WO1992014156A1 - Test d'immunofluorescence servant a detecter des antigenes mycobacteriens dans des fluides biologiques - Google Patents

Test d'immunofluorescence servant a detecter des antigenes mycobacteriens dans des fluides biologiques Download PDF

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Publication number
WO1992014156A1
WO1992014156A1 PCT/US1992/001165 US9201165W WO9214156A1 WO 1992014156 A1 WO1992014156 A1 WO 1992014156A1 US 9201165 W US9201165 W US 9201165W WO 9214156 A1 WO9214156 A1 WO 9214156A1
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Prior art keywords
solid phase
mycobacteria
mai
nitrocellulose
complex
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PCT/US1992/001165
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English (en)
Inventor
Nicolae Istrate
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Dynagen, Inc.
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Publication of WO1992014156A1 publication Critical patent/WO1992014156A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran

Definitions

  • This invention pertains to. the field of immunology, and in particular, solid phase immunoassays for detecting the presence of members of the bacterial genus Mycobacterium.
  • the mycobacteria are a diverse assemblage of acid-fast, Gram-positive bacteria, some of which are important disease-causing agents in humans and animals, Bloom et al. , Rev. Infect ⁇ Pis. , j>:765-780 (1983); Chaparas, CRC Rev. Microbiol. , 9:139-197 (1982).
  • the two most common diseases caused by mycobacteria are tuberculosis and leprosy, which result from infection with Mycobacterium tuberculosis and Mycobacterium leprae, respectively.
  • mycobacterial species are capable of causing tuberculosis or tuberculosis-like disease, Wallace, R.J., et aJL. , Review of Infectious Diseases, ⁇ :657-679 (1984).
  • Mycobacterium avium for example, causes tuberculosis in fowl and in other birds.
  • Members of the M. avium-intracellulare (MAI) complex are pathogenic among individuals with acquired immuno-deficiency syndrome (AIDS) , as well
  • tuberculosis is the result of respiratory infection with M. tuberculosis ⁇ Infection may often be asymptomatic, but could lead to disease producing pulmonary or other lesions which might result in severe debilitation or death.
  • tuberculosis remains a significant health problem especially in developing countries.
  • an estimated 11 million people are affected by the disease and about 3.5 million new cases occur each year.
  • U.S. Congress, OTA "Status of Biomedical Research and Related Technology for Tropical Diseases", OTA-H-258, Washington, D.C. 1985.
  • certain groups of individuals with AIDS have a markedly increased incidence of tuberculosis.
  • Early diagnosis of tuberculosis is particularly important because the disease is preventable, treatable and curable.
  • bacilli may be detected by microscopy, Shoemaker, S.A., et al., Am. Res. Respir. Pis. , 131:760-763 (1985), intact bacilli are required and sensitivity is low. Mycobacteria are therefore cultured to allow for more accurate diagnosis as well as to permit definitive species identification. Vestal, A.L., HEW Publ. No. (CDC)77-8230 Atlanta, 1975; Bates, J.H., Am. Rev. Respir. Pis. , 132: 1342 (1985). However, M. tuberculosis is difficult to culture and has a generation time of 15-20 hours. Wayne, L.G., Am. Rev. Respir. Pis. , 125 (Suppl.) 31-41 (1982). A delay of up to 6 weeks before results of laboratory tests are available is not unusual.
  • a further object of the invention is to provide
  • the invention pertains to a solid phase immunoassay for determining the presence of mycobacteria in a sample of biological fluid.
  • the immunoassay does not require chemical fixation of mycobacteria to the solid phase.
  • chemical fixatives are not used and lipid-containing antigenic components of the mycobacteria are preserved.
  • This permits the use of highly specific monoclonal antibodies, which is not practical when chemical fixatives are used since antigenic alteration caused by chemical fixation adversely affects monoclonal antibody-antigen binding to an unpredictable extent.
  • a biological fluid suspected of containing mycobacteria is applied to a solid phase.
  • the biological fluid is processed.
  • the solid phase is characterized by having the ability to bind mycobacteria without the need for chemical fixation so that the antigenic components of the bound mycobacteria are preserved.
  • Preferred solid phases are porous nitrocellulose membranes. The presence of bound mycobacteria is determined by applying to the solid phase an antibody capable of
  • binding to an antigenic component of mycobacteria and then detecting the presence of the antigen-antibody complex.
  • a fluorescent-labelled, anti-immunoglobulin is used, and detection is conducted by microscopic examination.
  • the invention also permits distinguishing between a particular member of the M. avium - M. intracellulare complex (MAI-complex) and other mycobacteria.
  • MAI-complex M. avium - M. intracellulare complex
  • a plurality of samples of a biological fluid are applied to discrete locations of a solid phase.
  • the presence of a first antigenic component of mycobacteria then is detected at a first location using a monoclonal antibody capable of binding to an antigenic component that is common to all mycobacteria.
  • the presence or absence of a second antigenic component of mycobacteria then is detected at a second location using a monoclonal antibody selectively specific for antigenic components found in a particular MAI-complex mycobacterium, and not in other mycobacteria.
  • Identifying MAI serovars 4 and 8 is clinically important since these are the two most common serovars found among AIPS patients and other immune compromised individuals. If both antigenic components are detected, the sample contains
  • Preferred monoclonal antibodies useful in methods of the invention are selectively specific for the lipoarabinomannan (LAM) and glycopeptidolipid (GPL) components of mycobacterial cell walls.
  • LAM lipoarabinomannan
  • GPL glycopeptidolipid
  • the LAM component is present in all tested strains of mycobacteria.
  • Certain GPL components (GPL4 and GPL8), on the other hand, are present only in serovars 4 and 8 respectively of the MAI complex mycobacteria.
  • test kits and kit components are provided.
  • a test kit for determining the presence of mycobacteria in a biological fluid includes a solid phase capable of binding mycobacteria without the need for chemical fixation.
  • the solid phase is porous and is constructed and arranged so that mycobacteria will be bound in a concentrated ring when applied to the porous solid phase.
  • the porous solid phase is porous nitrocellulose attached to a glass microscope slide.
  • Antibodies selectively specific for antigenic components of mycobacteria also may form part of the kit.
  • the kit also may include positive and negative controls, as well as slide mounting fluid compatible with the solid
  • Blocking solution capable of binding to the solid phase to prevent non-specific binding of antibodies to the solid phase also may be included.
  • Products of the invention thus include glass microscope slides that have affixed onto them one or more porous nitrocellulose solid phases as well as porous nitrocellulose solid phases to which are (noncovalently) bound mycobacteria.
  • Fig. 1 is a plan view of a test strip of the invention.
  • Fig. 2 is a view in perspective of a kit of the invention.
  • Petailed Pescription of the Invention pertains to solid phase immunoassays for the detection of mycobacterium in biological fluids.
  • biological fluid includes but is not limited to pulmonary fluids, urine, genital fluids, fecal fluids, spinal fluid, sputum or blood.
  • Preferred methods of the invention include the processing of a biological fluid sample prior to application to a solid phase.
  • process or “processed” refers to methods of treating biological fluids in order to kill or reduce to negligible levels all bacteria except
  • mycobacteria to liquefy mucoid samples (e.g. sputum, pus, exudates), and/or to concentrate mycobacteria. Processing also may be designed to release whole mycobacteria from any cellular and/or non-cellular matrix.
  • sputum samples advantageously may be processed because the mycobacteria of interest are entrained within the gelatinous matrix of the sputum sample.
  • the processing methods employed vary with the biological fluid being assayed. Nevertheless, some specimens do not require substantial processing. Cerebrospinal fluid need only be centrifuged to concentrate the bacteria and the pellet resuspended. Pleural fluid can be collected in sterile anticoagulant, centrifuged and the pellet resuspended.
  • Blood can be collected in sterile anticoagulant and allowed to stand.
  • the leukocyte-rich plasma is withdrawn, lysated, centrifuged and resuspended in buffer.
  • Various processing methods appropriate for particular biological samples are well known to those of ordinary skill in the art.
  • Gastric lavage should be processed immediately or neutralized with a basic solution (e.g. 10% sodium bicarbonate) and
  • centrifugation may be appropriate (e.g. 3600 x g for 30 minutes) and only the sediment saved for processing in the decontamination and/or concentration steps.
  • Urine samples can be divided into about four volumetric aliquots, e.g. 50 ml, and centrifuged to form a sediment or pellet (e.g. 3600 x g for 30 minutes). The supernatant fluid may be decanted. The combined sediment then may be collected, brought up to 10 ml and used in the decontamination and/or concentration steps (preferably as in Example 5) .
  • one or two grams of formed stool or 5 ml of liquid stool can be transferred to a 50 ml centrifuge tube and distilled water added to bring the volume up to 10 ml.
  • the suspension is vortexed thoroughly.
  • the specimen then is filtered through gauze to remove particulate material.
  • Ten ml of NALC-NaOH can be added to the suspension and allowed to stand at room temperature for 45 to 60 minutes.
  • 25 ml of phosphate buffer is added, mixed thoroughly, and centrifuged for 20 minutes at 3,600 X g.
  • the supernatant fluid is decanted and the sediment resuspended and assayed according to the invention.
  • Pus and wound aspirates can be transferred to a
  • Pieces of tissue thought to be contaminated are finely minced using a tissue grinder tub and pestle. Ten ml of distilled water is added, vortexed vigorously, and then allowed to stand for 20 to 30 minutes. The material then is transferred to a 50 ml centrifuge tube. An equal volume of NALC-NaOH is added and then mixed vigorously and allowed to stand for 20 minutes. Twenty-five ml of phosphate buffer is added. The tube is mixed vigorously and centrifuged at 3,600 X g for 20 minutes. The supernatant fluid is decanted and the pellet is suspended in 100 to 200 ⁇ l Tris buffered saline and used in the assays of the invention.
  • Cerebrospinal fluid may be centrifuged to concentrate the bacteria and the supernatant fluid discarded.
  • the pellet containing the bacteria may be resuspended in distilled water or in an aqueous solution with thorough mixing and the mixed solution may be used in the agglutination methods of the invention.
  • Pleural fluid may be collected in sterile anticoagulant (e.g. in the presence of ethylene diamine tetraacetic acid or heparin), centrifuged to
  • the pellet may be resuspended in distilled water or an aqueous solution and the mixed solution may be assayed according to the invention. If the pleural fluid becomes clotted, it may be liquified using sputolysin and/or vigorous mixing. Preferably, a pellet from 20 ml of pleural fluid is resuspended in 50 to 100 microliters of buffer and then assayed according to the invention.
  • Blood may be collected in sterile anticoagulant.
  • the blood may be allowed to stand at room temperature until separation of the plasma and other blood components has occurred.
  • the leukocyte-rich plasma may be removed and centrifuged at 400 x g until a pellet is formed (e.g. 15 minutes).
  • the pellet may be resuspended under conditions that allow cell lysis (e.g. in a cell lysing agent such as ammonium chloride) .
  • the lysed cells may then be centrifuged (3600 x g) until a pellet is formed (e.g. 30 minutes).
  • the pellet may be resuspended in a buffer solution (preferably 100 - 200 ⁇ l) and used in the agglutination methods of the invention.
  • Sputum may be processed by mixing with a basic solution, often a NaOH-NALC solution.
  • a basic solution often a NaOH-NALC solution.
  • the combination of the basic solution and the sputum may be processed by mixing with a basic solution, often a NaOH-NALC solution.
  • the supernatant fluid may be decanted and a buffer having a pH of about 6.5 may be added to the pellet.
  • the pH of the sputum may be tested with pH paper to determine whether the sputum is substantially neutral. The details of processing sputum are set forth in detail in Examples 5, below.
  • the immunoassays of the invention permit enhanced sensitivity relative to those that require intact bacilli, such as the AFB smear method.
  • the immunoassays of the invention involve binding mycobacteria (or portions thereof) to a solid phase without chemically fixing the mycobacteria to the solid phase.
  • binding or “bound” refers to a combination of physical interactions including hydrophobic interactions and electrostatic interactions. In this manner, the antigenic properties of mycobacterial antigens derived from the sample are preserved.
  • solid phase is meant to include any
  • substantially solid material that is adapted to bind mycobacteria and associated antigenic components without destroying or otherwise compromising the ability of the mycobacteria or component thereof to bind to antibody.
  • the solid phase is adapted to bind mycobacteria when the mycobacteria are applied to the solid phase in a solution containing only saline as a buffer. It is important that the binding be strong enough such that the mycobacteria or portions thereof or even soluble antigenic determinant are not released from the solid phase during other steps of the immunoassay such as washing.
  • the preferred solid phase is porous nitrocellulose.
  • nitrocellulose refers to nitric acid esters of cellulose, which may be nitrocellulose alone, or a mixed ester of nitric acid and other acids. In particular, aliphatic carboxylic acids are commonly used with acetic acid being preferred.
  • Solid phases formed from cellulose esterified with nitric acid and/or a mixture of cellulose esterified with acetic acid are commonly referred to as nitrocellulose paper or nitrocellulose membranes. Nitrocellulose solid phases can be of any size or shape, i.e, square or round.
  • the invention is based in part on the discovery
  • a solid phase such as porous nitrocellulose imparts special advantages to certain immunoassays.
  • Nitrocellulose solid phases having a particularly defined pore size will capture, bind and concentrate mycobacteria deposited in solution onto the nitrocellulose solid phase. A variety of properties interact to achieve this, including charge and pore size.
  • the charged moieties of nitrocellulose are capable of interacting with moieties of mycobacteria to form bonds between the two, without further chemical assistance or modification.
  • the pore size should be such that the bound mycobacteria will be located both on the surface of the solid phase and within the interstices of the pores.
  • a pore size of about 0.22 microns or smaller will tend to act as a physical barrier to mycobacteria and will concentrate the mycobacteria only on the surface of the solid phase. Few, if any, mycobacteria will become lodged within the pores. Pore sizes of about 1.0 micron or larger will not effectively capture mycobacteria. Instead, the mycobacteria will pass through the pores.
  • the preferred pore size thus is between about 0.22 and 1.0 microns.
  • Use of a nitrocellulose membrane having a pore size of about 0.40 microns is particularly preferred.
  • prior art immunoassays were forced to employ polyclonal antibodies that reacted with a wide array of mycobacterial antigens in the hopes of forming an antibody-antigen complex with some, unknown antigenic determinant that had survived harsh treatment with chemical fixatives.
  • the solid phase immunoassays of the present invention preserve the structure of mycobacterial antigens. This permits use of monoclonal antibodies in the immunoassays of the invention. Such antibodies also can be manufactured in precise and reproducible quantities. This results in improved standardization of
  • immunoassays because some antigenic components of mycobacteria are specific for certain species of mycobacteria, monoclonal antibodies can be used to develop immunoassays capable of discriminating between species of mycobacteria.
  • nitrocellulose solid phase offers still another advantage. Normally opaque nitrocellulose solid phases will become semi-transparent when placed in contact with slide-mounting fluids such as glycerol. The semi-transparent nature of the resulting solid phase is important for microscopic examination under transmitted light and very convenient for examination under incident light. Thus, visually detectable assays (e.g. ultraviolet/fluorescent assays) may be performed according to the invention.
  • visually detectable assays e.g. ultraviolet/fluorescent assays
  • detection is a function of concentration, as well as absolute number of mycobacteria and enhancement of fluorescence near the edge of the circular pattern greatly improves detection.
  • this spatial separation is caused by a chromatographic process.
  • the components of the processed biological fluid are contained in the moving carrier phase (the fluid) and become attached to the stationary solid phase (i.e. nitrocellulose) as the fluid moves.
  • fluorescence enhancement near the edge may be due to soluble LAM antigen which tends to concentrate on that part of the circular pattern.
  • mycobacteria are detected by forming an antigen-antibody binding pair or "complex" and detection of the complex is indicative of the presence of mycobacteria.
  • antigenic components as used herein, is meant to include determinants contained within or on the surface of a mycobacterium that are capable of binding with an
  • Antigenic components can be species-specific or they can be common to all mycobacteria.
  • the antigenic components of mycobacteria to be detected are those components that define serovars of the Mycobacterium avium-Mycobacterium intracellulare complex (hereinafter "MAI complex").
  • Serovars of the MAI complex are differentiated from those of other mycobacteria by their own set of antigenic glycopeptidolipids (hereinafter "GPL's"), often referred to as polar C-mycosides.
  • GPL's antigenic glycopeptidolipids
  • Antigenic specificity of the MAI-complex serovars is due to the structural variability present at the nonreducing end of the oligosaccharide which is attached to the threonine unit of the lipopeptide.
  • the variable oligosaccharide unit whose distal segment is unique to the serotype, accounts for its antigenic specificity. See Brennan et al. , Jd.
  • mycobacterial antigens common to all mycobacteria i.e. both non-MAI members and MAI-members
  • mycobacterial antigens common to all mycobacteria i.e. both non-MAI members and MAI-members
  • lipoarabinomannan is a prominent component of the cell walls of all tested strains of mycobacteria, and has been implicated as a major B cell stimulant in tuberculosis and leprosy. Portions of LAM are exposed on the surface of intact mycobacteria.
  • the antibodies of this invention are preferably those directed towards mycobacteria. Pirected towards is intended to encompass antibodies capable of binding with an antigenic portion of a mycobacterium.
  • the term antibody is intended to include whole antibodies, antibody fragments, chimeric antibodies containing portions from two different species, and synthetic peptides identical to or functionally analogous to the antibody.
  • Antibody fragments such as F(ab') 2 , Fab and F v may be produced by standard techniques of enzyme digestion.
  • synthetic peptides representing Fab and F analogues can be produced by genetic engineering techniques. See e.g., Better, M. et al. (1988) Science 240:1041; Huston, J.S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. See also U.S. Patent No. 4,946,778 to Ladner, issued August 7, 1990.
  • the preferred form of antibody is whole monoclonal antibody.
  • a preferred monoclonal antibody capable of selectively binding to LAM is
  • monoclonal antibody ML9P3 produced by the ML9P3 cell line, ATCC Accession No HB 10684, Rockville, MP.
  • Preferred monoclonal antibodies selectively specific for serovars of the MAI complex include monoclonal an,tibody M24B1, selectively specific for serovar 4 of the MAI complex, and M85F7, selectively specific for serovar 8 of the MAI-complex. Together, these two anti-MAI complex antibodies recognize MAI serovars 4 and 8. These monoclonals are produced by the M24B1 cell line and the M85F7 cell line respectively.
  • Examples of monoclonal antibody-producing cell lines include hybridoma cell lines, myeloma cell lines, or viral or oncogenically transformed lymphoid cells.
  • Hybridoma cells which can produce the specific antibodies for use with this invention may be made by the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975) or similar procedures employing different fusing agents. Briefly, the procedure is as follows: the hybridoma which secretes the monoclonal antibodies are produced by fusion of myeloma cells and hyper-immunized B cells. Lymphoid cells (e.g. splenic lymphocytes) are obtained from an immunized animal and fused with immortalizing cells (e.g. myeloma or heteromyeloma) to produce hybrid cells. The hybrid cells are
  • the hybridoma cells producing the desired antibody can be subsequently expanded.
  • the hybridomas are expanded by injecting them intraperitoneally into mice under conditions which allow ascites fluid to develop.
  • the ascites fluid is collected from the mice, pooled together and centrifuged. The supernatant from this process is termed neat ascites.
  • the immunoassays of the invention can employ an immunological reagent to detect antigen-antibody binding pairs attached to the solid phase.
  • the reagent may include a substance capable of binding to the monoclonal antibody that is attached to the antigenic component of mycobacteria. This substance of course may be a second antibody (anti-immunoglobulin) .
  • Preferred anti-im unoglobulins of this invention are polyclonal anti-mouse IgG raised in rabbit, goat, or horse. While anti-immunoglobulins are commercially available, others can be prepared using conventional immunological techniques.
  • the anti-immunoglobulin is labeled.
  • Many types of labels can be employed for assaying mycobacteria according to this invention. For example, many types of labels can be employed for assaying mycobacteria according to this invention.
  • radioactive iodine (I131) or radiometals can serve as label. These materials are preferably detected using photo-sensitive film.
  • Other labels can include avidin-biotin, and enzymes (e.g. horseradish peroxidase) .
  • Particularly preferred labels for methods of this invention are fluorescent materials that can be visualized microscopically under ultraviolet light.
  • the fluorescent label is fluorescein isothiocyanate
  • Rhodamine B can also be used. Labelled anti-immunoglobulin reagents are commercially available or can be readily prepared using conventional techniques for linking the label to the anti-immunoglobulin.
  • the antibody-antigen component conjugate can also be made visible by directly labeling the monoclonal antibody. This avoids the need for a separate immunological reagent that is an anti-immunoglobulin.
  • Products useful in immunoassays of the invention include solid phases adapted to bind mycobacteria without chemical fixation and fixed to a substrate.
  • the solid phase is porous and is constructed and arranged such that it concentrates mycobacterial antigen in discrete locations when a fluid sample containing mycobacteria is applied to the solid phase.
  • the substrate preferably is compatible with the detection method used.
  • the substrate can be almost any solid material capable of being used with photographic emulsions or scintillation counting methods.
  • the substrate should have the same light transmission, reflective and refractive properties as glass.
  • the substrate need not have the same spectral properties as glass. Glass microscope slides, however, are generally preferred as the substrate to which the nitrocellulose solid phase is affixed when visual detection methods are employed.
  • Nitrocellulose solid phases to be affixed to glass slides can conveniently be cut into small pieces.
  • One side of the solid phase can be glued to the glass slide using, for example, a commercially available polyadhesive such as Stix-all (Bordon Co., Columbus, Ohio).
  • Stix-all a commercially available polyadhesive such as Stix-all (Bordon Co., Columbus, Ohio).
  • the solid phase then is capable of being moved away from the surface of the substrate, which facilitates washing and other assay steps as will be more apparent from reading the examples below.
  • phase 10 is attached along an edge 12 to a substrate, glass slide 14.
  • a kit for clinical or research purposes is shown in FIG. 2.
  • a typical kit can comprise a holder 16 with compartments that hold glass slides 14 having attached thereto nitrocellulose strips 10.
  • the slide may have a single strip or multiple strips attached thereto.
  • the kit also includes a container 18 containing an antibody capable of binding any mycobacteria (e.g., anti-LAM) and/or a container 20 containing a species specific monoclonal antibody (e.g. anti GPL4 and anti GPL8).
  • the kit also may include a container 22 containing a labelled anti-immunoglobulin, a container 24 containing a blocking reagent, a container 26 containing a wash solution, and a container 28 containing a mounting fluid, preferably mixed with an anti-bleaching agent.
  • the kit may contain instructions for conducting an immunoassay of the invention.
  • the assays and kits may be employed by hospitals or clinical laboratories to determine the presence or absence of mycobacteria in biological fluids and to determine whether a particular patient is infected with members of the MAI complex.
  • the assay may also be used to monitor the ability of a patient to respond to treatment primarily for mycobacterial diseases or during the course of other infectious
  • the assays can be of predictive value in managing the course of treatment in a variety of mycobacterial disease states.
  • a sample of a biological fluid is obtained.
  • sputum was collected and processed as follows:
  • the supernatant fluid is carefully decanted and 100-200 ⁇ l of O ⁇ ⁇ buffer (pH 6.8) is added to the pellet.
  • the processed sputum is neutralized, if necessary, with neutralizing reagent (1 N HCl).
  • the pH of the sputum to be used in the assay must be approximately
  • the solid phase was nitrocellulose, 0.4 micron pore size, millipore, Bedford, MA.
  • the nitrocellulose was prepared by cutting an approximately 2 x 0.5 inch piece and adhering that piece along an edge to one side of a microscope slide.
  • Various commercial adhesives may be employed, including the polyadhesive Stix-All., from Bordon Co. , of Columbus, OH. Using a pen, four points (30, Fig. 1) were marked on the nitrocellulose and 2-4 microliters of test sample was delivered to two points. (Alternatively, 4 discrete solid phases can be adhered to the microscope slide. Since all samples receive the same monoclonal antibody, the necessity to use physically separated solid phases is not as important as it is in embodiments using a plurality of different monoclonal antibodies.)
  • TMC 107 [Saranac Lake, NY] was used. This strain also is available from ATCC, No. 35801, Rockville, Md., USA. For the negative control, 2-4 microliters of 100 microgram E. coli/ml was delivered to a point.
  • the fluids were allowed to dry and then the slide was placed overnight on a slide warmer at a temperature in order to kill any mycobacteria present on the solid phase. Temperatures between about 65°C and about 70°C are preferred.
  • blocking reagent is to render inactive all binding sites on the nitrocellulose solid phase not previously bound by test or control materials. This is important to prevent non-specific binding of reagents added subsequently, which could obscure or effectively eliminate meaningful results.
  • the blocking reagent used was 50 microliters of a mixture of 10% non-immune serum of the animal species in which the immunological reagent (conjugated secondary antibody) (described below) had been raised (anti-mouse IgG raised in goat), combined with 2% bovine serum albumin in buffer (20mM tris-HCl and 0.15 M NaCl, pH 7.5).
  • the volume of 50 microliters is more than sufficient to provide for a layer of free-standing liquid on the surface of the nitrocellulose solid phase.
  • the blocking reagent was incubated for about 20 minutes at about room temperature in a humidified chamber. After the requisite incubation time, blocking reagent was removed by merely tilting the slide and blotting the rim of the slide with blotting paper as the free-standing liquid drained.
  • monoclonal antibody diluted murine ascites
  • the monoclonal antibody is selectively specific for an antigenic component of mycobacteria, preferably a cell wall component of mycobacteria, that is common to all mycobacteria.
  • the particular monoclonal antibody used was selectively specific for the LAM component of mycobacteria (ML9P3, as described above).
  • the concentration of anti-LAM monoclonal antibody employed can be determined routinely by those skilled in the art. The concentration employed was defined as the working dilution and was selected as the last dilution of the antibody which gave a strong positive reaction using two microliters of 100 icrogram/ml M. tuberculosis Erdman strain spotted on the solid phase, and the appropriate dilution of conjugate (described below).
  • the anti-LAM antibody was provided to the solid phase under conditions and for a time sufficient for the anti-LAM antibody to bind to the antigenic lipoarabinomannan mycobacterial component.
  • incubation was on the order of about one hour at room temperature in a humidified chamber.
  • the unreacted antibody then was removed from the surface of the solid phase, by blotting as described above.
  • Anti-LAM monoclonal antibody that is not bound to the LAM antigen was further removed from the solid phase by immersing the solid phase in saline and washing for about 20 minutes in a beaker under agitation. Excess wash solution (e.g., saline) was removed by blotting, as described above.
  • Evans Blue is preferred when FITC is the label because FITC fluoresces apple-green against a dark red background.
  • Other counterstains such as methylene blue can be used with labels such as Rhodamine B, which gives a red color under UV light.
  • the reagent and the solid phase were incubated under conditions and for a time sufficient for the anti-immunoglobulin of the reagent to bind to the anti-LAM monoclonal antibody affixed to the mycobacteria on the solid support. Incubation conditions will vary depending on the concentrations of reactions and amount of mycobacteria in the sample.
  • the reagent was incubated at room temperature for about 30 minutes in a humidified chamber. Unreacted reagent was then separated from the solid phase by blotting and the solid phase was then washed to remove all nonreacting materials and contaminants, as described previously.
  • the solid phase was then dried, mounting fluid applied, and the solid phase was covered with a cover-slip.
  • a clear solid phase is preferred for viewing under incident UV light, and is important for viewing the solid phase under transmitted light.
  • a preferred mounting fluid is 90% buffered glycerol pH 8.4. This fluid renders the nitrocelluose solid phase substantially transparent.
  • a substance is preferably added to the mounting fluid.
  • Preferred anti-photobleaching substances are l,4-diazobicyclo-[2,2,2]-octane or n-propyl gallate.
  • Preferred anti-photobleaching mounting fluids are 2.5% l,4-diazobicyclo-[2,2,2]-octane in 90% buffered glycerol, pH 8.4 or 5% n-propyl gallate in 90% buffered glycerol, pH 8.4.
  • a microscope or other imaging device equipped with incident UV light and the appropriate excitation and emission filters for FITC is used preferably wavelengths of 480 nm and 520 nm respectively.
  • the slide was then viewed at a total magnification of 400x or 600x using ultraviolet light.
  • the positive control was seen as whole and/or fragmented bacteria with bright, apple-green fluorescence on a dark red background. It is believed that since soluble LAM antigen tends to concentrate near the leading edge of the circular pattern, the fluorescence was enhanced in that region surrounding the marked point of the positive control. In the negative control, only a dark red background was seen.
  • the test sample looked
  • anti-LAM monoclonal antibody was delivered to one of the pieces containing the test samples and anti-GPL monoclonal antibodies were delivered to another.
  • the anti-GPL monoclonal antibodies used were selectively specific for MAI serovars 4 (M24B1) and 8 (M85F7).
  • MAI complex is commonly isolated from individuals with underlying acquired immune deficiency syndrome (AIPS) and MAI serotype 8 is the most common MAI organism isolated from patients with non-tuberculous mycobacteria, although it is also commonly found in patients with AIPS.
  • AIPS acquired immune deficiency syndrome
  • a mixture of monoclonal anti-GPL 4 and anti-GPL 8 was added to the other of the test samples on the solid phase.
  • the amount of monoclonal antibody added was determined as described above. Like amounts of antibody were added to the positive and negative controls. After incubation, free-standing liquid containing antibody was removed from the surface of the solid phase, most preferably by tilting the slide and blotting, as above. Care should be taken in this procedure to avoid accidentally contaminating subsamples to be tested for GPL with anti-LAM antibody. Since LAM is also found in MAI-complex mycobacteria, such contamination will yield a false positive result if mycobacteria containing the GPL specific (i.e. MAI-complex serovars 4 and 8) antigens are not. actually- present. Physically separating the solid phases, as described above, eliminated this problem. The remaining steps were carried out as described above.
  • the immunoassays described above provide rapid, highly sensitive, inexpensive and reproducible methods for detection of mycobacteria in biological fluids, as well as for differentiating between particular MAI-complex mycobacteria and other non
  • MAI-complex mycobacteria Except for the overnight heating at 65-70°C (which is optional), the entire assay can be performed in under three hours and can detect mycobacteria at concentrations of less than

Abstract

On décrit des procédés et un appareil servant à détecter rapidement des mycobactéries dans des fluides biologiques. Les procédés consistent à appliquer un fluide biologique, un ou plusieurs anticorps monoclonaux agissant de manière sélective et spécifique contre un constituant antigénique des mycobactéries, et un réactif immunologique marqué sur une phase solide. Les mycobactéries contenues dans le fluide biologique sont liées à la phase solide sans qu'il soit nécessaire de recourir à des techniques de fixation chimique. La phase solide se compose de préférence de nitrocellulose présentant une grosseur de pores supérieure à 0,22 microns et inférieure à 1,0 microns environ. Des kits servant à effectuer ce test sont aussi décrits.
PCT/US1992/001165 1991-02-12 1992-02-12 Test d'immunofluorescence servant a detecter des antigenes mycobacteriens dans des fluides biologiques WO1992014156A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028420A1 (fr) * 1993-05-22 1994-12-08 Rhone Poulenc Diagnostics Ltd Procede de detection de bacteries
WO1997034149A1 (fr) * 1996-03-12 1997-09-18 Stefan Svenson Methode diagnostique de mycobacteriose et trousse d'essai immunologique
EP1329718A2 (fr) * 2002-01-10 2003-07-23 Becton, Dickinson and Company Methodes et appareils pour rassembler et preparer des echantillons afin de detecter des mycobacteries et leurs antigenes

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US4774177A (en) * 1985-07-15 1988-09-27 Vet-Test Partners Immunoassay method for detection of antibodies and antigens

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US4774177A (en) * 1985-07-15 1988-09-27 Vet-Test Partners Immunoassay method for detection of antibodies and antigens

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AYOUB et al., Microbial Determinants of Virulence and Host Responce, published 1990 by AMERICAN SOCIETY FOR MICROBIOLOGY (WASHINGTON, D.C.), see pages 55-75. *
CLINICAL AND EXPERIMENTAL IMMUNOLOGY, Volume 58, issued 1984, KOLK et al., "Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae", see pages 511-521. *
INFECTION AND IMMUNITY, Volume 55, No. 11, issued November 1987, GAYLORD et al., "Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan", see pages 2860-2863. *
INFECTION AND IMMUNITY, Volume 57, No. 10, issued October 1989, RIVOIRE et al., "Generation of monoclonal antibodies to the specific sugar epitopes of Mycobacterium avium complex serovars", see pages 3147-3158. *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028420A1 (fr) * 1993-05-22 1994-12-08 Rhone Poulenc Diagnostics Ltd Procede de detection de bacteries
WO1997034149A1 (fr) * 1996-03-12 1997-09-18 Stefan Svenson Methode diagnostique de mycobacteriose et trousse d'essai immunologique
EP1329718A2 (fr) * 2002-01-10 2003-07-23 Becton, Dickinson and Company Methodes et appareils pour rassembler et preparer des echantillons afin de detecter des mycobacteries et leurs antigenes
EP1329718A3 (fr) * 2002-01-10 2004-06-02 Becton, Dickinson and Company Methodes et appareils pour rassembler et preparer des echantillons afin de detecter des mycobacteries et leurs antigenes
EP1780547A1 (fr) * 2002-01-10 2007-05-02 Becton, Dickinson and Company Methodes et appareils pour rassembler et preparer des echantillons afin de detecter des mycobacteries et leurs antigenes

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