WO1998016829A1 - Organism-specific and allergen-specific antibody capture assay and compositions for detection of disease-causing organisms and allergens - Google Patents

Abstract

A new capture assay method employs novel compositions of reformulated antigens including epitopes specific for an organism that is a target of the assay, and epitopes specific for an allergen, wherein each antigen is present in equivalent amounts, and to which non-specific epitopes are added to remove non-specific binding as a confounding factor in the assay. The assay is suitable for detection of immunoglobulins directed to specific organisms, such as micro-organisms and parasites, and for allergens. For example, specific IgG in combination with IgE levels are used to detect Helicobacter pylori and Chlamydia pneumoniae and to monitor response to therapy.

Description

ORGANISM-SPECIFIC AND ALLERGEN-SPECIFIC ANTIBODY

CAPTURE ASSAY AND COMPOSITIONS FOR DETECTION

OF DISEASE-CAUSING ORGANISMS AND ALLERGENS

This invention relates to a new capture assay for detecting

immunoglobulins (antibodies) directed to specific epitopes, in particular epitopes

derived from organisms such as microorganisms and small parasites, and epitopes

specific for an allergen, to novel antigen compositions for use in such assays, and

to methods of preparing the compositions.

Assays to detect IgA, IgD, IgE, IgG and IgM antibodies directed to

specific epitopes have used either an entire organism, an entire antigen molecule,

mixtures of antigenic molecules, or antigenic portions of individual molecules as

part of the assays. Various degrees of purification of antigens have been

employed. Methods to detect antigen-antibody complexing from which the

presence of specific epitopes are inferred, are well known and include sandwich-

type immunoassays, capture-type immunoassays, competition-type

immunoassays, homogeneous-type immunoassays and others. (Tijssen, 1985)

However, significant problems with interspecies cross-reactivity and binding

among antibodies, prevent the specificity and sensitivity of immunoassays for

disease causing organisms from reaching acceptable levels.

Antibody capture assays are sometimes referred to as "reverse" ELISAs.

Antibody capture assays have some advantages over indirect enzyme-linked

immunosolvent assay (ELISA) methods, but the problem of cross-reactivity and

non-specific reactions has not been solved satisfactorily. Attempts were made in

some studies to block common epitopes by incubating an antigen on a solid phase with antiserum to related organisms, or by adding heterologous, heat denatured

organisms to consume antibodies reacting with common epitopes.

Immunological methods (assays) have been reported to detect the presence

of specific disease-causing or allergy causing organisms in biological fluids.

Diagnostic kits comprising antigen, antibody, label, and anti-antibodies have been

described based on some of the assays. Superficially, some of the assays seem

similar to each other and to methods of the present invention. However, more

sophisticated scrutiny reveals major differences in components of the assays,

specifically, the compositions of antigens used, mechanisms for blocking binding

sites on antigens or antibodies to increase sensitivity and specificity of the assays,

configuration of the elements of the assay relative to a support, and which element

is labeled, wherein "element" includes antigen, antibody, and an agent to capture

the antibody. An additional difference among immunological detection methods

is the type of antibody used or detected, e.g. type of immunoglobulin. Finally,

previous immunoassays designed to detect organisms causing disease or allergy,

to detect the presence of the associated disease, and to monitor the effects of

treatment, have not been clinically successful and have not been applicable in

general to a variety of organisms.

H. pylori, an organism associated with gastrointestinal disease including

gastric cancer, is one specific target of such assays. With regard to antigenic

compositions, "antigens" selected from H. pylori (initially termed C. pylori) have

included the entire organism, only the surface antigens, the outer membrane

proteins, and various fractions of antigenic proteins, wherein certain fractions are selected and/or pooled for use in an assay. Kits comprising antigen, antibody,

label, and anti-antibodies have been described. None of the reported assays are

acceptable for clinical use.

Alemohammad (1993) reviewed problems in developing non-invasive

methods to detect H. pylori. Major problems with ELISA were identified as "the

source, type and characteristics of the antigen used." Col. 3, lines 7-9. For

example, Evans et al. (1992) relate an antigen which is derived from C. pylori and

is said to be "purified," "high molecular weight," (300-700 kD) and "cell-

associated." In the Evans method, the antigens are immobilized on a solid

support, the antibodies in a biological sample to be tested complex with the

immobilized antigens, the antibody detected is IgG, and labeled anti-IgG is added

to detect the antigen-antibody complexes. Blocking of the non-specific binding to

the solid support is accomplished by providing excess of BSA (bovine serum

albumin). The "antigen" used in the assay is prepared by pooling fractions

selected on the basis of high molecular weight, and in a preferred embodiment,

urease activity. Evans erroneously suggested that mere presence of H. pylori

reactive IgG is indicative of disease. However, elevated levels of IgG, IgA or IgM

directed to H pylori in a biological fluid sample from a subject indicates only that

a subject is carrying the bacteria, not necessarily that the individual is the affected

with a disease related to the infection.

Hirschl (1990) attempted to detect H. pylori using crude, impure antigen

preparations (ultracentrifuged cell sonicates or acid glycine extracts) with a 120

kD protein for serodiagnosis. Only IgG was used in the assay. He acknowledged a theoretical advantage for using whole cell sonicates to expose a maximum

number of surface antigens, but stated that such compositions "increased the risk

of non-specific binding of immunoglobulins and of cross reactions with related

species." (page 512)

Newell (1990) prepared antigens from C. pylori by separating proteins by a

chromatographic technique, removing fractions responsible for non-specific cross-

reactions and optionally combining other fractions. Blaser (1989) proposed a

variety of fractional components of C. pylori as antigenic compositions, including

components from many strains. Cover (1995) advocated use of the tag A protein

or fragments thereof in an immunoassay for H. pylori.

Reid used an immunoassay configuration in which antigen is immobilized

on a support, then antibodies are added from a serum to be tested, than a labeled

anti-IgE is added. Reid believed an improvement was the use of a mouse

monoclonal antibody to human IgE. The problem of IgG interfering with the IgE

assay was addressed by "dilution, serial transfer, and cumulative counting."

(Abstract)

Even methods that focused on use of a library of isolated and purified

antigens specific for an organism, although an improvement on previously

reported methods, were not completely satisfactory. Calenoff (1996) did not

employ all, or even many, specific epitopes present in the organism because not

all the epitopes were included in the antigenic molecules selected. Also, some

methods of antigen preparation were not easily reproducible, e.g. if strains of

microorganism mutated, causing the appearance or loss of specific epitopes, new libraries may be needed and these were not easy to prepare.

With regard to immunoassays to detect agents causing other diseases or

deleterious conditions, Calenoff (1989) taught a method of assaying for allergen

specific IgE wherein the presence of specific IgE indicated the presence of an

allergy. In the assay, an allergen is bound to a solid support, then contacted with

serum from a subject. If allergen (e.g. mold) specific IgE is present in the serum,

the binding of IgE to the mold antigen is detected by labeled anti-E antibody.

Despite some progress in development of immunoassays for specific

diseases or conditions, or specific allergens, significant levels of misclassification

still plague the reported assays and make them suspect for clinical use to replace

more invasive and expensive diagnostic procedures (such as

esophagogastroduodenoscopy and biopsy for H. pylori detection). Therefore,

better methods are needed to increase the sensitivity and specificity of antigen-

antibody complexing assays to detect the presence of organism-specific or

allergen specific antibodies in a biological fluid, in particular microorganism-

specific and small parasite-specific antibodies. Improved assays for detecting the

presence of infecting or colonizing organisms and their deleterious effects require

different antigenic compositions, blocking or quenching mechanisms, and/or assay

configurations such as organization of elements in the assay relative to a support.

The assays also need to be accurate, reproducible, and relatively easy to prepare

and use.

SUMMARY OF THE INVENTION The present invention attacks existing problems in clinical immunoassays

by presenting new antigen compositions and a novel and effective solution to the

problem of cross-reactivity. A new capture antibody assay for immunoglobulins

is directed to epitopes that are specific to a target organism or allergen and are

immunoreactive. Novel antigen compositions for use in such assays; methods of

preparing the compositions; and methods for blocking reactions that would

decrease accuracy of the assay, are also aspects of the invention.

The present invention relates compositions neither taught nor suggested in

the art-compositions that include substantially all the antigenic sites (epitopes)

present in an organism, or allergen, reformulated with a quantitative factor so that

the epitopes are present in the compositions in approximately equivalent amounts,

so that specific epitopes that are present in relatively small quantities in the

organism or allergen have as good a chance of being discriminated as those

present in greater proportions. Labeling of antigens is indiscriminate, that is, all

antigenic molecules prepared from the target organism or allergen are labeled.

The improved performance of these compositions in immunoassays reflects a

greater number of specific epitopes available compared to other reported

compositions used for similar purposes. Compositions of the present invention

are also easier to prepare than other reported antigenic compositions because

extensive purification is not required, and the specificity of the epitopes does not

need to be predetermined.

The specific epitopes included in a mixture of antigens derived from the

target organism or allergen are expected to bind specifically to the antibodies in a biological sample, if indeed, the biological sample being tested has antibodies

which react with the specific epitopes. If the sample comes from a subject

infected or colonized with the target organism, and the subject has produced an

immunological response to the organism, immunoglobulins that recognize

epitopes on the organism should be present. For some diseases or conditions, the

mere presence of a target organism may not be sufficient to establish presence of

an associated disease or condition. In the case of gastric ulcers, for example, if H.

pylori has not produced an IgE-mediated immune response, the organism may be

present and detectable by specific IgG, but associated disease is absent as

evidenced by lack of IgE specific for H. pylori.

In addition to novel antigen compositions, another novel aspect of the

invention is the means by which non-specific epitopes present in a target organism

or allergen are prevented from diminishing the accuracy of the assay to detect

antibodies specific for the target organism or allergen. Nonlabeled reformulated,

or non-reformulated antigen is used to quench (block) the non-specific, antibody-

based recognition of a non-specific epitope or plurality of epitopes on a labeled

antigen of interest. To be effective, the non-labeled antigen shares non-specific

epitopes with the labeled antigens, i.e. has homologous epitopes. The presence of

non-specific epitopes on non-labeled antigen ties up non-specific binding sites and

reduces the chance of non-specific epitope binding of immunoglobulin to the non¬

specific epitopes on the labeled antigens. Recognition of non-specific epitopes is

thereby removed as a confounding factor in an assay for a specific organism.

Homologous non-specific epitopes are those that are structurally similar enough to successfully compete with non-specific epitopes on molecules from the target

organism for binding with antibodies in a biological sample. Whether to

reformulate the non-specific epitope mixture or use it without reformulation is

based on convenience and cost. It generally takes more material to achieve the

same quenching effect if the mixture is not reformulated. Also, adding too much

non-reformulated antigen may confound clear, reliable performance of the assays

of the present invention because some molecules may be "sticky" and when in

excess may act like glue attracting labeled molecules to the solid support.

The detection method of this invention comprises attaching an anti-

immunoglobulin to a support, generally an insoluble support, and exposing a

biological fluid sample to the support so that some or all antibodies of the fluid

can be captured by the anti-immunoglobulin of the support. Preferably the

quantity of anti-Ig applied is known. Monoclonal antibodies or polyclonal

antibodies that are affinity purified are suitable as anti-immunoglobulins, as are

other suitable materials such as protein A or G. Cross recognition of other Ig

types is minimized because only the Ig type corresponding to the binding agent on

the support is detected e.g. if anti-IgE is on the support, IgG will wash out before

the assay is evaluated. A preferred capture assay depends on a covalent linkage

between the capture antibody and the surface so washing the system does not

disrupt the binding.

After removing the uncaptured antibodies by washing away the biological

fluid sample, a mixture containing the labeled reformulated antigen and non-

labeled reformulated or non-reformulated antigen, the latter usually extracted from an antigen source or sources which is(are) closely related taxonomically to the

source of the labeled antigen, is added to the assay. The captured antibodies bind

the labeled antigen and unlabeled antigen of the antigen mixture. After allowing

non-bound antigen solution to wash away, only the labeled antigen is detected,

thereby identifying the presence of antibodies from the biological fluid sample

which are specific for epitopes characteristic of a particular target organism.

Another aspect of the invention is that a combination of values from

different immunoglobulin types is a preferred type of diagnostic algorithm, e.g.

combining IgG and IgE values. A preferred type of combination is to multiply

target specific levels of IgG by target specific levels of IgE.

To elaborate, aspects of the present invention include:

1. antigen extraction;

2. reformulating of the extracted antigen; and

3. a novel capture assay to detect antibodies specific for an individual

target organism, or allergen, an assay that employs non-specific epitopes from

organisms or allergens related to the target organism to address problems of non-

specificity that plague other immunoassays.

An "antigen" is a molecule containing one or more epitopes. As used

herein, antigen refers to proteins or protein-containing molecules. Antigens may

have a plurality of epitopes, that is, sites that individually complex with different

antibodies. Some epitopes are specific for an individual microorganism or other

antigen source; others are non-specific, that is, may appear on molecules provided

by other antigen sources. The invention uses all available specific epitopes on an antigen of interest, and blocks from the specific reaction, non-specific epitopes

which are detrimental to high sensitivity and specificity of the assay. However,

most of the antigens from a target organism have no specific epitopes or have only

a few, yet when an antigen (protein) mixture is reformulated in accordance with

the present invention so that the varieties of antigens (the various epitopes) are

present in approximately equivalent amounts, maximum strength of the antigen

compositions is achieved.

The specific antibody capture assay of the present invention employs a

labeled reformulated antigen and reliably and reproducibly quenches, by a novel

means, non-specific, antibody-based recognition. In addition, the reformulated

antigen mixture of the present invention has a larger specific epitope repertoire

than used in other methods. This large repertoire greatly increases assay

sensitivity while maintaining high specificity of an assay for these compositions.

The antigenic components are not required to be "purified" as known in the

art. Substantially all of an organism or allergen is used, where "substantially"

means that no parts or fractions of parts are routinely or necessarily removed. The

components are merely fractionated, and fractions pooled to enrich for proteins in

smaller concentrations. Because of the ease of preparing the compositions of the

present invention, rapid and reliable antigen adjustment is possible in case the

strain of microorganism changes. An aspect of the invention is that the

manufacturing process is standardized, resulting in batch to batch uniformity.

Antigen extraction and reformulation are achieved by the following steps: 1. extracting all soluble proteins from an antigen source;

2. fractionating the proteins;

3. mapping the fractionated proteins on SDS-P AGE;

4. determining relative amounts of individual proteins; and

5. combining sufficient and approximately equivalent amounts of the proteins into a composition. (The quantitative combining of fractionated proteins

to achieve approximately equivalent amounts of each component in the

combination is referred to as "reformulation").

More specifically, reformulating an antigen mixture is achieved by:

1. obtaining an antigen source, e.g. by culturing a microorganism of

interest;

2. extracting all or a representative fraction of the constituent proteins

of the antigen source;

3. fractionating the extracted proteins individually or in small groups;

4. determining the concentration of each protein within each fraction,

e.g. by analyzing bands resulting from SDS-polyacrylamide gel electrophoresis;

5. combining a sufficient quantity of each protein either by adding

individually purified proteins or by adding a predetermined quantity of a

fractionated protein mixture, wherein the quantity of the desired protein or

proteins within the mixture has been determined to afford an approximately

equivalent end concentration of each constituent protein after all proteins

originally extracted from the specific antigen source are remixed together, wherein

constituent is defined as belonging to or derived from the target organism/microorganism or allergen; and

6. labeling the reformulated antigen mixture.

The improved detection method of this invention is partially a result of

reformulating the protein composition of the microorganism to be used in an assay

so that all antigenic molecules or proteins of the microorganism are present in

approximately equivalent amounts in the reformulation. The proteins in this

reformulated composition are then labeled and used in the assays of this invention.

Improvement is also due to the method by which non-specific epitopes are

prevented from reducing assay accuracy.

In an embodiment, labeled, e.g. biotinylated, reformulated antigen mixture

is spiked with non-biotinylated quenching elements, that is, non-labeled, non¬

specific epitopes. If the spiked antigen mixture is used, the captured

immunoglobulins being held on the support bind protein molecules at both

specific and non-specific epitopes. But because the non-specific epitopes are also

found on the non-biotinylated (non-labeled) quenching proteins, and because the

quenching proteins exist in far greater quantities within the antigen mixture than

the corresponding labeled proteins, the quenching proteins preferentially bind to a

subgroup of the captured immunoglobulin molecules and are unrecognized by

analysis of the label. Conversely, those biotinylated molecules which possess

specific epitopes are bound to the captured immunoglobulin molecules, and are

recognized in later steps because they are labeled.

This invention provides a means for identifying recent or concurrent

presence of immunoglobulins in a biological fluid which react with epitopes that are specific or highly specific for an organism, as well as a method for measuring

and quantifying the biological response to the organism in a subject. The

biological fluid sample is selected from the group consisting of whole blood,

plasma, serum, sputum, urine, cerebrospinal fluid, intra-abdominal fluid,

intrathoracic fluid, pericardial fluid, joint space fluid, pustular fluid, tear fluid,

nasal secretions, sinus fluid, and abscess fluid. Specific immunoglobulins that are

detected by this method are IgA, IgD, IgE, IgG and IgM. Types of organisms and

allergens suitable to practice the invention include bacteria, chlamydia,

mycoplasma, protozoa, rickettsia, viruses, pollens, epidermal agents, mold spores,

foods, venoms and allergenic pharmaceutical agents. Helicobacter pylori and

Chlamydia pneumoniae are examples of specific suitable organisms from which

compositions of the present invention are prepared. Small multicellular organisms

such as intracorporal parasites also qualify as antigen sources for which there are

many antigens and many epitopes per antigen. Helminths such as flukes

(Trematodes), tapeworms (Cestodes), and roundworms (Nematodes) are examples

of suitable parasites. Pollens comprise Orchard Grass Pollen, Brome Grass

Pollen, Giant Ragweed Pollen, Pigweed Pollen, Smooth Alder Pollen and River

Birch Pollen.

Individually, and in combination, each of the aspects of this invention

increases the sensitivity and specificity of the assay for the presence of organism-

specific antibodies in a biological fluid. Benefits of this new assay include

detecting antibodies in the biological fluid of patients exhibiting symptomatology

and/or diseases of infectious origin in order to determine the source of the infection and the level of organism-specific immunoglobulin response in the

patient's fluid sample. Ease of implementation and uniformity of antigen assays

are other aspects of the invention.

A diagnostic kit useful for the practice of the present invention includes a

capture antibody coated microtiter plate; and, in separate containers: a calibrator

solution containing varying concentrations of analyte e.g. one with none and 4

with positive calibrator solutions, e.g. see Table 1 ; labeled (e.g. biotinylated)

reformulated antigen mixture with unlabeled quenching proteins; streptavidin-

alkaline phosphatase; 4-methylumbelliferyl phosphate; a wash buffer; a serum

diluent; a conjugate diluent; and a substrate buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of anti-immunoglobulin antibody

molecules (anti-Ig) coupled to a support, wherein the anti-immunoglobulin

antibody molecules are capable of capturing antibodies from a sample of

biological fluid antibodies that may carry both antigen specific (SBR) and non¬

specific epitope (NSBR) binding sites (receptors).

FIG. 2 is a diagrammatic representation of biological fluid sample

antibodies captured by anti-immunoglobulin antibody molecules coupled to a

support (FIG. 1). The captured antibodies depicted have either epitope-specific

(SBR) or non-specific epitope binding sites (receptors) (NSBR).

FIG. 3 is a diagrammatic representation of an immunoassay system

including labeled (*) antigen molecules obtained from a complex antigen source

or organism such as a bacterium. The antigen molecule (diagonal lines) may contain both specific (SE)(0) and non-specific (NSE) (dotted square) epitopes, or

all SE or all NSE. Mixed in with the labeled antigen molecules are antigen

molecules from other organisms or other complex antigen sources (solid) which

display non-specific epitopes similar to (homologous to) the non-specific epitopes

on the labeled antigen molecules. Both specific and non-specific epitopes from a

target organism can be bound by the immunoglobulin-anti-immunoglobulin

complex attached to the support, said binding taking place at the specific epitope

binding receptors (SBR) or the non-specific epitope binding receptors (NSBR)

respectively.

FIG. 4 is a diagrammatic representation of an embodiment of the

immunoassay of FIG. 3 in which an antigen molecule is labeled by biotin (*), and

the means to detect the label is via streptavidin (SA)/alkaline phosphatase (AP)

conjugated to the biotin and also indirectly to the antigen. S symbolizes the

substrate which when processed by the labeling reactions, produces fluorescence

(F) which is detectable by a fluorescent scanning device.

FIG. 5 is a graphical representation of the quantitative effects of various

non-specific epitope sources (NSE) on detection of H. pylori specific serum IgG.

The symbol D refers to non-biotinylated reformulated C. jejuni antigen; to non-

biotinylated reformulated Giant Ragweed antigen; O to non-biotinylated

reformulated C. jejuni and Giant Ragweed antigens; and M to non-biotinylated

reformulated H. pylori antigen.

FIG. 6 is a calibration (standard) curve derived by using sera with known

quantities in "Enteron units" of IgG specific to H. pylori. FIG. 7 is a graphical representation of joint levels of H. pylori (HP)

specific serum IgG (Y-axis) and IgE (X-axis) in "Enteron units" in serum obtained

from 96 subjects with HP related disease as determined by current standard

methods (esophagogastroduodenoscopy and biopsy). All subjects showed

evidence of H. pylori. The symbol M refers to subjects with duodenal ulcers, r to

subjects with gastric ulcer, and F to subjects with chronic gastritis.

FIG. 8 is a graphical representation of HP specific serum IgG levels

(Y-axis) and IgE levels (X-axis) in "Enteron units" in serum from 34 subjects

without the diseases defined in FIG. 7, although some subjects have evidence of

H. pylori infection based on IgG levels.

FIG. 9 is a graphical representation of the percent change of IgE times IgG

level [reduction (-), increase (+), or no change (0) (Y-axis)] a number of days after

cessation of anti-microbial therapy (X-axis) used in an attempt to eradicate H.

pylori. M represent subjects in which no HP organisms were detected

post-therapy by gastroscopy with biopsy or a urease assay; F represent subjects in

which HP was still detected post-therapy.

FIG. 10 shows IgE changes in the same subjects shown in FIG. 9.

FIG. 11 shows IgG changes in the same subjects shown in FIG. 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates novel antigen compositions and means for dealing

with the problem of non-specific epitopes reacting in an assay for a specific

organism or allergen, thereby reducing accuracy of tests for specific target

organisms or allergens. The compositions are used in the specific antibody capture assay of the present invention which comprises the steps of (i) attaching

an anti-immunoglobulin to a support, generally an insoluble support; (ii) capturing

antibodies of a biological sample fluid by complexing with the anti-

immunoglobulin antibodies (immunoglobulins) adhered to the support, said

complexing is achieved by exposing a biological fluid sample to the support for a

sufficient period of time to allow the isotype-specific antibodies within the

biological fluid sample to complex with the specific anti-immunoglobulin

molecules attached to the support, and then removing the uncomplexed fluid;

(iii) exposing a mixture comprising labeled reformulated antigen molecules

described herein, and nonlabeled reformulated or non-labeled non-reformulated

antigen molecules containing non-specific epitopes homologous to the non¬

specific epitopes of the labeled reformulated antigen, to the support for a period of

time sufficient to allow the antigen molecules to be bound by the matching type of

the captured immunoglobulins; (iv) removing uncomplexed antigen molecules;

and (v) detecting the presence of labeled antigen remaining on the support, from

which the presence of antibodies in the biological fluid sample that are specific for

the labeled antigen is determined, and the presence of the target organism or

allergen is inferred. The amount of antigen-specific antibody in the biological

fluid sample is determined.

FIGS. 1-4 illustrate a specific antibody capture assay. FIG. 1 depicts the

anti-immunoglobulin antibody coupled to a support (immobilized, complexed).

The anti-immunoglobulin antibody is capable of capturing both antigen-specific

immunoglobulin with specific binding sites for antigens of the target organisms or allergens and non-specific immunoglobulin with non-specific binding sites. A

single isotype or several isotypes originating in a particular mammal are suitable,

wherein immunoglobulin includes IgA, G, M, D, E antibodies. Several isotypes

may be analyzed together if their binding to specific epitopes is distinguishable.

Suitable anti-Ig includes a monoclonal antibody or affinity purified polyclonal

antibody specific for any type of Ig, e.g. A, G, E, M, or D, or immunoglobulin

binding proteins such as protein A and protein G. (Harlow and Lane, 1988) Each

anti-Ig antibody or immunoglobulin binding protein binds at the Fc portion of the

captured antibody from the biological fluid sample, leaving antibody Fab sites

available for antigen binding.

FIG. 2 is a schematic presentation of the anti-Ig of FIG. 1 complexed with

immunoglobulins that may have binding sites (receptors) specific for an epitope of

an antigen being assayed, (SBR) and binding sites (receptors) that are present in,

but non-specific for, the antigen of interest (NSBR). FIG. 2 depicts the binding

(capture) of mammalian antibodies which occurs when a biological fluid sample

containing antibodies capable of complexing with the anti-antibody molecule is

added to the support. Biological fluids are generally obtained from a mammal,

and include whole blood, plasma, serum, sputum, urine, cerebrospinal fluid, intra-

abdominal fluid, intrathoracic fluid, pericardial fluid and other fluids found in

various body cavities and spaces. FIG. 2 depicts a captured antibody capable of

binding an organism-specific epitope (SBR) and another captured antibody

capable of binding a non-specific epitope (NSBR).

FIG. 3 is a schematic presentation of the anti-Ig of FIG. 1 bound to a solid support, complexed with immunoglobulin molecules in the biological fluid sample

to be assayed, and also depicts the captured antibodies binding antigenic

molecules either by their specific epitopes (SE) or their non-specific epitopes

(NSE). Molecules may have epitopes specific for an organism or allergen that is

the target of an assay, as well as non-specific epitopes. Molecules from sources

other than the organism or allergen of interest are selected to contain non-specific

epitopes that are the same in terms of binding abilities, as the non-specific

epitopes on molecules from the target organisms or allergens. Shown both

floating free and attached to the immunoglobulin binding sites are antigen

molecules e.g. from H pylori, with both specific (SE)(0) and non-specific (NSE)

(dotted square) epitopes. One labeled molecule is shown only with NSE.

Unlabeled molecules with only non-specific epitopes are shown both floating free

and attached to immunoglobulin binding sites. If the NSE on these molecules tie

up the non-specific binding sites (NSBR), the NSE on the labeled molecules of the

target organism will not be able to bind. Therefore, binding by the molecules of

interest will be due to binding of the specific epitopes (SE). Because more non¬

specific epitopes are presented on unlabeled antigen molecules, the unlabeled

antigens are preferentially captured by the antibody having NSBR. Labeled

antigens which have no competition are bound via their specific epitopes to the

captured antibody. The quantity of non-specific antigen required for successfully

preventing the labeled, reformulated antigen from binding to captured antibody

when the two are mixed together is 10 to 1, 000-fold greater in quantity than the

corresponding labeled antigen. A preferred embodiment is a factor of about 70:1 for reformulated non-specific epitopes.

FIG. 4 is a schematic presentation of elements shown in FIGS. 1-3 with the

addition of the fluorescent labeling system fluorescent label (F) effected by biotin

(*), streptavidin (SA), alkaline phosphatase (AP), and substrate(S). Other labeling

systems are also suitable, such as radionuclides.

FIG. 5 is a graphical illustration of the quantitative effects of non-specific

antibody sources (NSE) that are useful for "spiking." Shown are curves for three

antigen sources: D represents values for non-biotinylated, reformulated C. jejuni

antigen; 0 for non-biotinylated, reformulated Giant Ragweed antigen; O for non-

biotinylated reformulated C. jejuni and Giant Ragweed antigens, and non-

biotinylated, reformulated H. pylori antigen. The X-axis relates the ratio of

spiking antigen to biotinylated reformulated H. pylori antigen.

The Y-axis depicts an H pylori specific IgG sample with an initial FSU

reading of 3300. As can be seen from the separate plots, the most distantly related

antigen source, Giant Ragweed, reaches a plateau sooner than the more similar

(more homologous) sources. This is because after the NSE in Ragweed occupy

matching antibody sites, addition of more Ragweed antigen has no further

quenching effects while more related sources to HP will tie up more NSBR

because they are likely to share more epitopes with HP.

The values on the curve for C. jejuni and Giant Ragweed antigen is not the

sum of the values for the two antigen sources because they share some non¬

specific epitopes that compete for some sites. FIG. 6 illustrates calibration of the fluorescent measuring system of the

capture antibody assay for H. pylori - specific serum IgG. The regression equation

is shown in the upper left hand corner of the figure. Similar calibration is used for

other Ig types. For IgE, undiluted ("neat") serum is generally suitable. Dilutions

are preferred for IgG, A, D and M.

The steps of the invention are further defined as follows:

I. Attaching Anti-Immunoglobulins To

A (Generally Insoluble) Support

An initial step in the assay of the present invention is adhering an anti-

immunoglobulin to a support; preferably a support that is insoluble in any of the

reagents used in the assay. Various types of insoluble surfaces provide a suitable

support for the anti-immunoglobulin. Considerations in selecting the insoluble

support are the ability of the support to bind the anti-immunoglobulin to the

surface, the absence of interference with the labeled antigen, substrate and

reagents, ease of use, and cost.

Organic and inorganic polymers, both natural and synthetic, are suitable as

a support. Examples of suitable polymers include polyethylene, polypropylene,

polybutylene, poly(4-methylbutylene), butyl rubber and other synthetic rubbers,

silicone rubbers and silastic polymers, polyesters, polyamides, cellulose and

cellulose derivatives (such as cellulose acetate, nitrocellulose and the like),

acrylates, methacrylates, vinyl polymers (such as polyvinyl acetate, polyvinyl

chloride, polyvinylidene chloride, polyvinyl fluoride, and the like), polystyrene

and styrene graft copolymers, styrene-acrylonitrile copolymers, rayon, nylon, polyvinylbutyrate, polyformaldehyde, and so forth. Other materials which are

suitable as a support are silica gel, silicon wafers, glass, paper, insoluble proteins,

metals, metaloids, metal oxides, magnetic materials, semi-conductive materials,

cements or the like. In addition are included substances that form gels, such as

proteins, gelatins, lipopolysaccharides, silicates, agarose, polyacrylamides,

polymers, or polysaccharides which form several aqueous phases such as dextrans,

polyalkylene glycols (alkylenes with 2 to 3 carbon atoms) or surfactants, e.g.

amphophilic compounds such as phospholipids, long chain (12-24 carbon atoms)

alkyl ammonium salts and the like.

Preferred supports of this invention comprise a polystyrene, styrene

copolymers including styrene-(vinyl monomer) copolymers such as styrene-

acrylonitrile copolymers, or polyolefins such as polyethylene and polypropylene,

and acrylate and methacrylate polymers and copolymers. The capture antibody or

antiserum is bound thereto by adsorption, ionic bonding, van der Waals

adsorption, electrostatic bonding, other non-covalent bonding. The antibody can

also be bound to the support by covalent bonding. A particularly advantageous

support for this invention comprises a polystyrene microtiter plate having a

plurality of wells. The well surface or plastic cup inserts therein can constitute the

antibody support. Most advantageously, the microtiter plate or the well inserts are

opaque to light so that excitation light applied to a well or fluorescence generated

in response thereto does not reach or influence contents of the surrounding wells.

With this system each well can be employed as a test system independent of the

other wells. II. Capturing Test Fluid Antibodies by the Anti-immunoglobulin Antibody Adhered to the Support

After coupling the anti-immunoglobulin to the insoluble support, e.g. a

microtiter plate well, the test subject's biological fluid sample (test fluid) is

exposed to the insoluble support. Table 1 shows an example of the layout of a

microtiter plate wherein the numbers in each square refer to samples, and "Calib."

and "Neg.cont." are internal standards.

Each test fluid is diluted with an appropriate immunoglobulin Assay

Diluent. The degree of dilution depends upon the total quantity of the type of

immunoglobulin sought within the biological fluid sample. In the microtiter plate

format described in part I herein, it is usually important not to have more antibody

in the sample than can be captured by the capture antibody or capture antisera

adhered to the microtiter well or other solid surface (approximately 0.2μg per

lOOμL well sample). For human serum IgA, IgD, IgG, and IgM, an appropriate

assay serum diluent is 10 mM Tris-HCl, pH 7.5, containing 600 mM NaCl, 4.0

mg/mL casein, 30.0 mg/mL PEG-4000 (polyethyleneglycol 4000) and 0.2 mg/mL

thimerosal. For IgE, an appropriate assay serum diluent is 14.3 mM Tris-HCl, pH

7.5, containing 214.5 mM NaCl, 5.72 mg/mL casein, 42.9 mg/mL PEG-4000 and

0.28 mg/mL thimerosal, although a preferred embodiment calls for using neat

serum. Other formulations are suitable for specific applications. The amount of

dilution varies depending on the immunoglobulin being targeted. For IgA, a

dilution factor for the test serum (fluid) is about 2,200-fold. For IgD, a dilution

factor for the test serum (fluid) is about 90-fold. For IgE, a dilution factor for the test serum (fluid) is about zero to 4-fold. For IgG, a dilution factor for the test

serum is 10,000-fold, with a range of 0-10,000 fold. For IgM, a dilution factor for

the test fluid is about 1500-fold.

The microtiter plate is washed 3 times with 300μL of Immunoassay Wash

Buffer per well (the Wash Buffer is 10 mM Tris-HCl, pH 7.5, containing 600 mM

NaCl, 2.0 mg/mL casein and 0.2 mg/mL thimerosal). lOOμL of each diluted or

neat test serum is added to the appropriate wells of the plate. The monoclonal or

affinity-purified polyclonal anti-immunoglobulin coupled to well surfaces

captures all or a representative amount of total targeted immunoglobulin in the test

sample. In the microtiter plate embodiment described, about 0.2μg of capture

antibody per well provided the best results. The plate is covered and incubated at

25°C for between 5 minutes-24 hours; preferably between 2 to 18 hours, and more

preferably about 4 hours. Then, the biological fluid sample is aspirated and the

microtiter plate is washed five times with the Immunoassay Wash Buffer.

III. Exposing Labeled Reformulated Antigen And Non-Labeled Nonspecific Antigen To The Captured Antibody On The Support

Labeled reformulated antigen and nonlabeled, non-specific antigen which

share homologous epitopes with the labeled reformulated antigen, are added to the

microtiter plate wells. Preferably labeled and non-labeled antigen are mixed prior

to being added to the wells. The epitope-specific capture sites of the bound

isotype-specific antibodies bind with the labeled reformulated antigen-specific

epitopes, whereas the non-specific epitope capture sites bind with the non-specific

unlabeled antigen epitopes. A. Preparing the Reformulated Antigen Composition

An important and novel aspect of this invention is the use of a composition

which is an antigen mixture which has been reformulated so that all antigenic

molecules are equally represented in the antigen mixture. The preferred method

for making this reformulated antigen mixture involves obtaining (purchasing or

culturing) a specific organism, microorganism or microorganisms or parasite, or

allergen. All or a substantial fraction of soluble proteins are extracted from the

organism or allergen. The extracted proteins are then separated from the initial

mixture into smaller groups by fractionation based on their solubility, size, ionic

charge or other chemical characteristics. Finally, the fractionated proteins are

reformulated into a new mixture where the quantitative presence of each protein is

approximately equivalent to that of the other proteins in the mixture. The

resulting reformulated antigen mixture is labeled and used in the isotype-specific

antibody assays described herein. Preferably, the reformulated antigen mixture is

combined with the quenching proteins (non-specific antigen) described herein and

used in the assay of this invention. The non-specific antigen may be reformulated

by the same procedures from a different antigen source than the target organism or

allergen.

B. Labeling the Reformulated Antigen from the Target Organism or Allergen

There are many suitable labels to attach to the reformulated antigens from

a target organism. A preferred method of labeling reformulated antigen is

biotinylation. However, the proteins can also be labeled with, by way of example, enzymes, fluorophores, chromophores, and radionuclides.

Biotinylation is accomplished by selecting the appropriate quantity of

concentrated reformulated antigen extract and reacting with NHS-LC-biotin. A

2.5 x 16 cm Sephadex G-15 column is equilibrated with at least 800 mL

Biotinylated Antigen Storage Buffer (consisting of 10 mM Tris-HCl, pH 8.0 +

0.1% SDS + 0.02% NaN3 + 1 mM EDTA + 1 mM EGTA) at a flow rate of

approximately 10 mL/min. Reformulated antigen extract is centrifiiged at 100,000

x g for 30 minutes at 4°C. The supernatant is collected, the pellet(s) is discarded,

and the protein concentration of the collected supernatant is determined using the

Lowry protein assay. (Waterborg and Matthews, 1994)

The amount of NHS-LC-biotin required for the reaction is calculated.

Molar ratios of 5:1 to 70:1 biotin to protein are suitable, but a ratio of

approximately 20:1 is preferred. The average molecular weight of the protein

mixture is calculated by densitometry studies of PAGE gels containing the protein

mixture separated into individual protein bands ^Peck et al, 1988). For example,

the average estimated molecular weight for Helicobacter pylori proteins in the

reformulated protein mixture is about 60,000.

The pH of the antigen extract supernatant is adjusted to 8.70+0.10 with 0.1

M sodium hydroxide solution. The calculated amount of NHS-LC-biotin is added

to the antigen extract supernatant. The mixture is capped tightly and vortexed

immediately. The reaction mixture is placed on a rotating mixer and incubated at

ambient temperature for 45 minutes. At the end of the 45 minute reaction time,

solid Tris base is added to the reaction mixture to a final concentration of 0.5 M (60 mg Tris base per mL biotinylated antigen solution). The container is capped

tightly and vortexed immediately. The reaction mixture is placed on a rotating

mixer and incubated at ambient temperature for 10 minutes.

The reaction mixture is applied to the equilibrated Sephadex G-15 column

at a flow rate no greater than 5 mL/min, collecting fractions of approximately 4

mL. The OD280 of each fraction is determined and the elution profile is plotted.

The biotinylated antigen-containing fractions are pooled into a suitably sized

graduated cylinder. (These comprise the protein-positive, e.g. the OD280-

positive, fractions.) The protein concentration of the pool is determined using the

Lowry protein assay. The biotinylated antigen mixture is then aliquoted and a

sufficient quantity is added to the support to bind with the anti-immunoglobulin-

immunoglobulin complex. Alternatively, the labeled reformulated antigen can be

frozen at -20°C or lower for later use.

C. Applying the Labeled Reformulated Antigen To the Support To apply the labeled reformulated antigen to the support, wherein the

support is a microtiter plate, lOOμL of Biotinylated Antigen Mixture is added to

each test well on the plate, covered and incubated at 25°C for 75 minutes. For

IgA, IgD, IgG and IgM antibodies, the Immunoassay Antigen Mixture suitably

comprises about 1 -4μg/mL biotinylated reformulated antigen, with or without

quenching proteins, in a solution comprising 10 mM Tris-HCl, pH 7.5, 600 mM

NaCl, 4.0 mg/mL casein, 30.0 mg/mL PEG-4000 and 0.2 mg/mL thimerosal. For

IgE, the Immunoassay Antigen Mixture suitably comprises 2-10μg/mL

biotinylated reformulated antigen, with or without quenching proteins, in a solution comprising 10 mM Tris-HCl, pH 7.5, 600 mM NaCl, 4.0 mg/mL casein,

30.0 mg/mL PEG-4000 and 0.2 mg/mL thimerosal. The labeled antigen solution

is incubated for a time ranging from 2 minutes to 24 hours. A range of 2 to 180

minutes is suitable. In a preferred embodiment of this invention the incubation

time is 75 minutes.

When using a reformulated antigen mixture from a target organism with

quenching ingredients (generally proteins), some of the captured antibodies being

held on the support bind antigen molecules at their specific epitopes and others

bind antigens at their non-specific epitopes. Because the non-specific epitopes are

also found on non-biotinylated (unlabeled) quenching antigens, and the quenching

antigens exist in far greater quantity within the complex mixture than do the

labeled antigens, the non-labeled antigens preferentially bind to the captured

immunoglobulin molecules which have affinity for the non-specific epitopes. The

bound quenching antigens go unrecognized because they are not labeled. The

biotinylated reformulated antigens which possesses specific epitopes bind to other

captured immunoglobulin subgroups and are recognized in later steps because

they are labeled.

The microtiter plate is washed five times with the Immunoassay Wash

Buffer (comprising 10 mM Tris-HCl, pH 7.5, containing 600 mM NaCl, 2.0

mg/ml casein and 0.2 mg/mL thimerosal). lOOμL of a diluted

streptavidin/alkaline phosphatase conjugate mixture is added to each well. The

streptavidin/alkaline phosphatase binds to the available biotin. For IgA, IgD, IgG,

and IgM antibodies, the conjugate mixture comprises 0.75 ng/mL streptavidin/alkaline phosphatase conjugate, in a solution comprising 10 mM Tris-

HCl, pH 7.5, 600 mM NaCl, 4.0 mg/mL casein, 30.0 mg/mL PEG-4000 and 0.2

mg/mL thimerosal. About 75 ng of streptavidin/alkaline phosphatase conjugate is

added per well. For IgE, the conjugate mixture comprises 2.75 ng/mL

streptavidin/alkaline phosphatase conjugate, in a solution comprising 10 mM Tris-

HCl, pH 7.5, 600 mM NaCl, 4.0 mg/mL casein, 30.0 mg/mL PEG-4000 and 0.2

mg/mL thimerosal. About 275 ng of streptavidin/alkaline phosphatase conjugate

is added to each well. The plate is covered and incubated at 25°C for 5 to 60

minutes. A preferred embodiment is 15 minutes. The microtiter plate is washed

six times with an Immunoassay Wash Buffer.

IV. Adding The Non-Specific Antigens

Another aspect of this invention is the use of non-specific antigens to

quench non-specific epitopes being bound by the captured, biological fluid

immunoglobulin. The molecules with non-specific antigens to be added are non-

labeled and are selected to have epitopes in common with (that is, homologous to)

non-specific epitopes on the specific antigen(s) of interest. Homologous is

defined herein to mean alike or corresponding for characteristics relevant to the

present invention, e.g. derived from a single or taxonomically related individual so

that antigenic composition is similar, that is, there are some shared epitopes that

can compete successfully for binding sites on antibodies.

Choice of non-specific epitope depends on the antigen source. For

example, the non-specific epitope mixture ("sister proteins" or "matching

proteins") is determined for microorganisms e.g. by using taxonomic relationships to determine the best "quenching" in a known assay with appropriate controls.

The purpose of the non-labeled proteins is to block the binding of the non-specific

epitopes on the labeled antigens of interest. Antibodies in a subject's serum or

other biological fluid which could bind to these homologous epitopes and thereby

effect a non-specific recognition, instead bind to the corresponding non-specific

epitopes on the nonlabeled proteins and are thereby not recognized during the

assay procedure. Therefore, it is preferred that an antigen source for these proteins

is related to the antigen source for the labeled reformulated proteins, although

homologous molecular relationships can exist among sources of antigen which

have distant taxonomic relationships one to the other.

The most direct way to extract proteins from the related or distant antigen

source is in the same manner described for reformulating the target antigen

described herein. Nonlabeled proteins can then be used to quench the non¬

specific, antibody-based recognition of the specific microorganism. The quantity

of quenching proteins added to the assay varies depending on differences in

antigen composition between individual organisms or other complex antigen

sources. Antigen inhibition studies are the best way of determining how much

spiking or quenching unlabeled antigen must be added to the labeled antigen. A

suitable amount of spiking antigen(s) is that which provides maximum inhibition

of non-specific epitopes. A gradual drop in the assay signal is observed until no

more reduction is possible. The quantity of spiking antigen which gives the

lowest reduction possible is that which is added to the labeled antigen. FIG. 5

shows examples of antigen inhibition for H. pylori. V. Reading The Result

The final step of this assay is detecting the binding of labeled reformulated

antigen to the complex on the support, from which the presence of antibodies in

the biological fluid tested that are specific for the antigen, is inferred. If

biotinylated antigen and streptavidin-alkaline phosphatase has been used, 1 OOμL

4-methylumbelliferyl phosphate solution is added to each well, and the plate is

read in a fluorescence microtiter plate reader, using 365 nm excitation and 450 nm

emission, at 15 minute intervals for 1 hour. Standard curves are drawn using the

test results of calibrator reagents which are run in parallel with the test sera or

other biological fluid test samples. The unknown test results are plotted onto the

standard curve, thereby ascertaining individual test values. (FIG. 6 represents a

calibration curve for H. pylori IgG.)

If labels other than biotin are added to the reformulated antigen mixture,

then the steps for identifying and reading the labels or markers are modified as

appropriate and are known to those of skill in the art. These other labeling

methods are adequately described in Tijssen (1985) and can be used as an

alternative labeling method.

EXAMPLES

The following examples illustrate the method of extracting and

reformulating proteins from organisms such as microorganisms, parasites,

allergens and various other antigen sources. Those of ordinary skill in the art

would recognize that these examples can be modified to make and reformulate

proteins from other organisms and antigen sources. They would also recognize that other methods can be used to reformulate the protein composition of an

organism so that all antigenic molecules are present in approximately equivalent

amounts.

Example 1: Helicobacter pylori Antigen Extraction and Reformulation Procedure

The first step requires obtaining the source for the antigen. Generally,

Helicobacter pylori organisms are cultured to a desired quantity, generally about

150g. Techniques for culturing varying quantities of H. pylori are described by

Deshpande, et al. (1995). Organisms are washed and concentrated using cold

normal saline and a tangential flow concentrator. Organisms are then pelleted by

centrifugation and frozen until needed.

To extract the protein from the H. pylori, the desired quantity of frozen H.

pylori is added to an appropriately-sized, heat resistant container or beaker. For

each gram (wet weight) of frozen Helicobacter pylori, 10 mL of a solution

comprising 20 mM sodium phosphate buffer, pH 7.35, with 100 mg/mL octyl-

beta-D-glucopyranoside (or other functional detergent) is added. The beaker is

placed on a stirring hot plate, a stir bar is added and the mixture is stirred, and

suspension is slowly warmed at approximately 1°C per minute, to 25°C. The

heating element is then turned off but stirring continues while clumps of

organisms are broken up by using a handblender for 90 ± 10 seconds, until a

uniform suspension is achieved. The suspension is stirred for 1 hour at ambient

temperature. The homogenate is centrifuged at 100,000 rpm for 30 minutes at

4°C. The supernatant is collected and the pellet(s) are discarded. If multiple centrifuge runs are required, the collected supernatant is held at 4°C and stirring

continues for the remaining suspension at ambient temperature. After centrifuging

is completed, the total supernatant volume collected, measured and recorded.

A number of dialysis bags is prepared, using 3500 MWCO dialysis tubing,

which is sufficient to dialyze the accumulated supernatant. The H. pylori extract

is dialyzed against 20 mM sodium phosphate buffer, pH 7.35, at 4°C, using a

dialysate to bag volume ratio of at least 13:1, or no more than 300 mL bag volume

in 4 L of dialysate. The dialysate is changed four times allowing at least 4 hours

between changes. The dialyzed H. pylori antigen extract is collected in a suitably

sized graduated cylinder, and the volume is recorded.

The next step involves fractional acetone precipitation of the antigen

extracts. This step begins by measuring the tare weight of an appropriate number

of centrifuge bottles with caps. The weight is recorded on each bottle. The

dialyzed H. pylori antigen extract is transferred to a suitably sized screw capped

glass container; a stir bar is added and the mixture is stirred.

The amount of acetone to be added to yield a concentration of 20 %

acetone is calculated. The calculated amount of acetone is measured and added to

the H. pylori antigen extract. The mixture is capped tightly and stirred at 4°C for

15 minutes. An appropriate number of weighed centrifuge bottles is labeled with

the acetone percentage and an identification number, i.e. 20 % - 1, 20 % - 2, and

so forth. The H pylori antigen extract is transferred to the labeled centrifuge

bottles. The H pylori antigen extract is centrifiiged at 6800 rpm at 4°C for 30

minutes. The volume of the combined supernatants is collected and measured (if more than one container is used to centrifuge the acetone-supernatant suspension).

The pellets are held at 4°C. The steps of calculating the acetone and measuring it

into labeled centrifuge bottles are repeated, incrementally increasing the acetone

concentration as follows: 25, 30, 35, 40, 45, 50, 55, 60, 65, and 80 percent. The

80% acetone supernatant is discarded. All of the centrifuge bottles are transferred

to a chemical fume hood, the caps are removed and the residual acetone is allowed to evaporate at ambient temperature overnight.

Two 7.5% and two 12.5% SDS-polyacrylamide gels are prepared (Cooper,

1977).

The proteins found in the different acetone-precipitated fractions are

resolubilized and the constituent proteins in each fraction are identified and

quantified by SDS-Polyacrylamide Gel Electrophoresis (PAGE). Each centrifuge

bottle is capped and weighed. The net weight of each pellet and the volume of 20

mM sodium phosphate buffer, pH 7.35 required for reconstitution at a ratio of 20

mL buffer per gram of pellet are calculated. The calculated volume of 20 mM

sodium phosphate, pH 7.35 is added to each bottle. The capped centrifuge bottles

are placed on an orbital mixer and shaken vigorously at 4°C for 30 minutes. The

individual suspensions resulting from each acetone percentage are pooled into a

single centrifuge bottle. The capped centrifuge bottles are placed on an orbital

mixer and shaken vigorously at 4°C for 90 minutes.

Stacking gels for each of the previously prepared SDS-polyacrylamide gels

are cast and allowed to polymerize for at least 1 hour.

Each of the reconstituted solutions are centrifuged to remove any undissolved residue at 100,000 x g for 30 minutes at 4°C, in capped

ultracentrifuge tubes. For volumes greater than 240 mL, centrifuging is at 6800 x

g for 90 minutes at 4°C in capped bottles.

After centrifuging, the volume of the supernatant from each reconstituted

solution is measured and recorded. The supernatants are held at 4°C. The pellets

are discarded. The protein concentration of each supernatant is determined using

the Lowry protein assay.

An appropriate volume of each supernatant is prepared for SDS-

polyacrylamide gel electrophoresis by boiling the supernatant in sample buffer

containing 2-mercaptoethanol. A 10 to 50μL aliquot of each treated protein

solution, containing approximately 50μg of protein, is applied to individual lanes

of both 7.5% and a 12.5% SDS-polyacrylamide gels. Molecular weight standards

are added and electrophoresis is performed. The gels are stained and dried.

The final steps involve pooling and concentrating the proteins. Using gel

densitometry, the approximate protein concentration is determined for each visible

protein band seen on the SDS gel lane corresponding to each supernatant attained

through the process described above. A sufficient volume from each supernatant

is mixed to achieve approximately equivalent concentration of each of the

different proteins, visualized in the SDS gels, in the resulting protein mixture.

[Supernatants giving sharp clear bands on SDS-polyacrylamide gels are included

while supernatants giving diffuse, blurred bands (generally 35 to 45 % acetone-cut

fractions) are generally excluded from the pool.] A 5000 MWCO tangential flow

concentrator unit is equilibrated with 20 mM sodium phosphate buffer, pH 7.35, and the pool of reconstituted supernatants is concentrated using the equilibrated

concentrator unit until a protein concentration of 15-30 mg/mL is attained. The

protein concentration of the concentrate is determined using the Lowry protein

assay. The concentrated H. pylori protein mixture can be frozen until needed.

Example 2: Campylobacter Jejuni Antigen Extraction And Reformulation

Procedure

The method of making the reformulated H. pylori antigen mixture of

Example 1 can be used to make a reformulated Campylobacter jejuni antigen

mixture. A method for culturing C. jejuni organisms to the quantity desired for

this extraction and reformulation procedure is described by Rollins et al. (1983).

Example 3: Orchard Grass Pollen Antigen Extraction And Reformulation Procedure

Defatted Orchard Grass Pollen (raw antigen) used in this extraction and

reformulation procedure can be purchased from a variety of reliable sources, such

as Crystal Laboratories in Luther, Oklahoma. A sufficient quantity of raw

allergen is transferred to a suitably sized screw cap container containing of 50 mM

sodium phosphate buffer, pΗ 8.0 or other suitable buffer. The ratio of buffer to

defatted pollen is 10 mL buffer to 1 gram dry weight pollen. The container is

securely attached to an orbital mixer and the suspension is agitated vigorously for

48 hours at 4°C. Upon completion, the resulting suspension of crude extract is

transferred to a suitable centrifuge bottle(s), and the crude extract is centrifuged at

6500-6800 x g for 1 hour at 4°C. The supernatant is transferred to a suitably sized

graduated cylinder. A small amount of pellet material may be included with the

supernatant. The pellet is discarded and the supernatant volume is recorded. The supernatant is transferred to dialysis bags prepared from pretreated 45

mm 3500 MW cutoff dialysis tubing. The supernatant is dialyzed against 50 mM

sodium phosphate buffer, pH 8.0, at 4°C at a ratio of not less than 20 mL of

dialysate per mL supernatant. The dialysate is changed four times allowing a

minimum of four hours between changes.

The dialyzed supernatant is transferred to a suitably sized graduated

cylinder so that it can be easily transferred to a suitable number of ultracentrifuge

tubes. The supernatant is centrifuged in the tubes at 100,000 x g for 1 hour at 4°C.

If multiple runs are required, the unused portion of the dialyzed supernatant is held

at 4°C. After each run, the supernatant is collected in a suitably sized graduated

container and the pellet is discarded. The collected supernatant should be free of

pellet residue. If the pellet is disturbed during supernatant collection, the tube

should be centrifuged again. The collected supernatant is held at 4°C until all

ultracentrifuge runs are completed. The total supernatant volume is recorded.

A 5000 MWCO tangential flow concentrator cassette is flushed with

purified water. The cassette is equilibrated with 50 mM sodium phosphate buffer,

pH 8.0. The ultracentrifuged supernatant collected above is diluted to a

concentration of 25-30 mg/mL. This concentrate may be frozen until needed or it

can be frozen after fully processed.

The next step involves fractional acetone precipitation of the antigen

extracts and can be performed in the same manner as described in Example 1 for

fractional acetone precipitating of the H pylori antigen extracts, and therefore, is

not repeated here. After fractional acetone precipitation of the Orchard Grass pollen antigen

extracts, the proteins are resolubilized in different acetone-precipitated fractions

and the protein constituents in each fraction are determined by SDS-

Polyacrylamide Gel Electrophoresis (PAGE). These steps can be performed in the

same manner as described in Example 1 , and therefore, are not repeated here.

The final steps involving pooling and concentrating the proteins. Using

gel densitometry, the approximate protein concentration of each visible protein

band seen on the SDS gel lane corresponding to each supernatant attained through

the work described above is determined. A sufficient volume from each

supernatant is mixed so as to achieve equivalent concentrations of each of the

different proteins visualized in the SDS gels in the resulting protein mixture. A

5000 MWCO tangential flow concentrator unit is equilibrated with 20 mM sodium

phosphate buffer, pH 7.35. With equilibration buffer circulating in the

concentrator unit, the system pressure is adjusted to 40 psi. The pool of

reconstituted supernatants is concentrated using the equilibrated concentrator unit

until a protein concentration of 25-30 mg/mL is attained. The protein

concentration of the concentrate is determined using the Lowry protein assay.

After preparing this concentrated Orchard Grass Pollen protein mixture, it can be

frozen until needed.

Example 4: Extraction and Reformulation Procedure For Other Allergen

Source Proteins

The procedure described in Example 3 for extracting and reformulating the

proteins in Orchard Grass Pollen works equally well with other allergen sources, such a Brome Grass Pollen, Giant Ragweed Pollen, Pigweed Pollen, Smooth

Alder Pollen, and River Birch Pollen, and is suitable to reformulate the proteins of

these allergens.

Example 5: Composing a Nonlabeled Antigen To Quench Nonspecific Epitope Binding On Labeled II. pylori Antigen

An embodiment of the method of combining nonspecific antigens with the

labeled reformulated H pylori antigen to make a antigen solution capable of

quenching nonspecific binding on the labeled antigen is as follows. Those of

ordinary skill in the art would recognize that this example can be modified to

make mixtures that would be effective in quenching nonspecific binding of other

labeled proteins. They would also recognize that other methods can be used to

make this mixture of labeled specific antigen and nonlabeled nonspecific related

antigen which has homologous epitopes to those nonspecific epitopes on the

labeled antigen.

To compose a reformulated antigen mixture, where non-biotinylated

nonspecific proteins serve to block out or quench non-specific epitopes on H

pylori proteins, add 70 mg of reformulated Campylobacter jejuni proteins and 30

mg of each reconstituted allergen mixture (comprising Orchard Grass Pollen,

Brome Grass Pollen, Giant Ragweed Pollen, Pigweed Pollen, Smooth Alder

Pollen, and River Birch Pollen reformulated proteins) to each mg of reformulated

biotinylated H. pylori protein mixture. This combination can then be used

effectively in the assay of this invention. Example 6: Using the Assay of the Present Invention to Detect Conditions Associated With H. pylori

The methods of the present invention were used to detect and to monitor

the course of diseases and conditions associated with H. pylori. The methods

proved particularly useful for monitoring the effects of anti-microbial therapy, and

the course of diseases associated with H. pylori.

Current methods of diagnosis, esophagiogastroduodenoscopy (EGD) and

biopsy were used on subjects. Some subjects were found to have chronic

gastroduodenal disease, e.g. gastric ulcers, duodenal ulcers, gastritis, duodenitis,

esophagitis. Some subjects had more than one condition.

Among those subjects who tested positive for the disease, some were

infected with H pylori as determined by histochemical tests and/or the urea breath

test. Other subjects did not evidence H. pylori infection.

Serologic testing was done to determine whether a subject had organism-

mediated disease. There are two parts to a complete answer to this question:

1. Is the subject infected with H. pyloril

2. If the subject is infected with H. pylori, is the infection causing a

disease?

Detection of IgG specific for H. pylori (HP) in a subject is indicative of HP

infection. Detection of IgE specific for H. pylori (HP) is indicative of the

presence of HP associated disease in a subject. (Calenoff, 1996) An aspect of the

present invention is that it is information on a combination of both IgG and IgE

levels that provides more accurate determination of organism-specific disease than is provided by information on the level of either immunoglobulin alone.

FIG. 7 is a graphical representation of serum levels of HP-specific IgG (Y-

axis) and IgE (X-axis) in "Enteron units" an arbitrary unit; serum is from 96

subjects with HP related disease.

FIG. 8 is a graphical representation of HP specific-serum IgG levels

(Y-axis) and IgE levels (X-axis) in "Enteron units"; serum is from 34 subjects

without HP-related diseases. The subjects forming the basis of FIG. 8 were also

without evidence of H. pylori organisms from standard tests, but are shown to the

carriers of HP by elevated specific IgG levels.

Example 7: Using the Assay of the Present Invention to Monitor the Effect of Treatment on H. pylori Eradication

Subjects who had evidence of H pylori infection and disease were treated

with standard antibiotic therapy to eradicate the organism, thereby alleviating the

disease. Using the methods and compositions of the present invention, levels of

IgG and IgE specific for HP were determined immediately prior to initiation of

treatment, and at various times after treatment.

FIG. 9 is a graphical representation of the percent change of IgE times IgG

level reduction or increase (Y-axis) days after cessation of anti-microbial therapy

(X-axis) used in an attempt to eradicate H. pylori. (Antibiotics and acid lowering

agents are used.) • represents subjects in which no HP organisms were detected

post-therapy by gastroscopy with biopsy; O represent subjects in which HP was

still detected post-therapy.

FIGS. 10 and 11 show the IgE and IgG values respectively for the same subjects shown in FIG. 9. The conclusion from a comparison of FIGS. 9, 10 and

11 is that the value of (IgE X IgG) presents a clearer delineation of effects of

antimicrobial treatment at various times after cessation of therapy. The first

reading was taken before administering treatment; the second at various times post

treatment as shown. A limitation of using IgG values alone is that they do not

seem to fall as fast as IgE levels in the same patients. The combined formula is a

more useful measure for monitoring the effects of treatment.

Example 8:A Diagnostic Kit

A diagnostic kit includes a support and, in separate containers, calibrator

solutions and a labeled reformulated antigen mixture containing quenching

antigens. Table 1 shows an example of a support with a matrix of wells (a

microtiter plate) with calibration solutions and a negative control. Sample

numbers are indicated in each well. An example of a set of calibrator solutions is

as follows:

The support is scanned for presence of label in each well; e.g. if a

fluorescent label was used, a fluorescence scanning device is used.

Example 9: Using the Assay of the Present Invention to Detect Conditions Associated with Chlamydia pneumoniae

Antigens were extracted and reformulated from Chlamydia pneumoniae by

the methods of the present invention. These are essentially similar to the methods

of Example 1. The reformulated mixtures were used to detect conditions

associated with the presence of Chlamydia. A particular condition associated with

Chlamydia is accelerated atherosclerotic plaque formation. IgE and IgG levels

were measured and multiplied. Subjects with the condition were differentiated

from normal using this value.

MATERIALS AND METHODS

I. Ultraviolet Light Activation and Covalent Coupling Procedure A procedure to covalently attach various proteins to polystyrene microtiter

plates is as follows:

Materials

Polystyrene Microtiter Plates, for example: Dynatech Microtite 1, cat. no.

011-010-7416; Dynatech Immulon 4, cat. no. 011-010-3855; or equivalent. Succinic anhydride, Sigma cat. no. S-7626 or equivalent.

l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, Sigma cat. no. E- 11769

or equivalent.

A protein to be covalently coupled, for example: A purified monoclonal

antibody; a purified polyclonal antibody; a protein with a specific binding capacity

such as NeutrAvidin (Pierce cat. no. 31000); a purified antigen for an antibody of

choice.

Buffers and solutions

A. 50 mM sodium borate, pH 8.10 ± 0.05

B. 50 mM sodium borate, pH 8.80 ± 0.05

C. 10 mM sodium phosphate, pH 7.20 ± 0.05

5M sodium hydroxide

100 g/L Sucrose with 200 mg/L sodium azide

Equipment

Ultraviolet (UV) light box, Fotodyne model 3-3000, or equivalent,

equipped with bulbs delivering light at 254 nm.

Multichannel pipettors

Procedure

1. Place a suitable number of polystyrene microtiter plates face down

on a UV light box. Expose the plates to the 254 nm UV light for 40 minutes.

(The exposure period may require optimization for specific applications.) During

the activation time the plates may be rotated or repositioned occasionally to

equalize the exposure of the internal surfaces of the wells of the light. 2. Approximately 15 minutes before the end of the UV exposure time,

prepare a 5 mg/mL solution of succinic anhydride in Buffer A. At the end of the

UV activation period, adjust the pH of the succinic anhydride solution to 8.40 ±

0.20 with 5M sodium hydroxide. Immediately add 200μL succinic anhydride

solution to each well of the activated microtiter plates. Fresh succinic anhydride

solution must be prepared for each set of activated plates.

3. Incubate the succinic anhydride solution in the wells for 10

minutes.

4. Wash the wells three times with deionized water. If multiple sets

of plates are being prepared, the succinylated wells may be held in deionized

water at ambient temperature.

5. Prepare a fresh solution of the protein to be coupled in Buffer B at

the desired protein concentration. For example, a lOμg/mL solution of a

monoclonal antibody and a coating volume (step 8) of 1 OOμL per well would yield

lμg of protein for coupling in each well. The coupling protein concentration must

be optimized for each application.

6. Prepare a fresh solution containing 100 mM l-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC) in Buffer C.

7. Aspirate the deionized water from the succinylated microtiter plate

wells. Add 11 OμL EDC solution to each well. Incubate at ambient temperature

for 10 minutes.

8. Aspirate the EDC solution from the wells and immediately add lOOμL of the coupling protein solution (step 5) to each well. Cover the plates and

incubate at ambient temperature overnight.

9. Aspirate the protein solution from the wells. Add 300μL sucrose

solution to each well. Incubate at ambient temperature for 20 minutes.

10. Aspirate the sucrose solution and transfer the plates to a laminar

flow hood to dry completely, approximately 4 hours.

11. Cover the dried plates and store at 4°C.

II. Antigen Biotinylation Procedure

The following extraction procedure is used to prepare biotinylated

antigens.

Material

Antigen extract.

Sulfosuccinimidyl-6-(biotinamido)hexanoate, Pierce cat. no. 21335 or

equivalent

2-Amino-2-(hydroxymethyl)-l,3-propanediol (TRIS base)

Lowry protein assay reagents

Buffers

A. 50 mM sodium phosphate, pH 8.0 ± 0.20 with 150 mM sodium

chloride and 1 mg/mL sodium dodecylsulfate

B. 50 mM sodium phosphate, pH 6.6 ± 0.1 with 150 mM sodium

chloride, 1 mg/mL sodium dodecylsulfate, and 200 mg/L sodium azide

Equipment Sephadex G-15 or G-25 desalting column

Vortex mixer

Tip plate or orbital mixer

Spectrophotometer

Procedure

Note: The antigen to be biotinylated must be dissolved in Buffer A at a

suitable concentration, generally 10 to 30 mg/mL. If the antigen is not in Buffer

A, perform a buffer exchange by dialysis or other suitable means.

1. Determine the protein concentration of the antigen to be

biotinylated. The Lowry method using the BioRad Dc assay, cat. no. 500-01 16, is

recommended.

2. If required, concentrate the antigen using either a tangential flow or

a pressure cell system equipped with a suitable membrane.

3. Equilibrate a suitably sized desalting column in Buffer B using at

least 5 column volumes of buffer.

4. Calculate the amount of Sulfosuccinimidyl-6-

(biotinamido)hexanoate (NHS-LC-biotin) required for the reaction. A 5 to 70 fold

molar excess of NHS-LC-biotin over antigen is generally used. The appropriate

ratio must be optimized for each application.

5. Weigh out the calculated amount of NHS-LC-biotin and add it to

the antigen solution. Vortex gently to dissolve the NHS-LC-biotin.

Note: If the amount of NHS-LC-biotin required is too small to be weighed

accurately, a stock solution of 2.5 mg/mL NHS-LC-biotin in Buffer A may be prepared and an appropriate amount of this solution added to the antigen. This

stock solution must be prepared fresh and used immediately.

6. Cover the reaction mixture tightly and mix gently on a tip plate or

orbital mixer for 45 minutes at ambient temperature.

7. Add solid 2- Amino-2-(hydroxymethyl)- 1,3 -propanediol (Tis base)

to a final concentration of 0.5M to the reaction mixture. Vortex gently to dissolve

the Tris base. Incubate on a tip plate or orbital mixer for 10 minutes.

8. Apply the reaction mixture to the equilibrated desalting column

(step 3) collecting suitably sized fractions.

9. Determine the OD280 of each collected fraction.

10. Pool the antigen containing fractions in a sterile screw capped

container.

11. Determine the protein concentration of the biotinylated antigen by

the Lowry method.

12. Store the biotinylated antigen at 4°C.

* * *

Although the methods and compositions of this invention have been

illustrated by the foregoing examples and embodiments, the invention is not

limited to only these examples and embodiments and modifications may be made

without departing from the essence or essential characteristics of this invention.

The scope of the invention is defined by the appended claims, and all

compositions and methods that come within the meaning of the claims, either

literally or by equivalence, are intended to be embraced therein. Documents Cited

ALEMOHAMMAD, U.S. Patent No. 5,262,156.

BLASER NJ. (1989) Antigenic Compositions and Methods for their Production and Use, EPO 329570A2 CALENOFF, E. (1996) U.S. Patent No. 5,567,594.

CALENOFF, E. (1989) U.S. Patent No. 4,849,337.

COOPER, T.G. (1977) Chapter 6, "Electrophoresis", in The Tools of Biochemistry, Wiley and Sons, N.Y., 1977

COVER (1995) U.S. Patent No. 5,403,924. DESHPANDE, et al. (1995) "Rapid large-scale growth of Helicobacter pylori in flasks and fermentors," Applied and Environmental Microbiology, Vol. 6, pp. 2431-2435.

EVANS, D.G., EVANS, D.J. AND GRAHAM, D.Y. (1992) U.S. Patent No. RE. 34,101. HARLOW AND LANE, 1988. Antibodies: A Laboratory Manual, Cold

Spring Harbor Laboratory.

HIRSCHL, A.M. et al. (1990) "Comparison of ELISA antigen preparations alone or in combination for serodiagnosing Helicobacter pylori infections," J. Clin. Path. 43:511-513. NEWELL, R.G. (1990) WO 90/03575.

PECK, K. et al. (1988) "Rapid scanning thermal lens/laser transmission densitometer." Applied and Theoretical Electrophoresis, l(l):41-5.

REID, M.J. (1985) U.S. Patent No. 4,539,292.

ROLLINS, D.M., COOLBAUGH, J.C., WALKER, R.I., and WEISS, E. (1983) "Biphasic culture system for rapid Campylobacter cultivation," Applied and Environmental Microbiology, 45(1); 284-9.

TUSSEN, P. (1985) "Practice and theory of enzyme immunoassays." In Laboratory Techniques in Biochemistry and Molecular Biology, eds. Burdon, R.H. and van Knippenberg, P.H. WATERBORG, J.H. and MATTHEWS, H.R. ( 1994) "The Lowry method for protein quantitation (Review)," in Methods in Molecular Biology. 32:1-4.

Claims

I CLAIM:

1. A composition comprising:

a. labeled molecules containing epitopes specific for an organism and non-specific proteins having epitopes that are found in the same organism; and

b. unlabeled molecules containing non-specific epitopes that compete with the non-specific epitopes on the labeled molecules for epitope binding sites on antibody molecules.

2. The composition of claim 1, wherein the epitopes specific for the organism are prepared by:

a. extracting proteins from a substantial portion of an organism;

b. fractionating the extracted proteins;

c. determining the concentration of each protein within each fraction; and

d. combining the proteins in quantities sufficient to produce an approximately equivalent end concentration of each protein in the composition.

3. The composition of claim 1, wherein the organism is selected from the group consisting of bacteria, chlamydia, mycoplasma, protozoa, rickettsia and viruses.

4. The composition of claim 1, wherein the organism is capable of infecting or colonizing a mammalian host.

5. The composition of claim 1, wherein the organism is a small multicellular organism.

6. The composition of claim 5, wherein the small multicellular organism is an intracorporal parasite.

7. A method for preparing a reformulated protein mixture from a specific organism, said method comprising:

a. extracting proteins from a substantial portion of the entire organism;

b. fractionating the extracted proteins;

c. determining the concentration of each protein within each fraction; and

d. combining all the proteins in quantities sufficient to produce an approximately equivalent end concentration of each protein in the mixture.

8. The method of claim 7, further defined as:

e. labeling the reformulated protein mixture; and

f. adding to the mixture unlabeled non-specific proteins having epitopes homologous to the non-specific epitopes found on the labeled proteins.

9. A capture assay method to detect organism-specific immunoglobulins in a biological fluid sample, the capture assay method comprising:

a. attaching anti-immunoglobulin molecules to a support;

b. exposing the biological fluid sample to the support so that the immunoglobulins in the fluid can be captured by the anti-immunoglobulin molecules;

c. exposing the composition of claim 1 to the captured immunoglobulins under conditions suitable for formation of antigen immunoglobulin complexes; and

d. measuring complexes of labeled molecules with the captured immunoglobulins, from which the presence in the biological fluid of immunoglobulins which are specific for the organism is inferred.

10. The capture assay method of claim 9, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum, sputum, urine, cerebrospinal fluid, intra-abdominal fluid, intrathoracic fluid, pericardial fluid, joint space fluid, pustular fluid, tear fluid, nasal secretions, sinus fluid, abscess fluid.

1 1. The capture assay method of claim 9, wherein the anti- immunoglobulin is anti-IgA.

12. The capture assay method of claim 9, wherein the anti- immunoglobulin is anti-IgD.

13. The capture assay method of claim 9, wherein the anti- immunoglobulin is anti-IgE.

14. The capture assay method of claim 9, wherein the anti- immunoglobulin is anti-IgG.

15. The capture assay method of claim 9, wherein the immunoglobulins in the fluid are IgE and IgG, and wherein the individually measured quantities of both are combined.

16. The capture assay method of claim 9, wherein the organism is Helicobacter pylori.

17. The capture assay of claim 9, wherein the organism is Chlamydia pneumoniae.

18. The capture assay method of claim 15, wherein the organism is H. pylori.

19. The capture assay method of claim 9, wherein serum IgE and IgG are measured in a biological sample obtained both before and after treatment of a subject to eradicate the organism.

20. The capture assay method of claim 19. further defined as combining the measured IgE and IgG values and comparing them to determine whether the organism is present.

21. The capture assay method of claim 19, wherein the organism is H. pylori.

22. The capture assay method of claim 19, wherein the organism is Chlamydia pneumoniae.

23. The capture assay method of claim 15, wherein the IgE and IgG quantities are combined by multiplying the quantity of specific IgE by the quantity of IgG corresponding to each biological fluid sample tested; and comparing the multiplied IgE x IgG values with a standard value to determine whether the organism is present.

24. The capture assay method of claim 23, wherein the organism is H. pylori.

25. A capture assay method to determine effects of treatment to eradicate an organism in a subject, said method comprising from a biological fluid sample obtained from the subject:

a. obtaining a value for a quantity of organism-specific IgG before treatment;

b. obtaining a value for the quantity of organism-specific IgG after treatment;

c. obtaining a value of organism-specific IgE before treatment;

d. obtaining a value of organism-specific IgE after treatment;

e. combining the values obtained in step a with those obtained in step c;

f. combining the values obtained in step b with those obtained in step d; and

g. inferring whether treatment is successful by determining whether the combined value in step f is less than in step e.

26. The capture assay method of claim 25, wherein the combined values are IgG multiplied by IgE.

27. The capture assay method of claim 26, wherein the value in step f is not more than 40% of the value of step e from which treatment is inferred to be successful.

28. A kit comprising a support and, in separate containers, calibrator solutions and a labeled reformulated antigen mixture containing quenching antigens.

29. The kit of claim 28, further defined as having a means to detect a complex between the labeled reformulated antigen mixture and an antibody in a biological fluid sample.

30. A composition comprising:

a. labeled molecules containing epitopes specific for an allergen and non-specific epitopes that are found in the same allergen; and b. unlabeled molecules containing non-specific epitopes that compete with the non-specific epitopes on the labeled molecules for epitope binding sites on antibody molecules.

31. The composition of claim 30, wherein the epitopes specific for the allergen are prepared by:

a. extracting proteins from a substantial portion of an allergen;

b. fractionating the extracted proteins;

c. determining the concentration of each protein within each fraction; and

d. combining the proteins in quantities sufficient to produce an approximately equivalent end concentration of each protein in the composition; and

e. using the composition to provide specific epitopes.

32. The composition of claim 30, wherein the allergen is selected form the group consisting of bacteria, chlamydia, mycoplasma, protozoa, rickettsia, viruses, pollens, epidermal agents, mold spores, foods, venoms and allergenic pharmaceutical agents.

33. The composition of claim 32, wherein the pollens comprise Orchard Grass Pollen, Brome Grass Pollen, Giant Ragweed Pollen, Pigweed Pollen, Smooth Alder Pollen and River Birch Pollen.

34. The composition of claim 30, wherein the allergen is a small multicellular organism.

35. The composition of claim 34, wherein the small multicellular organism is an intracorporal parasite.

36. A method for preparing a reformulated protein mixture from a specific allergen, said method comprising:

a. extracting proteins from a substantial portion of the entire allergen;

b. fractionating the extracted proteins;

c. determining the concentration of each protein within each fraction; and

d. combining all the proteins in quantities sufficient to produce a mixture comprising an approximately equivalent end concentration of each protein.

37. The method of claim 36, further defined as:

e. labeling the reformulated protein mixture; and

f. adding to the mixture an unlabeled mixture of non-specific proteins having epitopes homologous to non-specific epitopes found on the labeled proteins.

38. A capture assay method to detect allergen-specific immunoglobulin molecules in a biological fluid sample, the capture assay method comprising:

a. attaching anti-immunoglobulin molecules to a support;

b. exposing the biological fluid sample to the support so that the immunoglobulin molecules in the fluid can be captured by the anti- immunoglobulin molecules;

c. exposing the composition of claim 30 to the captured immunoglobulin molecules under conditions suitable for formation of antigen immunoglobulin complexes; and d. measuring the complexes of labeled molecules with the captured immunoglobulins, from which the presence in the biological fluid of immunoglobulins which are specific for the allergen is inferred.

39. The capture assay method of claim 38, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum, sputum, urine, cerebrospinal fluid, intra-abdominal fluid, intrathoracic fluid, pericardial fluid, joint space fluid, pustular fluid, tear fluid, nasal secretions, sinus fluid, and abscess fluid.

40. The capture assay method of claim 38, wherein the anti- immunoglobulin is anti-IgA.

41. The capture assay method of claim 38, wherein the anti- immunoglobulin is anti-IgD.

42. The capture assay method of claim 38, wherein the anti- immunoglobulin is anti-IgA.

43. The capture assay method of claim 38, wherein the anti- immunoglobulin is anti-IgG.

44. The capture assay method of claim 38, wherein the immunoglobulins in the biological fluid sample are IgE and IgG, and wherein the individually measured quantities of both are combined.

45. A capture assay method to detect allergen-specific immunoglobulin molecules in a biological fluid sample, the capture assay method comprising:

a. covalently attaching anti-immunoglobulin molecules to a support;

b. exposing the biological fluid sample to the support for a period of time so that the immunoglobulin molecules in the fluid can be captured by the anti-immunoglobulin molecules; c. exposing a biotinylated antigen composition to the captured immunoglobulin molecules minutes so that suitable antigen-immunoglobulin complexes are formed;

d. contacting the antigen-immunoglobulin complexes with a labeled avidin-like molecule so that the labeled molecule can specifically attach to any biotin on the antigen-immunoglobulin complex; and

e. measuring the quantity of label within the captured antigen- immunoglobulin complex, from which the presence in the biological fluid of immunoglobulin molecules which are specific for a disease causing organism or molecule is inferred.

46. The capture assay method of claim 45, wherein the biological fluid is exposed to the support for a period of time between 5 minutes and 24 hours.

47. The capture assay method of claim 45, wherein the biotinylated antigen composition is exposed to the captured immunoglobulin molecules for a period of time between 5 minutes and 180 minutes.

48. The capture assay method of claim 45 wherein the complexes are contacted with a labeled avidin-like molecule for a period of time between 2 and 180 minutes.

49. The capture assay method of claim 43, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum, sputum, urine, cerebrospinal fluid, intra-abdominal fluid, intrathoracic fluid, pericardial fluid, joint space fluid, pustular fluid, tear fluid, nasal secretions, sinus fluid, and abscess fluid.

50. The capture assay method of claim 45, wherein the anti- immunoglobulin is anti-IgA.

51. The capture assay method of claim 45, wherein the anti- immunoglobulin is anti-IgD.

52. The capture assay method of claim 45, wherein the anti- immunoglobulin is anti-IgE.

53. The capture assay method of claim 45, wherein the anti- immunoglobulin is anti-IgG.

54. The capture assay method of claim 45 wherein the biotinylated antigen is derived from a bacteria, chlamydia, mycoplasma, protozoa, rickettsia, virus, pollen, epidermal agent, mold spore, food, venom and allergenic pharmaceutical agent.

55. A capture assay method to detect allergen-specific immunoglobulins in a biological fluid sample, the capture assay method comprising:

a. attaching strongly anti-immunoglobulin molecules to a support;

b. exposing the biological fluid sample to the support so for a period of time between 5 minutes and 24 hours so that the immunoglobulins in the fluid can be captured by the anti-immunoglobulin molecules;

c. exposing a biotinylated antigen composition to the captured immunoglobulin molecules for a period of time between 5 minutes and 180 minutes so that suitable antigen-immunoglobulin complexes are formed;

d. contacting the antigen-immunoglobulin complexes with a labeled avidin-like molecule for a period of time between 2 and 180 minutes so that the labeled molecule can specifically attach to any biotin present on the antigen- immunoglobulin complex; and

e. measuring the quantity of label within the captured antigen- immunoglobulin complex, from which the presence in the biological fluid of immunoglobulin molecules which are specific for a disease-causing organism or molecule is inferred.

56. The capture assay method of claim 55, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum, sputum, urine, cerebrospinal fluid, intra-abdominal fluid, intrathoracic fluid, pericardial fluid, joint space fluid, pustular fluid, tear fluid, nasal secretions, sinus fluid, and abscess fluid.

57. The capture assay method of claim 55, wherein the anti- immunoglobulin is anti-IgA.

58. The capture assay method of claim 55, wherein the anti- immunoglobulin is anti-IgD.

59. The capture assay method of claim 55, wherein the anti- immunoglobulin is anti-IgE.

60. The capture assay method of claim 55, wherein the anti- immunoglobulin is anti-IgG.

61. The capture assay method of claim 55 , wherein the biotinylated antigen is derived from a bacteria, chlamydia, mycoplasma, protozoa, rickettsia, virus, pollen, epidermal agent, mold spore, food, venom and allergenic pharmaceutical agent.

62. A capture assay method to detect allergen-specific immunoglobulins in a biological fluid sample, the capture assay method comprising:

a. covalently attaching anti-immunoglobulin molecules to a support;

b. exposing the biological fluid sample to the support for a period of time between about 5 minutes and 24 hours so that the immunoglobulins in the fluid can be captured by the anti-immunoglobulin molecules;

c. exposing a biotinylated antigen composition to the captured immunoglobulins for a period of time between about 5 minutes and 180 minutes so that suitable antigen-immunoglobulin complexes are formed;

d. contacting the antigen-immunoglobulin complexes with an avidin- like molecule conjugated to a chemiluminogenic, chromogenic or fluorogenic enzyme for a period of time between about 2 and 180 minutes so that the labeled molecular complex can specifically attach to any biotin present on the antigen- immunoglobulin complex;

e. contacting the bound molecular complexes, for a time between 2 and 240 minutes, with a solution of substrate which undergoes reaction in the presence of the enzyme to yield a colored, chemiluminescent or fluorescent product;

f. measuring the color, chemiluminescence or fluorescence product; and

g. determining the amount of organism-specific or antigen-specific immunoglobulin in the biological fluid by comparing the color, chemiluminescence or fluorescence level determined in step (f) with those of control solutions.

63. The capture assay method of claim 62, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum, sputum, urine, cerebrospinal fluid, intra-abdominal fluid, intrathoracic fluid, pericardial fluid, joint space fluid, pustular fluid, tear fluid, nasal secretions, sinus fluid, and abscess fluid.

64. The capture assay method of claim 62, wherein the anti- immunoglobulin is anti-IgA.

65. The capture assay method of claim 62, wherein the anti- immunoglobulin is anti-IgD.

66. The capture assay method of claim 62, wherein the anti- immunoglobulin is anti-IgE.

67. The capture assay method of claim 62, wherein the anti- immunoglobulin is anti-IgG.

68. The capture assay method of claim 62, wherein the biotinylated antigen is derived from a bacteria, chlamydia, mycoplasma, protozoa, rickettsia, virus, pollen, epidermal agent, mold spore, food, venom and allergenic pharmaceutical agent.