WO2008067497A2 - Enriched antibody for detecting mycobacterial infection, methods of use and diagnostic test employing same - Google Patents

Abstract

The disclosed technology provides an enriched antibody population, specific for an surface polysaccharide antigen from a mycobacterium. In a related embodiment, the antibody is enriched by having been raised in an environment that maintains antigenically active antigen. These enriched antibodies may be used in conjunction with an immunoassay, particularly an immunoassay in an ICT format, for detecting the presence of a mycobacterial infection in a sample from a subject, wherein the antibody is selected to be unaffected by the presence of HIV in the sample.

Description

Attorney Docket: 2912/112WO

Enriched Antibody for Detecting Mycobacterial Infection, Methods of Use and

Diagnostic Test Employing Same

Cross Reference to Related Applications

The present application claims priority from provisional patent application U.S.

Application Serial Number 60/861 ,661 , filed November 29, 2006. For purposes of national phase in the United States, this application is also a continuation-in-apart of U.S. Application Serial Number 11/186,933, filed July 20, 2005, which claims priority from provisional patent application U.S. Application Serial Number 60/589,419, filed July 20, 2004. Each of the foregoing applications is hereby incorporated herein by reference in its entirety.

Technical Field

The present invention relates to diagnostic tests for detecting microbial-based diseases and conditions, and more particularly for diagnostic tests and methods for detecting tuberculosis.

Background

During the last decades TB has evolved from a predominantly pulmonary infection into a multifaceted pathology with a growing rate of extrapulmonary cases. Until to date effective TB prevention programs are hampered by the absence of a rapid and field adapted screening assay. In high-income countries mycobacterial culture remains the diagnostic standard, but it is time-consuming and relatively expensive. Ideally, sputum microscopy based on three sputum smears can identify up to 67% of culture positive cases. HIV co- infection has been reported to impair the demonstration of Mycobacterium tuberculosis in sputa, although some investigators do not report any influence of the HIV serostatus on the AFB (Acid-fast bacillus smear, culture, and sensitivity) diagnosis. The higher percentage of extrapulmonary TB in HIV positive TB patients additionally increases the rate of AFB- negative TB cases. This renders tuberculosis an increasing diagnostic challenge and underlines an urgent need for improved laboratory tools for its diagnosis.

Current approaches for diagnosing TB are not satisfactory. The sputum test for pulmonary TB is not always effective, particularly if there are no detectable bacteria in the sputum, or no sputum sample can be obtained. In addition, this diagnostic test requires microscopy and/or culture of the bacteria to confirm the diagnosis, neither of which is especially suitable to diagnosis in the field. Using cerebrospinal fluid for diagnosis of TB- meningitis is also problematic, particularly in the field since, once again, microscopy and/or culture of the bacteria and/or an ELISA test is usually required to confirm the diagnosis. Blood tests for TB are also known, but have a poor track record, being complex and unreliable. Urine tests are simpler and more reliable, but current methods require processing of the urine before performing the diagnostic test - such processing usually involving concentration of the urine.

Among the newly developed methods antibody tests against a number of mycobacterial antigens have been developed, but none of these tests has so far reached the needed specificity for routine diagnostic purpose. The drop of sensitivity in HIV positive cases is also a major constraint. A different approach is to measure immune responses to Mycobacterium tuberculosis specific antigens like ESAT-6, but so far the differentiation between latent TB infection and TB disease is not possible. Tuberculosis is an extremely complex pathology existing in multiple forms, but always starting as an airborne infection. Pulmonary tuberculosis occurs immediately at the entry point of the microorganism and extrapulmonary tuberculosis is the result of further penetration into the body of the patient with the most widespread examples of tuberculous meningitis and bone tuberculosis. Complexity of the pathology determines multitude of various approaches tried during this century of modern medicine. Furthermore clinical and radiographic manifestations of HIV -related pulmonary tuberculosis are dramatically altered by immunodeficiency. These factors severely limit our capability of early symptomatic recognition of tuberculosis in HIV/TB patients and also increase the danger of TB transmission to relatives and caregivers of such patients. Mycobacteria can potentially be recovered from a variety of clinical specimens, including upper respiratory collections (sputum, bronchial washes, bronchoalveolar lavage, bronchial biopsies and such); urine, feces, blood, cerebrospinal fluid (CSF), tissue biopsies, and deep needle aspirations of virtually any tissue or organ. Bacterial culture remains the gold standard in the diagnosis of tuberculosis, but it can take up to 6-8 weeks to make a conclusive diagnosis. There are three major technologies used for rapid (faster than bacterial culture) diagnosis of the mycobacterial infections:

• Direct microscopy of sputum smears;

• PCR-based assays;

• Immunodiagnostic methods.

Direct microscopy of sputum smears. More than a century ago, Robert Koch identified the etio logic agent of tuberculosis by staining it and culturing it from clinical specimens. Today, the diagnosis of tuberculosis is usually established using staining and culturing techniques that do not differ substantially from those that Koch used. Direct microscopy of sputum is the norm for the diagnosis of tuberculosis in developing countries today and it is the benchmark against which the efficiency of any new test must be assessed. It is applied to pulmonary tuberculosis, but is not very useful for children or for patients with initial stages of pulmonary tuberculosis.

PCR-based assays.

A comparative study of the performance of PCR tests in seven laboratories has shown high levels of false-positive PCR-results, ranging from 3% to 20% (with an extreme of 77% in one laboratory). This relatively poor performance resulted from lack of monitoring of each step of the procedure and underscores the necessity for careful quality control during all stages of the assay.

Immunodiagnostic methods.

The Tuberculosis Skin Test.

This is the probably oldest immunological test for tuberculosis. A small amount of substance called PPD Tuberculin is placed just under the top layer of the skin on the forearm with a small needle. The test is read 48 to 72 hours after it has been given. Generally, a swelling of 10 mm. or more is considered positive. Many developing countries use BCG vaccination to protect against TB. After BCG vaccination, the PPD skin test usually becomes positive. Results of the skin test vary dependent on the quality of the PPD antigen, reactivity of the immune system and probably even race of the individual. This test also does not provide an unequivocal indication about the stage and location of the infection.

Serological tests for M. tuberculosis. This approach, based on the detection of antibody immune response to mycobacterial antigens is one of the most widely used in research and clinical environments. All serological tests have approximately the same sensitivity and specificity if they use purified antigens. The sensitivity of the best tests is in a range of 80% for smear-positive cases and 60-70% for smear negative cases. The reported specificity is generally high and is in a range of 95-100%. Currently existing technologies are limited in their performance in several ways.

Most widely accepted rapid microscopic test requires several hours to complete, skillful technician and clinical laboratory environment. Test interpretation is far too difficult compared to current standards of rapid POC (point of care) testing in the infectious diseases area. Real cost of one analysis per one patient runs in the range of $100-150 for a US hospital. Clinical specificity of the test is very good, but any improvements in sensitivity will be more than welcome.

Skin test has sufficient sensitivity, but takes a long time and does not provide information about stage of pathological process and does not sufficiently differentiate infected and vaccinated individuals. Serological tests usually do not have sufficient sensitivity. Test results vary with variations in the individual immune response to TB antigens. These tests practically do not work in HIV patients infected by M. tuberculosis. This factor severely limits their applicability in Africa and many Asian countries. In the US this group of patients constitutes the majority of TB infected patients as well. PCR tests are widely used in developed countries, but are complex, expensive and are not sensitive enough to justify their use as a screening test in developing countries. A preferred method for rapid diagnosis of infectious diseases is based on the detection of a bacterial antigen in the patient sample, that provides unequivocal proof of active infectious process caused by specific pathogen. The concept of using a direct antigen test for detection of mycobacterial infections was described in several publications.

For example, the development of one of the first direct antigen assay for M. tuberculosis was reported in 1982 - a radioimmunoassay for the detection M. tuberculosis antigens in sputum of patients with active pulmonary tuberculosis, using a rabbit antibody specific to the whole cells of M. bovis (BCG vaccine). Autoclaved and sonicated sputum was used as a sample. The assay detected antigen in 38 of 39 sputum samples from patients with active tuberculosis pulmonary tuberculosis.

Later studies reported the development of the ELISA system for the detection of mycobacterial antigens in the cerebrospinal fluid of patients with tuberculous meningitis, also using antibodies specific to the whole cells of M. bovis. Both systems showed surprisingly high specificity. Despite the fact that LAM was the major antigen responsible for the detection, it was reported that M. kansasii showed 5% cross-reactivity, and M. intracellulare, M. avium, M. fortitum, and M. vaccae cross-reacted only at 2%. Others reported detection, by ELISA, of mycobacterial antigen in the CSF of nine of 12 patients with tuberculous meningitis, corresponding to the sensitivity of 81.25%. Specificity of the test was equal to 95%.

Practically all previous attempts to develop a test for diagnosis of tuberculosis have focused on the detection of the pulmonary form of the disease. Extrapulmonary forms, which are notoriously difficult to diagnose, attracted relatively little attention due to low prevalence rate compared to pulmonary forms. Until the 1950s and 1960s, extrapulmonary TB cases comprised only around 10% of all tuberculosis cases. The onset of the HIV/ AIDS pandemic has changed the situation completely. These two diseases eventually merged into a new complex public health problem. Now fully 60 % of untreated HIV patients develop active TB during their lifetime and up to 70% of TB patients are HIV infected in sub-Saharan Africa and Asia. Superimposition of HIV and TB changed not only the epidemiology of tuberculosis, but also the course of the disease itself. During the last decades TB has evolved from predominantly a pulmonary infection into a multifaceted pathology with an ever growing prevalence of extrapulmonary forms. It is estimated that extrapulmonary TB cases currently comprise up to 30% of all cases of tuberculosis; this number might even be an underestimation due to the lack of tools for rapid screening and diagnosis of extrapulmonary forms of tuberculosis. Moreover, even pulmonary tuberculosis in HIV patients frequently exhibits atypical symptoms. For example, such patients typically do not produce sputum. These factors severely limit our capability of early symptomatic recognition of tuberculosis in HIV/TB patients and also increase the danger of TB transmission to relatives and caregivers of such patients. An easy to use screening test, capable of detecting a broad spectrum of pathologies due to M. tuberculosis infection, is urgently needed, including a test for extrapulmonary forms of TB. Such a need has long been discussed with no progress towards realising goal. Today the need has became a public health care emergency.

In other cases of pulmonary bacterial infections, the current screening process of choice is based on the detection of polysaccharide antigens secreted in the patient's urine. Bacterial polysaccharides are composed of monosaccharides uncommon to humans and therefore resistant to cleavage by human enzymes. This enables their secretion in urine in immunochemically intact forms suitable for detection by a polysaccharide-specific immunoassay. Extremely low concentrations of bacterial polysaccharides secreted in urine require very high sensitivity of the immunoassay in order to use it as a screening procedure. Collaborating research groups from Sweden and Norway attempted development of a

LAM-specific ELISA system detecting LAM antigen in patient urine. The system used antigen capture for detecting tuberculosis from urine based on lipoarabinomannan, a polysaccharide present on the surface of Mycobacterium tuberculosis, the organism responsible for causing tuberculosis in humans, as disclosed in PCT application no. WO97/34149 to Svenson, hereby incorporated by reference herein. The disclosed diagnostic procedure detected the presence of LAM in patient urine in 81.3% of AFB-positive patients and 57.4% of AFB-negative patients and demonstrated utility of the detection of mycobacterial LAM antigen for diagnosis of mycobacterial infections. At the same time the system failed to demonstrate utility of the disclosed process for screening purposes. Despite use of the affinity purified rabbit polyclonal antibody specific to LAM antigen, the procedure lacked sufficient sensitivity to be used on non-processed un-concentrated urine samples. The diagnostic procedure required approximately 24-48 hrs of sophisticated manipulations in a biochemical lab focused on concentrating patient urine and preparing it for analysis by ELISA test. Overall, the sensitivity of the Svenson assay is not sufficient for practical use of the disclosed method. The complexity and length of the immunoassay also prevents its practical use as a screening test for detection of mycobacterial infections because it proved too cumbersome for use in a clinical setting, where speed, ease of use, and high sensitivity are all critically important for diagnostic tests used to detect disease conditions.

Summary of the Invention

In a first embodiment of the invention there is provided an antigenically active isoform of lipoarabinomannan from mycobacterium tuberculosis, prepared by mild, partial oxidation of LAM using controlled concentrations OfNaIO₄. In other embodiments, the antigenically active isoform of LAM, generated by controlled and mild oxidation methods, is used to prepare highly specific, highly pure antibodies raised to inactivated mycobacterium, more particularly raised to surface polysaccharides such as the lipopolysaccharide lipoarabinomannan (LAM), for use in the detection of lipopolysaccharides in urine, sputum, blood, tissue or other samples from patients of interest. Other embodiments use the specific, isolated antibody raised to the antigenically active form of a surface polysaccharide antigen, such as the lipopolysaccharide LAM, to diagnose tuberculosis and other mycobacterial infections in patients of interest. In another particular embodiment, there is provided an enriched antibody population specific for an antigen of a surface polysaccharide from a mycobacterium. In this embodiment, the enriched antibody population may be enriched by having been raised in an environment that maintains antigenically active antigen. Alternatively or in addition, the antibody is enriched by exclusion of antibodies that recognize relatively inactive antigen, such as those rendered less antigenically active by modification with an agent, such as the oxidizing agent sodium periodate (NaIO₄) . In other embodiments, the mycobacterium may be Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, or Mycobacterium tuberculosis. In other embodiments, the surface polysaccharide antigen may be a lipopolysaccharide antigen such as lipoarabinomannan (LAM).

In one embodiment there is provided an enriched antibody population specific for a mycobacterial surface polysaccharide antigen, wherein the antibody specific for the mycobacterial antigen is enriched by exclusion of antibodies that recognize modified mycobacterial surface polysaccharide antigen that have been rendered less antigenically active, wherein the modified antigen is modified by oxidation with NaIO₄. In such embodiments, the mycobacterium may be Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, or Mycobacterium tuberculosis, more particularly Mycobacterium tuberculosis or

Mycobacterium paratubercuolsis, and the surface polysaccharide may be a lipopolysaccharide, more particularly lipoarabinomannan. In other embodiments, the enriched antibody population may be a polyclonal antibody population.

One particular embodiment provides a process for producing an isolated enriched antibody specific to an surface polysaccharide antigen of a mycobacterium, the process comprising isolating antigenically active surface polysaccharide antigen from mycobacteria under NaIO₄ oxidation conditions sufficient to maintain antigenic activity in a population of surface polysaccharide antigen so as to produce isolated antigenically active antigen, and raising and isolating antibody to the isolated antigenically active antigen, so as to produce isolated enriched antibody specific to the surface polysaccharide antigen of the mycobacterium.

Another particular embodiment provides a process for producing an isolated enriched antibody specific to a surface polysaccharide antigen of a mycobacterium, the process comprising exposing surface polysaccharide antigen, isolated from the mycobacterium, to NaIO₄ oxidation conditions so as to produce an antigen population including antigens that remain antigenically active and antigens that have been rendered less antigenically active, raising and isolating antibody to the antigen population so as to produce a population of isolated antibody, and removing, from the population of isolated antibody, antibody that is specific to the less antigenically active antigen, so as to produce isolated enriched antibody specific to the surface polysaccharide antigen of the mycobacterium. Yet another particular embodiment provides a process for producing an isolated enriched antibody specific to a surface polysaccharide antigen of a mycobacterium, the process comprising, exposing surface polysaccharide antigen, isolated from the mycobacterium, to NaIO₄ oxidation conditions so as to produce an antigen population including antigen that remains antigenically active and antigen that has been rendered less antigenically active, isolating antigenically active surface polysaccharide so as to produce isolated antigenically active antigen, isolating antigen that has been rendered less antigenically active so as to produce isolated less antigenically active modified antigen, applying sera from a mammal inoculated with mycobacteria to a first affinity matrix prepared with the isolated antigenically active antigen, such that antibody specific to the antigenically active antigen is retained by the first affinity matrix, isolating antibody specific to the isolated antigen from the first affinity matrix, applying the isolated antibody to a second affinity matrix prepared with the isolated modified antigen, such that antibody specific to the modified antigen is retained by the second affinity matrix, and isolating enriched antibody specific to the antigenically active antigen by collecting effluent from the second affinity matrix, so as to produce isolated enriched antibody specific to the surface polysaccharide antigen of the mycobacterium.

In such processes, the mycobacterium may be Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis, more particularly Mycobacterium tuberculosis or Mycobacterium paratubercuolsis, and the surface polysaccharide may be a lipopolysaccharide, including the surface polysaccharide lipoarabinomannan (LAM). More particularly, the surface polysaccharide may be isolated from Freund's adjuvant. In related embodiments, the modified antigen is rendered less antigenically active with NaIO₄.

Another particular embodiment provides a method for detecting a mycobacterial infection in a urine sample from a subject of interest, by detecting mycobacterial surface polysaccharide antigen in the sample, the method comprising providing an ICT device, such device (i) having an arrangement for receiving a sample, (ii) providing a visual test result, and (iii) utilizing an antibody according to any of those described or produced by the processes described; contacting the sample with the arrangement in the device for receiving a sample, so as to cause the device to provide a visual test result that is positive if the antibody in the test device binds to a mycobacterial surface polysaccharide antigen in the sample. In other embodiments, the antibody is selected so that the visual test result is unaffected by the presence of HIV infection in the subject. In such embodiments, a positive immunoassay result compared to an appropriate control is considered positive for a mycobacterial infection. Additional embodiments provide a method wherein the appropriate control may be a positive control, a negative control, or any combination thereof, and the mycobacterial infection may be Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae,

Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis, more particularly M. tuberculosis ox Mycobacterium paratubercuolsis.

For still other embodiments, the surface polysaccharide is a lipopolysaccharide, such as lipoarabinomannan (LAM), and the sample may be a concentrated urine sample or a non- processed unconcentrated urine.

Other particular embodiments provide a kit for detecting a mycobacterial infection in a sample, the kit comprising an assay for detecting a surface polysaccharide antigen from a mycobacterial infection, wherein the assay comprises an enriched antibody as described above or as produced by a method described above. Another embodiment provides a kit as described, wherein the assay is an ELISA or an ICT format. In particular embodiments of the kit, the mycobacterial infection detected may be Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis, more particularly Mycobacterium tuberculosis or Mycobacterium paratubercuolsis. In some embodiments of the kit, the surface polysaccharide antigen is a lipopolysaccharide, such as lipoarabinomannan.

Brief Description of the Drawings

The foregoing features of the invention will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which: Figures IA and IB show a structural model of mycobacterial ManLAM, PILAM, and

AraLam. MTP corresponds to 5-methylthiopentose described up to date (*) in M. tuburculosis strains H37Rx, H37Ra, CSU20 and MT K3. 5' corresponds to succinyl residues located on arabinan domain of ManLAM of M. bovis BCG. One to four succinyl groups, depending on the M. bovis BCG strain, were shown to esterify the 3,5-α-Araf units at position 0-2. MPT, mannosyl-phosphotidyl-myo-inositol; Manp, mannopyranose; Araf, arabinofuranose; Ins, myo-inositol; Ra, fatty acyl groups. ManLAM contain approximately 60 Araf and 40 Manp units. Manp units are distributed among the mannose caps and the mannan core. Figure 1C shows an attempt to present a composite structure of ManLAM from M. tuberculosis (Erdman strain) based on the recognition of certain small motifs and on the relative amounts of the carious arabinose and mannose units in their different linkages. Figure ID shows a chemical model of the mycobacterial cell wall. The three-dimensional and spatial arrangement of the key molecules are largely unknown. It is thought that most mycobacterial cell walls conform to this model with mAGP and LAM as the two principal constituents. The surface glycolipids include a variety of species- and strain-specific glycopeptidolipids, lipo-oligosaccharides, and phenolic glycolipid, the chemical identity and amount of which varies from one species to another.

Figure 2 shows a comparison of serological activity for LAM experiments.

Figure 3 shows the efficiency of LAM-specifϊc Ab preparations in capture ELISA. Figure 4A Sensitivity of the LAM ELISA for different concentrations of LAM in urine.

Solid circles represent ELISA results using LAM from M. tuberculosis, and open circles represent the control ELISA results. The cut off was the Optical Density of the Negative

Control + 0.1, resulting in a minimal detection limit of 0.25 ng/ml.

Figure 4B Binding of LAM-specific antibodies in the ELISA to non-mycobacterial antigens was excluded for the following bacterial species:

Klebsiella pneumoniae, Streptococcus agalactiae, Streptococcus pneumoniae 14/12F,

Pseudomonas aeruginosa, Staphylococcus aureus 25923/43300, Proteus vulgaris, E. coli

8739, Neisseria meningitidis A/B/13102, Haemophilus influenzae A/B/D. Open triangles represent ELISA results for binding of LAM-specific antibodies to antigens of M. tuberculosis, whereas the remaining symbols (e.g. solid triangles, open and solid diamonds, open and solid circles.) represent ELISA results using the same LAM-specific antibodies with other bacterial species described above.

Figure 4C Sensitivity of the LAM ELISA for various mycobacterial strains. LAM of M. bovis and M. tuberculosis are detected most sensitively. Figure 4D MTB-ELISA results for urine tests with LAM in the urine compared to negative control with no LAM in urine.

Figure 4E MTB-ELISA results for urine tests with LAM in the urine compared to negative control with no LAM in urine, for lower concentrations of LAM (0 to 1 ng/niL).

Figure 5 Correlation between the microscopic mycobacterial density of AFB positive patients and their antigen concentration measured by the LAM ELISA in unprocessed urine.

AFB + (light microscopy 1000 x magnification: 4-90 acid fast bacilli/100 fields) 28 cases.

AFB ++ (1-9/field) 23 cases. AFB +++ (~10/field) 20 cases. Box plot showing 10^th, 25^th, 50^th,

75^th, 90^th percentile and the mean antigen concentration.

Figure 6 shows a schematic of an antigen purification process in accordance with particular embodiments of the claimed invention.

Figure 7 shows a schematic for preparing affinity columns in accordance with particular embodiments of the claimed invention. Figure 8 shows a schematic of an antibody purification process in accordance with particular embodiments of the present invention.

Figure 9 shows a schematic of a conjugate preparation, in accordance with particular embodiments of the present invention. Figure 10 shows MTB-ELISA signals were determined in the urine of 96 HIV(-) Tanzanian bar workers.

Figure 11 shows the kinetics of the secretion of LAM antigen in the urine of TB patients.

Figure 12 shows a comparison of the detection of TB in patients from Group A (LJ positive),

Group B (radio logically diagnosed with TB), Group C (no clinical proof of TB) and Group D (healthy Tanzanians) using a cut-off value of 0.1 OD unit above the negative control (Group

D).

Figure 13 shows a graph of the cumulative distribution of TB(+) patients as a function of urinary LAM concentration.

Figure 14 shows the correlation between urinary LAM concentration and smear AFB signal. Figure 15 shows the results of the LAM-ELISA tests on various patient groups in the

TS2005 study.

Figure 16 shows the results of the LAM-ELISA tests on various patient groups in the T2006 study.

Figure 17 shows an MBT-ICT analysis of PBS/BSA spiked with LAM-4 antigen - a comparison of ICT lots 080602NH, 080603 VD, and 06160105.

Figure 18 shows an MBT-ICT analysis of healthy urine spiked with LAM-4 antigen - a comparison of ICT lots 080602NH, 080603 VD, and 06160105.

Figure 19 shows a picture of actual LAM-ICT strip tests treated with urine samples.

Figure 20 shows a picture of actual LAM-ICT strips showing the visual sensitivity of the LAM-ICT test.

Figure 21 shows a calibration graph for LAM-ICT strip test (lot #080602NH).

Detailed Description of Specific Embodiments

Definitions The following terms shall have the meanings indicated unless the context otherwise requires:

"Immunoreactive environment" as used herein means, an environment supportive of immunoassays, immunoreactions, immunochemistry, and any process, assay, methodology or system which involves, relates to or relies on an immunological reaction to achieve a desired result. Examples of immunoreactive environments are those detailed in US Patent No. 5,073,484 to Swanson et al.; and US Patent Nos. 5,654,162 and 6,020,147 to Guire et al, incorporated by reference herein, disclosing method and apparatus for quantitatively determining an analyte in a liquid, wherein particular embodiments employ immunochemical reactions in which the analyte and the reactant represent different parts of a specific ligand

(antigen) - antibody (anti -ligand) binding pair. These patents relate to technology that has been implemented as what we call in this description and the following claims as a "strip test." "Freund's adjuvant" is from Sigma, USA. "Antigenically active" as use herein means, a given isolated antigen is capable of producing an antibody capable of binding to a similar, related or naturally occurring, non-isolated antigen presented by an organism such as a mycobacterium, or is capable of provoking an immunoresponse or immunoreaction or provoking a binding interaction with an antibody raised against a similar, related, or naturally occurring, non-isolated antigen presented by an organism such as a mycobacterium, such that the antigenic activity of the given antigen is comparable to the naturally occurring, similar or related antigen presented by the mycobacterium. As used herein, "sufficient to maintain antigenically active antigen" means that the treatment conditions, environment, or circumstances related to the antigen do not destroy the capability of the isolated antigen to be antigenically active, as defined and understood herein.

We have developed a high-sensitivity method for detecting the presence of mycobacterium antigens, particularly M. tuberculosis antigens, such as the surface polysaccharides lipoarabinomannan (LAM) and related species, in bodily fluids including but not limited to urine. Heretofore, tests of this nature lacked sensitivity and were not operable for unprocessed urine samples or for detecting extrapulmonary TB infections. In particular, we have developed enriched antibodies raised to antigen from mycobacteria wherein the antibody is enriched by having been raised in an environment that maintains antigenically active antigen using controlled, milder NaIO₄ oxidation conditions. We call the method for producing this first class of enriched antibody the "direct method," which is described in further detail below.

We have also developed antibody that is enriched by exclusion of antibodies that recognize relatively inactive antigen rendered less antigenically active by treatment using controlled, stronger NaIO₄ oxidation conditions. The method for producing this class of antibodies begins by following the "direct method" to obtain enriched antibodies, but then also operates by excluding antibodies that recognize relatively inactive antigen. We call the method for producing this second class of enriched antibody the "enhanced method," which is also described in further detail below. Figure 8 is a schematic depiction showing the steps involved in practicing an embodiment of the enhanced method. Because the enhanced method builds on the direct method, Figure 8 also illustrates the direct method, if one stops after the first affinity column. Below we show how these enriched antibodies of either or both classes can be used to detect pulmonary and extrapulmonary infections of TB in a variety of samples, including but not limited to untreated (i.e. non-concentrated) urine samples. (Other potential sources of sample include sputum, cerebrospinal fluid, blood, tissue, lavages.) In the examples which follow, the enriched antibodies are raised to an epitope of lipoarabinomannan (LAM) in an environment which maintains its antigenic activity.

Prior methods for detecting surface polysaccharides (LAM) using different body fluids such as serum, urine or sputum have been investigated, but have proven problematic.

In serum, the detection of LAM seems to be disturbed by immune complex formation. Detection of LAM in sputum is possible only in the samples of the patients with pulmonary TB because extra-pulmonary infections often do not provide sputum containing mycobacterial antigens. Prior studies with urine required extensive sample processing and manipulation, limiting such methodologies in the field. None were effective for diagnosing extra-pulmonary mycobacterial infections such as those on the rise in HIV-positive subjects.

Embodiments of the present invention overcome difficulties in the prior art by providing enriched antibodies that may be used for detecting mycobacterial antigens in a wide range of sample types from a subject. These sample types include sera, blood, sputum, lavages, tissue, and unprocessed, non-concentrated urine, among others.

Lipoarabinomannan (LAM) is a 17500 mol wt lipopolysaccharide specific for the genus mycobacterium. Lipoarabinomannan is a complex polysaccharide antigen composed of mannose and arabinose residues forming a highly branched and complex structure. Despite more than four decades of structural studies of polysaccharide antigens of mycobacteria, those in the art still speak only about fragments of the structure or structural motifs and composite models. Two composite models of the LAM structure are presented in Figs. IA and IB, and in 1C. Figures IA and IB show the structural model of mycobacterial ManLAM, PILAM, and AraLAM, where MTP corresponds to 5-methylthiopentose described up to date (*) in M. tuburculosis strains FB 7Rx, FB 7Ra, CSU20 and MT K3 and 5' corresponds to succinyl residues located on arabinan domain of ManLAM of M. bovis BCG. One to four succinyl groups, depending on the M. bovis BCG strain, were shown to esterify the 3,5-α-Ara/units at position 0-2. MPT represents mannosyl-phosphotidyl-myo-inositol; Man/? represents mannopyranose; Ara/represents arabinofuranose; Ins represents myoinositol; and R_a represents fatty acyl groups. ManLAM contain approximately 60 Ara/and

40 Man/? units. Man/? units are distributed among the mannose caps and the mannan core. In Fig. 1C, a composite structure of ManLAM from M. tuberculosis (Erdman strain) is presented (Ann. Rev. Biochem. 64, 29-63 (1995)), which is based on the recognition of certain small motifs and on the relative amounts of the carious arabinose and mannose units in their different linkages.

Figure ID shows a chemical model of the mycobacterial cell wall (Glycobiol. 8 (2),

113-120 (1998)). The three-dimensional and spatial arrangement of the key molecules are largely unknown. It is thought that most mycobacterial cell walls conform to this model with mAGP and LAM as the two principal constituents. The surface glycolipids include a variety of species- and strain-specific glycopeptido lipids, lipo-oligosaccharides, and phenolic glyco lipid, the chemical identity and amount of which varies from one species to another. As part of the outer cell wall of mycobacteria, LAM is released from metabolically active or degenerating bacterial cells. It is assumed that in active TB infection LAM leaks into the circulation, passes through the kidneys and can therefore be detected in the urine reflecting the level of mycobacterial burden. Since LAM is a carbohydrate antigen with glycosidic linkages for which no human degrading glycosidases exist, the antigen occurs in the urine in intact form.

LAM antigen of mycobacteria is composed of three major structural domains: the mannosyl-phospahtidyl-myo-inositol (MIP) anchor, containing variable number of fatty acids with variable chain length; mannan core polysaccharide variable in number of mannose residues; and branched arabinan polysaccharide chains connected to mannan core. Despite many efforts, the attachment site(s) for arabinan chains on the mannan core remain unknown. Arabinan polysaccharide chains are capped by mannose oligosaccharides, consisting of mono-, (αl-2)-di- and (αl-2)-tri-mannosyl units variable in their length (capping motifs). Capping degree is variable from strain to strain and possibly is also dependent from growth conditions.

Extremely high structural complexity and variability of mycobacterial LAM lead to very complex spectrum of antigenic epitopes. Complexity of the selected diagnostic antigen forces us to use affinity purified polyclonal antibody as a main immunoassay reagent. Only use of polyclonal antibody allows one to cover the full spectrum of antigenic specificities potentially associated with LAM present in clinical samples. In order to achieve the highest possible assay sensitivity of sandwich immunoassay, we use the highest concentration of antigen-specific antibody in the capture zone and also as the labeled antibody. Antigen- specific affinity purification is known to produce such an antibody.

To prepare the antigen-based affinity column, we developed a process for antigen isolation and coupling to the solid phase support. The process of LAM antigen isolation is based, with some minor modifications, on the methods of isolation of other bacterial polysaccharides described in the literature and well-known to those in the art, and described below.

Previous LAM-based direct antigen immunoassay described in the literature used polyclonal antibody purified by antigen-specific affinity chromatography using a LAM-

Sepharose column. The prior art approach to the synthesis of the affinity matrix was based on the partial Nalθ₄-oxidation of LAM polysaccharide with subsequent coupling to NH₂- Sepharose. Surprisingly, our experiments have shown that standard Nalθ₄-oxidation conditions reduce antigenic activity of LAM polysaccharide, as can be seen from the Fig.2. Because coupling efficiency of oxidized polysaccharide to NH₂-SO lid support is proportional to the degree of oxidation, we coupled LAM antigen oxidized with 50 mM NaIO₄ to Sepharose support via functionalized BSA-spacer molecule. At this level of oxidation LAM polysaccharide still retains some antigenic activity, as described below, but provides high coupling efficiency. Application of the immune serum to such affinity matrix resulted in the isolation with high yield of the fraction of rabbit antibody. Testing of such antibody in the plate ELISA immunoassay format as a capture antibody showed some functional activity, but not at the level sufficient to be used in the high sensitivity immunoassay necessary for screening applications using non-concentrated urine samples. These data explain results obtained in the literature previously, where LAM-specific affinity purified antibody was used, but it was still necessary to concentrate urine samples in order to detect Lam present in the samples.

Unexpectedly, by changing the LAM coupling chemistry to a milder non-destructive process, based on polysaccharide activation with cyanogen bromide (CNBr) resulted in the purification of a much better quality of LAM specific antibody, as can be seen in Fig.3. Then, surprisingly, passing the antibody purified on the column with intact LAM (CNBr- activation process) through a column with LAM antigen after deep, strong NaIO₄ oxidation (see below) produced a relatively small fraction of antibody, approximately 7-10% of the applied amount, with very high activity in the LAM-specific direct antigen immunoassay.

Fig.3 shows the efficiency of such antibody as a capture antibody. When such antibody was labeled with horse radish peroxidase (HRP) and used as a labeling antibody, it also demonstrated activity higher than any other antibody tested or known. The ELISA system based on such antibody has shown extremely high sensitivity and proven to be useful in testing non-concentrated urine samples. This enabled us to produce a screening LAM- specific immunoassay with performance characteristics suitable for rapid screening, in the field, or both pulmonary and extra-pulmonary TB cases, a feat unattainable by others before. Thus, although LAM has been described in the frozen urine of TB patients, the assay for such reports requires an extensive sample preparation and therefore is not field adapted.

PROTOCOLS

In this section we describe protocols suitable for practicing the "direct method" and the "enhanced method" defined above. This discussion is not sorted strictly according to the direct method and the enhanced method per se, but describes specifically methods of preparing columns suitable for use in either or both methods, depending upon the context.

Figure 8 shows a schematic of the direct and enhanced methods.

Isolation of dry cells of M. tuberculosis from Freund's Adjuvant

First, allow cells with Freund's Adjuvant to settle for a minimum of 1 week at room temperature before use. Remove caps from adjuvant vials and without disturbing cells settled on the bottom of the vial, pull off the bulk mineral oil. A small amount of mineral oil may be left in the vial as a precaution to avoid drawing cell precipitate. Discard mineral oil and then mix 6.0 L of ethanol and 6.0 L diethyl ether, and add 5mL ethanohdiethyl ether mixture to each vial.

Next, close the vial, vortex and quickly transfer the suspension into a 1-L Erlenmyer flask. Avoid letting cells resettle in the vial during this step. When the 1-L Erlenmeyer flask is filled to the 1-L line, let the cells settle for 1-1.5 hr. Next, gently decant solvent from the

Erlenmeyer flask into a clean 1-L beaker. Avoid disturbing settled cells and moving them with solvent. If solvent decanted into beaker is clear, discard it. If a significant amount of cells were decanted with the solvent, return the decanted solvent to the Erlenmeyer flask and repeat settling step. Using 20-30 ml aliquots of ethanol: diethyl ether mixture, transfer cells onto a glass sintered filter.

Wash the cells with 500 mL of an ethanol- diethyl ether mixture, then wash with 200 ml of diethyl ether. Next, air-dry cells on a filter using a vacuum of 100 mmHg +/- 10 (low vacuum) Occasionally mix and homogenize the cell mass, then cover the filter with a porous material (such as a Kim-wipe) and leave in hood until dry (approximately 15 hours, i.e. overnight).

Weigh and record the total weight and calculate the dry weight of the cells. Then tightly seal with rubber lined cap and store at 15-30 ⁰C.

Phenol extraction of crude LAM antigen Place the dry cells of M. tuberculosis into a 250-mL Pyrex media bottle and add warm deionized water to the cells. Vortex and pulse sonicate (~ 20 second pulses) the suspension in the ultrasonic water bath until suspension is homogeneous.

Phenol extract the cells, then ethanol precipitate and place the precipitated cells in the refrigerator (2-8 ⁰C) overnight (~ 16 hours) to allow the precipitate to settle. Being very careful not to disturb precipitate, gently draw off the supernatant until about 100 mL of supernatant is left covering the precipitate. Gently swirl to mix, then transfer the remaining suspension into teflon centrifuge tubes and centrifuge at 12,000rpm for 20 minutes. Draw off as much supernatant as possible from all tubes with out disturbing the pellet, add 5 ml of deionized water to each tube and, using vortexing and pulse sonication, dissolve pellet in water. Combine all the fractions with the dissolved pellet and place in a 500-mL flask (Note: do not exceed 1/10 of the flask capacity/volume). Rotary evaporate to minimal volume, but avoid caramelizing the sample. Redissolve film with approximately 50 mL of water and repeat drying and redissolving until sample has been dried 3 times. Redissolve in 50 mL of water and lyophilize.

Purification of LAM antigen by Sephadex G-25 chromatography Dissolve 800 mg of crude LAM Ag in 15 mL of 0.25% acetic acid solution. Vortex and sonicate in ultrasonic bath to achieve complete dissolution. Centrifuge in a microcentrifuge at 5000 rpm for 5 min. Collect the supernatant in a 20-mL glass vial, divide the supernatant into 3 equal parts for separate chromatographic runs, and then gently apply l/3^rd of the LAM Ag supernatant collected above onto the chromatographic column. After a volume of 100 mL has flowed through the column, begin collecting fractions. Continue collecting fractions until 350 mL of mobile phase has passed since the start of chromatography. Cover all fractions and store at 2-8 ⁰C. Rotary evaporate in a 250-mL evaporation flask (no volumes greater than 25 mL). Evaporate to minimal volume, but avoid caramelizing the sample. Dilute evaporated material in 20 mL of water, sonicate, vortex until complete dissolution and then lyophilize (approximately 8 hours). Scrape dried material with a spatula into a tared glass vial and weigh.

The foregoing steps are depicted schematically in Figure 6.

Coupling LAM antigen to BSA-spacer by CNBr activation. First, prepare 0.5 M sodium bicarbonate and 1 M potassium carbonate solutions.

Then dissolve 30.0 mg of purified LAM Ag in 1.5 mL of deionized water. Use pulse sonication (10-20 sec pulses) and vortexing to completely dissolve the LAM Ag.

Dissolve 300 mg of BS A-hydrazine ligand in 15 mL of deionized water. Pulse sonicate (10-20 sec pulses) and vortex to dissolve completely, then place in micro fuge tubes and centrifuge in a microcentrifuge for 10 minutes at 10,000 rpm. Using a Pasteur pipette carefully collect and pool the clear supernatant from each tube and transfer into a 20-mL vial. Avoid disturbing any pellet that may form. Add 1.0 mL of 0.5M sodium bicarbonate to the vial and mix well by shaking. Add 150 μL of chilled IM potassium carbonate to the LAM solution and mix well by brief vortexing. Place obtained solution in ice/water bath. Prepare 5 mg/mL CNBr in acetonitrile for immediate use and add 180 μL of the cyanogen bromide solution to the LAM solution. Mix by vortexing and place on ice for approx. 15 minutes. Add this solution to the BSA-Hydrazine ligand solution (above) with a Pasteur pipette. Mix well and incubate overnight (16 -24 hours), at 2-8 ⁰C, in tightly sealed vial.

Coupling of LAM antigen to BSA-spacer by NaIO₄ activation Dissolve the LAM antigen in 1.25 rnL of deionized water in a 3-4-mL vial. Pulse sonicate and vortex to dissolve completely. Prepare a 0.1 M sodium periodate solution: (in NaOAc buffer, pH 4.0). Add 1.25 mL of the 0.1M NaIO₄ solution to the 1.25 mL LAM solution. Vortex to mix. Cover the vial with aluminum foil; place it on the rocking platform and mix for 1 hour +/- 5 minutes at ambient temperature. Dissolve 250 mg of BS A-hydrazine ligand in 12.5 mL of deionized water in a 25-40 mL glass serum vial. Use pulse sonication and vortexing to dissolve completely, then centrifuge for approx. 10 minutes at 10,000 rpm. Using a Pasteur pipette collect the supernatant from each tube and pool into a 25-40 mL glass vial. Avoid disturbing the pellet. Add 12.5mL of 0.1M sodium phosphate (pH 6.8) to the vial and mix well by brief vortexing.

Coupling process:

To the BS A-hydrazine solution add the oxidized LAM solution and vortex. Add 100 mg of sodium cyanoborohydride and seal. Sample 10 μL of the final solution and dilute with 90 uL IX PBS buffer (QC solution) and retain for further analysis (LAM concentration will be approximately 0.75 mg/mL).

Activation of Sepharose by NaIO₄

Measure an aliquot of suspension of Sepharose 4B-CL corresponding to 80 ml of settled gel and transfer onto a sintered glass filter. Wash with 500 mL water and drain using low vacuum (approx 300 mmHg) until the granular structure of the gel surface becomes visible. Avoid formation of the air cracks in the gel layer. Prepare a 0.1 M sodium acetate buffer, pH 4.0 solution and use to prepare a 30 mM solution OfNaIO₄ in 0.1 M NaOAc.

Add 250 mL of 30 mM NaIO₄ to the gel and thoroughly mix. Cover the mixture with aluminum foil and place at a 45° angle on a rocker platform at medium speed for 1.5 hours ± 10 minutes at ambient temperature. Transfer to the sintered glass filter and wash with 1 L of water using low vacuum (approx 300 mmHg). The activated gel must be prepared within a maximum of 4 hours of use. Coupling of BSA-LAM ligand to activated Sepharose (For Synthesis of first and second affinity columns)

Preparation of matrix

Prepare a 0.1% sodium azide solution in IX PBS (phosphate buffered saline). Measure a suspension of activated Sepharose corresponding to 60 ml of the settled gel (or other suitable matrix) and transfer it onto a sintered glass filter. Drain gel using low vacuum (300 mm Hg) until the gel packs and granular structure becomes visible, but avoid formation of cracks on the gel surface.

BSA-LAM Ligand Solution:

In a 250-mL media bottle dilute approximately 17 to 20 mL of the solution of BSA- LAM ligand to 90 mL with sodium phosphate buffer (pH 6.8). Add 90 mg of crystalline sodium cyanoborohydride to the solution. Tightly close the bottle using the supplied plastic cap. Mix well by vortexing briefly. The solution may appear opalescent but there should be no precipitate. Microscopic gas bubbles formed by the sodium cyanoborohydride may be visible.

Coupling step:

To the LAM solution prepared above add the drained activated Sepharose gel. Tightly close and thoroughly mix the suspension using gentle vortexing. Incubate for approx 4 hours at 37⁰C+/- 2°C, mixing (by inversion) the reaction mixture every hour. Add 4.5 mL of 1.5 M Tris buffer and tightly close cap again. Continue incubating at 37⁰C +/- 2°C for approximately 16 hours (overnight).

Transfer the reaction mixture onto a sintered glass filter and collect the liquid phase into a clean 100 - 200 mL Bunzen flask by applying low vacuum (300 mm Hg). Wash the

LAM - Sepharose gel on the filter with 400 ml of deionized water and continue washing with

60O mL of IX PBS.

Packing and storage of Column:

In a 250 mL beaker add 100 mL of IX PBS to the prepared gel. Stir manually into a slurry. Pack into a column according to standard procedures, using IX PBS. Equilibrate the column with IX PBS plus 0.1% sodium azide. Generic Coupling of LAM ligand to activated Sepharose (for Preparation of affinity columns I and II)

Measure suspension of Activated Sepharose corresponding to 100 ml of the settled gel and transfer it onto a sintered glass filter. Drain gel using low vacuum (300 mm Hg) until the gel packs and granular structure becomes visible, but avoid formation of cracks on the gel surface. Retain drained gel for later use.

BSA-LAM ligand Solution:

In 250 mL Pyrex media bottle dilute approx. 27.5mL solution of BSA-LAM ligand to 100 mL with the sodium phosphate buffer (pH 6.8). Add 100 mg of crystalline sodium cyanoborohydride to the solution. Tightly close the bottle using the supplied plastic cap. Mix well by briefly vortexing. The solution may appear opalescent but there should be no precipitate. Microscopic gas bubbles formed by sodium cyanoborohydride may be visible.

Coupling step:

To the LAM solution prepared above add the drained activated Sepharose gel. Tightly close with the supplied plastic cap. Thoroughly mix the suspension using gentle vortexing (medium speed) and incubate for approx 4 hours at 37⁰C ± 2°C, mixing the reaction mixture (by inversion) every hour. Add 7.5 mL of 1.5 M Tris buffer and tightly close. Continue incubating at 37⁰C ± 2°C for approximately 16 hours (overnight).

Transfer the reaction mixture onto a sintered glass filter and collect the liquid phase into a clean 100-200 mL Bunzen flask by applying low vacuum (300 mm Hg). Wash the LAM- Sepharose gel on the filter with approx 800 ml of deionized water. Continue washing with approx 1.2L of IX PBS.

Packing and storage of Column:

In a 250-mL beaker add approximately 160 mL of IX PBS to the gel above. Stir manually (with spatula/glass rod) into a slurry. Pack into a column according to standard procedures using IX PBS. Equilibrate the column with IX PBS with 0.1% sodium azide. The foregoing steps involving use of purified LAM and preparation of affinity columns I and

II are depicted schematically in Figure 7. Isolation of antibody by affinity chromatography-I (the "Direct Method").

Prepare the following stock solutions:

1 liter of 0.1M glycine buffer and adjust the pH to 2.5 with IM HCl. 1 liter of 3x PBS solution (dilute a 1Ox PBS stock solution with deionized water) and check the pH, and re-adjust to 7.2 to 7.4, if needed, with IM HCl or IM NaOH.

200 mL of a IX PBS plus 0.1% sodium azide solution. 100 mL of a 0.5 M disodium hydrogen phosphate (Na₂HPO₄) solution.

Serum Preparation Slow-thaw frozen serum in the refrigerator (approx 16 hours/overnight) until completely thawed. Measure sera volume and weigh 2.9g of sodium chloride for every 100 mL of serum and add to the sera. Swirl gently until completely dissolved: the final concentration will be 0.5M NaCl.

Centrifuge (4-8 ° C) at ~ 8000 g for 20 minutes. Draw off supernatant from all centrifuge tubes with Pasteur pipette. Do not to disturb the pellet. Filter supernatant through a cotton-plugged funnel and collect the filtrate. Collected filtrate should be slightly opalescent, but should not contain any particulate materials. Place filtered serum in the refrigerator until the beginning of the affinity chromatography step.

Serum Application:

Prepare column I (non-modified LAM coupled to column material) for serum application by equilibrating with IX PBS. Adjust the flow rate to 2.0 mL/min and continue applying IX PBS until the baseline remains stable for at least 1 hour. Adjust zero for the recorder and detector as needed. Once the baseline is stable, adjust the flow rate to 0.5 to 0.6 mL/min. and then apply the serum prepared above to the LAM Affinity Column I at the 0.5 -

0.6 ml/min flow rate. Collect void volume eluant (it will be approximately 30% of the column volume). Monitor fractions by UV detection at 260-280 nm and when an increase in signal occurs, begin collecting serum passed through the column in a 500- 1000 mL serum. After the entire volume of serum has been applied to the column, briefly stop the column flow, apply 3X PBS buffer, and then resume liquid flow. Continue to wash column with 3X PBS until the signal decreases to approximately 50% of baseline. At this point stop collection of serum and save all collected fractions. Change the flow rate to 2.0mL/min and continue washing the column with 3X PBS until baseline is approximately 10-15%. Discard flow-through. Replace 3X PBS buffer with IX PBS buffer and wash with approx 2 - 2.5 column volumes at a flow rate of 2.0 mL/min. Discard flow-through.

Elution of Antibodies Step: Adjust flow rate to 1.0 mL/min. Replace IX PBS with cold 0. IM GIy-HCl buffer prepared above, and start elution of the adsorbed antibody. When the signal increases rapidly and gains about 10-15% of the full scale, begin collecting eluent column into 15 ml conical tubes placed in ice-water bath (0⁰C ). Collect 5-ml fractions.

Continue collecting antibodies in GIy-HCl buffer until the signal begins to decrease rapidly. Stop fraction collection when the signal drops to the signal level of the beginning of collection (10-15% of full scale). Neutralize the collected antibody solution by to each 5-mL fraction 0.5 mL of 0.5M Na ₂HPO₄ in 0.1 -ml increments. The total volume added should be equal to 10% of the fraction volume before neutralization.

Gently mix solution during addition of Na ₂HPO₄ buffer and pool the neutralized fractions. Measure the O. D. of antibodies at 280 nm against a blank containing only 0. IM

GIy-HCl buffer and calculate the antibody concentration. Place the antibody collected at 2-8° C for a minimum of 3 days to allow crashing and shedding.

Column Care: Equilibrate the column with IX PBS until neutral (pH 7). During non-use, equilibrate the column with the IX PBS plus 0.1% sodium azide solution and store the column at 4-8 ⁰C until future use.

Dialysis Centrifuge prepared antibodies at 10,000G for a minimum of 5 minutes. Transfer the supernatant to 12-14 mol. wt. cut-off dialysis tubing and dialyze against IX PBS for 2-3 days with a minimum of 4 changes of buffer, with a ratio of Ab solution to total volume of > 1 :20. Remove antibodies from dialysis. Measure volume of antibody solution using glass graduated cylinder. If there is any additional crashing/shedding (in the form of a precipitate) centrifuge the antibody solution again at 10,000G for a minimum of 5 minutes. Measure the

O. D. of antibodies at 280 nm after blanking the spectrophotometer with IX PBS buffer.

Calculate the concentration in mg/mL and place for storage at 4-8⁰C. Isolation of antibody by affinity chromatography-II (the "Enhanced Method")

Purification of Highly Specific Antibodies

Apply IX PBS to the LAM Affinity Column 2 prepared above, (LAM modified by strong oxidation, coupled to column material using NaIO₄), at a 2.0 niL/min flow rate until the baseline remains stable for at least 15 minutes. Adjust the recorder and detector to Zero, as required. Continue to monitor the baseline for the next 30 minutes and once stable, apply antibody to the column. Adjust the flow rate to 0.5-0.6 niL/min and apply a volume of antibody, as prepared above, corresponding to -100-150 mg of Ab to the LAM Affinity Column 2 using an Econo pump or similar device. Collect void volume eluate (It will be approximately 30% of the column volume) at 280 nm. Begin collecting antibodies as the signal increases to about 10-15% above baseline in a clean serum bottle. When the total antibody volume has been applied, briefly stop the liquid flow, apply IX PBS buffer and resume liquid flow at 0.5-0.6 niL/min. Continue to collect material flowing through column at 280 nm. When the signal drops to 10-15% above the start of collection (30-50% above baseline), stop collecting the solution.

Measure the O. D. of highly specific antibodies at 280 nm after and calculate the antibody concentration. Immediately place antibody solution at 4-8° C for temporary storage.

Column Wash Continue to wash the column with IX PBS at a flow rate of 2.0-2.5 niL/min. Pass minimum 3 column volumes of IX PBS. Elute material absorbed onto column with cold 0.1M GIy-HCl buffer, prepared above. Collect material eluted in glass vials. When the monitor/signal drops to -10-15% of baseline, stop collection. Neutralize the collected Antibody solution by adding 10% of total volume of 0.5M sodium phosphate, prepared above, by adding in 0.5 mL increments. Measure the O. D. of antibodies at 280 and calculate the antibody concentration. Immediately place the collected antibodies solution at 4-8 ⁰C and retain until the analysis of antibodies collected in step above is complete. If the concentration of antibodies above is less than 0.3 mg/mL, concentrate.

Wash the column with a minimum of 3 column volumes of IX PBS at a 2.0-2.5 niL/min flow rate. Wash the column again with 1 column volume of IX PBS plus 0.1% sodium azide, and store at 4-8 ⁰C until future use. The foregoing steps showing isolation of enriched antibodies from affinity columns I and II using the direct and enhanced methods are depicted schematically in Figure 8.

ELISA Plate coating process.

Set-up of the Moduline 300 System.

The Ab coating must be completed within maximum 8 hours from end of preparation of the coating solution M815. The Antibody coating solution must be kept in on ice (O⁰C) during the coating process. Step One

Pre -weigh and inspect empty plates and discard any broken plates. Dispense 100 μl of MTB-LAM specific Ab coating solution into each well of each strip plate using a Moduline 300 System. Visually check all the 96 wells in each plate for uniformity of well filling during coating process. Save unused Ab solution and store at (2-8 ⁰C) until the complete lot of plates are processed and passed for use. Stack plates with dispensed Ab in stacks of 10 plates each and cover the top plate with an empty plate used as a cover. Label each stack cover plate from 1 to 18. Refrigerate the stacked plates at 2-8 ⁰C and incubate overnight (14-18 hrs).

Step Two: Set-up of the Moduline 300 System to perform 3-times wash cycles followed by immediate dispense cycle of 312 uL Block Solution. Block Solution must be used within maximum 24 hours from end of preparation. Remove plates from refrigerator and remove the covering plates from stacks as they are being placed on the Moduline and place them aside. Set the timer for 6 hours. Set blocked plates coming from the conveyor, on sequentially numbered trays and block for 5 to 6 hours at ambient temperature(20-28 ⁰C).

Place the plates on trays in the Drying Chamber and incubate at 20-23 ⁰C and 20-22% relative humidity for 24 -72 hr. Remove dry plates from drying chamber.

MTB-Ab Preparation for Conjugation to HRP

(MTB-Ab solution preparation should be performed at least 7 days before conjugation procedure.) Dialysis:

Dialyze the necessary amount of MTB-LAM-Ab solution against IX PBS for minimum 48 h with minimum 4 changes, at 2-8°C . Use dialyzing tubing with MWCO 12- 14,000. After dialysis centrifuge Ab solution at 12,000 rpm for 10 min. and carefully aspirate supernatant into the 15 ml graduated centrifuge tube.

Measure optical density of Ab solution after dialysis at 280 nm andCalculate Ab concentration after dialysis. IfAb solution after dialysis has OD _{280 nm} > 2.8, make a 1 :7 dilution of Ab solution in IXPBS.

Concentration:

Prewash an Amicon Ultrafree-15 centrifugal filter device with IX PBS. Place approx. 15 mL of IXPBS solution into device and centrifuge at 3500 rpm for approximately 5 min. Discard all the solution from device units. Concentrate the above Ab solution after dialysis with Ultrafree-15 centrifugal filter devices to 4.5 -5.5 mg/ml by centrifugation on

Bench-top centrifuge (bucket rotor) at 3500 rpm for approx. 5 min. x 3. Carefully aspirate the concentrated Ab solution from the filter unit of the Amicon device into a 15 mL tube. To maximize recovery, remove concentrated sample immediately after centrifugation and resuspend concentrate volume several times with a pipette to ensure proper mixing before Ab aspiration.

Centrifuge the concentrated Ab solution at 10000 rpm for approx. 15 min. and aspirate the Ab supernatant into a 15 mL tube. Measure the OD₂So of Ab solution at 1 :20 dilution in IXPBS and calculate concentration of Ab. Sample 0.1 ml of Ab solution for ELISA analysis. Store at 2-8°C.

MTB-LAM-Ab-HRP Conjugate Preparation

Wash all glass vials and stir bars for conjugation steps and glass vials for conjugate storage with H₂SO₄ solution and thoroughly rinse them with tap water and deionized H₂O.

Preparing the Sephadex G-25 column for chromatography:

Obtain a column (1.5 x 30 cm) with approximately V = 50 ml packed with Sephadex G-25 (Fine). Pack the column as described above. Set the following chromatography conditions to equilibrate the column with the 1 mM Sodium • Acetate Buffer, pH 4.4.

• UV Monitor wavelength for 280 nm

• Monitor Sensitivity: 0.2 OD

• Chart recorder speed: 2 mm/min. • Pump Flow rate for column washing: 60 ml/h

Wash the column with approx 100-150 ml of 1 mM Sodium Acetate, pH 4.4 and adjust the UV monitor baseline to 0-position. Make sure that established base line is stable for approx. 30 min. Calculate the amount of MTB-LAM-Ab solution necessary for conjugation and centrifuge Ab at 12,000 rpm for approx. 10 min. Carefully aspirate the Ab supernatant into a clean glass vial. Measure the OD_2So _nm of Ab solution at 1 : 20 dilution in

IXPBS and Calculate concentration of the undiluted Ab solution. Store the Ab solution at 2- 8°C until use.

Oxidation of HRP (horse radish peroxidase) with NaIO₄: Weigh 8 mg of HRP in V-shaped glass vial. Add 2.0 ml of deionized H₂O. Gently stir the solution for approx. 2-3 min. until all the HRP has dissolved. Make sure there are no undissolved HRP particles on the glass vial walls left.

Prepare a fresh solution of 0.1 M NaIO₄, pH 4.4 for use within a maximum of 5 minutes and protect from light. Add 0.4 ml of 0.1 M NaIO₄ to the HRP solution prepared above, while stirring. Cover the vial with aluminum foil to protect the mixture from light.

Incubate the mixture for 20 min. with stirring at ambient temperature. Add 4 drops of ethylene glycol to the reaction mixture and stir for approximately 2 min.

Chromatography and Concentration of Oxidized HRP: Immediately after completing the above step purify the oxidized HRP by gel-filtration on Sephadex G-25 (Fine) column. Set the pump flow rate for sample elution to approximately 50 ml/h. Carefully apply the total volume of the oxidized HRP prepared above onto the dry gel bed but take care not to disturb the gel bed. Do not over dry gel. Collect all oxidized HRP (colored solution) into one 15 ml tube. - 1^st peak on the chromatography Chart (OD₂8o_nm >0.05). After chromatography is completed, empty the column of Sephadex G-25 and discard the gel and record the volume of HRP solution after chromatography.

Concentrating Oxidized HRP Prewash Ultrafree-15 centrifugal filter devices with 1 rnM Sodium Acetate, pH 4.4 with approximately 15 mL of 1 mM Sodium Acetate, pH 4.4, and centrifuge the filter unit for approx. 5 min. at 3500 rpm using a bench-top centrifuge (bucket rotor). Then discard all solutions from the filter unit. Immediately after chromatography, concentrate the oxidized HRP solution (from above) to approx. 2 + 0.2 ml with an Ultrafree-15 centrifuge filter unit

(Biomax-1 OK membrane) by centrifugation at 3500 rpm for approx. 5 min. Carefully aspirate the concentrated HRP solution from the filter unit of the device into the clean glass vial, measure and record the volume, and store at 2-8 ⁰C.

Conjugation HRP to MTB-LAM-Ab:

Calculate the amount of MTB-LAM-Ab solution necessary for conjugation to HRP. Place the MTB-LAM-Ab (from above) into a V-shaped glass vial with triangular stir bar, without leaving drops of the Ab solution on the vial walls. Add Vi volume of oxidized HRP solution (above) to the MTB-LAM-Ab solution, cover the vial with aluminum foil to protect reaction mixture from the light and stir reaction mixture in the glass vial for 30 min at room temperature. Avoid foaming.

Add 1 M Carbonate-HCl, to pH 9.5 and stir at room temperature for two hr. Protect from the light and avoid foaming.

Prepare 4 mg/ml Sodium Borohydride (NaBH₄) immediately before use and protect from the light with the aluminum foil. Immediately add the calculated amount OfNaBH₄ required to the MTB-LAM-Ab solution prepared above, and incubate the reaction mixture at approx. 2-8°C for 2 hr. Dialyze reaction mixture against 1 X PBS for minimum 48 h at 2-8 ⁰C with a minimum of 4 buffer changes at 8-16 hours intervals. Use 12-14kDa cut-off dialyzing tubing for dialysis.

Conjugate Storage and Analysis:

After dialysis, centrifuge the conjugate solution at 4000 rpm for approx. 4 min. Carefully withdraw supernatant and place conjugate solution into the clean 6 ml glass vial. Measure 18 ml of Gardian Peroxidase Conjugate Stabilizer/ Diluent into the 50 ml glass bottle with magnetic stir bar. Add 2 ml of MTB-LAM-Ab-HRP conjugate and stir the mixture for approx. 10 min. Store at 2-8°C, and protect from light. The foregoing steps relating to MTB conjugate preparation are depicted schematically in Figure 9.

RESULTS Below we present data from the evaluation of a direct antigen ELISA which detects

LAM in unprocessed, non-concentrated urine using the "direct method" for enriched antibody production. (It is believed that even better data will result by using enriched antibodies produced using the "enhanced method" described above.) The studies producing these data were carried out in the Mbeya region that is located in the Southwestern highlands of Tanzania in collaboration with the Regional TB and Leprosy Programme and the Mbeya

Medical Research Project (MMRP). In the Mbeya Region approximately 3,500 new TB cases are diagnosed annually and treatment is conducted according to the national DOTS strategy. Initiation of every therapy is initiated at a central facility at the Mbeya Referral Hospital. The TB cure rate was 72.3% in 2002. The aim of the study was to evaluate the performance of a commercially available LAM-capture ELISA in clinical practice and to compare the results with the gold standard for TB diagnosis: Sputum microscopy, TB-culture, chest radiography and clinical investigation.

MATERIAL AND METHODS LAM-ELISA Description

The MTB-ELISA direct antigen sandwich immunoassay (MTB-ELISA, Chemogen, So. Portland, ME, USA) is a LAM-ELISA similar to an assay developed by others. The immune sera were harvested from white New Zealand rabbits that were immunized with inactivated whole cells of M. tuberculosis H37Rv. Polyclonal LAM- specific antibodies were isolated by affinity chromatography using immobilized LAM as a ligand. The test kit consists of an 96-well ELISA plate pre-coated with LAM-specific antibody, blocked and sealed in a plastic pouch with desiccant; a vial with LAM-specific HRP -conjugated LAM- specific polyclonal antibody; a vial with TMB (3,3',5,5'-tetramethylbenzidine) single component chromogenic substrate; a vial with the negative control solution, and three vials with calibrators corresponding to 0.5 ng/ml, 1.5 ng/ml and 4.5 ng/ml of LAM in urinary samples.

Urine samples were considered positive in the ELISA when the obtained optical density at 450 nm was at least 0.1 above signal of the negative control (>2SD). A patient urine sample of 0.1 ml is placed in duplicates on the ELISA plate, incubated for 1 hour and washed with 0.05% Tween-20/ PBS (PBST) solution. 0.1 ml of LAM-specific HRP-conjugate are added. After 1 hour incubation the plate is washed with PBST solution and 0.1 ml of TMB substrate are added. After 10 minutes of incubation time the substrate reaction is stopped by adding 0.1 ml of IM H₂SO₄ and the color development is read at 450 nm.

In other embodiments, the specific isoform of lipoarabinomannan (LAM) determined to contain the antigenic activity is used to generate highly specific, highly pure polyclonal antibodies for use in the detection of mycobacterium lipoarabinomannan in the urine of patients to be screened for active tuberculosis, using protocols similar to that described above. The antigenically active isoform of LAM was identified using selective controlled mild NaIO₄ oxidation of LAM, wherein two iso forms were readily identifiable and distinguishable (data not shown).

One contained isoform wherein the serological activity of the LAM was lessened or destroyed. The other isoform maintained serological activity. A comparison of two methods of oxidation of LAM, using either mild oxidizing agents or low concentrations OfNaIO₄ (i.e. less than 50 mM) preserved the antigenic activity of the LAM. Oxidation by high concentrations OfNaIO₄ (50 mM or higher), however, resulted in reduced or loss of antigenic activity of the LAM. Therefore, only LAM activated with CNBr, or oxidized with mild oxidizing agents or low concentrations OfNaIO₄ is used to generate highly antigenic LAM for use in the preparation of highly specific, highly pure polyclonal antibodies for use in detecting LAM in urine samples for diagnosing TB in patients of interest.

These results are completely unexpected compared to the detection methods disclosed by Svenson et al. (see e.g. WO97/34149) which used only high concentrations OfNaIO₄ to oxidize the mycobacterial LAM, and consequently destroyed antigenic activity of the LAM used to generated the antibodies. Not knowing that there was more than one isoform of the LAM to be detected, it was not possible in the earlier disclosure to prepare highly specific antibodies to the antigenically active form of LAM, because no one prior to these studies even knew that a separate isoform existed that contained the antigenic activity, or that such activity was lost during standard means of oxidation, namely, treatment with high concentrations OfNaIO₄. Clinical Site Description.

Within eight weeks 242 suspected TB patients were recruited at the outpatient departments of 5 clinical centers in Mbeya, Tanzania. The standard protocol of investigation included clinical assessment, chest radiography, ESR, white blood cell x count and HIV test, 3 x AFB staining (Ziehl Neelson) of sputum at day 1, 2 and 3, 2 sputum culture on

Loewenstein Jenssen medium and LAM-ELISA in urine and serum.

All patients had clinical signs of TB (cough > 4 weeks, night sweats, weight loss, loss of appetite). One hundred thirty-seven of these had laboratory confirmed pulmonary TB (PTB), 9 had high radiological suspicion of PTB (pleural effusions or enlarged hilar lymph nodes), and 8 showed clinical and radiological signs of military TB. Consenting patients were tested for their HIV status and 70% were confirmed as HIV-positive. Data were handled confidentially. The study was approved by the local Institutional review board and the national ethical committee of the Republic of Tanzania.

All laboratory procedures were performed in the laboratory facilities of the Mbeya Medical Research Project.

Stability of the LAM-ELISA plates

Plates were found to retain 95% of the original activity after 72 days at 5O⁰C, and open plates kept without packaging retained 82% of the original activity after 3 days at 4O⁰C and 80% relative humidity. The Ab conjugate retained 100% of the original activity after 72 days at 22⁰C, and 70% of its original activity after 72 days at 5O⁰C. Moreover, during the summer of fall of the T2003 study, kits with LAM-ELISA survived without loss of activity after being shipped at ambient temperature from the United States to Germany to Tanzania back to Germany and back to the United States.

Microscopy and Culture of Sputum Samples

Ziehl Neelson staining and microscopy was done by an experienced and well qualifiedlab technician. After decontamination sputum samples were cultured on Loewenstein Jenssen medium in duplicates. Cultures were examined weekly for growth for 8 weeks.

Urine Specimens 27

From each patient 30 ml of urine were collected in a sterile plastic container, which was labeled with the code number of the respective patient's data form. 100 μl of fresh and unprocessed urine was added to the wells of the ELISA plate in duplicate. Negative controls, low, medium and high positive controls were also added to each plate in duplicates. Specimens were processed within 24 h and then stored at -20⁰C for future testing in

Germany.

Control Groups from Tanzania and USA

Urine samples of 23 staff members of the Mbeya Referral Hospital, of 20 staff members of Chemogen, Inc. and of 200 patients from 2 clinics in New York were tested in the LAM ELISA. All of them appeared healthy in clinical examination and did not have any signs of respiratory infections.

RESULTS

Preclinical Evaluation of the ELISA System.

Fig. 4 A shows the dose response curve using different concentrations of LAM in urine, wherein solid circles represent ELISA results using LAM from M. tuberculosis, and open circles represent the control ELISA results. The optimal cut off value was defined according to this curve as LAM concentration producing an optical density (OD) exceeding

OD of negative control by 0.1 OD, that corresponds to more than 2 standard deviations above the signal of the negative control sample. All samples with an optical density above this cut off were considered as ELISA positive. The cut off was equal to approximately 0.25 ng/ml of LAM in untreated fresh urine. The MTB-ELISA was evaluated for cross-reactivity with other species and genera of various Gram-positive and Gram-negative bacteria typical for urinary tract infections and bacterial pneumonia. None of the tested species has shown any reactivity in the evaluated LAM-ELISA system even at the highest tested concentrations, as can be seen by comparing the ELISA results for M. tuberculosis (open triangles) with the ELISA results for other bacterial species tested (solid triangles, open and solid diamonds, open and solid circles) depicted in Fig. 4B. An analysis of whole cells of various species of mycobacteria in the LAM-ELISA system shows cross-reactivity with all tested species of mycobacteria (M.) (Fig. 4C), however, M. tuberculosis H37Rv and M. bovis are detected most sensitively. Both species are very close from the immunochemical standpoint, but M. bovis is rarely a cause of mycobacterial infection in humans.

Additional studies in Figures 4D and 4E show MTB-ELISA results for urine tests with LAM in the urine compared to negative control with no LAM in urine. Fig. 4D is for LAM concentrations of 0 to 6 ng/mL, and Fig. 4E is for LAM concentrations from 0 to 0.8 ng/mL.

Study Participant Data

According to Table 1 the 242 TB suspects were divided into 3 major categories: (1) pulmonary TB patients with confirmed microscopic and/or culture diagnosis, (2) patients with typical clinical and radiographic signs and (3) patients with clinical symptoms of TB, that were not considered TB patients as all available diagnostic tools (radiography, sputum microscopy and culture) were negative.

Group one included 137 patients that had a laboratory confirmed pulmonary TB. 132 were confirmed by Loewenstein Jenssen culture and five had a negative culture but positive

AFB-stain. Out of the 132 culture positive cases 62.12 % were AFB positive.

Group two comprised an additional 17 patients that were enrolled into the DOTS therapy program based on radiographic and clinical findings (Table 1). The 88 patients of group three were sputum negative and did not present specific radiological signs of pulmonary TB and were therefore not enrolled in the DOTS program.

The mean age of the participants was 34 years. The female male ratio was 41 :59. The overall HIV prevalence among the 223 patients that agreed to be tested for HIV was 69.1 % (see Table 2). The HIV prevalence was 73.2% among patients with and 60.8% among patients without confirmed TB.

Clinical Evaluation of the ELISA

Of the 137 patients with confirmed pulmonary TB (culture or AFB positive) 111 were LAM-ELISA positive (sensitivity 81.02 %) for the predefined cut off (optical density (OD) of negative control + 0.1). The mean OD increment (= absolute mean OD - OD of negative control) for the smear and culture positive group (82) was 0.604. For smear negative and culture positive cases (50) the mean OD increment was 0.293 and for smear positive, but culture negative cases (5) 0.249.

Of the 17 patients in group two that were culture and AFB negative, but had typical radiological and clinical signs for TB 13 (76.47%) had a positive LAM-ELISA test results with a mean OD increment of 0.183. 13 (76.47%) of them were HIV positive.

The remaining 88 patients that came to the special TB clinic with clinical signs suggestive of pulmonary TB were culture and AFB negative and had no specific radiographic findings for TB. Of these 13 (14.77%) had a positive LAM-ELISA test (mean OD increment

0.184).

Based on the known concentration in the low, medium and high positive control that were included on each plate, it was possible to determine the approximate LAM concentration of each urine sample based on the OD value of the ELISA. Whether the LAM concentration correlates to the individual burden of tubercle bacteria was assessed in AFB positive patients. While patients with a low density of tubercle bacteria in microscopy (AFB +) had a mean LAM antigen concentration of 0.93 ng/ml in the urine, patients with an intermediate density of acid fast bacilli (AFB ++) had a mean antigen concentration of 1.74 ng/ml in their urine and AFB +++ patients 2.02 ng/ml (Fig. 5). The later value is lower than the real concentration of LAM in urine of AFB+++ patients because the ELISA reader used in the Tanzania lab could not read signals above two corresponding to about 4 ng/ml.

The HIV serostatus did not influence the sensitivity of the LAM-ELISA in confirmed pulmonary TB patients. Of 124 patients with known HIV serostatus and positive TB culture and/or AFB stain 73 of 89 HIV infected patients (82.0%) were positive in the LAM-ELISA compared to 26 out of 35 uninfected individuals (74.3%). Similarly, the sensitivity of the

AFB was not compromised by HIV serostatus. The sensitivity was 61.2% and 58.8% in HIV infected and negative individuals, respectively. Similarly, MTB-ELISA signals were determined in the urine of 96 HIV(-) Tanzanian bar workers, the results of which are shown in Figure 10. As can be seen, 94 of the 96 workers fell along a normative response, but there were two outliers among the group - patient Hl 13 and patient H354 being substantially above and below the norm for the other subjects, respectively.

The specificity of the assay was assessed using the urine of healthy Tanzanian and US volunteers. The urine of 23 healthy hospital staff members of Tanzanian origin was analyzed. None of the samples was tested positive in LAM-ELISA (-0.047 mean relative OD, specificity 100%). Urine samples of 220 healthy volunteers from US were collected and analyzed. All but 4 had an optical density below the cut-off value of 0.1 ng/mL (specificity 98.18%). Also, the kinetics of the secretion of the LAM antigen was investigated in the urine of TB patients, the results of which are shown in Figure 11. As can be seen, there is a lag-time with low levels (~0.1 to 0.2 OD) of LAM antigen detectable in the urine, followed by a steady increase, then a drop, and then a subsequent increase in LAM secretion in the urine. Further analysis shows the T2003 results (see Figure 12) comparing the detection of

TB in patients from Group A (LJ positive), Group B (radiologically diagnosed with TB), Group C (no clinical proof of TB) and Group D (healthy Tanzanians) using a cut-off value of 0.1 OD unit above the negative control (Group D), wherein essentially all of the patients in Groups A and B are at or above the cut-off value, and all of the patients in Groups C and D are below the cut-off value. Figure 13 shows a graph of the cumulative distribution of TB(+) patients as a function of urinary LAM concentration, showing that a cut-off value of 0.1 above negative control, corresponding to ~0.2 ng/mL LAM concentration gives 84% inclusion of measurable LAM for such patients.

Figure 14 shows the correlation between urinary LAM concentration and smear AFB signal. As can be seen, the AFB(+) patients were further subdivided into AFB (+), AFB (++) and AFB (+++) patients, and AFB (-) patients were categorized as either LV(+) or LJ(-) with no proof of TB.

DISCUSSION The classical tools for the diagnosis of TB, sputum culture and smear microscopy, have obvious limitations. Both methods only detect cases of open pulmonary TB. This significantly impairs the possibility of the detection of all cases of active TB regardless of the organ manifestation. Therefore multiple new methods have been evaluated in the past that could supplement the classic tools, especially in resource poor settings. The criteria a-that were set for such a new assay are a) a higher sensitivity than microscopy, b) comparable specificity, c) a limited additional work load, d) the possibility to diagnose sputum-negative TB and e) a sensitivity that is not impaired by HIV co-infection.

In the first evaluation, the sensitivity of the LAM-ELISA (81% of culture positives) was superior to AFB-stain (69%). Sensitivity can be further improved by concentrating fresh urine, which would however result in an additional effort for a lab technician. The detection rate of the LAM-ELISA for cases with radiological confirmed military TB (87.5%) as well as for sputum negative cases with typical radiological signs of pulmonary TB (67%) was encouraging, although the case numbers were not high enough to allow a final conclusion. For healthy individuals the specificity of the ELISA was high (98.18% in US and 100% in Tanzania). HIV co-infection in culture positive TB cases did not influence the sensitivity of the LAM-ELISA.

In comparison to previous published results of the LAM-ELISA the new test detects LAM at lower concentrations (0.2 ng/ml) than former tests. The sensitivity of the new test was 82.9 % (of AFB +) for unconcentrated and fresh urine compared to a sensitivity of 81.3 % for the previous test using processed and frozen urine. The test specificity was 98.36% in this study compared to 86.9% in the previous study.

The limitation of this cross sectional TB study was the fact that a certain proportion of TB suspected patients remained ambiguous in terms of their TB status (group 2 and 3). To acknowledge this problem we have created three major categories for analysis: Group 1 : laboratory confirmed TB, Group 2: clinically and radiological diagnosed TB, Group 3: no laboratory or radiological proof of TB. While we are confident that participants in category 1 are true TB cases, we cannot exclude that category 2 and 3 contain some wrongly categorized patients. We therefore excluded them from our sensitivity and specificity calculation.

Diagnosis of TB often requires the longitudinal follow-up of patients. Especially sputum negative patients with unusual radiological features would have needed several follow-up consultations in order to re-question their TB status. In a longitudinal study clinical as well as diagnostic reevaluation and TB treatment outcome would have given important additional information to classify group 2 and 3 in TB and non TB patients.

Of major interest is the question if there is a quantitative correlation between the bacterial burden of M. tuberculosis and the amount of LAM detected in urine. The only way to address this question in a cross sectional study format was to correlate the AFB sputum staining score with concentration of LAM in urine. As shown in Figure 5 there was an obvious positive correlation of antigen concentration in urine and tubercle bacteria density in sputum. Such a correlation opens up several additional applications for the LAM assay. The monitoring of treatment success and the early recognition of relapses after completion of treatment are of immediate practical relevance. The combination of a sensitive urine assay with the capacity to detect extrapulmonary and AFB-negative TB renders the LAM assay a potent tool in an environment with a growing prevalence of extrapulmonary forms of TB and pulmonary forms with atypical clinical symptoms. The LAM-ELISA could not only be used for the diagnosis of patients with clinical symptoms, but also for screening HIV positive patients and other high risk groups. Early case detection of active TB and effective treatment are the two pillars in a successful fight against TB. To further explore the role of the LAM assay in this fight additional studies, including prospective multicenter studies, were carried out.

Additional patients from Tanzania were evaluated in a 2005 study (the T2005 project), and data for a subset of a group of 600 patients, selected as described above, are shown below in Table 3. In that subset of the 600 patients, 78 patients were assigned to Group A, defined as those determined to be true TB patients, based on AFB-microscopy and analysis. Another two groups, group C and group CC, comprise patients with sputum cultures that are free of mycobacteria. This means that they are free of mycobacterial colonization or that any such colonization is below the sensitivity threshold of the microbiological procedures used in the T2005 project. For the purposes of this analysis, all group C and CC patients are presumed to be true TB-negative patients. The results for these groups for all the patients in the subset of the 600 patient from the T2005 project are summarized in Figure 15 below. As seen in Figure 15, the LAM- ELISA had a sensitivity of 67.1% compared to AFB-microscopy for detecting mycobacterium, but this number was higher - 82.7%, for HIV co-infected patients. These numbers are in agreement with other numbers and conclusions analyzing a larger group of patients. In addition, as can be seen in Fig. 15, the specificity of the LAM-ELISA among the

C group, defined as those patients being TB-symptomatic, but proven TB-negative, was also quite high - 95.9%. Finally, the sensitivity for those patients that are TB asymptomatic was found to be 99.1%.

Two false positive patients in the C group were enrolled at the very end of the enrollment period for the whole group, where changes in the demographics of the study group were an issue, and thus may have affected this number. Potentially, the actual specificity of the LAM-ELISA for TB-negative patients, whether symptomatic or not, may be even higher. This suggestion finds some support in the data on group CC patients (those that are TB asymptomatic and TB negative by culture and AFB analysis), where specificity of the assay was found to be 99.1%.

Evidence for the high specificity of LAM-ELISA in both TB-symptomatic and TB- asymptomatic patients in the T2005 and T2006 projects provides strong support, coupled with results from the T2003 project, that a positive LAM-ELISA result using the technology of the present invention indicates positive mycobacterial colonization in a patient. Analysis of the entire group of patients in the T2005 project shows that the LAM-ELISA detected 140 patients as TB positive (data not shown), almost twice as many identified as AFB-positive patients in the same group by the AFB microscopy/culture test. These results are consistent with the results of the T2003 project (results shown above in Tables 1 and 2, and Figures 2-

6), and so are representative of the true sensitivity of the LAM-ELISA and show its superior utility over other tests for the detection of mycobacterial colonization in the field and elsewhere.

EVALUATION OF THE NOVEL LAM-ELISA URINE TB TEST USING AN ICT FORMAT.

Evaluation of the LAM-ICT test was done using non-concentrated and concentrated urinary samples wherein the antibody is selected so that the visual ICT result is unaffected by the presence of HIV infection in the subject.

Sample groups

All patients enrolled into TB T2006 study were categorized into four major study groups: group A - AFB(+)/culture (+); group B - AFB (-)/culture(+); group C - AFB(-)

/culture (-) and group CC - negative control group comprised of healthy volunteers. Samples from group A were used for the estimation of the sensitivity of the ICT-format modification of the urinary LAM-assay. Urinary samples from the TB-suspected patients confirmed by AFB microscopy and bacterial culture to be TB-negative (group C) were used as a negative control samples. Group C served for the estimation of the clinical specificity of the urinary LAM assays. This group was important for the evaluation of the assay performance during diagnosis of TB in symptomatic patients (passive case finding process).

Because even bacterial culture tests do not provide a gold standard in the epidemiological setting with patients having a high TB-HIV co-infection rate, there is no 100% guarantee that group C patients do not have some form of tuberculosis or other mycobacterial infection. Therefore, samples from additional healthy patients, defined as group CC (see above), were used extensively as a supplementary negative control for the evaluation the specificity of the urinary LAM assay. Data obtained from this group of samples are important for the evaluation of the assay performance in the screening for TB in asymptomatic patients not seeking medical help (active case finding process). The samples of the group B patients were not used in evaluating results from the T2006 project.

Tested assays.

During T2006 project two assays were developed:

> Urinary LAM direct antigen test.

> Direct antigen test for 38-kDa protein antigen from M. tuberculosis.

The urinary antigen test was the primary focus of these studies, and protein-specific test was tested on a second priority basis. Urinary LAM test was used in two formats - the 96-well

ELISA format and the ICT format.

Both formats were tested using both non-concentrated urine samples and samples concentrated by centrifugal ultrafiltration process. Addition of the urine concentration step is technically simply a form of sample preparation, but it significantly changed the test outcomes compared to the original procedure in the absence of urine concentration.

Therefore, LAM-ELISA tests for concentrated urine samples represent a second preferred embodiment for the presently claimed invention, with the first preferred embodiment being LAM-ELISA tests for non-concentrated urine samples, as described above. For the ICT strip format for the urinary LAM-ELISA, three different lots of strips made at different times and with different materials were tested. One lot was made in advance using BBI-made gold conjugate and low-porosity nitrocellulose membrane. Two other lots were produced during the study from components made by the researchers, but with a higher porosity nitrocellulose membrane. Comparison of these three lots of ICT strips was informative for determining how to optimize the assay as needed, depending on, among other factors, where and how the test was to be performed, how the samples would be collected and prepared, for example.

T2006 38-KDa PROTEIN ELISA & ICT FORMAT. A sandwich immunoassay specific to a 38-kDa protein from M .tuberculosis had been developed as part of the T2003 study, because of the knowledge that human saliva samples sometimes give false positive results in apparently TB-negative individuals. Both assays, ELISA and ICT, were manufactured prior to the T2006 study and stored at 4⁰C. Immediately prior to beginning the T2006 study, the ELISA and ICT kits were re-tested for efficacy, and both demonstrated performance similar or identical to freshly manufactured kits used in the T2006 project.

The assay format study was designed to allow estimation of the analytical sensitivity for ELISA and ICT formats using pure protein antigen and quantitative estimation of the analytical assay specificity using bacterial cultures of pathogenic mycobacteria.

RESULTS.

Experiments with culture isolates from clinical samples and standard cultures confirmed data, obtained previously, regarding positive and negative TB samples. The analytical sensitivity estimate obtained before this study was at the level of 0.5-1.0 ng/ml of pure protein or approximately 10⁵ cfu/ml of cells of M .tuberculosis H37Ra strain both for the ELISA and ICT assays.

Experiments with culture isolates from clinical samples and standard cultures confirmed the data previously obtained for the ELISA and ICT format test. Analytical sensitivity was confirmed in this study to be at the level of 0.5-1.0 ng/ml of pure protein or approximately 10⁵ cfu/ml of cells of M. tuberculosis for both test formats, although experiments with live sputum samples of group A patients from the T2006 study indicates further optimization is needed when using samples from the field. Nonetheless, the 38-kDa protein specific ICT test represents a viable alternative to the LAM-ELISA format, following optimization for use with samples from the field.

T2006 LAM-ELISA STUDY - Non-concentrated Urine Samples

This study was undertaken to confirm the results observed in the T2005 study (see above), to investigate the effects of urine concentration, and to continue exploring and optimizing use of the ICT format.

Samples

Patient group A (110 patients -220 samples) and group C (53 patients - 106 samples). Group CC patients (100 samples/patients).

Procedural comments. ELISA was performed as described above in the T2003 study with the following TB- LAM ELISA kit components:

1. Plates:

2. HRP Conjugate: 3. TMB Substrate:

4. TB LAM Positive Control - 20 ng/ml:

5. PBST: (Sigma)

6. Urine Negative Control

As a control for the TB-LAM ELISA kit performance, double serial dilutions of TB LAM positive control in Urine Negative Control were made throughout the first two rows

(rows A, B; wells 1-8) of each running ELISA plate.

Outcomes

Results obtained using LAM-ELISA strips prepared previously for the T2005 study were compared with results obtained using LAM-ELISA strips produced just prior to the

T2006 study. Not surprisingly, the newly-prepare strip kits appeared to have a somewhat higher analytical sensitivity at low concentrations of LAM standard as compared to results using the previously prepared strip kits, although the older strips were still quite sensitive. Nonetheless, all samples from group A patients were tested using new kits of LAM-ELISA and the obtained results were compared with data from the T2005 database.

Data comparison between the T2005 and T2006 projects showed good correlation, although all three samples from one patient yielded discrepant results, probably due to human error. There was also some difference in signals observed between the T2005 and T2006 plates, but this did not effect the assay outcome. Overall, it appeared that the T2006 strip kits yielded data with lower background, but the differences between the 2005 and 2006 lots did not have any apparent systematic character. It is possible that the observed difference was merely due to variations in sample preparation and handling.

Despite any variability in actual numerical values, both kits showed identical clinical sensitivity for the selected samples of group A patients - 60%, with some minor differences. These results indicate that the LAM-ELISA technology of the present invention shows stable clinical performance and delivers reproducible results.

Analytical sensitivity of the T2006 LAM-ELISA strips was found to be at the level of 0.05 -0.1 ng/ml of purified LAM antigen spiked in urine or PBS/BSA solutions. The current cut-off for ELISA using non-concentrated urine corresponds approximately to 0.1 ng/ml, a remarkable performance, especially for an assay detecting bacterial polysaccharide. The T2006 results also provide much useful data regarding test kit performance, including signal reproducibility, operator to operator variability and such. The specificity of the LAM-ELISA, as applied to TB-symptomatic but confirmed culture-negative patients (Group C patients) was re-evaluated using samples from group C patients. A randomly-selected group of 53 patients (105 samples) was used, such selected samples covering more or less evenly the entire study group of 873 patients. The T2006 strip data are in very good correlation with the T2005 strip data. Additional re-validation of clinical specificity of the LAM-ELISA was done on the selected samples of the group CC patients. This group represents a demographic control, because it was composed of subjects that were asymptomatic and presumed to be healthy from within the local population. Retesting of 100 group C samples produced 3 positive results, confirming the high specificity of the assay- 97%.

Concentrated Urine Samples.

This study examined the effect of urine concentration of the sample on assay sensitivity and on assay specificity.

Ultrafiltration was used to concentrate urine samples using two types of centrifugal ultrafiltration devices with sample capacity of 0.5 ml and 2 ml, although any suitable centrifugal filtration device would work, including those with different sample capacity, as would with other means for concentrating (e.g. evaporation). Devices with a capacity of 0.5 ml allow one to use the same microcentrifuge for sample concentration and the sample preparation. Devices with capacity of 2 ml are also useful, but require an additional centrifuge, a consideration that may be important when conducting testing in the field.

Comparison of the efficiency of both types of concentration devices (0.5 ml and 2 ml sample) using 10 samples of group A patients showed that both were equivalent. For logistical reasons, samples of group A and group CC patients were concentrated using 2-ml devices and samples of group C patients were concentrated using 0.5-ml devices. The same concentration factor was maintained for both types of devices - with a concentration fact in the range of from 4 to 5-fold the degree of concentration typically used.

Samples. Patient group A (110 patients - 153 samples concentrated out of 220 total), group C (53 patients - 106 samples), Group CC patients (40 samples/patients concentrated from the set of 100 samples). It was decided to concentrate For group A samples, only samples meeting a cut-off of OD>1.0 above background, based on T2006 strip measurements, were concentrated. This minimized work load while also allowing concentration of all negative and low positive samples. All 106 samples of the group C patients were concentrated, because all met the selection criteria (OD > 0.1) used for group A samples. This group was also instrumental for investigating assay specificity. Only 28 out of 100 group CC samples were concentrated because of equipment limitations.

Outcomes

Figure 16 shows the number of the new positive results gained after concentrating urine sample in the different patient groups and at the different cut-off values. The objective of this analysis was to find cut-off value at which LAM-ELISA with concentrated urine has the same specificity as LAM-ELISA with non-concentrated urine samples.

In group A, as was expected, the majority of the concentrated urine samples gave a higher signal than the signal of the non-concentrated samples. As can be seen in Figure 16, 64 positive results from non-concentrated urine samples corresponded to a sensitivity of 59% for this subset of patients. This compared to 85 positive results (78%) based on a cut-off 0.1; 81 positive results (74%) based on a cut-off 0.15; and 76 positive results (70%) based on a cut-off 0.2.

The cut-off value for the concentrated urine samples was determined by analyzing the results of the limited set of concentrated urine samples from group CC patients. The use of a cut-off value equal to 0.2 OD above negative control retained the initial specificity of the assay as was seen for non-concentrated samples. Although the selected set of 28 samples from CC patients is limited in size, it nonetheless provided much insight. Analyzing the data on the 100 concentrated urine samples from the presumed to be TB negative US residents, the same conclusion was reached for a cut-off value of 0.2 ng/mL. Interestingly, lowering the cut-off value to 0.15 OD above negative control brought in an additional positive result in the group of CC patients, with essentially no change in specificity of the LAM-ELISA. That one additional positive patient gained in the 28-group CC samples represented a 3.8% increase of additional presumed false positive results, which does not necessarily compromise approach the approach, so cut-off values lower than the chosen 2 ng/mL could be used without compromising specificity and selectivity. Concentrated urine samples of US patients remained negative using the cut-off of 0.15. Based on this limited data it was concluded that a cut-off 0.15 ng/mL above the negative control was appropriate, so this lower cut-off value was used in the later experimental studies.

Application of the 0.15 ng/mL cut-off to the LAM-ELISA data resulted in an increase of clinical sensitivity among the group A patients by about 30%, which is significant. Even more significant was the change observed in the results of the group C patients, where use of the concentrated urine samples lead to a three-fold gain in the number of TB-suspected patients. Data from the group CC patients essentially ruled out any argument that such gain was due to experimental artifacts originated by changes in the sample matrix composition. It also should be noted that group C represented TB-symptomatic patients able to produce sputum, but who tested negative in sputum AFB microscopy and culture. With the sensitivity less than 100% for both the AFB and LAM-ELISA methods, it was therefore not possible to rule out TB in this group based on the negative results of these two tests.

Conclusions

The T2006 project results re -confirmed results of the T2005 project, as related to the performance of the LAM-ELISA. In addition, two studies using two different lots of test kits produced identical clinical results, confirming the stability of the manufacturing process for the strips used in the LAM-ELISA protocols.

Analysis of the T2005 data obtained on 600 TB-suspected patients and 226 negative control patients demonstrated that the LAM-ELISA had a sensitivity of 69% compared to the sputum samples analyzed by AFB-microscopy. In the HIV-positive subgroup of patients, the

LAM-ELISA had a sensitivity of 84% compared to that observed with AFB-microscopy (see Figure 15). In such experiments, the antibody was selected so that the test result was unaffected by the presence of HIV infection in the subject. Specificity of the LAM-ELISA among TB-suspected but proven to be TB-negative patients was 96%. Specificity of the LAM-ELISA among the negative control group was 99%.

The detection rate among the whole study subset of 600 TB suspected patients was 1.8-times higher (180%) compared to the detection rate of AFB-microscopy. Based on the high specificity with the LAM-ELISA, we reasoned that this represented the true detection rate for TB in the tested patients.

It was also shown that concentrating urine sample did increase sensitivity of the LAM-ELISA without negatively impacting clinical specificity of the assay. On the samples of group A patients specifically, the increased sensitivity approached 30-40% without any loss of assay specificity, and results are not affected by the presence of HIV infection in the subject.

T2006 LAM-ICT - Non-concentrated urine samples This study was designed to evaluate the projected sensitivity of the LAM-ICT devices. It was estimated that the LAM-ICT assay had a sensitivity, estimated using model samples, in the range of 0.25-0.5 ng/ml of the LAM-4 standard, which is close to the sensitivity of the LAM-ELISA. The ICT part of the T2006 study focused on evaluating the performance of the ICT devices using a large number of clinical samples. One lot of ICT devices was made a year prior to the study using BBI-produced gold particles conjugated to LAM-specific Ab (MTB-I CNBr-50 type) at BBI as well. This lot was used in the course of the T2005 project in experiments with sputum samples. Two other lots of the ICT devices were made using only in-house made gold particles and conjugate just prior to the T2006 study. These two lots were prepared from the same reagents, but with some differences in the process of striping the Ab on the nitrocellulose membrane, so each was designated as a different lot. Comparison of these three lots was thus evaluated in the T2006 project.

Samples T2005 study group A (I lO patients -218 samples), group C (selection of 106 samples) and Group CC (100 patients/samples). Prior to be used with ICT devices, all samples were tested with the LAM-ELISA. The ELISA data were used for the estimation of analytical sensitivity of the ICT devices, and for the comparison of the sensitivity of LAM- ICT to LAM-ELISA.

Model samples

ICT devices used in the project did not have a sample pad or a clear protective overlabel. A sample pad was not used to avoid its effect on sample volume, assay timing and ultimately assay sensitivity. Without a sample pad, the ICT devices were thus run only in a vertical position, corresponding to the "dip-stick" concept.

The loose structure of the glass fiber material used for construction of the gold pad allowed easy reconstitution of gold conjugate and did not introduce any additional artifact. In all devices, the gold pad, as well as the nitrocellulose strip, did not retain any traces of conjugate. This confirmed the correctness of the compositions used in the gold conjugate manufacturing, and that appropriate parameters for the conjugate drying process had been determined.

Sample volume varied within a range of 100-300 μl. Standard sample volume was equal to 150 μl with 150 μl of PBST wash. In some experiments it was possible to use samples with volumes up to 250-275 μl with 100-150 μl wash. Such volumes did not compromise structural integrity of the ICT device itself. The ICT devices made in 2005 were optimized for lower capacity, though with higher hydrophilicity, and therefore had somewhat higher "pulling power". The use of large sample volumes with the devices of the 2005 ICT lot was more problematic and so 150-μl sample size was the largest volume used. This allowed direct comparison of the results between three lots.

All three lots of ICT devices tested were compared using standards, and all demonstrated similar sensitivity - approximately 0.25-0.5 ng/ml of LAM-4 standard antigen in urine, corresponding to an ELISA cut-off of 0.2-0.3 OD above negative control. ICT signals were quantified using QuadScan reflectance reader and data are presented in Figures

17 and 18.

The ICT devices of lot #061605 made for the T2005 project had somewhat better sensitivity, especially at the low end of the curve. As mentioned earlier, these strips were made by BBI and used 40 nm gold particles, as compared to the two lots made in the T2006 study. The T2005 lot was also made using a different lot of Ab. This difference in sensitivity was visible not only by instrument, but also visually. Sensitivity of 0.25 ng/ml corresponds to the ELISA signal equal to approximately 0.2 OD above negative control for the current lot of the test. This was also better than sensitivity of ELISA plates used in T2005 clinical trials. A sensitivity in the range of 0.25-0.5 ng/ml for ICT devices is expected to correspond to the sensitivity of the T2005 lot of the ELISA plates.

Clinical samples The sensitivity of ICT devices was evaluated with clinical urine samples of different optical densities (ELISA data). The wash step was an important procedural step that impacted results. ICT of all three lots under the study demonstrated similar sensitivity: in general, the devices were able to see some positive samples with 0.2-0.25 AU above negative control in ELISA. However, the sample composition (salt concentration, color, pH, sample density etc.) can influence the visibility of ICT signal in clinical samples with similar readings in ELISA format. Washing strips with PBST (0.02 % - Tween 20) helped to reduce the background (color) of the strip. This made the test line more visible. At the same time some samples with higher ELISA readings, like 0.4-0.6 AU above negative control, were not as clearly visible as were expected. Figure 19 shows actual LAM-ICT strips that were treated with urine samples spiked with decreasing concentrations of LAM-2 (25, 6.25 and 1.56 ng/ml) for 4 different preparations of strips.

Figure 20 shows the visual sensitivity of various lots of ICT strips treated with urine spiked with decreasing concentrations of LAM ranging from 25 down to 0.125 ng/mL. The lower limit of visual detection for these ICT strips appears to be at -6.25 ng/mL LAM, which is comparable to that observed with the ICT strips shown in Figure 19 although some strips in Figure 19 appear to show faint visual detection of LAM -4 as a concentration in urine as low as 1.56 ng/mL.

The reaction kinetics in the lateral flow format of ICT showed quite complicated character, depending not only on Ag interaction with Ab on the solid phase, but also on the flow of each individual component of the reaction inside of the membranes and pads, GoId- Ab-Ag interaction, etc. All these factors explained why the intensities of ICT signal were not always proportional to the antigen concentration in the sample. In general, there was no possibility to discriminate ICT signals for the low positive (by ELISA) samples by their intensity, especially, if their ELISA OD readings were in the close proximity to the Negative sample values. In our case this meant that optimal signals for low positive sample were difficult to obtain, while some samples with the signal of 0.07-0.09 above negative control were visible, which would have been ruled-out as negative by ELISA. Nonetheless it was known, based on the concentration experiments, that such samples represented a very high probability of being true positives.

Concentration of the low positive samples (initial sample OD equal approx. to 0.1 - 0.4 AU above negative control) using centrifugal ultrafiltration devices (membranes with 10- kDa exclusion limits) gave not only the proportional increase in the ELISA results, but also positively affected ICT results by increasing the test sensitivity. It was demonstrated with the low positive samples of group A patients that ICT signals could become much stronger after concentrating the urine sample. After concentrating it was possible to see as a positive, samples with ELISA signals in the range of 0.07-0.1 OD above negative control before concentrating.

At the same time the concentration of the true negative samples (CC group - healthy controls) in most cases did not affect ICT signals values (if there were any), thus confirming the fact that the concentration of the urine itself does not produce false positive signals in the test. Consequently, urine samples of the Group C patients (AFB negative, culture negative, clinical TB manifestation and positive chest X-ray) might either stay negative after concentration or became positive, depending on the extent of the concentration and initial LAM-antigen presence in the sample. Based on the urine concentration experiments, we presumed such samples were true positives, but the final evaluation of the sensitivity/specificity combination for ICT based on the concentrated urine samples was not done to confirm this.

Outcomes -Non-concentrated samples

Estimation of the clinical sensitivity of the ICT devices was done based on the correlation of the positivity of ICT strip and reading of the ELISA data for the same sample. If an ELISA cut-off of 0.25 above negative control was used as a sensitivity threshold for

ICT devices, projected clinical sensitivity of ICT should be equal to approximately 80% of the sensitivity of ELISA test applied to the same group of patients. In our T2006 project group A samples this cut-off should thus correspond approximately to 45-50% of clinical sensitivity compared to AFB-microscopy. These estimations are made using non-optimized ICT devices. Optimized ICT devices containing a sample pad are expected to have better sensitivity, so it is expected that non-centrifuged samples can be used with POC devices, with only minimal sacrifice in sensitivity.

Some of the ICT devices were used for testing liquid cultures for the presence of mycobacteria. All AFB positive culture samples were positive using the LAM-ICT test. This result corresponded to data (not shown) on the sensitivity of LAM-ICT as applied to pure culture isolates.

In addition, testing of standard bacterial cultures confirmed selectivity of certain LAM-assays towards M. tuberculosis, compared to M.fortuitum and M. kansasii. Outcomes - Concentrated Samples.

Patient group A (110 patients -220 samples) and group C (selection of 30 samples), group CC patients (selection of 28 samples). Use of concentrated urine samples significantly increased the sensitivity of the LAM-

ICT assay. If optimized ICT devices containing a sample pad are able to detect samples corresponding to ELISA signal of 0.2 OD above negative control, then such tests will be more sensitive than even the current LAM-ELISA without concentration. As the technology now stands, for the selected subset of group A samples, the sensitivity of the LAM-ICT used with sample concentration approached 70%, compared to the current sensitivity of 60% for

ELISA without concentration.

Because concentrating urine does not impact clinical specificity of the assay in a negative way, it is expected that an ICT assay using a concentrated urine sample will have even lower NSB and higher analytical specificity. Figure 20 shows a picture of actual LAM-ICT strips showing the visual sensitivity of the

LAM-ICT test.

As can be seen in Figure 21, a calibration graph for the LAM-ICT strip test (using lot #080602NH) was done which shows LAM-4 concentration, in ng/mL plotted against the relative units (reflectance).

Conclusions

The T2006 project demonstrated that the LAM-ICT device is functional and can be used with clinical urinary samples for the detection of LAM antigen present in both unconcentrated and concentrated urine samples. The analytical sensitivity of the LAM-ICT devices is at the level of 0.25-0.5 ng/ml of LAM antigen in human urine. All three tested lots of ICT device demonstrated comparable analytical and clinical sensitivity, while the Lot of devices made in 2005 using BBI Ab-Au conjugate demonstrated stronger signal in visual and instrumental detection modes on the model samples, although on clinical samples this lot did not have a strong advantage due to the lower capacity of the adsorbent pad optimized specifically to match Ab-Au conjugate. The clinical sensitivity of all three lots was comparable. There is also no strong linear correlation between LAM-ELISA readings and strength of the visual ICT signal for the same clinical sample. The observed clinical sensitivity of the LAM-ICT prototype is equal to approximately 80% of the sensitivity of LAM-ELISA achieved on the same clinical samples. Use of a concentrated urine sample improved sensitivity of ICT -based assay, so it is anticipated that an ICT kit using concentrated urine will equal or exceed the sensitivity level of current the LAM-ELISA using a non- concentrated urine sample. In addition, it is expected that optimized ICT product will allow use of a non-centrifuged urine sample, and use of gravity settling of the proteins coagulated by a heating step might be sufficient to obtain a sample usable with the ICT device. Thus, if performance level comparable to 70-80% of the LAM-ELISA is sufficient for purposes of identifying patients, it is expected that an ICT kit that requires no lab equipment for implementation can be developed and used in the field for screening patients for TB infection.

In summary, the LAM-ELISA and the LAM-ICT device can be easily integrated in the routine diagnostic procedures of laboratories of both developed and developing countries.

Both systems represent easy to use and robust assays. Completion of the ELISA requires only 2 ¹A hr and many samples can be analyzed at the same time. Use of the ICT should allow screening of patients in the field with no requirement for laboratory equipment. And, since the antigen Lipoarabinomannan is stable, it was shown that urine samples could be kept stable for 3 days if refrigerated without a significant drop in optical density detection of

LAM. The newly developed MTB-ELISA or LAM-ICT device for detection of LAM in unprocessed urine have the potential to become screening tests to be used also under field conditions in developing countries.

49

Table 1. Analysis of urinary LAM excretion in the 242 patients coming to the OPD with clinical suspicion for TB and the 243 clinically healthy controls. The cut off value for LAM- ELISA positivity is 0.1 above the mean optical density of the negative control on the plate. + = positive.

Table 2. Proportion of HIV positive patients in the different groups. 223 of 242 patients consented to be tested for HIV.

Claims

What is claimed is:

1. An enriched antibody population specific for a mycobacterial surface polysaccharide antigen, wherein the antibody specific for the mycobacterial antigen is enriched by exclusion of antibodies that recognize modified mycobacterial surface polysaccharide antigen that have been rendered less antigenically active, wherein the modified antigen is modified by oxidation with NaIO₄.

2. An enriched antibody population according to claim 1, wherein the mycobacterium is selected from the group consisting of Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium par atubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis .

3. An enriched antibody population according to claims 1-2, wherein the mycobacterium is Mycobacterium tuberculosis.

4. An enriched antibody population according to claims 1-2, wherein the mycobacterium is Mycobacterium paratubercuolsis .

5. An enriched antibody population according to claims 1-4, wherein the surface polysaccharide is a lipopolysaccharide.

6. An enriched antibody population according to claims 1-4, wherein the surface polysaccharide is lipoarabinomannan.

7. An enriched antibody population according to claims 1-6, wherein the antibody population is a polyclonal antibody population.

8. A process for producing an isolated enriched antibody specific to an surface polysaccharide antigen of a mycobacterium, the process comprising: isolating antigenically active surface polysaccharide antigen from mycobacteria under NaIO₄ oxidation conditions sufficient to maintain antigenic activity in a population of surface polysaccharide antigen so as to produce isolated antigenically active antigen; and raising and isolating antibody to the isolated antigenically active antigen, so as to produce isolated enriched antibody specific to the surface polysaccharide antigen of the mycobacterium.

9. A process for producing an isolated enriched antibody specific to a surface polysaccharide antigen of a mycobacterium, the process comprising: exposing surface polysaccharide antigen, isolated from the mycobacterium, to NaIO₄ oxidation conditions so as to produce an antigen population including antigens that remain antigenically active and antigens that have been rendered less antigenically active; raising and isolating antibody to the antigen population so as to produce a population of isolated antibody; and removing, from the population of isolated antibody, antibody that is specific to the less antigenically active antigen, so as to produce isolated enriched antibody specific to the surface polysaccharide antigen of the mycobacterium.

10. A process for producing an isolated enriched antibody specific to a surface polysaccharide antigen of a mycobacterium, the process comprising: exposing surface polysaccharide antigen, isolated from the mycobacterium, to NaIO₄ oxidation conditions so as to produce an antigen population including antigen that remains antigenically active and antigen that has been rendered less antigenically active; isolating antigenically active surface polysaccharide so as to produce isolated antigenically active antigen; isolating antigen that has been rendered less antigenically active so as to produce isolated less antigenically active modified antigen; applying sera from a mammal inoculated with mycobacteria to a first affinity matrix prepared with the isolated antigenically active antigen, such that antibody specific to the antigenically active antigen is retained by the first affinity matrix; isolating antibody specific to the isolated antigen from the first affinity matrix; applying the isolated antibody to a second affinity matrix prepared with the isolated modified antigen, such that antibody specific to the modified antigen is retained by the second affinity matrix; and isolating enriched antibody specific to the antigenically active antigen by collecting effluent from the second affinity matrix, so as to produce isolated enriched antibody specific to the surface polysaccharide antigen of the mycobacterium.

11. A process according to any of claims 9-10, wherein the mycobacterium is selected from the group consisting of Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis .

12. A process according to any of claims 8-11, wherein the mycobacterium is Mycobacterium tuberculosis .

13. A process according to any of claims 8-12, wherein the mycobacterium is Mycobacterium paratubercuolsis .

14. A process according to any of claims 8-13, wherein the surface polysaccharide is a lipopolysaccharide.

15. A process according to any of claims 8-14, wherein the surface polysaccharide is lipoarabinomannan (LAM).

16. A process according to any of claims 9-15, wherein the modified antigen is rendered less antigenically active with NaIO₄.

17. A process according to any of claim 12, wherein the surface polysaccharide is isolated from Freund's adjuvant.

18. A method for detecting a mycobacterial infection in a urine sample from a subject of interest, by detecting mycobacterial surface polysaccharide antigen in the sample, the method comprising: providing an ICT device, such device (i) having an arrangement for receiving a sample, (ii) providing a visual test result, and (iii) utilizing an antibody according to any of claims 1-7 or produced according to any of claims 8-17; contacting the sample with the arrangement in the device for receiving a sample, so as to cause the device to provide a visual test result that is positive if the antibody in the test device binds to a mycobacterial surface polysaccharide antigen in the sample.

19. A method according to claim 18, wherein the antibody is selected so that the visual test result is unaffected by the presence of HIV infection in the subject.

20. A method according to claims 18-19, wherein a positive immunoassay result compared to an appropriate control is considered positive for a mycobacterial infection.

21. A method according to claim 20, wherein the appropriate control is selected from a positive control, a negative control, or any combination thereof.

22. A method according to claims 18-21 , wherein the mycobacterial infection is selected from the group consisting of Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium par atubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis .

23. A method according to claims 18-22, wherein the mycobacterial infection is M. tuberculosis.

24. A method according to claims 18-22, wherein the mycobacterial infection is Mycobacterium par atubercuolsis .

25. A method according to claims 18-24, wherein the surface polysaccharide is a lipopolysaccharide.

26. A method according to claims 18-25, wherein the surface polysaccharide is lipoarabinomannan (LAM).

27. A method according to claims 18-22, wherein the mycobacterial infection is a pulmonary Mycobacterium tuberculosis infection.

28. A method according to claims 18-22, wherein the mycobacterial infection is an extra-pulmonary Mycobacterium tuberculosis infection.

29. A method according to claims 18-28, wherein the sample is a concentrated urine sample.

30. A method according to claims 18-28, wherein the sample is non-processed unconcentrated urine.

31. A kit for detecting a mycobacterial infection in a sample, the kit comprising: an assay for detecting a surface polysaccharide antigen from a mycobacterial infection, wherein the assay comprises an enriched antibody according to any of claims 1-7 or produced according to any of claims 8-17.

32. A kit according to claim 31 , wherein the assay is an ELISA or an ICT format.

33. A kit according to claims 31-32, wherein the mycobacterial infection is selected from the group consisting of Mycobacterium bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium paratubercuolsis, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, and Mycobacterium tuberculosis.

34. A kit according to claims 31-33, wherein the mycobacterial infection is Mycobacterium tuberculosis .

35. A kit according to claims 31-33, wherein the mycobacterial infection is Mycobacterium paratubercuolsis.

36. A kit according to claims 31-35, wherein the surface polysaccharide antigen is a lipopolysaccharide.

37. A kit according to claims 31-36, wherein the surface polysaccharide is lipoarabinomannan.

OOOOl/REFILEBJC 776602.1