. The sensor of Claim 18 wherein the active indicator material has sufficient size to form the tagged antigen of sufficient size.

22. The sensor of Claim 21 wherein the active indicator material is selected from fluoresceinthiocarbamyl ethylenediamine, rhodamine B isothiocyanate, eosin-5-isothiocyanate, malachite green isothiocyanate, rhodamine X isothiocyanate, Lissamine™ rhodamine B sulfonyl chloride, 6-carboxyrhodamine 6G hydrochloride, 5-(and-6) -carboxy-X-rhodamine, 6-(fluorescein-5-(and -6)-carboxamido)hexanoic acid, succinimidyl ester, Texas Red® sulfonyl chloride.

23. The sensor of Claim 18 wherein the active indicator material is attached to the antigen through a long chain high molecular weight compound to form the tagged antigen of sufficient size.

24. The sensor of Claim 18 having a long chain high molecular weight compound attached to the antigen in addition to the active indicator material.

25. The sensor of Claim 18 further comprising an optical isolation layer formed on the waveguide.

26. The sensor of Claim 18 further comprising a plurality of different antibodies, each specific to a different antigen, and having a different active indicator material attached to each different antigen, immobilized on the waveguide.

27. The sensor of Claim 18 wherein the waveguide is an optical chip.