WO2000073345A2

WO2000073345A2 - Antibodies specific for mycobacterial polypeptides and uses thereof

Info

Publication number: WO2000073345A2
Application number: PCT/US2000/014546
Authority: WO
Inventors: Mary Haak-Frendscho; Christopher Landowski; Scott Lesley
Original assignee: Promega Corporation
Priority date: 1999-05-28
Filing date: 2000-05-26
Publication date: 2000-12-07
Also published as: WO2000073345A3; AU5294000A

Abstract

The invention provides antibodies specific for mycobacterial polypeptides, and methods which employ the antibodies.

Description

ANTIBODIES SPECIFIC FOR MYCOBACTERIAL POLYPEPTIDES AND USES THEREOF

Background of the Invention

The mycobacteria are a diverse collection of acid fast, gram-positive bacteria, some of which cause significant human and animal diseases. In humans, the two most common mycobacteria-caused diseases are tuberculosis (TB) and leprosy. TB is caused by Mycobacterium tuberculosis, M. bovis, M. africanum, and microti. TB displays all of the characteristics of a global epidemic disease. A third of the world's population is estimated to be infected with M. tuberculosis, and TB is the most common cause of death of adults from infectious disease throughout the world (Kochi, 1991). Similar infections are produced by other mycobacteria widely distributed in the natural environment, e.g., "atypical" mycobacteria such as the M. avium-intracellular complex of M. kansasii (Wolinski, 1979).

The majority of patients never develop symptomatic primary TB because of the cellular immunity induced by M. tuberculosis (Collins, 1993). They are, however, at risk for developing reactivation (reinfection) TB, and it is estimated that about 10% do so (Sutherland, 1976). Much of the latest increase in TB both in the United States and worldwide has been linked to reactivation of previously acquired mycobacterial disease in patients with HIV infection and AIDS along with social changes that have contributed to substandard living conditions among certain populations, especially in large cities (Barnes et al., 1991). It has now been shown that one group of mycobacteria which consists of M. avium, M. intracellular and M. scrofulaceum, jointly known as MAIS complex, is responsible for disseminated disease in a large number of persons with AIDS (Cantwell et al., 1994). Since Koch identified M. tuberculosis as the causative agent of TB in

1882, many scientific studies and public health efforts have been directed at diagnosis, treatment and control of this disease. However, characteristics of M. tuberculosis have hampered research to improve diagnosis and to develop more effective vaccines. In addition, the biochemical composition of the organism has made identification and purification of the cellular constituents difficult, and many of these materials once purified, lack sensitivity and specificity as diagnostic reagents. While early definitive detection of active infection with M. tuberculosis is a key to effective treatment and full recovery, diagnostic and immunoprophylactic measures for mycobacterial diseases have changed little in the past half century.

The conventional methods for the diagnosis of M. tuberculosis include bacteriologic detection and morphological detection. Immunoassay is not generally accepted as a detection method because of low sensitivity and/or poor specificity (Ivanyi et al., 1988; Miorner et al., 1995). The bacteriologic detection of the organism in culture takes 2-13 weeks (Ford et al., 1994). While morphologic identification of acid-fast bacilli in sputum smears is more rapid, it is less sensitive as it requires a much larger number of organisms (roughly 50% of cases which are true positives test positive in sputum smears) and is labor intensive. Moreover, positive sputum smears are significantly less likely to be found in patients with AIDS or AIDS-related complex and TB than in non-HIV- infected patients with TB (Klein et al., 1989). In general, organisms can be difficult to detect morphologically and hard to culture in cases of paucibacillary TB. Only 90-95% of TB cases are confirmed by positive culture of M. tuberculosis; the remainder, especially those in children, are based on clinical criteria (CDC, 1992; Smith et al., 1996).

Although delayed-type hypersensitivity (DTH) to PPD is a major element in the diagnosis of TB (Bass et al., 1990), tuberculin anergy, i.e., the absence of delayed skin response to PPD, occurs in 15-25% of non-HIV-infected TB patients (McMurray, 1980; Daniel et al., 1981), and is significantly higher in populations infected with both M. tuberculosis and HIV (Okwera et al., 1990). For example, 50% or more of patients with AIDS and TB are skin test negative (Chaisson et al., 1989). This lack of PPD reactivity in an appreciable number of TB patients creates a need for other tests which accurately indicate mycobacterial infection and which do not depend on the integrity of a host immune response.

Therefore, what is needed is a method for detecting active mycobacterial infection, e.g., mycobacterial products directly in patient sera. What is also needed is a mycobacterial detection method that is quantitative, specific, sensitive, and/or not dependent on the host immune response.

Summary of the Invention The invention provides isolated and purified immunogenic mycobacterial-specific peptides or variants thereof. The peptides are useful to prepare mycobacterial-specific antibodies, and as a reagent to detect antisera specific for the peptide or the corresponding mycobacterial polypeptide. Preferred peptides of the invention are those which correspond to a mycobacterial polypeptide which is expressed during active infection, e.g., the mycobacterial Ag85 complex, and 38 kDa and 14 kDa mycobacterial polypeptides. Peptides of the invention are preferably at least about 7 to about 100, more preferably at least about 9 to about 50, and even more preferably at least about 9 to about 30, amino acid residues in length. Preferred peptides useful in preparing the antibodies of the invention include, but are not limited to, a peptide comprising an amino acid sequence corresponding to a Ag85B peptide, e.g., LRAQDDYNGWD (SEQ ID NO:l), NGTPNELGGAN (SEQ ID NO:2), VRSSNLKFQ (SEQ ID NO:3), SSDPAWERNDPT (SEQ ID NO:4), or LNAMKGDCQSSL (SEQ ID NO:5); a peptide of the 14 kDa mycobacterial polypeptide, e.g., TLPVQRHPRSL (SEQ ID NO:6), TIKAERTEQKDFDGR (SEQ ID NO:7), ADEDDIKATYDKGI (SEQ ID NO:8), SEGKPTEKH (SEQ ID NO:9), or RLEDEMKEGRYE (SEQ ID NO.10); or a peptide of the 38 kDa mycobacterial polypeptide, e.g., GSKPPSGSPETGAG (SEQ ID NO:l 1), LDQASQRGLGE (SEQ ID NO: 12), TIKWDDPQI (SEQ ID NO: 13), YLSKQDPEGWGKS (SEQ ID NO: 14), or VNNRQKDAAT (SEQ ID NO: 15); a variant thereof, or an immunogenic portion thereof.

As described hereinbelow, each of five fusion proteins were prepared and employed as an immunogen. The fusion proteins include mycobacterial-specific sequences, i.e., a peptide of the invention, as well as other amino acid sequences which may be present in the immunogen or may be removed prior to immunization. The resulting antisera was shown to bind to polypeptide products of M. tuberculosis, e.g., the Ag85 complex, a 38 kDa polypeptide, and a 14 kDa polypeptide, in culture filtrates, serum, sputum, and/or to the corresponding recombinant mycobacterial polypeptide. The antibodies are highly selective and sensitive antibodies and, thus, are useful as a diagnostic for active TB infection. Moreover, the antibodies may also be useful to detect other strains of Mycobacterium, e.g., M. avium, M. bovis BCG, M. fortuitum, M. gordonae, M. kansasii, M. phlei, M. smegmatis, M. tuberculosis, and/or M. xenopi. The antibodies of the invention may be used singly, or in combination.

Thus, the present invention provides an isolated and/or purified antibody, or a preparation of antibodies, that specifically reacts with, or binds to, a polypeptide expressed during active mycobacterial infection, e.g., during infection with M. tuberculosis. The isolated antibody may be a monoclonal antibody or a polyclonal antibody. Likewise, the preparation of antibodies may comprise a mycobacterial-specific monoclonal antibody or polyclonal antibody. A preferred antibody preparation of the invention is a preparation of polyclonal antibodies that specifically binds to a polypeptide expressed during active infection of a mammal, e.g., a human, with M. tuberculosis. Preferably, the antibodies of the invention are substantially free of antibodies that do not react with a mycobacterial polypeptide.

The invention also provides an expression cassette comprising a first DNA segment encoding an immunogenic mycobacterial-specific peptide, e.g., a peptide comprising the amino acid sequence corresponding to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof, which is operably linked to a promoter functional in a host cell. Host cells include prokaryotic or eukaryotic cells, e.g., yeast, plant or mammalian host cells. A preferred expression cassette comprises a promoter functional in a prokaryotic cell or in yeast. Preferably, the expression cassette further comprises a second DNA segment encoding a carrier protein, wherein the first and second DNA segments are linked so as to encode a fusion protein. The carrier protein provides T helper cell help and, preferably, has low immunoreactivity alone or in combination with the peptide of the invention. Preferably, the fusion protein is less than about 30 kDa, more preferably less than about 25 kDa, and even more preferably about 10 to about 12 kDa, in size. For example, a fusion protein of the invention which is between 25 to 30 kDa may be useful to generate antibodies that are reactive across mycobacterial species. In contrast, smaller fusion proteins, e.g., those between 10 to 12 kDa, are likely to generate antisera that are mycobacterial species- specific. The expression cassettes can be incoφorated into expression vectors which can be employed to transform prokaryotic or eukaryotic host cells, so as to result in expression of an immunogenic fusion protein, preferably a fusion protein comprising the amino acid sequence corresponding to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof. As used herein, the term "immunogenic mycobacterial peptide" means those regions of a mycobacterial polypeptide which are capable of eliciting an immune response, preferably an immune response which results in antibodies that are capable of specifically reacting with the mycobacterial peptide and/or the corresponding mycobacterial polypeptide. For methods to determine whether an antibody reacts with a particular peptide or polypeptide, see, for example, Bentley-Hibbert et al., 1999, and Drowart et al., 1992.

The invention also provides an immunogenic composition comprising a peptide of the invention, preferably a fusion protein of the invention, in combination with a pharmaceutically acceptable carrier. It is preferred that an immunogenic composition of the invention comprises a peptide or fusion protein comprising the amino acid sequence corresponding to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO.l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof. While it is preferred that the fusion protein comprises a peptide of the invention linked through a peptide bond to a carrier protein, the invention includes any covalent or non-covalent association of a peptide of the invention with a carrier protein. The administration of the immunogenic composition to a vertebrate, e.g., an avian such as a chicken, turkey or other domestic or wild fowl, or a mammal, e.g., a rabbit, rat, mouse, guinea pig, bovine, equine, ovine, swine, or primate such as a human or a non- human primate, induces the production of antibodies specific for a mycobacterial polypeptide which comprises the amino acid sequences of the peptide employed in the composition.

Also provided is a method for detecting a mycobacterial polypeptide in a mammalian sample, e.g., a physiological sample. The method comprises contacting an amount of isolated and/or purified antibodies of the invention which bind to a mycobacterial polypeptide or peptide with the sample to be tested for a sufficient time to form binary complexes between at least a portion of the antibodies and a portion of the mycobacterial polypeptide in the sample. The presence or amount of the binary complexes is then detected, optionally, by means of a second antibody which binds to the binary complex. The presence of binary complexes is indicative of the presence of the mycobacterial polypeptide in the sample. Mammalian samples include, but are not limited to, those from a rabbit, rat, mouse, guinea pig, bovine, equine, ovine, swine, or human. For example, the samples to be tested include culture filtrates or extracts, non-human mammalian tissue samples and physiological fluids, and human tissue or physiological fluid, such as serum, plasma, sputum, or urine.

The invention further provides a diagnostic method. The method comprises contacting an amount of isolated and/or purified antibodies of the invention, which specifically react with a mycobacterial polypeptide, with a physiological sample obtained from a mammal suspected of being at risk of, or having, a mycobacterial infection, for a sufficient time to form binary complexes between at least a portion of the antibodies and a portion of the polypeptide in the sample. The amount of the binary complexes is then determined or detected. The presence or the amount of the complexes is indicative of a mammal infected with Mycobacterium. The detection of complexes may be the result of employing antibodies that are labeled or bind to a detectable label. Alternatively, binary complex formation is detected by a second agent, such as an antibody, comprising a detectable label, e.g., an antibody labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP), or which binds to a detectable label, to form a detectable ternary complex. The second agent may bind to the same polypeptide as the first antibody, or may bind to the first antibody, e.g., to the Fc region of the first antibody. Thus, the methods of the invention do not rely on the host immune response, e.g., the presence of anti- mycobacterial human antibodies in serum. The methods of the invention are useful to detect early stages of mycobacterial infection, and infection in immunocompromised hosts. Further, the direct detection of mycobacterial polypeptides can discriminate between active and past infections, immunizations, and/or infection with atypical mycobacteria.

The invention also provides a kit for detecting mycobacterial infection. The kit comprises packaging, containing, separately packaged: (a) an amount of at least a first antibody which binds to a mycobacterial polypeptide; and (b) instruction means. Preferably, the antibody is labeled or is bound by a detectable label or a second antibody that is labeled. The kit may comprise a mixture of antibodies, each of which binds to a different epitope on the same mycobacterial polypeptide, e.g., a mixture of antibodies comprising antibodies that bind to SEQ ID NO:4 and antibodies that bind to SEQ ID NO:5, or a mixture of antibodies, each of which binds to a different mycobacterial polypeptide, e.g. comprising antibodies that bind to SEQ ID NO:4 and antibodies that bind to SEQ ID NO: 12. The kit may also comprise a blocking agent, e.g., BSA, which may be contacted with a sample to be tested before contacting the sample with the antibody, or may be contacted concurrently with the antibody. In one embodiment of the invention, the kit further comprises a known amount of a second antibody which is detectably labeled or binds to a detectable label. The second antibody may bind to the same polypeptide as the first antibody, or may bind to the first antibody. Preferably, the kit is a diagnostic kit.

Also provided is a kit useful to detect a mycobacterial polypeptide in a sample. The kit comprises a solid substrate on which the sample to be tested is placed and a preparation of antibodies. Preferably, the antibodies are labeled or bind to a detectable label.

Brief Description of the Figures Figure 1. Western blot analysis of culture supernatants and recombinant M. tuberculosis polypeptides with exemplary antibodies of the invention. A) Polyclonal antibody 85-CPL-4 (1 μg/ml, not affinity purified) directed against Ag85 detects purified recombinant Ag85B and purified Ag85 complex from M. bovis culture filtrate (left to right). The Ag85 complex consists of three subunits (A, B, and C) and appears as a doublet around 35 kDa. The upper band consists of A and C subunits, while the lower band is the B subunit. The recombinant Ag85B (far left) has a different molecular weight compared to that of the native B subunit. B) Monoclonal antibody from clone 240.9 (1 μg/ml) directed against Ag85 detects subunits A and/or C from purified Ag85 complex from M. bovis culture filtrate but notpurified recombinant or native Ag85B (right to left).

Figure 2. A) Polyclonal antibody 85-CPL-5 (1 μg/ml) directed against Ag85 detects 100 and 200 ng/lane (left to right) purified Ag85 complex from M. bovis culture filtrate. B) Polyclonal antibody 38-CPL-3 (1 μg/ml) directed against a 38 kDa mycobacterial polypeptide detects 12.5, 25, 50, and 100 ng/lane (left to right) purified recombinant 38 kDa mycobacterial polypeptide. C) Polyclonal antibody 38-CPL-2 (1 μg/ml) directed against a 38 kDa mycobacterial polypeptide detects 25, 50, and 100 ng/lane (left to right) purified recombinant 38 kDa mycobacterial polypeptide. Figure 3. A) Polyclonal antibody 38-CPL-2 (1 μg/ml, affinity purified) directed against a 38 kDa mycobacterial polypeptide detects 50, 25, 12.5, 6.25, and ng/lane (right to left) purified recombinant 38 kDa mycobacterial polypeptide. B) Polyclonal antibody 85-CPL-4 (1 μg/ml, affinity purified) directed against Ag85 detects 50, 25, 12.5, 6.25, 3, and 1.5 ng/ml (right to left ) purified recombinant Ag85. C) Monoclonal antibody from clone 240.9 (1 μg/ml) directed against Ag85 detects 25, 50, 100, and 200 ng/lane (left to right) purified Ag85 complex from M. bovis culture filtrate. Figure 4. Codons.

Figure 5. Exemplary amino acid substitutions. Detailed Description of the Invention

Definitions

As used herein, "isolated and/or purified" refer to in vitro preparation, isolation and/or purification of a nucleic acid molecule, a nucleic acid segment, a nucleic acid sequence, an expression cassette, a vector, peptide, or polypeptide, e.g., antibody, preferably so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. Thus, the term "substantially purified", when referring to a nucleic acid molecule, peptide, polypeptide, or antibody of the invention, means a chemical composition which is essentially free of other cellular components. A "preparation" is not substantially purified, i.e., it is a mixture of cellular components. With respect to an antibody, essentially free of other components includes free of ligand, e.g., the immunogen or antigen, or a portion thereof, to which the antibody binds. Such a composition is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography (for peptides or polypeptides), or A₂₆₀ A_2g0 ratios (for nucleic acids). A peptide which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified polypeptide, peptide or nucleic acid molecule comprises more than 80% of all macromolecular species present in the preparation. Thus, with respect to an antibody, a substantially purified preparation of antibodies may be obtained by well known methods, e.g., the use of protein A or protein G. Preferably, the polypeptide, peptide or nucleic acid molecule is purified to represent greater than 90% of all macromolecular species present. More preferably, the polypeptide, peptide or nucleic acid molecule is purified to greater than 95%, and more preferably, the polypeptide, peptide or nucleic acid molecule is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.

The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. The phrase "a nucleic acid molecule, sequence or segment encoding" refers to a nucleic acid, i.e., DNA or RNA, which directs the expression of a specific polypeptide, protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into proteins. The nucleic acid sequences include both full length nucleic acid sequences as well as shorter sequences derived from the full length sequences, e.g., those encoding a peptide or a fusion protein comprising a peptide of the invention. It is understood that a nucleic acid molecule includes the degenerate codons of the native sequence (Figure 4) or sequences which may be introduced to provide codon preference in a specific host cell. The nucleic acid molecule includes both the sense and antisense strands as either individual single strands or in the duplex form.

An isolated "variant" nucleic acid molecule of the invention is a nucleic acid molecule which has at least 80%, preferably at least about 90%, and more preferably at least about 95%, but less than 100%, contiguous nucleotide sequence homology or identity to a reference nucleotide sequence, e.g., a nucleic acid sequence encoding a peptide of the invention comprising the native (wild- type) sequence of a mycobacterial polypeptide. For example, a variant of a nucleic acid molecule encoding SEQ ID NO: 1 has at least 80%, preferably at least about 90%, and more preferably at least about 95%, but less than 100%, contiguous nucleotide sequence homology or identity to the nucleotide sequence encoding SEQ ID NO: 1. Moreover, a variant nucleic acid molecule of the invention may include nucleotide bases not present in the corresponding non- variant nucleic acid molecule, as well as deletions relative to the corresponding wild-type nucleic acid molecule.

An isolated "variant" of a peptide of the invention is a peptide which has at least about 50%, preferably at least about 80%, and more preferably at least about 90%, but less than 100%, contiguous amino acid sequence homology or identity to a reference amino acid sequence, i.e., the native or wild-type sequence of the peptide, such as the amino acid sequence comprising SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, or an immunogenic portion thereof. It is preferred that a variant peptide, or an immunogenic portion thereof, has at least about 1%, more preferably at least about 10%, and even more preferably at least 100% of the activity, e.g., the immunogenic activity, of the corresponding non-variant peptide, such as peptide comprising SEQ ID NO: 1. The activity of a peptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the peptide to elicit a sequence-specific immunologic response when the peptide is administered to an organism, e.g., to chicken, cattle, goat, sheep, donkey, rat, guinea pig, rabbit, or mouse, or to induce the production of antibodies that bind to the inducing peptide and preferably to the corresponding native form of the polypeptide from which the sequences of the peptide were derived.

The phrase "binding specificity", or "specifically immunoreactive with," refers to a binding reaction which is determinative of the presence of a polypeptide or peptide in the presence of a heterogeneous population of proteins and other biologies. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular polypeptide or peptide, e.g., Western blot, dot blot, and ELISAs. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein. See Harlow et al. (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

The terms "recombinant DNA", "recombinant nucleic acid" or "recombinantly produced DNA" refer to DNA which has been isolated from its native or endogenous source or synthetically synthesized, and modified either chemically or enzymatically by adding, deleting or altering naturally occurring flanking or internal nucleotides. Flanking nucleotides are those nucleotides which are either upstream or downstream from the described sequence or subsequence of nucleotides, while internal nucleotides are those nucleotides which occur within the described sequence or sub-sequence.

The term "labeled antibody" as used herein refers to an antibody bound to a label such that detection of the presence of the label (e.g., as bound to a biological sample) indicates the presence of the antibody, i.e., it is a reporter molecule. As used herein, the terms "label" or "labeled" refer to incorporation of a detectable molecule, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide such as an antibody. Various methods of labeling are known in the art. Examples of labels include, but are not limited to, the following: radioisotopes (e.g., ³H, ¹ C, ³⁵S, ¹²⁵O, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide, phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, e.g., groups that can be detected by avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity (that can be detected by optical or colorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels may be attached by spacer arms of various lengths to reduce potential steric hindrance.

As used herein, the term "antibody" refers to a polypeptide consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and includes intact immunoglobulins, e.g., anti-idiotypic antibodies, or modified antibodies including, for example, fragments of intact antibodies, FabFc₂, Fab, Fv, Fd, (Fab')₂, an Fv fragment containing only the light and heavy chain variable regions, a Fab or (Fab)'₂ fragment containing the variable regions and parts of the constant regions, a single-chain antibody (e.g., ScFv). The heavy and light chain of a Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Avian IgY corresponds to IgG.

The antibody may be of animal (e.g., chicken or rat) or human origin or may be chimeric or humanized. A "chimeric" antibody indicates that the antibody is derived from two different sources, e.g., an antibody having murine heavy chains and human light chains, or an antibody that comprises a murine Fv region and a human Fc region, are examples of chimeric antibodies. A humanized antibody is a chimeric antibody in which non-human antibodies have been genetically manipulated to replace most or all non-human portions (> 80%) of the antibody with the human equivalent. Antibody variable domains have been humanized by various methods, such as CDR grafting (Riechmann et al, 1988), replacement of exposed residues (Padlan, 1991) and variable domain resurfacing (Roguska et al., 1994). The CDR grafting approach has also been successfully used for the humanization of several antibodies either without preserving any of the mouse framework residues (Jones et al., 1986, and Verhoeyen et al., 1988) or with the preservation of just one or two mouse residues (Riechmann et al., 1988; Queen et al., 1989). More specifically, humanization is accomplished by aligning the variable domains of the heavy and light chains with the best human homolog identified in sequence databases such as GENBANK or SWISS-PROT using the standard sequence comparison software as described above. Sequence analysis and comparison to a structural model based on the crystal structure of the variable domains of a monoclonal antibody (Queen et al., 1989; Satow et al., 1986; Protein Data bank Entry IMCP) allows identification of the framework residues that differ between the mouse antibody and its human counterpart. Thus, as used herein, the term "antibody" includes these various forms. Using the guidelines provided herein and those methods well known to those skilled in the art which are described in the references cited above and in such publications as Harlow et al. (1988), the antibodies of the present invention can be readily made. The term "antigenic determinant" designates the structural component of a molecule that is responsible for specific interaction with corresponding antibody (immunoglobulin) molecules elicited by the same or related antigen or immunogen, e.g., polypeptide or peptide.

The term "immunogenic determinant" or "epitope" designates the structural component of a molecule that is responsible for the induction in a host of an antibody containing an antibody combining site (idiotype) that binds to the determinant or epitope.

The term "antigen" means an entity that is bound by an antibody. The term "immunogen" describes an entity that induces antibody production in the host animal. In some instances, the antigen and immunogen are the same entity, while in other instances, the two entities are different, e.g., a peptide immunogen may yield an antibody that recognizes a polypeptide antigen. The term "synthetic" means that the polypeptide molecule or peptide has been prepared by chemical means, i.e., chemically synthesized, rather than being prepared by biological means, as by genetic engineering techniques.

Peptides and Variants Useful to Prepare Antibodies of the Invention. Candidate peptides which have amino acids sequences which are unique to mycobacteria and which have favorable antigenic characteristics are employed as immunogens. While other immunogenic peptides may be useful to prepare antibodies of the invention, it is preferred that a peptide of the invention comprises amino acid sequences of mycobacterial polypeptides that are expressed during active infection, e.g., Ag85, 38 kDa, 45/47 kDa, and 14 kDa, as well as KATG, MPT51 , MPT64, MTC28, ES AT-6, and 19 kDa (see Table 1 of Colangeli et al. (1998), which is specifically incorporated by reference herein). Only actively dividing mycobacteria efficiently generate protective cell- mediated immunity to M. tuberculosis (Orme, 1988). Secreted or cell-associated mycobacterial proteins may be important for host protective response. Produced early in the growth phase, they can account for up to 30% of secreted proteins in culture, and are also present on the cell surface. Proteins of the antigen 85 complex (Ag85A, Ag85B, and Ag85C) are major secretory proteins of actively replicating M. tuberculosis (Wiker et al., 1992; Borremans et al., 1989, DeWit et al., 1994; Harm et al., 1996)). Ag85B is the major secretory product of M. tuberculosis, while Ag85 A is the major secretory product of M. bovis BCG

(Wiker et al., 1992). They share high sequence homology at the nucleotide and protein level both with each other and with Ag85 from other mycobacterial species (Wiker et al., 1986). This high degree of homology results in a particular Ag85 protein containing common epitopes found in many Ag85, in addition to unique species- and subtype-specific epitopes (Drowert et al., 1992; Rinkle de Wit et al., 1993). Ag85 complex proteins are mycolyltransferases (Belisle et al., 1997). As such, they play an essential role in the final stages of mycobacterial cell wall synthesis, since inhibitors of this activity inhibit both the transfer and the deposition of mycolates into the mycobacterial cell wall and cell growth (Belisle et al., 1997). The function of Ag85 complex in mycobacterial physiology and pathogenesis of tuberculosis is otherwise incompletely understood (Godfrey et al., 1992; Hetland et al., 1994; Ratliff et al, 1985). Ag85 complex proteins induce delayed hypersensitivity, protective immune responses, and specific antibodies in infected mice and guinea pigs (Baldwin et al., 1998; Denis et al., 1997; Haslov et al., 1995; Horwitz et al., 1995, Huygen et al., 1990; Lozes et al., 1997). They also induce readily elicitable cellular immune responses in cultured peripheral blood mononuclear cells of most healthy purified protein derivative of tuberculin (PPD)-positive people and a few patients with clinically active tuberculosis (Havlir et al., 1991; Huygen et al, 1988). While levels of anti-Ag85 antibodies are often low in healthy PPD-positive subjects, they increase in patients with active tuberculosis (Havlir et al., 1991 ; Turneer et al., 1988). Similar patterns of response are exhibited by healthy lepromin-positive subjects and patients with lepromatous leprosy (Launois et al., 1991).

Ag85 proteins bind to plasma and cellular fibronectins (Abou-Zeid et al., 1988; Godfrey et al., 1992), high-molecular-weight glycoproteins found in plasma and tissues that play important roles in cell motility and adhesion, development, phagocytic function, wound healing, and inflammation (Hynes, 1990). Although microgram doses of Ag85 elicit delayed hypersensitivity reactions in sensitive guinea pigs, nanogram doses of these proteins inhibit local in vivo expression of delayed hypersensitivity by binding to and inactivating a specialized T-cell fibronectin produced after antigenic stimulation (Godfrey et al., 1992). Thus, patients with active tuberculosis may have high levels of circulating Ag85 proteins that could possibly play a role in the systemic anergy these patients often exhibit.

Although species-specific epitopes of Ag85 have been suggested for immunodiagnostic reagents (Matsuo et al., 1990), polyclonal anti-Ag85 antibodies are directed to both groups of epitopes. Moreover, many of the available MAb specific for Ag85 recognize cross-reactive epitopes present on Ag85 of many mycobacterial species (Drowart et al., 1992; Anderson et al., 1986; Salata et al., 1991).

Ag85 protein can be detected in the sera of patients with active TB who are skin test negative (Bentley-Hibbert et al., 1999). This, coupled with the observed rise in immune complexes during TB, implies that other mycobacterial antigens are likely to be found in the circulation. High levels of antibody directed against 38 kDa and 45/47 kDa complex antigens are present in the serum of infected individuals. The 38 kDa protein also is actively secreted from M. tuberculosis and accounts for about 10% of secreted proteins in culture.

Indeed, it is believed to be one of the most important antigens of M. tuberculosis (Harboe et al., 1998). The 38 kDa polypeptide is both a major constituent of M. tuberculosis culture fluid and its presence is highly correlated with active M. tuberculosis infection. Several investigators have developed serological tests to facilitate the diagnosis of TB, especially in those individuals with smear negative disease, employing 38 kDa protein as a probe for measuring specific antibody in infected individuals (Bothamley et al, 1992; Bothamley et al., 1994; Thybo et al., 1995; Zhou et al., 1996; Bassay et al., 1996; Cole et al., 1996). Their ELISA results ranged from 90% or greater sensitivity and specificity (Bothamley et al., 1992; Bothamley et al., 1994; Zhou et al., 1996) to about 50% sensitivity and specificity (Thybo et al., 1995), depending on the patient population being examined. Although testing for anti-38 kDa protein antibodies is the most sensitive serological test to date for M. tuberculosis, tests for smear-negative and HIV-related TB remain insufficient in their sensitivity and specificity (Bothamley et al., 1995). However, combinations of antigens have been shown to yield more sensitive and specific assays than those targeting only the 38 kDa protein, especially in samples from patients who have a poor humoral response (Bothamley et al., 1992; Bassay et al., 1996; Bisetti et al., 1995).

The 45/47 kDa complex has been shown to be an important secreted protein that has specificity for replicating M. tuberculosis. Thus, it may also be useful to prepare mycobacterial specific antisera. Although M. tuberculosis Ag85 and 45/47 kDa complexes are cross-reactive with antibodies generated to other mycobacterial species, likely due to conserved epitopes between species, they both are likely to also possess M. tuberculosis-τestήcted epitopes that would confer specificity to antibodies raised to those epitopes.

The 14 kDa polypeptide is of interest as it is an abundant mycobacterial polypeptide, and may play a role in pathogenesis. The 14 kDa polypeptide is membrane bound and not soluble, and so is not likely to be present in culture filtrates.

Therefore, elevated levels of these polypeptides might be expected to be present in serum of patients with active TB, and detection of these elevated levels could be the basis of a diagnostic assay for active infection with M. tuberculosis. In particular, certain peptides, e.g., those derived from a 38 kDa M. tuberculosis polypeptide, may result in antibodies that are specific for M. tuberculosis. Thus, preferred peptides of the invention include those based on sequences of secreted or cell-associated mycobacteoal polypeptide Exemplar ' peptides include, but are not limited to, peptides composing SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5. SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, a variant thereof or an immunogenic portion thereof Vaoant peptides have at least one amino acid substitution relative to SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, or an immunogenic portion thereof In particular, ammo acids are substituted in a relatively conservative manner For example, hydrophobic residues are substituted for hydrophobic residues (norleucme, met, ala, val, leu, lie) neutral hydrophilic residues for neutral hydrophilic residues (cys, ala, gly, ser, thr), acidic residues for acidic residues (asp, glu), basic residues for basic residues (asn, gin, his, lys, arg), and aromatic residues for aromatic residues (trp, tyr, phe) Other prefeoed substitutions are shown in Figure 5 The invention also envisions variants with non-conservative substitutions Non-conservative substitutions entail exchanging a member of one of the classes descobed above for another For example, for SEQ ID NO 4, the se ne (S) at position 1 may be replaced with lysine (K), the serme at position 2 with glutamic acid (E) or threonme (T), the glutamic acid (E) at position 7 with glutamine (Q) or lysine (K), and the threonme (T) at position 12 with leucme (L), methionme (M) or seone (S) For SEQ ID NO 5, the asparagine (N) at position 2 may be replaced with valme (V), the alanine at position 3 with aspartic acid (D), the glycine (G) at position 6 with alanme (A) or prolme (P), the cysteme (C) at position 8 with leucme (L) or isoleucine (I), the seone at position 10 with the arginme (R), glutamine (Q), alanine (A) and histidine (H), and the seone at position 1 1 with alanme (A), tyrosme (Y), threonme (T), or va ne (V) After the substitutions are introduced, the variant peptides are screened for biological activity, e g , ability to generate mycobacteoal-specific antibodies or to specifically react with mycobacteoal-specific antibodies To substitute an ammo acid residue for another amino acid residue, at least one nucleotide base in the codon encoding the amino acid is substituted with a different nucleotide base so as to encode the selected amino acid residue (see Figure 4) Methods to substitute one nucleotide base for another are well known to the art

One of skill would recognize that modifications can be made to the peptide or fusion protein of the invention without diminishing the biological activity Some modifications may be made to facilitate the cloning, expression, or incorporation of the mycobacteoal portion into the expression cassette, e g , encoding a fusion protein Such modifications are well know n to those of skill in the art and include, for example, a methionme added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restoction sites or termination codons

One of skill will recognize that other modifications may be made Thus, for example, ammo acid substitutions may be made that increase specificity or binding affinity Alternatively, non-essential regions of the molecule may be shortened or eliminated entirely Thus, where there are regions of the molecule that are not themselves involved in the activity of the molecule, they may be eliminated or replaced with shorter segments that serve to maintain the correct spatial relationships between the active components of the molecule

Preparation of Peptides of the Invention Methods which are well known to those skilled m the art can be used to construct expression cassettes and vectors containing a coding sequence and appropoate transcoptional/translational control signals These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic techniques See, for example, the techniques descobed m Sambrook et al (1989)

Generally, the expression cassette is in the form of chimeoc DNA, and composes plasmid DNA that can also contain coding regions flanked by control sequences which promote the expression, or stop the expression, of the DNA segment once the expression cassette is introduced into host cell Aside from DNA sequences that serve as transcoption units for mycobacteoal peptides, a portion of the DNA molecule may be untranscobed, serving a regulatory or a structural function Other elements functional in the host cells, such as introns. enhancers, polyadenylation sequences and the like, may also be a part of the DNA. Such elements may or may not be necessary for the function of the DNA. but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

Thus, depending on the host/vector system utilized, any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like can be used in the expression vector (see, e.g., Bitter et al., 1987; WO 97/1 1761 and WO 96/06167). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ; plac, ptrup, ptac (ptφ-lac hybrid promoter) and the like may be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for controlled and high level transcription of the inserted coding sequence.

The expression cassette may encode other peptides. For example, an expression cassette comprising an isolated DNA molecule, which encodes a fusion protein, operably linked to a promoter may be employed to prepare an immunogen. The isolated DNA molecule comprises a first DNA segment encoding an immunogenic mycobacterial peptide and a second DNA segment encoding a carrier protein. The carrier protein facilitates purification of the resulting fusion peptide and activates T helper cells. The carrier protein preferably possesses low immunoreactivity. Carrier proteins include, but are not limited to, KLH, GST, and the carrier protein encoded by SSNAP (Promega Coφ., Madison, Wisconsin, see U.S. patent application Serial No. 09/174,060, which is specifically incoφorated by reference herein).

"Control sequences" is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotic cells, for example, include a promoter, and optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. "Operably linked" is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader can be operably linked to DNA coding for a polypeptide, and expressed as a prepolypeptide that participates in the secretion of the polypeptide; a promoter or enhancer can be operably linked to a coding sequence and affect the transcription of the sequence; or a ribosome binding site can be operably linked to a coding sequence and positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used to provide them in accord with conventional practice.

The expression cassette to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956). Reporter genes are used for identifying transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable polypeptides are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity.

Preferred genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E. coli, and the luciferase gene (luc) from firefly Photinus pyrali . Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

The recombinant DNA can be readily introduced into host cells, e.g., mammalian, plant, bacterial, yeast or insect cells by transfection with an expression cassette by any procedure useful for the introduction into a particular cell, e.g., calcium phosphate precipitation, lipofection, microinjection, electroporation, and the like, to yield a transformed cell, so that the peptide, e.g., fusion protein, of the present invention is expressed by the host cell.

The general methods for isolating and purifying a recombinantly expressed protein from a host cell are well known to those in the art. For example, the culture medium or lysate can be centrifuged to remove particulate cell debris. The insoluble and soluble polypeptide fractions are then separated. The peptide of the invention may then be purified from the soluble fraction or the insoluble fraction, i.e., refractile bodies (see, for example, U.S. Patent No. 4,518,526, the disclosure of which is incoφorated by reference herein). The peptide can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, Scopes, 1982; Deutscher 1990). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred. Examples of the isolation and purification of recombinant polypeptides and proteins are given in Sambrook et al., cited supra.

Alternatively, the immunogenic peptides or fusion proteins can be synthesized by in vitro transcription translation reactions or the solid phase peptide synthetic method (Stewart et al., 1969; Merrifield, 1963; Meienhofer , 1973; and Bavaay and Merrifield, 1980).

Preparation of Antibodies. The antibodies of the invention are prepared by using standard techniques, preferably, techniques for preparing polyclonal antibodies. To prepare polyclonal antibodies or "antisera," an animal is inoculated with an immunogen, i.e., a purified immunogenic peptide or fusion protein, and immunoglobulins are recovered from a fluid, such as blood serum, that contains the immunoglobulins, after the animal has had an immune response. For inoculation, the immunogen is preferably bound to a carrier peptide and emulsified using a biologically suitable emulsifying agent, such as Freund's incomplete adjuvant.

Although a variety of mammalian or avian host organisms may be used to prepare polyclonal antibodies against the peptides of the invention, chickens are a preferred host organism.

Moreover, avian IgG, also known as IgY (Leslie et al., 1969), is deposited in large quantities into the egg yolk and can be easily purified by sequential precipitation (Jensenius et al., 1981 ; Akita et al.,1992). Furthermore, IgY does not react with protein A (Langone et al., 1983; Katz et al., 1985), protein G (Guss et al., 1986), rheumatoid factor (Larsson et al, 1988) and other human Ig (Larsson et al., 1992) and does not activate the human complement system (Larsson et al., 1992), all of which reduce the problem of non-specific reactivity. Thus, the use of IgY may provide advantages in some immunologic assays. Following immunization, Ig is purified from the immunized bird or mammal. For certain applications, particularly certain pharmaceutical applications, it is preferable to obtain a composition in which the antibodies are essentially free of antibodies that do not react with a mycobacterial peptide or polypeptide. This composition is composed virtually entirely of the high titer, monospecific, purified polyclonal or ohgoclonal antibodies to the mycobacterial peptide. Purification of avian Ig may be accomplished with Eggstract™ purification system (Promega, Madison, WI). Alternatively, antibodies may be purified by affinity chromatography, using purified mycobacterial peptide or polypeptide which is bound to a chromatographic support. Purification of antibodies by affinity chromatography is generally known to those skilled in the art (see, for example, U.S. Patent No. 4,533,630). Briefly, the purified antibody is contacted with the purified mycobacterial peptide or polypeptide bound to a solid support for a sufficient time and under appropriate conditions for the antibody to bind to the mycobacterial peptide or polypeptide. Such time and conditions are readily determinable by those skilled in the art. The unbound, unreacted antibody is then removed, such as by washing. The bound antibody is then recovered from the mycobacterial peptide by eluting the antibodies, by methods well known to the art, so as to yield purified, monospecific polyclonal antibodies.

Monoclonal antibodies against the mycobacterial peptide can be also prepared, using known hybridoma cell culture techniques. In general, this method involves preparing an antibody-producing fused cell line, e.g., of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species, such as a murine species, from which the myeloma cell line used was derived or is compatible. Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and sensitive and relatively "pure" immunochemically. Immunologically active fragments of the present antibodies are also within the scope of the present invention, e.g., the F(ab) fragment, as are partially humanized monoclonal antibodies.

It will be understood by those skilled in the art that the hybridomas herein referred to may be subject to genetic mutation or other changes while still retaining the ability to produce monoclonal antibody of the same desired specificity. The present invention encompasses mutants, other derivatives and descendants of the hybridomas.

It will be further understood by those skilled in the art that a monoclonal antibody may be subjected to the techniques of recombinant DNA technology to produce other derivative antibodies, humanized or chimeric molecules or antibody fragments which retain the specificity of the original monoclonal antibody. Such techniques may involve combining DNA encoding the immunoglobulin variable region, or the complementarily determining regions (CDRs), of the monoclonal antibody with DNA coding the constant regions, or constant regions plus framework regions, of a different immunoglobulin, for example, to convert a mouse-derived monoclonal antibody into one having largely human immunoglobulin characteristics (see EP 184187A, 2188638A, herein incoφorated by reference). Uses of Anti-Mycobacterial Antibodies of the Invention. The antibodies of the invention are useful for detecting or determining the presence of a mycobacterial polypeptide in a physiological sample, e.g., a mammalian tissue or physiological fluid sample. The antibodies are contacted with the sample for a period of time and under conditions sufficient for antibodies to bind to the mycobacterial polypeptide so as to form a binary complex between at least a portion of antibodies and a portion of the mycobacterial polypeptide. Such times, conditions and reaction media can be readily determined by persons skilled in the art.

For example, the physiological sample may be obtained from a mammal, e.g., a human. Preferably, the sample is a fluid sample or comprises cells. For secreted mycobacterial polypeptides, a fluid sample is preferred. For a sample comprising cells, the cells may be lysed to yield an extract which comprises cellular proteins. Alternatively, intact cells, e.g., a tissue sample such as paraffin embedded and/or frozen sections of biopsies, may be contacted with the antibody or the cells maybe permeabihzed in a manner which permits macromolecules, i.e., antibodies, to enter the cell. The anti-mycobacterial antibodies are then incubated with the protein extract, e.g., in a Western blot, fixed cells or permeabihzed cells, e.g., prior to flow cytometry, so as to form a complex. The presence or amount of the complex is then determined or detected, e.g., through determination or detection of a label.

The antibodies of the invention may also be coupled to an insoluble or soluble substrate. Soluble substrates include proteins such as bovine serum albumin. Preferably, the antibodies are bound to an insoluble substrate, e.g., a solid support such as a dipstick format (see, e.g., Laidoueu et al, 1998; Buhrer et al.,1998; Yersin et al., 1999; and Jelinek et al., 1999). The antibodies are bound to the support in an amount and manner that allows the anti-mycobacterial antibodies to bind mycobacterial polypeptide (ligand). The amount of the antibodies used relative to a given substrate depends upon the particular antibody being used, the particular substrate, and the binding efficiency of the antibody to the ligand. The antibodies may be bound to the substrate in any suitable manner. Covalent, noncovalent, or ionic binding may be used. Covalent bonding can be accomplished by attaching the antibodies to reactive groups on the substrate directly or through a linking moiety.

The solid support may be any insoluble material to which the antibodies can be bound and which may be conveniently used in the assay of the invention. Such solid supports include permeable and semipermeable membranes, glass beads, plastic beads, latex beads, plastic microtiter wells or tubes, agarose or dextran particles, sepharose, and diatomaceous earth. Alternatively, the antibodies may be bound to any porous or liquid permeable material, such as a fibrous (paper, felt and the like) strip or sheet, or a screen or net. A binder may be used as long as it does not interfere with the ability of the antibodies to bind the ligands.

The invention also comprises reagents and kits for detecting the presence of mycobacterial polypeptide in a sample. Preferably, the reagent or kit comprises the purified antibodies of the invention in a liquid that does not adversely affect the activity of the antibodies in the intended assay. Preferably, the liquid is saline solution. Alternatively, the reagent or kit may comprise the purified antibodies attached to a substrate as discussed above. Preferably, the substrate is an insoluble solid support, e.g., the well of a microtiter plate. An alternative preferred substrate is solid particles, such as latex beads. The diagnostic kit comprises, in a container or packaging, one or more of the antibodies or peptides of the invention and a means for detecting or measuring the formation of complexes created by the binding of a mycobacterial polypeptide and the antibodies, or antibodies present in a physiological sample and the peptide. The detecting or measuring means is preferably an immunoassay, such as a radioimmunoassay, ELISA, or an immunofluorescence assay. Most preferably, the detecting or measuring means is a reagent capable of binding to the complexes formed by the mycobacterial polypeptide and the antibodies, or antibodies present in a physiological sample and the peptide, and contains a detectable moiety. Such a reagent may be an antibody of the invention conjugated with a detectable moiety. Alternatively, the antibody can be a second antibody, which is an antibody which binds to the antibodies of the invention, conjugated to a detectable moiety. An example of such a second antibody is anti-IgY-FITC conjugate, anti-IgY-AP conjugate, or anti-IgY-HRP conjugate. Thus, once antibodies having suitable specificity have been prepared, a wide variety of immunological assay methods are available for determining the formation of specific antibody-antigen complexes. Numerous competitive and non-competitive protein binding assays are known to the art. Exemplary immunoassays which are suitable for detecting a serum antigen include those described in U.S. Patent Nos. 3,791,932; 3,817837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Suitable detection means include the use of labels such as radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes and the like. Radioisotopes include ¹²⁵I and ¹³ 'I, enzymes include such as alkaline phosphatase, horseradish peroxidase, beta- D-galactosidase and glucose oxidase, and fluorescers including fluorochrome dyes such as fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), tetramethylrhodamine isothiocyanate (TRITC), 4, 4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). See, for example, U.S. Patent Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.

In a preferred embodiment, peptides, fusion proteins and antibodies of the invention are useful in the methods of the present invention, e.g., in diagnostic tests, such as immunoassays. Such diagnostic techniques include, for example, enzyme immune assay, enzyme multiplied immunoassay technique (EMIT), ELISA, radio-immune assay (RIA), fluorescence immune assay, either single or double antibody techniques, and other techniques in which either the peptide or antibody is labeled with some detectable tag or indicating means. See generally Maggio, 1981 ; and Goldman, 1980.

An illustrative diagnostic system in kit form embodying one aspect the present invention that is useful for detecting mycobacterial polypeptides present in an aliquot of a sample contains antibodies, substantially whole antibodies, such as antibodies raised to a single peptide of the invention or antibodies raised to a plurality of peptides of the invention, or antibody combining sites like Fab and F(ab')₂ antibody portions raised to a peptide of the invention, in one package. This system also includes an indicating means for signaling the presence of a complex between the antibody and the mycobacterial polypeptide in the sample. The kit may also comprise a blocking agent which is contacted with the sample prior to contacting the sample with the primary antibody. The blocking agent and the primary antibody may also be concurrently contacted with the sample. The indicating means permits the complex to be detected, and is packaged separately from the antibody when not linked directly to an antibody of the invention. When admixed with a body sample, such as serum, the antibody binds to the mycobacterial polypeptide in the serum to form a binary complex, and the presence of the indicating means signals the formation of the complex. Optionally, a blocking agent is employed prior to the addition of the antibodies, with the antibodies, or after rinsing away any un-bound antibodies, to block any non-specific binding sites with a protein such as bovine serum albumin (BSA), if desired. A second reagent, e.g., an antibody which binds to the Fc of the primary antibody, can then be added. After this second incubation, any unreacted second antibody is removed as by rinsing leaving only that which is bound to the binary complex. The second antibody may be labeled, e.g., by being linked to a fluorochrome dye, or bind to a detectable label.

A preferred diagnostic system, preferably in kit form, useful for carrying out the above assay method includes, in separate packages, (a) an amount of antibodies that specifically bind to a mycobacterial polypeptide, and (b) a second antibody which is labeled or binds to a detectable label, that reacts with either the first antibody or which binds to the mycobacterial polypeptide but which antibody binds to different epitope than the antibodies of (a). The antibodies may be provided in solution, as a liquid dispersion, or as a substantially dry powder, e.g., in lyophilized form.

The packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such packages include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like.

The use of whole, intact antibodies is not necessary in many diagnostic systems such as the immunofluorescent assay described above. Rather, only the antigen binding site of the antibody molecule may be used. Examples of such antibody combining sites are those known in the art as Fab and F(ab')₂ antibody portions that are prepared by proteolysis using papain and pepsin, respectively, as is well known in the art.

The invention will be further described by the following examples. Example 1

Antibodies Specific for Mycohacterium tuberculosis Sequences for mycobacterial polypeptides may be obtained from any source, e.g., by cloning and sequencing DNA encoding a mycobacterial polypeptide, or by using known nucleic acid or amino acid sequences, e.g., those in a database such as GenBank. Peptide immunogens are then selected for each polypeptide by homology searches, e.g., to identify sequences which are specific for Mycobacteriwn, and algorithms useful to predict surface probability, hydrophilicity and antigenicity (Protean™ Program, DNAstar, and a BLAST search). From each of the peptide sequences, oligonucleotide sequences encoding the peptide or a portion thereof are prepared. For example, an oligonucleotide encoding one of the five peptides shown in Table 1 was cloned into the SSNAP system (Promega Coφ., Madison, WI) for expression of a fusion protein in E. coli. These fusion proteins were used to immunize chickens. The polyclonal antibodies (pAb) were then evaluated by Western blot analysis using culture filtrates (from Dr. Kris Huygen, Institute Pasteur, Brussels) and recombinant mycobacterial antigens (from Dr. Marila Gennaro, NY Public Health Institute), ELISA, and dot blot. An Ag85 MAb (Dr. Henry Godfrey, NY Medical College) was used as a positive control and for benchmarking puφoses. The IgG fraction of the antibodies was specific for the Ag85 complex

(antibodies raised to SEQ ID NO:4 and SEQ ID NO:5), 38 kDa polypeptide complex (antibodies raised to SEQ ID NO: l 1 and SEQ ID NO: 12), and 14 kDa polypeptide (antibodies raised to SEQ ID NO: 10), of M. tuberculosis. Moreover, the pAb was specific for and reacted against both recombinant and native proteins. Further, the antibodies detected smaller amounts of the cognate polypeptide than Ag85 MAb. 85- CPL-4, which was very reactive based on Western analysis, is unique for tuberculosis when the sequence of the peptide used to prepare the antibodies was compared to the corresponding Ag85 sequence of M. bovis, leprae, βavescens, scrofulaceum, intracellular, kansasii, and avium. In addition, the results indicated that the Ag85 MAb is likely directed against Ag85A and not Ag85B, which explains why the MAb does not react against rAg85B, but only native Ag85 complex. Thus, the anti-peptide antibodies are highly selective and sensitive antibodies which are directed against mycobacterial polypeptide. Therefore, the antibodies of the invention may be useful as a diagnostic for active TB infection and may be used singly, e.g., in a dipstick format, and/or in combination, to detect active infection with other mycobacterial species. Furthermore, these antibodies may prove useful even with individuals co-infected with HIV or other pathogens.

Table 1 Antigen Epitope Sequence Ab Name

14 kDa 40-RLEDEMKEGRYE-53 14-CPL-l

(SEQ ID NO: 10) 38 kDa 25-GSKPPSGSPETGAG-38 38-CPL-2

(SEQ ID NO: 11) 38 kDa 253-LDQASQRGLGE-263 38-CPL-3

(SEQ ID NO: 12) Ag85 223-SSDPAWERNDPT-234 85-CPL-4

(SEQ ID NO:4) Ag85 31 1-LNAMKGDCQSSL-322 85-CPL-5 (SEQ ID NO:5)

Example 2

Reactivity of the Antisera on Clinical Samples Further analysis of the antisera includes the reactivity of the antibodies for individuals with M. tuberculosis (Mtb) infection, Mtb and atypical mycobacteria infection, atypical mycobacteria infection, HIV infection, infants accidentally infected (immunized) with adult doses (100^χ) of BCG, non- tuberculous pulmonary disease, and no apparent disease. In addition, the reactivity of the antibodies with the following patient groups is assessed: patients with active TB and positive PPD reactions, patients with active TB and negative PPD reactions, patients with inactive TB or no apparent disease and positive PPD reactions, patients with no apparent disease and negative PPD reactions, patients with non-tuberculous pulmonary disease and negative PPD reactions, and general population controls, with or without positive PPD reactions.

For example, fivefold serial dilutions of 100 μl aliquots of coded samples (diluted in phosphate-buffered saline [PBS], pH 7.2) are analyzed by dot blot with a filtration manifold and affinity purified antibody. Briefly, serum samples and a half-log serial dilution series of purified antibody are adsorbed to nitrocellulose, and the nitrocellulose dried for 15 minutes and blocked overnight at 4°C in PBS (pH 7.2) containing 5% nonfat dried milk. Blots are developed with detection antibodies, e.g., horseradish peroxidase-conjugated second antibodies, enhanced chemiluminescence (ECL or ECL-Plus; Amersham

Pharmacia Biotech, Arlington Heights, IL), and standard X-ray film (Kodak). Using the Ig fraction of 85-CPL-5, the profile of reactivity (+/-) of 85- CPL-5, e.g., for sera from patients with active mycobacterial disease, paralleled that of the control MAb (Dr. Henry Godfrey, NY Medical College).

References

Abou-Zeid et al., Infect. Tmmun.. 50:3046 (1988). Akita et al., J. Food Science. 51:629 (1992). Andersen et al., J. Clin. Microhiol.. 2.3:446 (1986). Balwin et al., Infect. Tmmun.. 66, 2951 (1998). Barnes et al., N. Rngl. I. Med.. 3-24: 1644 (1991 ). Barnes et al., Infect. Tmmun.. 61:3482 (1993). Bass Jr. et al., Am. Rev. Resp. Pis.. 142:725 (1990). Bassay et al., Tuber. T , ting Pi s.. Z--:136 (1996). Bavaay and Merrifield, The Peptides, (eds.) E. Gross and F. Meienhofer, Vol. 2 Academic Press, pp. 3-285 (1980). Belisle et al., S-c nce, 226: 1420 (1997). Bentley-Hibbert et al., Tnfect. Immunity. 67:581 (1999). Bird et al., S iencs, 242:424 (1988). Bisetti et al, Fur. Resp. J.. £:2008 (1995).

Bitter et al, Methods in F.nzymol.. 153-:516 (1987). Borremans et al.. Tnfect. Tmmun.. 57:3123 (1989). Bothamley et al., Ihorax, 42:270 (1992). Bothamley et al., Eur. Respir.. 7:240 (1994V

Bothamley, Fur. Respir. J. Snppl.. 20:676 ( 1 995V

Buhrer et al., Am J. Trop. Med. Hyg.. 5£: 133 (1998).

Calmette, Masson et Cie, Paris (1936). Cantwell et al, IΔMΔ 222:535 (1994).

Centers for Disease Control, Morbid. Mortal. Week. Rep.. 41:39 (1992).

Chaisson et al., J. Tnfect. Pis.. 152:96 (1989).

Chan et al., Tnfect. Tmmun.. 52: 1755 (1991).

Chaparas et al, Am. Rev. Respir. Pis.. 122:533 (1980). Clo.ss et al.. Scand. J. Immunol.. 1 2:249 . 1980).

Cole et al., Tuber. Lung Pis-, 22:363 (1996).

Collins et al., CRC Crit. Rev. Microbiol.. 12: 1 (1993).

Daniel et al., Am. Rev. Resp. Pis._: 123-:556 (1981 ).

Daniel et al., Microbiol. Rev.- 42:84 (1978). Penis et al.. Tnfect. Tmmun., 65:676 (1997).

Deutscher, Methods in Enzymology, Vol. 182, Academic Press, Inc., New York (1990).

De Wit et al., DNA Sequence, 4:267 (1994).

Drowart et al., Scand. J. Tmmtmol., 3-6:697 (1992). Rllner et al.. Rev. Tnfect. Pis._: 1 1 :455 (1989V

Fine, Tubercle, 65: 137 (1984).

Fine et al., Lancet, (ii):499 (1986).

Ford et al., Occup. Med., 2:561 (1994).

Godfrey et al., Tnfect. Immun., 60:2522 (1992). Goldman. Academic Press, New York. N.Y. (1980V

Guss et al., EM----Q--L,, 5: 1567 (1986).

Harboe et al., J. Infect. Pis.. 1 6:874 (1992).

Harth et al., Infect. Immun., 64:3036 (1996).

Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

Hasley et al, Tnfect. Tmmun. 63-:804 (1995).

Havlir et al., Tnfect. Tmmun. 52:665(1991 ).

Hetland et al., Tmmimology 82:445 (1994). Holliger et al., Proc. Natl. Acad.. Sci. USA. 2-3:6444 ( 1993).

Horwitz et al, Proc. Natl., Acad. Sci. USA. 22: 1530 (1995).

Huston et al., Proc. Natl., Acad. Sci. USA, £5:5879 (1988)

Huygen et al., Infect. Tmmun. 5£:2192 (1990). Huygen et al.. Scand. J. Tmmunol. 27: 1 87 (1 988V

Hynes, Sponger Verlag, New York. N.Y. (1990).

Jelinek et al., J. Clin. Micro, 3-2:721 (1999).

Jensenius et al., J. Immunol. Methods. 46:63 (1981).

Jones et al., N------U---, 321, 522-525 (1986). Kiehn et al.. J. Clin. Microbiol.. 21 : 1 68 ( 1985V

Katz et al, J. Virol. Methods. 12:59 (1985).

Klein et al., Chest, 25: 1 190 (1989). Kochi, T^U2£----le- 22: l (1991). Krebber et al., FFBS Lett., 3-22:227 (1995). Kuwabara. S. J. Biol. Chem., 250:2556 (1 75). Laidoueu et al, Trop. Poc, 2£:85 (1998). Langone et al., J. Immunol. Methods, 63: 145 (1983) Larson et al., Comp. Immunol. Microbiol. Tnfect. Pis.. 12: 199 (1990) Launois et al., Clin. Rxp. Tmmunol. £6:286 (1991). Larsson et al., J. Immunol. Methods. K>£:205 (1988). Larsson et al., Hyhridoma, 11:33 (1992). Larsson et al., J. Tmmunol. Methods, 15-6:79 (1992). Leslie et al., J. Exp. Med.. 13-Ω: 1337 (1969). Lozes et al., Vaccine 15:830 (1997). Luelmo. Am. Rev. Respir. Pis.. 1 25:70(1 982).

Maggio, Enzyme Immunoassay, CRC Press, Cleveland, Ohio ( 1981). Manca et al., Infect. Tmmun., 52:503 (1991). Matsuo et al, Tnfect. Tmmun, 5£:550 (1990).

Meienhofer, Hormonal Proteins and Peptides, ed.; CH. Li, Vol. 2 (Academic Press), 48-267 (1973).

McMurray, Chest, 22:4 (1980). Merrifield, J. Am. Chem. Soc, £5:2149 (1963). Minden et al., Infect, Immun,, 46:519 (1984). Morrison et al, Proc. Natl. Acad. Sci. USA. £1:6851 ( 1984).

Orme- Tnfect. Tmmun., 56:3310 (988).

Okwera et al, Morbid. Mortal. Week. Rep.. 12:638 ( 1990).

Padlan, Mol. Tmmunol.. 28, 489-498 (1991). Queen et al., Proc. Natl. Acad. Sci. USA. £6, 10029-10033 ( 1989).

Ratliff et al., J. Gen. Microbiol., 134: 1307 (1988).

Riechmann et al., Nature. 332, 323-327 (1988).

Rinke, et al., Infect. Immun.. 61:3642 (1993).

Roguska et al., Proc. Natl. Acad. Sci. USA. 21, 969-973 (1994). Salata et al.. .1. Lab. Clin. Med.. 1 1 8:589 (1 991

Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed.) (1989).

Satow et al., J. Mol. Bio!., 190, 593-604 (1986).

Scopes, Protein Purification, Springer- Verlag, N.Y. (1982). Smith et al.. Pediatrics. 97: 155 (1996).

Stanford et al., Tubercle. 55: 143 (1974);

Stewart et al., Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco (1969).

Sutherland. Adv. Tuberc. Res.. 19: 1 (1976). Thybo et al.. Tuber Lung Pis.. 76: 149 (1995V

Turneer et al.. Microbiol.. 26: 1714 (1988).

Verhoeyen et al., S-cienc-e, 232, 1534-1536 (1988).

WHO, Tech. Rep. Ser.. 651: 15 (1980).

Wiker et al., Microbiol. Rev. 56:648 (1992). Wiker et al.. Tnt. Arch. Allergy Appl. Tmmunol. 81 :307 (1 86V

Wolinski, Am. Rev. Resp. Pis.. 12: 107 (1979).

Wong et al., Amer. J. Med., 2£:35 (1985).

Yersin et al., Trop. Med Int. Health, 4:38 (1999).

Zhou et al., Clin. Piagn. T ab. Tmmunol.. 3:337 (1996).

All publications, patents and patent applications are incoφorated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for puφoses of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. An isolated polyclonal antibody which specifically binds mycobacterial Ag85 complex, a variant of mycobacterial Ag85 complex, or an immunogenic portion thereof.

2. The antibody of claim 1 which binds to a polypeptide comprising SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, a variant thereof, or an immunogenic portion thereof

3 . An isolated monoclonal antibody which specifically binds mycobacterial Ag85 complex, a variant of mycobacterial Ag85 complex, or an immunogenic portion thereof, wherein the antibody binds to a polypeptide comprising SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, a variant thereof, or an immunogenic portion thereof.

4. An isolated polyclonal antibody which specifically binds a 38 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

5. The antibody of claim 4 which binds to a polypeptide comprising SEQ ID NO: l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof.

6. An isolated monoclonal antibody which specifically binds a 38 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof, wherein the antibody binds to a polypeptide comprising SEQ ID NO: l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof.

An isolated polyclonal antibody which specifically binds a 14 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

8. The antibody of claim 7 which binds to a polypeptide comprising SEQ ID NO: 10, a variant thereof, or an immunogenic portion thereof.

9. An isolated monoclonal antibody which specifically binds a 14 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof, wherein the antibody binds to a polypeptide comprising SEQ ID NO: 10, a variant thereof, or an immunogenic portion thereof.

10. The antibody of claim 1 , 4, or 7 which is obtained from a non-human source.

1 1. The antibody of claim 1, 3, 4, 6, 7 or 9 which is labeled.

12. A preparation of non-human polyclonal antibodies comprising antibodies that specifically bind mycobacterial Ag85 complex, a variant of mycobacterial Ag85 complex, or an immunogenic portion thereof.

13. A preparation of non-human polyclonal antibodies comprising antibodies that specifically bind a 38 kDa mycobacterial polypeptide, a variant of the 38 kDa polypeptide, or an immunogenic portion thereof

14. A preparation of non-human polyclonal antibodies comprising antibodies that specifically bind a 14 kDa mycobacterial polypeptide, a variant of the 14 kDa polypeptide, or an immunogenic portion thereof.

15. A preparation of monoclonal antibodies comprising antibodies that specifically bind mycobacterial Ag85 complex, a variant of mycobacterial Ag85 complex, or an immunogenic portion thereof, wherein the antibodies bind to a polypeptide comprising SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, a variant thereof, or an immunogenic portion thereof.

16. A preparation of monoclonal antibodies comprising antibodies that specifically bind a 38 kDa mycobacterial polypeptide, a variant of the 38 kDa polypeptide, or an immunogenic portion thereof, wherein the antibodies bind SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof.

17. A preparation of monoclonal antibodies comprising antibodies that specifically bind a 14 kDa mycobacterial polypeptide, a variant of the 14 kDa polypeptide, or an immunogenic portion thereof, wherein the antibodies bind to SEQ ID NO: 10, a variant thereof, or an immunogenic portion thereof.

18. The preparation of antibodies of claim 12, 13, 14, 15, 16 or 17 which is labeled.

19. An immunogenic mycobacterial peptide comprising amino acid residues of mycobacterial Ag85B complex, a variant thereof, or an immunogenic portion thereof.

20. The peptide of claim 19 wherein the peptide comprises SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, a variant thereof, or an immunogenic portion thereof.

21. An immunogenic mycobacterial peptide comprising amino acid residues of a 38 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

22. The peptide of claim 21 wherein the peptide comprises SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof.

23. An immunogenic mycobacterial peptide comprising: amino acid residues of a 14 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

24. The peptide of claim 23 wherein the peptide comprises SEQ ID NO: 10, a variant thereof, or an immunogenic portion thereof.

25. The peptide of claim 19, 21 or 23 wherein the peptide is a fusion peptide.

26. An immunogenic composition comprising the peptide of claim 19, 21 or 23 or a combination thereof, in combination with a pharmaceutically acceptable carrier, wherein the administration of the composition to an animal induces production of antibodies to the mycobacterial polypeptide.

27. The composition of claim 26 wherein the animal is an avian or a mammal.

28. An expression cassette comprising: a recombinant DNA molecule comprising a first DNA segment encoding an immunogenic mycobacterial peptide, a variant thereof, or an immunogenic portion thereof, which is operably linked to a promoter functional in a host cell.

29. The expression cassette of claim 28 wherein the first DNA segment encodes a peptide of Ag85 complex, a 38 kDa polypeptide, a 14 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

30. The expression cassette of claim 29 wherein the first DNA segment encodes a peptide of Ag85 complex comprising SEQ ID NO: l, SEQ ID

NO:3, SEQ ID NO:4, SEQ ID NO:5, a variant thereof, or an immunogenic portion thereof.

31. The expression cassette of claim 29 wherein the first DNA segment encodes a peptide of the 38 kDa mycobacterial polypeptide comprising SEQ ID NO: l 1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a variant thereof, or an immunogenic portion thereof.

32. The expression cassette of claim 29 wherein the first DNA segment encodes a peptide of the 14 kDa mycobacterial polypeptide comprising SEQ ID NO: 10, a variant thereof, or an immunogenic portion thereof.

33. The expression cassette of claim 28 further comprising a second DNA segment encoding a carrier protein.

34. The expression cassette of claim 28 wherein the host cell is a prokaryotic cell.

35. The expression cassette of claim 28 wherein the first and the second DNA segments are linked so as to encode a fusion peptide comprising the carrier protein and the mycobacterial peptide.

36. A method to detect a mycobacterial polypeptide in a sample, comprising:

(a) contacting the sample with an amount of the antibody of claim 1, 3, 4, 6, 7 or 9 so as to yield a binary complex; and

(b) detecting or determining binary complex formation.

37. A method to detect a mycobacterial polypeptide in a sample, comprising:

(a) contacting a sample with an amount of the preparation of antibodies of claim 12, 13, 14, 15, 16 or 17 so as to yield a binary complex; and

(b) detecting or determining binary complex formation.

38. The method of claim 36 or 37 wherein the sample is a physiological fluid sample.

39. The method of claim 36 or 37 wherein the sample is a mammalian sample.

40. The method of claim 36 or 37 further comprising detecting the complex with a second antibody.

41. The method of claim 40 wherein the antibody is labeled, or binds to a detectable label.

42. The method of claim 36 wherein the antibody is labeled, or binds to a detectable label.

43. The method of claim 39 wherein the mammal is a human, a domestic ungulate, a feral ungulate, or a laboratory test animal.

44. The method of claim 43 wherein the domestic ungulate is a bovine or swine.

45. The method of claim 43 wherein the feral ungulate is an elk or deer.

46. The method of claim 43 wherein the laboratory test animal is a mouse.

47. A diagnostic assay comprising:

(a) contacting a sample obtained from a mammal at risk of, or suspected of having, a mycobacterial infection with an amount of the antibody of claim 1, 3, 4, 6, 7 or 9 so as to form binary complexes; and

(b) detecting or determining binary complex formation, wherein complex formation is indicative of mycobacterial infection.

48. A diagnostic assay comprising:

(a) contacting a sample obtained from a mammal at risk of, or suspected of having, a mycobacterial infection with an amount of the preparation of antibodies of claim 12, 13, 14, 15, 16 or 17 so as to form binary complexes; and (b) detecting or determining binary complex formation, wherein complex formation is indicative of mycobacterial infection.

49. A kit which comprises packaging, containing, separately packaged: the preparation of antibodies of claim 12, 13, 14, 15, 16 or 17, or a combination thereof, wherein the antibody is labeled or binds to a detectable label.

50. A kit which comprises packaging, containing, separately packaged: the antibody of claim 1, 3, 4, 6, 7 or 9, or a combination thereof, wherein the antibody is labeled or binds to a detectable label.

51. A diagnostic kit for detecting mycobacterial infection in mammalian physiological sample which comprises packaging, containing, separately packaged:

(a) the antibody of claim 1, 3, 4, 6, 7 or 9; and

(b) a second antibody.

52. A diagnostic kit for detecting mycobacterial infection in mammalian physiological sample which comprises packaging, containing, separately packaged:

(a) the preparation of antibodies of claim 12, 13, 14, 15, 16 or 17; and

(b) a second antibody.

53. The kit of claim 51 or 52 wherein the second antibody binds to the same mycobacterial polypeptide as the antibody of (a) but does not bind to the same epitope on the polypeptide as the antibody of (a).

54. The kit of claim 51 or 52 wherein the second antibody binds to the antibody of (a).

55. The kit of claim 33 or 34 wherein the second antibody is labeled or binds to a detectable label.

56. The kit of claim 49, 50, 51, or 52 further comprising instruction means.

57. A host cell comprising the expression cassette of claim 28.

58. An animal exposed to the immunogenic composition of claim 26.

59. A composition comprising a first anti-mycobacterial antibody and a second anti-mycobacterial antibody, wherein each antibody binds to a different mycobacterial epitope.

60. The composition of claim 59 wherein the first antibody is a polyclonal antibody.

61. The composition of claim 59 wherein the second antibody is a polyclonal antibody.

62. The composition of claim 59 wherein the first antibody is a monoclonal antibody.

63. The composition of claim 59 wherein the second antibody is a monoclonal antibody.

64. The composition of claim 59 wherein the first antibody specifically binds mycobacterial Ag85 complex, a variant of mycobacterial Ag85 complex, or an immunogenic portion thereof.

65. The composition of claim 59 wherein the second antibody specifically binds mycobacterial Ag85 complex, a variant of mycobacterial Ag85 complex, or an immunogenic portion thereof.

66. The composition of claim 59 wherein the first antibody specifically bind a 14 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

67. The composition of claim 59 wherein the second antibody specifically binds a 14 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

68. The composition of claim 59 wherein the first antibody specifically bind a 38 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.

69. The composition of claim 59 wherein the second antibody specifically binds a 38 kDa mycobacterial polypeptide, a variant thereof, or an immunogenic portion thereof.