EP1352241A1

EP1352241A1 - Absorbance multiplex technology

Info

Publication number: EP1352241A1
Application number: EP01992259A
Authority: EP
Inventors: Bassam Damaj; Ali-Samer Al-Assaad
Original assignee: Biosignature Diagnostics Inc
Current assignee: Biosignature Diagnostics Inc
Priority date: 2000-12-21
Filing date: 2001-12-21
Publication date: 2003-10-15
Also published as: WO2002050537A1; CA2432398A1; AU2002232722A1

Abstract

A method of simultaneously detecting and quantitating multiple target molecules in a single reaction well of a reaction surface. Antibodies to different target molecules are bound to a reaction surface. A sample solution believed to contain the target molecules in added to the reaction surface and incubated. During incubation, the target molecules from complexes with the bound antibodies. After a washing step, an enzyme-bound antibody mixture is added to the reaction surface. The enzyme-bound antibodies have an affinity for the target molecule/bound antibody complexes. The sequential addition of specific enzyme substrates permits the detection of multiple target molecules without poisoning any of the enzymes. A kit for the simultaneous detection of multiple target molecules is also disclosed.

Description

TITLE OF THE INVENTION ABSORBANCE MULTIPLEX TECHNOLOGY

CROSS REFERENCE TO RELATED APPLICATION Related application: Under the provisions of 35 U.S.C. § 119(e), priority is claimed from Provisional Patent Application Serial No. 60/258,286 filed December 21,

2000.

TECHNICAL FIELD The present invention relates to immunodiagnostics. More specifically, the invention relates to an immunoassay method for simultaneously detecting and quantifying multiple target molecules from the same sample, in the same reaction well, and at the same time.

BACKGROUND The usefulness of solid-phase heterogeneous enzyme-linked immunosorbent assay ("ELISA") is well documented as a method of detecting single antigen or antibody measurements using enzyme-linked ligands to detect an antibody-antigen reaction. Briefly, in ELISA, a solid phase support is coated with an antibody having affinity for a target molecule. Alternatively, a test antigen having affinity for the target molecule is bound to the solid phase support. A solution suspected of containing the target molecule is introduced to the solution surrounding the bound antibody or antigen. After incubation, a wash is performed to remove unbound target molecules. Then, an enzyme-labeled ligand, which will bind to the target molecule/ bound antibody complex, is incubated with the sample. Typically, the enzyme-labeled ligand is an enzyme-labeled antibody. Commonly used enzymes include alkaline phosphatase, horseradish peroxidase, β-galactosidase, β- glucuronidase, luciferase, and urease. During incubation, the enzyme-labeled ligand binds the target molecule/bound antibody complex. Once a labeled ligand has been attached to the target molecule/bound antibody complex, a substrate, specific for the enzyme, is added to the solid phase support. Commonly used substrates include 3,3', 5,5'-tetramethyl benzidine ("TMB"), para- nitrophenyl phosphate ("PnPP"), o-phenylene diamine ("OPD"), 2, 2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) ("ABTS") o-nitrophenyl-D-galatopyranoside ("ONGP") and 5-bromo-4-chloro-3 indolyl phosphate ("BCIP"). When the substrate is added, the enzyme label converts the substrate, causing a color change that can be visualized with light microscopy. The presence of a color change indicates the presence of the target molecule and allows, for example, a health care provider to determine, assess and diagnose the disease level and severity.

ELISA has been used in a wide variety of immunodiagnostic applications such as serodiagnositics to detect antigens from a wide range of specific viruses, bacteria, fungi, and parasites. ELISA is also used to monitor factors involved in non-infectious disease such as hormone levels, hematological factors, serum tumor markers, drug levels and antibody levels. ELISA can be used to monitor treatment for an allergy or measure antibodies that develop in response to an infectious disease. Further, ELISA can be used to screen for chemicals in a biological sample or to screen for pesticides or microorganisms in soil or water samples.

Current absorbance immunoassay techniques are limited as they are only capable of detecting one target molecule at a time within any particular reaction well. Simultaneous detection of more than one target molecule in a single well using absorbance immunoassays is not well taught in scientific or patent literature. However, it is common for several antigenic substances or markers to be associated with a pathological or physiological disorder. Thus, in order to accurately diagnose or monitor a condition, a different sample and different immunoassay would have to be prepared for each target molecule to be detected. Similarly, scientific researchers are often concerned with more than one molecule of interest. The multitude of tests and samples increases the cost, time, and the possibility of analytical error. Thus, it would be advantageous to have an immunoassay for simultaneous detection of multiple target molecules within the same sample, in the same reaction well and at the same time.

DISCLOSURE OF THE INVENTION

The current invention detects and quantitates multiple target molecules from the same sample, in the same reaction well and at the same time. In general, antibodies to different target molecules may be bound to a reaction surface. A sample solution believed to contain the target molecules, for example, a biological specimen, may then be added to the coated wells and incubated. During incubation, the target molecules form complexes with the bound antibodies. After a washing step, an enzyme-bound antibody mixture may be added to the wells. The enzyme-bound antibodies have an affinity for the different target molecule/bound antibody complexes. The sequential addition of specific enzyme substrates permits the measurement and analyses of color changes using one wavelength, without poisoning any of the enzymes.

The invention may also be performed by coating the antigens directly to a reaction surface such as a coated or noncoated polystyrene well, or to a solid or semisolid phase support. After a washing step, an enzyme-bound antibody mixture may then be added to a single reaction surface. The enzyme-bound antibodies have an affinity to existing antigens. The mixture may be developed sequentially by the addition of specific enzyme substrates where changes in colors are measured and analyzed using one wavelength, without poisoning any of the enzymes.

A goal of the present invention is to provide a method for determining the level of several target molecules in the same sample, in the same reaction well and at the same time by a single absorbance immunoassay technique.

Another aim of the present invention is to provide a new, enhanced and modified enzyme-linked immunosorbent assay (ELISA) or EIA as a kit or individual reagents for the rapid diagnosis and/or prognosis of diseases or the evaluation of responses that involve the measurement of more than one target molecule which can be used on a routine basis in clinical or non-clinical laboratories. The same reaction may be used to identify unrelated target molecules from one individual to diagnose multiple diseases. The invention thus includes methods of making the kit according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention allows simultaneous detection of multiple target molecules within the same reaction well. As used herein, the terms "target molecule" or "marker" refer to substances detectable by the present invention. Target molecules include, but are not limited to, antigens, or fragments thereof, including antigens derived from microorganisms and other pathogens, antibodies or fragments thereof, including antibodies produced in response to antigens derived from microorganisms and other pathogens, tumor markers, oligonucleotides, nucleic acids, carbohydrates, proteins, chemicals, drugs, receptors, haptens, hormones, gamma globulins, allergens, viruses, virus subunits, bacteria, toxins such as those associated with tetanus and with animal venoms, enzymes, nucleic acid molecules including DNA fragments, RNA fragments, and artificial nucleic acid fragments, and self-antibodies generated in autoimmune disease, or any mix thereof.

Generally, absorbance immunoassays are only capable of detecting one target molecule at a time because the buffers and substrates used to react with one enzyme are often toxic to another enzyme. Several antigenic substances or markers are often associated with a pathological or physiological disorder and can be early indicators of disease. Hence, the accurate diagnosis of infection or disease may require several different specimens and several different screening processes. For example, a toxicology screening may search for several different chemicals or drugs in a blood or urine sample. Further, researchers are often interested in more than one target molecule. Thus, it would be advantageous to be able to test and detect several target molecules, including chemicals or drugs, within the same sample and in the same reaction well.

Although the invention is described herein using detection antibodies, or fragments thereof, those skilled in the art will appreciate that it is also applicable to any antagonist that irreversibly binds the target molecule, including antigens, primers, nucleic acids, or fragments thereof, that recognize specific proteins, markers, receptors or antibodies, or fragments thereof, to be detected. For example, if a particular nucleic acid sequence is the target molecule, an artificial or naturally occurring sequence having affinity for the target nucleic acid sequence may be used. The detecting sequence need not necessarily be complementary to the target sequence. The detecting sequence should have sufficient affinity for the target sequence so that the two sequences remain bound during the detection process.

When using antigens to detect target molecules, several antigens, each specific for a different target molecule of interest, are bound to a reaction surface. The antigen may be bound to any reaction surface, solid or semisolid phase support, including, but not limited to, polystyrene wells of microtitre plates, glass or plastic strips, glass or plastic beads, membranes, microplates, magnetic particles, latex particles, nitrocellulose particles, polyacrylamide beads, magnetic beads, polystyrene, polyurethane, agarose, collagen, gelatin, SEPHAROSE, SEPHADEX, SEPHARON, nylon, rayon, and test tubes. Preferably, the antigen is bound to a single reaction well of the reaction surface. The term "reaction well" is intended to include reaction wells, for example, in a microtitre plate, as well as any localized area on a reaction surface such that the multiple markers are detected and quantitated on the same general section. The present invention does not require that the antigen be bound to separated areas of a reaction surface based on the specificity of the antigen.

The reaction well having bound antigen may be washed and a sample believed to contain the target molecules may be introduced to the reaction well. The target molecules bind to the bound antigen in the reaction well. After a wash, a first enzyme-conjugated antibody having an affinity for a first target molecule and a second enzyme-conjugated antibody having an affinity for a second target molecule are added to the reaction well. After sufficient incubation and wash, an enzyme substrate specific for the first enzyme may be added and any enzymatic activity, for example, a color change, may be measured. After sufficient incubation and a wash, a second enzyme substrate specific for the second enzyme may be added and any color change may be measured. The changes in color that are visualized, for example, using dip sticks or light microscopy, indicate the presence of the target molecules.

An embodiment of the invention includes a method of simultaneously detecting and quantitating multiple markers in a single reaction well of a reaction surface. The reaction well may be coated with at least three molecules, each having an affinity for different markers. The molecules can be any antagonist having a binding affinity for the target molecules, including, but not limited to, single chain antibody fragments, antibody, antibody fragments, artificial antibodies, peptides, chemical entities, lipids, carbohydrates, artificial nucleic acid sequences, DNA, RNA, antigen or fragments thereof. Preferably, the molecules are antigen or antibody or fragments thereof. The reaction well or reaction surface may be coated with the molecules by any method known in the art. Optionally, the reaction surface having molecules bound thereon may be purchased commercially. For example, attachment to the reaction surface may be non-covalent wherein the molecule binds to the reaction surface through adsorption. Alternatively, the attachment may be covalent wherein the molecule is chemically coupled to the reaction surface with a linker molecule. A wash step may be performed after the reaction well is coated. A sample suspected of containing at least one marker may be added to the reaction well. As used herein, "sample" can be any sample of interest, including, but not limited to, a biological sample, including, but not limited to, serum, blood, urine or saliva, a soil sample, or any liquid sample, including water. Biological samples may be of either human or non-human origin. After incubation, the markers bind different, specific bound molecules. Preferably, a wash step may be performed after the bound molecule/marker complexes are formed to remove unbound molecules.

A solution of ligands may be introduced to the reaction well. The solution comprises a first ligand having an affinity for a first marker, a second ligand having an affinity for a second marker, and a third ligand having an affinity for a third marker. The first ligand may be conjugated to a first reactant, the second ligand may be conjugated to a second reactant and the third ligand may be conjugated to a third reactant. Preferably, the ligands are secondary antibodies, the reactants are enzymes, and the first reactant, second reactant and third reactant are different. However, the ligand may be any antagonist having a binding affinity for the reactant, including, but not limited to, single chain antibody fragments, antibody, antibody fragments, artificial antibodies, peptides, chemical entities, lipids, carbohydrates, artificial nucleic acid sequences, DNA, RNA, antigen or fragments thereof. The sample and solution are incubated for a sufficient time to allow the first marker to bind with the first conjugated ligand, the second marker to bind with the second conjugated ligand and the third marker to bind with the third conjugated ligand.

In a preferred embodiment, the first reactant is β-galactosidase and the first substrate is ONGP, the second reactant is horseradish peroxidase ("HRP") and the second substrate is ABTS, and the third reactant is alkaline phosphatase ("AP") and the third substrate is PnPP.

Preferably, a wash step occurs before the first marker is detected. The presence of the first marker may be detected by adding a first substrate, specific for the first reactant, to the reaction well. After incubation for a sufficient time to allow a reactant/substrate reaction, the reaction well can be assayed for reactant activity. When the reactant is an enzyme, the reactant activity is preferably a color change detectable with light microscopy. Preferably, a wash step occurs before the second marker is detected. The presence of the second marker may be detected by adding a second substrate, specific for the second enzyme, to the reaction well. After sufficient incubation, the reaction well may be assayed for a color change. Similarly, a wash step may occur before the third marker is detected. The presence of the third marker may be detected by adding a third substrate, specific for the third enzyme, to the reaction well. After sufficient incubation, the reaction well may be assayed for a color change. Although the identification of three markers is described, the present invention can be used to identify two or more markers of interest.

The invention also includes a kit for the simultaneous identification of multiple markers in a biological sample. The kit may include a reaction surface having a coating of at least three molecules immobilized thereon. Each molecule may be specific for a different marker of interest. The kit may also include a conjugated antibody solution including a first antibody having affinity for a first marker, a second antibody having affinity for a second marker, and a third antibody having affinity for a third marker. The first antibody may be conjugated to a first enzyme, the second antibody may be conjugated to a second enzyme, and the third antibody may be conjugated to a third enzyme. The kit may also include reagents for detecting the labeled antibodies, a first substrate specific for the first enzyme, a second substrate specific for the second enzyme and a third substrate specific for the third enzyme. In a preferred embodiment, the first reactant is β-galactosidase and the first substrate is GNGP, the second reactant is HRP and the second substrate is ABTS, and the third reactant is AP and the third substrate is PriPP. The invention further includes a method of making the kit and a method of using the kit to diagnose a disease or identify the presence of multiple target molecules.

The invention may be further understood by reference to the non-limiting examples set forth below. EXAMPLES

General Description of the Absorbance Multiplex Technology for Multiple Concomitant Antigens:

1. Conjugation of antibodies. Detection antibodies to anti-human IgG (antibody 1), anti-human IgA (antibody 2), and anti-human IgM (antibody 3) were conjugated to different enzymes. Antibody 1 was conjugated to horseradish peroxidase (HRP), antibody 2 was conjugated to alkaline phosphatase (AP), and antibody 3 was conjugated to β- galactosidase (β-Gal). Conjugation of antibodies to the enzymes was performed as follows: 5 mg/ml of the antibody solution was dialyzed in 0.1M phosphate buffer at pH 6.8 (PBS) overnight at 4° C. 0.5 mg of dialyzed antibody was added to 1.5 mg of the enzyme in 10 ml of 10 mM PBS. 80 μl 25% glutaraldehyde was added and mixed gently. The solution was let stand at room temperature for 2 hrs. The reaction was stopped by adding an equivalent volume (10 ml) of PBSLE (10 mMPBS containing 100 mMlysine and 100 mM ethanolamine). The solution was desalted with a SEPHADEX G25 column in PBSN (10 mMPBS with 0.05MNaN₃). 20 ml of enzyme-antibody conjugates were mixed with 40 ml of blocking buffer (0.17Mborate buffer containing 2.5 mMMgCl₂, 0.05% TWEEN20, 1 mM EDTA, 0.25% BSA and 0.05% NaN₃). 60 ml of the conjugates were filtered through a low-protein binding filter, Millex HV 0.45 μm, for sterilization and stored at 4° C. 2. Preparation of antibody-coated microtiter plates. Using multichannel pipettes,

200 μl of a mixture of the three non-conjugated antibodies (antibodies 1, 2, and 3) solution was dispensed at 2 μg/well in coating buffer (1.6 gNa₂CO₃, 0.2 gNaN₃, 2.9 gNaHCO₃ in 1 L of distilled sterile water, pH 9.6) into each well of a microtiter plate (MICRO WELL) (the number of antibodies added is proportional to the number of antigens that will be added to the plate). The plates were wrapped in plastic wrap to seal for 30 minutes at room temperature and incubated for 30 minutes at 37° C. Alternatively, the plates may be incubated overnight at 4-8° C. The antibody-coated plate was rinsed by flooding with washing buffer (20 mM TrisHCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) a minimum of three times. Each well was filled with blocking buffer (0.2% BSA, 0.01% Gelatin in TBS, pH 7.5) dispensed using multichannel pipettes and incubated 15 min at 37° C. The plate was rinsed three times with washing buffer and any residual liquid was removed by gently flicking it face down onto paper towels.

3. ELISA. Step I. 200 μl aliquots of 1/50 serum dilution in PBS of the test antigen sample solutions (patient serum or plasma enzyme) or the standard antigen dilutions were added to the antibody-coated wells and incubated for 30 minutes at 37° C. The plates were rinsed three times in wash buffer and residual liquid was removed by blotting.

Step II. 200 μl of specific antibody-enzyme conjugate, IgA-AP aliquots of 1/500 dilution, IgG-HRP and IgM-β-Gal aliquots of 1/250 dilution in diluent buffer (0.1% BSA, 0.01% Gelatin in TBS, pH 7.5) were added and incubated for 15 min at 37° C. The plate was washed as in Step I. Step III. 200 μl of substrate solution was added sequentially to each well depending on the enzyme activity used. First, β-Gal substrate (ONGP™) solution was added to a final concentration of 2mg/ml and incubated for at least 15 minutes in darkness at room temperature. Absorbance from wells was read at 405 nm. The test plate was washed as described in Step I. Second, HRP substrate (ABTS™) was added as a single reagent and incubated for 5 minutes in darkness at room temperature and wells were read at 405 nm. The test plate was washed as described in Step I. Finally, AP substrate (PnPP™) solution was added to a final concentration of 4X and incubated for 10 minutes in darkness at room temperature and absorbance from the wells was read at 405 nm. All readings were done using the OP SYS MR™ (Dynex Technologies) microtiter plate reader.

Example I

Sequential Assay of the Absorbance Multiplex Technology for Multiple Concomitant Antigens for IgG, IgM and IgA

Procedure The assay involves the simultaneous measurement of three immunoglobulin markers

(IgG, IgM and IgA) from the same sample, in the same reaction well and at the same time. The assay procedure was as described in the ELISA section. The method described in this invention permits the use of a single marker or several different markers or antibodies without having any effect on the assay performance when compared to the measurement of one marker per well as seen in Table 1. Reacti Bind 96-well Pierce EIA microtiter plates were coated and incubated with a mixture of capture IgG, IgM and IgA antibodies overnight at 4° C. Human serum was diluted and incubated for 30 minutes at 37° C, which was followed by the addition of the mixture of the detection conjugated antibodies. The plate was washed and excess unbound reagents were removed. The OD obtained from the sequential addition of the substrates on the solid phase was determined as seen in Tables 2a-2f. The signal for β-Gal substrate was read at 15 minutes, wells were washed to remove the enzyme, HRP substrate was then added and read at 5 minutes, then wells were washed to remove the enzyme, and then AP substrate was added and read at 5-10 minutes.

Results

The assay illustrates the ability to detect and quantitate at least three markers from the same sample, in the same reaction well and at the same time on a solid phase support following the addition of the specific sequence of substrates. The sequence of addition as shown in Tables 2a to 2f are as follows:

In series 1 (Table 2a), ABTS, the substrate for HRP, was added first. OD reading at 405nm was followed by a washing step. ONGP, the substrate for β-Gal, was added second and followed by a washing step after reading the OD at 405nm. PnPP, the substrate for AP, was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.038, 0.174 and 1.883 for ABTS, ONGP, and PnPP respectively as compared with 0.992, 1.025 and 1.816 obtained from reading the OD of the same marker plated individually in single wells. ONGP's OD was quenched almost 10 fold from 1.025 to 0.174 due to interference from previous enzymes or their buffers. In series 2 (Table 2b), ABTS was added first. OD reading at 405nm was followed by a washing step. PnPP was added second and followed by a washing step after reading the OD at 405nm. ONGP was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.039, 1.800 and 0.175 for ABTS, PnPP and ONGP respectively as compared with 0.992, 1.816 and 1.025 obtained from reading the OD of the same marker plated individually in single wells. ONGP's OD was quenched almost 10 fold from 1.025 to 0.175 due to interference from previous enzymes or their buffers.

In series 3 (Table 2c), PnPP was added first. OD reading at 405nm was followed by a washing step. ONGP was added second and followed by a washing step after reading the OD at 405nm. ABTS was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.947, 0.940 and 0.465 for PnPP, ONGP and ABTS respectively as compared with 1.816, 1.025 and 0.992 obtained from reading the OD of the same marker plated individually in single wells. ABTS's OD was quenched almost 2 fold from 0.992 to 0.465.

In series 4 (Table 2d), PnPP was added first. OD reading at 405nm was followed by a washing step. ABTS was added second and followed by a washing step after reading the OD at 405nm. ONGP was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.938, 0.482 and 0.170 for PnPP, ABTS and ONGP respectively as compared with 1.816, 0.992 and 1.025 obtained from reading the OD of the same marker plated individually in single wells. ONGP's OD was quenched almost 10 fold from 1.025 to 0.170 and similarly the ABTS's OD was quenched by almost 2 fold from 0.992 to 0.482.

In series 5 (Table 2e), ONGP was added first. OD reading at 405nm was followed by a washing step. PnPP was added second and followed by a washing step after reading the OD at 405nm. ABTS was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.089, 1.962 and 0.266 for ONGP, PnPP and ABTS respectively as compared with 1.025, 1.816 and 0.992 obtained from reading the OD of the same marker plated individually in single wells. ABTS's OD was quenched almost 4 fold from 0.992 to 0.266.

In series 6 (Table 2f), ONGP was added first. OD reading at 405nm was followed by a washing step. ABTS was added second and followed by a washing step after reading the OD at 405nm. PnPP was added third and the OD was read at 405nm. This sequential addition of substrates worked, as the OD values obtained at 405nm were 1.069, 1.282 and 1.727 for ONGP, ABTS and PnPP respectively as compared with 1.025, 0.992 and 1.816. This series showed OD levels of absorbance of all the three markers compatible with the OD readings seen in Table 1. Table 1 : Single marker data from single wells.

Table 1 shows the results obtained from wells containing single markers. Results in Table 1 for A, B and C are single readings given in terms of relative absorbance (% OD) at 405 nm. Av.= Average; Std. = Standard Deviation.

Table 2a: Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.

Table 2b: Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.

Table 2c: Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.

Table 2d: Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.

Table 2e: Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.

Table 2f: Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.

Legends Table 2.

• Table 2a: ABTS was added first, read at 405 nm and followed by a washing step. ONGP was added next, read at 405 nm and followed by a washing step. PnPP was added last and read at 405 nm. ODs were 1.038, 0.174 and 1.883 for ABTS, ONGP, and PnPP respectively as compared to the 0.992, 1.025 and 1.816 obtained from reading the single marker OD in Table 1.

• Table 2b: ABTS was added first, read at 405 nm and followed by a washing step. PnPP was added next, read at 405 nm and followed by a washing step. ONGP was added last and read at 405 nm. ODs were 1.039, 1.800 and 0.175 for ABTS, PnPP and ONGP respectively as compared to the 0.992, 1.816 and 1.025 obtained from reading the single marker OD in Table 1.

• Table 2c: PnPP was added first, read at 405 nm and followed by a washing step. ONGP was added next, read at 405 nm and followed by a washing step. ABTS was added last and read at 405 nm. ODs were 1.947, 0.940 and 0.465 for PnPP, ONGP and ABTS respectively as compared to the 1.816, 1.025 and 0.992 obtained from reading the single marker OD in Table 1.

• Table 2d: PnPP was added first, read at 405 nm and followed by a washing step. ABTS was added next, read at 405 nm and followed by a washing step. ONGP was added last and read at 405 nm. ODs were 1.938, 0.482 and 0.170 for PnPP, ABTS and ONGP respectively as compared to the 1.816, 0.992 and 1.025 obtained from reading the single marker OD in Table 1. • Table 2e: ONGP was added first, read at 405 nm and followed by a washing step. PnPP was added next, read at 405 nm and followed by a washing step. ABTS was added last and read at 405 nm. ODs were 1.089, 1.962 and 0.266 for ONGP, PnPP and ABTS respectively as compared to the 1.025, 1.816 and 0.992 obtained from reading the single marker OD in Table 1.

• Table 2f: ONGP was added first, read at 405 nm and followed by a washing step. ABTS was added next, read at 405 nm and followed by a washing step. PnPP was added last and read at 405 nm. ODs were 1.069, 1.282 and 1.727 for ONGP, ABTS and PnPP respectively as compared to the 1.025, 0.992 and 1.816 obtained from reading the single marker OD in Table 1.

Example II

Sequential Assay of the Absorbance Multiplex Technology for multiple concomitant antigens for TNF-alpha, TL-l-ALPHA-alpha, and IL-8

Procedure The assay involves the simultaneous measurement of three cytokine markers (IL-

1- alpha, IL-8 & TNF-a) from the same sample, in the same reaction well and at the same time. The assay procedure was as described in the ELISA section. The method described permits the use of a single marker or several different markers or antibodies without having any effect on the assay performance when compared to the measurement of one marker per well as seen in Table 3.

Reacti Bind 96 well Pierce EIA microtiter plates were coated and incubated with a mixture of capture IL-1 -alpha, IL-8 & TNF-a antibodies overnight at 4°C. Human serum was diluted and incubated for 30 minutes at 37°C, which was followed by the addition of the mixture of the detection conjugated antibodies as explained in Example I. The plate was washed and excess unbound reagents were removed. The OD obtained from the sequential addition of the substrates on the solid phase was determined and the signal for b-Gal substrate was read at 30 minutes, wells were washed to remove the enzyme, HRP substrate was then added and read at 30 minutes, then wells were washed to remove the enzyme, and then AP substrate was added and read at 30 minutes. The results are listed in Table 4. Results

The assay detects and quantitates at least three markers from the same sample, in the same well and at the same time on a solid phase support following the specific sequential addition of substrates as illustrated in Example I.

Table 3. Single marker data from single wells.

Table 3 shows the results obtained from wells containing single markers. Results in Table 3 for A, B and C are single readings given in terms of relative absorbance (% OD) at 405 nm. Av. = Average; Std. = Standard Deviation.

Table 4: Triple marker data from single wells.

Replicates

Reading A J Reading B Av. Std.

IL-1 -ALPHA 2.086 2.232 2.159 0.103

IL-8 1.767 1.798 1.783 0.022

TNF-α 0.850 I 0.7660 0.808 0.059

Table 4 shows the results obtained from wells containing triple markers. Results in Table 4 for A and B are single readings given in terms of relative absorbance (% OD) at 405 nm. Av. = Average; Std. = Standard Deviation.

Example HI

Sequential Assay of the Absorbance Multiplex Technology for multiple concomitant antigens for Adult Respiratory Disease Syndrome (ARDS)

Procedure The assay involves the simultaneous measurement of two chemokine markers (IL- 8

& MCP-1) from the same sample, in the same reaction well and at the same time. The assay procedure was as described in the ELISA section. The method described permits the use of a single marker or several different markers or antibodies without having any effect on the assay performance when compared to the measurement of one marker per well as seen in Table 5.

Reacti Bind 96 well Pierce EIA microtiter plates were coated and incubated with a mixture of capture IL-8 & MCP-1 antibodies overnight at 4°C. Human serum was diluted and incubated for 30 minutes at 37°C, which was followed by the addition of the mixture of the detection conjugated antibodies as explained in Example I. The plate was washed and excess unbound reagents were removed. The OD obtained from the sequential addition of the substrates on the solid phase was determined and the signal for b-Gal substrate was read at 30 minutes, wells were washed to remove the enzyme, HRP substrate was then added and read at 30 minutes, then wells were washed to remove the enzyme, and then AP substrate was added and read at 30 minutes. The results are listed in Table 6. Results

The assay illustrates the ability to detect and quantitate at least three markers from the same sample, in the same reaction well and at the same time on a solid phase support following the addition of the specific sequential addition of substrates as illustrated in Example I. Table 5. Single marker data from single wells.

Table 5 shows the results obtained from wells containing single markers. Results in Table 5 for A, B and C are single readings given in terms of relative absorbance (% OD) at 405 nm. Av. = Average; Std. = Standard Deviation.

Table 6. Double marker data from single wells.

Table 6 shows the results obtained from wells containing triple markers. Results in Table 6 for A and B are single readings given in terms of relative absorbance (% OD) at 405 nm. Av. = Average; Std. = Standard Deviation.

Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some exemplary embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions, and modifications to the invention, as disclosed herein, which fall within the meaning and scope of the claims are to be embraced thereby.

Claims

CLAΓMSWhat is claimed:

1. A method of simultaneously detecting multiple markers in a single reaction well of a reaction surface, said method comprising: providing a single reaction well having at least two molecules immobilized thereon, each molecule of said at least two molecules having a binding affinity for different markers of said multiple markers; introducing a sample suspected of containing at least one marker of said multiple markers to said single reaction well; contacting said sample in said single reaction well with a solution comprising a first ligand having an affinity for a first marker of said multiple markers, said first ligand conjugated to a first enzyme, a second ligand having an affinity for a second marker of said multiple markers, said second ligand conjugated to a second enzyme, wherein said first enzyme and said second enzyme are different; incubating said sample and said solution for a sufficient time to allow said first marker to bind with said first conjugated ligand and said second marker to bind with said second conjugated ligand; and detecting a presence of said at least one marker of said multiple markers, said detecting comprising sequentially adding to said single reaction well a first substrate specific for said first enzyme and then a second substrate specific for said second enzyme.

2. The method according to claim 1, wherein said solution further comprises a third ligand having an affinity for a third marker of said multiple markers, said third ligand conjugated to a third enzyme wherein said third enzyme is different from said first enzyme and said second enzyme, and wherein said detecting further comprises adding a third substrate specific for said third enzyme to said single reaction well after adding said first substrate and said second substrate.

3. The method according to claim 2, wherein said detecting further comprises : incubating said sample after adding said first substrate; assaying said sample for a change in enzymatic activity and then washing said sample before adding said second substrate; incubating said sample after adding said second substrate; assaying said sample for a change in enzymatic activity and then washing said sample before adding said third substrate; incubating said sample after adding said third substrate; and assaying said sample for a change in enzymatic activity after incubating said sample with said third substrate.

4. The method according to claim 3, wherein said assaying comprises reading absorbance from said single reaction well.

5. The method according to claim 2, wherein said solution further comprises a fourth ligand having affinity for a fourth marker of said multiple markers, said fourth ligand conjugated to a fourth enzyme.

6. The method according to claim 5, further comprising adding a fourth substrate specific for said fourth enzyme after adding said third substrate.

7. The method according to claim 3 , wherein said enzymatic activity comprises a detectable color change.

8. The method according to claim 2, wherein said first enzyme is β- galactosidase, said second enzyme is horseradish peroxidase and said third enzyme is alkaline phosphatase.

9. The method according to claim 8, wherein said first substrate is ONGP, said second substrate is ABTS and said third substrate is PnPP.

10. A method of simultaneously determining a level of several target molecules in a single biological sample, said method comprising: providing a single reaction surface with at least two different antibodies immobilized thereon, said at least two different antibodies each specific to a different target molecule of said several target molecules; incubating said at least two immobilized antibodies and a biological sample; introducing an enzyme-conjugated antibody mix including a first antibody having an affinity for a first target molecule of said several target molecules, said first antibody conjugated to β-galactosidase, and a second antibody having an affinity for a second target molecule of said several target molecules, said second antibody conjugated to horseradish peroxidase; and adding ONGP and determining a level of said first target molecule and then sequentially adding ABTS and determining a level of said second target molecule.

11. The method according to claim 10, wherein said enzyme-conjugated antibody mix further comprises a third antibody having an affinity for a third target molecule of said several target molecules, said third antibody conjugated to alkaline phosphatase.

12. The method according to claim 11, further comprising adding PnPP and determining a level of said third target molecule after determining said level of said second target molecule.

13. The method according to claim 12, wherein said determining comprises detecting enzymatic activity.

14. The method according to claim 13, wherein said enzymatic activity comprises a detectable color change.

15. A kit for the simultaneous identification of multiple markers in a biological sample, said kit comprising: a single reaction surface having a coating of at least three molecules immobilized thereon, each molecule of said at least three molecules specific for a different marker of said multiple markers; a conjugated antibody solution including a first antibody having affinity for a first marker of said multiple markers, said first antibody conjugated to a first enzyme, a second antibody having affinity for a second marker of said multiple markers, said second antibody conjugated to a second enzyme, and a third antibody having affinity for a third marker of said multiple markers, said third antibody conjugated to a third enzyme; and reagents for detecting said enzyme-conjugated antibodies, said reagents comprising a first substrate specific for said first enzyme, a second substrate specific for said second enzyme and a third substrate specific for said third enzyme.

16. The kit of claim 15, wherein said at least three molecules are antibodies or antigens.

17. The kit of claim 15, wherein said first antibody is conjugated to β- galactosidase, said second antibody is conjugated to horseradish peroxidase, and said third antibody is conjugated to alkaline phosphatase.

18. The kit of claim 17, wherein said first substrate is ONGP, said second substrate is ABTS and said third substrate is PnPP.

19. The kit of claim 15, further comprising one or more positive or negative control reagents.

20. The kit of claim 15, further comprising one or more buffer solutions.

21. A method of making a kit for use in simultaneously identifying multiple markers in a biological sample, said method comprising: providing a single reaction surface having a coating of at least three molecules immobilized thereon, each molecule of said at least three molecules specific for a different marker of said multiple markers; providing a conjugated antibody solution including a first antibody having affinity for a first marker of said multiple markers, said first antibody conjugated to a first enzyme, a second antibody having affinity for a second marker of said multiple markers, said second antibody conjugated to a second enzyme, and a third antibody having affinity for a third marker of said multiple markers, said third antibody conjugated to a third enzyme; and providing reagents for detecting said enzyme-conjugated antibodies, said reagents comprising a first substrate specific for said first enzyme, a second substrate specific for said second enzyme and a third substrate specific for said third enzyme.

22. The method according to claim 21, wherein said providing a single reaction surface having a coating of at least three molecules immobilized thereon comprises providing a single reaction surface having said coating of at least three antibodies or antigens thereon.

23. The method according to claim 21, wherein said providing a conjugated antibody solution comprises providing said conjugated antibody solution wherein said first antibody is conjugated to β-galactosidase, said second antibody is conjugated to horseradish peroxidase, and said third antibody is conjugated to alkaline phosphatase.

24. The method according to claim 23, wherein said providing reagents for detecting said enzyme-conjugated antibodies comprises providing ONGP as said first substrate, ABTS as said second substrate, and PnPP as said third substrate.

25. The method according to claim 21, further comprising providing one or more positive or negative control reagents.

26. A method of diagnosing a presence of multiple markers for at least one disease in a sample, said method comprising: providing a kit for simultaneously performing multiple simultaneous immunoassays, said kit comprising a single reaction surface having a coating of at least three molecules immobilized thereon, each molecule of said at least three molecules specific for a different marker of said multiple markers; introducing a sample to said single reaction surface, said sample suspected of containing said multiple markers; introducing a conjugated antibody solution to said single reaction surface, said conjugated antibody solution comprising a first antibody having affinity for a first marker of said multiple markers, said first antibody conjugated to a first enzyme, a second antibody having affinity for a second marker of said multiple markers, said second antibody conjugated to a second enzyme, and a third antibody having affinity for a third marker of said multiple markers, said third antibody conjugated to a third enzyme; and detecting a presence of said multiple markers.

27. The method according to claim 26, wherein said detecting comprises introducing reagents for detecting said enzyme-conjugated antibodies to said single reaction surface, said reagents comprising a first substrate specific for said first enzyme, a second substrate specific for said second enzyme and a third substrate specific for said third enzyme.

28. The method according to claim 27, wherein said detecting comprises reading absorbance from said single reaction surface.

29. The method according to claim 27, wherein said conjugated antibody solution further comprises a fourth antibody having affinity for a fourth marker of said multiple markers, said fourth antibody conjugated to a fourth enzyme.

30. The method according to claim 29, further comprising adding a fourth reagent of said reagents, said fourth reagent specific for said fourth enzyme.

31. The method according to claim 26, wherein said detecting said presence of said multiple markers comprises detecting a color change on said single reaction surface.

32. The method according to claim 27, wherein said first enzyme is β- galactosidase, said second enzyme is horseradish peroxidase and said third enzyme is alkaline phosphatase.

33. The method according to claim 32, wherein said first substrate is ONGP, said second substrate is ABTS and said third substrate is PnPP.