Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi

Definitions

• the present invention relates to a diagnostic method for detecting the presence of Pneumocystis carinii antigens in the blood.
• Pneumocystis carinii commonly subclinically infects healthy individuals, but also causes severe infections in malnourished infants and other im unocompromised patients, such as those receiving transplants or therapy for various malignancies, and persons with acquired immunodeficiency syndrome or certain congenital abnormalities.
• carinii is not a reliable indication of clinical disease given that a majority of normal individuals have antibodies to P. carinii antigens and immunocompromised patients may not have sufficient immune function to produce detectable amounts of antibodies to P. carinii antigens. See L. Pifer et al.. Pediatrics, 61:35-41, 1978.
• P. carinii The antigenic characteristics of P. carinii have been explored using both polyclonal and monoclonal antibodies.
• a direct comparison of the major rat and human P. carinii antigens detectable by Western blotting with polyclonal antisera has been reported by P. Walzer et al., J. Immunology. 138:2257-2267, 1987.
• P. carinii The disease caused by P. carinii is almost entirely limited to the lungs of the infected animal. Definitive diagnosis of infection has been dependent upon detection of P. carinii organisms in lung tissue, in sputum, in a lung aspirate, or respiratory tract aspirate. P. carinii organisms have been detected in pharyngeal smears, tracheal aspirates, sputum, gastric aspirates, bronchopulmonary lavages and in rare cases, bone marrow. However, aside from the unreliable detection of P. carinii antigens using polyclonal sera or anti-P. carinii antibodies described above, there have been no reports of
• the present invention therefore provides an assay method and diagnostic system in kit form useful for detecting the presence of a clinical P. carinii infection in a mammal.
• the present invention contemplates a method of assaying for the presence of a clinical P. carinii infection in a mammal comprising forming an immunoreaction admixture by admixing a vascular fluid sample obtained from the mammal with an antibody immunospecific for a 116 kD P. carinii antigen or an or antigenically cross- active fragments thereof.
• the admixture is maintained for a time period sufficient for any of the 116 kP P. carinii antigen present in the blood sample to immunoreact with the anti-116 kD antibodies and form a 116 kP P. carinii antigen- containing immunoreaction product.
• Detecting the presence of any 116 kP P. carinii antigen- containing immunoreaction product formed thereby provides a measure of the presence of a P. carinii infection in said mammal.
• a diagnostic system in kit form useful for performing the contemplated assay methods.
• the system comprises a package that includes the monoclonal antibody, Ca-3, which is immunospecific for a P. carinii antigen having a reduced apparent molecular weight of about 116 kP and antigenically crossreactive fragments thereof having apparent molecular weights 23 kP and 56 kD.
• the diagnostic method and systems of this invention provides several benefits and advantages.
• One benefit provided by the diagnostic method and systems of this invention is the ability to serologically screen an individual for the presence of a clinical P. carinii infection thus eliminating the need to perform invasive procedures such as a lung biopsy.
• Figure 1 illustrates the detection of a P. carinii antigen in the serum according to Example 3. A distinct band is observed in the serum of a patient with P. carinii pneumonia. Lane 1. P. carinii antigen is not present in the serum of a patient without P. carinii pneumonia. Lane 2.
• Figure 2 illustrates the direct detection of the P. carinii 116 kD antigen in induced sputum from a patient with P. carinii pneumonia.
• the arrows indicate the P. carinii 116 kP antigen and the two 23 kP antigenically crossreactive fragments that are detected, Lane 1.
• lymphosenchymal infection is used herein to refer to a subclinical P. carinii infection, i.e., the presence of a low number of organisms and no significant clinical or histological evidence of disease.
• the methods and systems of the present invention detect the presence of a P. carinii antigen in a vascular fluid sample, such as blood, serum, plasma and the like, and thereby indicate the presence of a P. carinii clinical infection.
• a vascular fluid sample such as blood, serum, plasma and the like.
• a preferred P. carinii antigen that acts as a blood-born marker for clinical infection is the 116 kP protein expressed by P. carinii trophozoites, and the antigenically crossreactive fragments thereof.
• 116 kD P. carinii antigen and “P. carinii 116 kP antigen” are used herein to refer to the proteinaceous antigen present on the surface of P. carinii trophozoites and having an apparent molecular weight of about 116 kD as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SPS-PAGE) under mild reducing conditions, e.g. less than about 0.2 M 2- mercaptoethanol.
• SPS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
• the phrase "antigenically crossreactive" refers to two polypeptides of differing overall a ino acid residue sequence that share a substantial portion of one antigenic determinant (epitope) and therefore are capable of immunoreacting with a given antibody as evidenced by competitive inhibition.
• Methods for making antigenically crossreactive fragments of a protein are well known in the art and include enzymatic cleavage, chemical cleavage, chemical synthesis and the like.
• Useful proteolytic enzymes include trypsin, chymotrypsin and the like.
• Agents useful for chemically cleaving proteins include cyanogen bromide and the like. Where the amino acid residue sequence of a portion of the protein is known, that portion can be synthesized by methods such as the solid phase technique of Merrifield. See J.M. Steward and J.D. Young, “Solid Phase Peptide Synthesis” (W.H. Freeman Co., San Francisco, 1969).
• the present invention therefore contemplates a method of immunoassaying for the P. carinii 116 kP antigen using an antibody, either polyclonal or monoclonal antibody to form an immunoreaction product whose presence relates, either directly or indirectly, to the presence of the 116 kP antigen in the sample.
• an antibody either polyclonal or monoclonal antibody to form an immunoreaction product whose presence relates, either directly or indirectly, to the presence of the 116 kP antigen in the sample.
• an antibody can be used to form an immunoreaction product whose presence relates to the presence of P. carinii 116 kP antigen present in a vascular fluid sample.
• exemplary assay methods are described herein, the invention is not so limited.
• Various heterogeneous and homogeneous protocols either competitive or noncompetitive, can be employed in performing an assay method of this invention. Particularly preferred protocols are described in U.S. Patents No. 4,233,401,
• the present invention contemplates an immunoassay method comprising the steps of: (a) Forming an immunoreaction admixture by admixing a vascular fluid sample with an antibody, perferably a monoclonal antibody, immunospecific for an epitope expressed by the P. carinii 116 kP antigen, or an antigenically crossreactive fragment thereof. Methods for making such an antibody are well known in the art.
• the vascular fluid sample is provided as a known amount of blood derived product such as serum or plasma.
• the immunoreaction admixture is maintained under biological assay conditions for a time period sufficient for any of the P.
• Bio assay conditions are those that maintain the biological activity of the immunochemical reagents of this invention and the 116 kP P. carinii antigen sought to be assayed. Those conditions include a temperature range of about 4 degrees C to about 45 degrees C, a pH value range of about 5 to about 9 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride. Methods for optimizing such conditions are well known in the art. Under biological assay conditions, typical maintenance time periods range from about 10 minutes to about 16-20 hours at a temperature of about 4 degrees C to about 45 degrees C.
• step (c) Petermining the presence of any P. carinii 116 kP antigen-containing immunoreaction product, and thereby the presence of a P. carinii clinical infection.
• Methods for determining the presence of an immunoreaction product are well known in the art. Those methods typically include attaching a labeled product and detecting the presence of labeled product. Exempary labels are discussed in the diagnostic systems section herein, as are "detection means" that do not require a label.
• the first immunoreaction product is further prepared for, i.e., labeled prior to, determining to step (c) above by:
• the present invention contemplates a double antibody or "sandwich" immunoassay comprising the steps of:
• a first immunoreaction admixture by admixing a vascular fluid sample with a first antibody, preferably a monoclonal antibody, wherein the antibody and any P. carinii 116 kD antigen present in the sample are capable of forming a first immunoreaction product that can immunoreact with a second antibody, preferably a monoclonal antibody, and more preferably MAB Ca-3.
• a second antibody preferably a monoclonal antibody, and more preferably MAB Ca-3.
• the first antibody is operatively linked to a solid matrix.
• the immunoreaction admixture is maintained under biological assay conditions for a time period sufficient for any P. carinii 116 kD antigen present in the sample to immunoreact with (immunologically bind) a portion of the anti-116 kD antibody combining sites present to form a P. carinii 116 kD antigen-containing immunoreaction product.
• the first immunoreaction product is then separated from the sample.
• a second immunoreaction admixture by admixing the first immunoreaction product with a second antibody, preferably a labeled second antibody, which is capable of binding the P. carinii 116 kP antigen when it has been bound by the first antibody.
• the second antibody is a monoclonal antibody, and more preferably it is MAB Ca-3 when the first antibody is not MAB Ca-3..
• step (d) Maintaining the second immunoreaction admixture formed in step (C) under biological assay conditions for a time period sufficient to form the second or "sandwich" immunoreaction product.
• the second antibody of step (c) is labeled, preferably with an enzyme, and the second immunoreaction product formed is a labeled product.
• the amount of immunoreaction product determined in step (e) is related to the amount of immunoreaction product similarly formed and determined using a control sample in place of the vascular fluid sample, wherein the control sample contains a known amount of P. carinii 116 kD antigen.
• the present invention contemplates an immunoassay method comprising the steps of:
• step (d) Maintaining the immunoreaction admixture of step (c) under biological assay conditions for a time period sufficient for any solid matrix affixed P. carinii 116 kD antigen or an antigenically crossreactive fragment thereof to immunoreact with the antibody.
• step (c) Maintaining the immunoreaction admixture of step (c) under biological assay conditions for a time period sufficient for any solid matrix affixed P. carinii 116 kD antigen or an antigenically crossreactive fragment thereof to immunoreact with the antibody.
• P. carinii 116 kP antigen-containing immunoreaction product formed, and thereby the presence of any P. carinii 116 kP antigen or an antigenically crossreactive fragment thereof in the sample. Petecting the presence of the P. carinii
• 116 kP antigen-containing immunoreaction product can be accomplished by assay techniques well known in the art, and typically depend on the type of indicating means used.
• antibody in its various grammatical forms refers to a composition containing immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
• an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
• antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
• Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v) .
• Fab and F(ab') 2 portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous and Oixon.
• Fab 1 antibody portions are also well known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
• An antibody containing intact antibody molecules are preferred, and are utilized as illustrative herein.
• a polyclonal antibody of the present invention is immunospecific for a P. carinii antigen having an apparent molecular weight of about 116 kD or an antigenically crossreactive fragment thereof.
• the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody molecule containing composition having only one species of antibody combining site capable of immunoreacting with a particular antigen.
• a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
• a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody.
• a monoclonal antibody (MAB) of the present invention (subject MAB) is characterized by its idiotype and as being immunospecific for an epitope expressed by a P. carinii 116 kD antigen or an antigenically cross reactive fragment thereof.
• immunoreactivity in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of 116 kD P. carinii antigen. That is, immunoreactivity is the concentration of antigen required to achieve a B/B 0 value of 0.5, where B 0 is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both B 0 and B have been adjusted for background. See, Rodbard, Clin. Che .. 20:1255- 1270 (1974).
• a subject monoclonal antibody typically containing whole antibody molecules can be prepared using the hybridoma technology described in Antibodies A Laboratory Manual, Harlow and Lane, eds.. Cold Spring Harbor Laboratory, New York, (1988) , which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with P. carinii organisma partially purified from P. carinii infected lungs. It is preferred that the myeloma cell line be from the same species as the lymphocytes.
• a mouse of the strain 129 G1X + is the preferred mammal.
• Suitable mouse myelomas for use in the present invention include the hypoxanthine- aminopterin-thy idine-sensitive (HAT) cell lines P3X63-Ag8.653, and Sp2/0-Agl4 that are available from the American Type Culture Collection, Rockville, MD, under the designations CRL 1580 and CRL 1581, respectively.
• HAT hypoxanthine- aminopterin-thy idine-sensitive
• Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody of this invention are identified using the radioimmunoassay (RIA) described in Example 2.
• a monoclonal antibody of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well known techniques.
• DMEM Dulbecco's minimal essential medium
• the monoclonal antibody produced by the above method can be used, for example, in diagnostic modalities wherein formation of a P. carinii 116 kD antigen-containing immunoreaction product is desired.
• hybridoma useful in producing a subject monoclonal antibody i.e., MAB Ca-3
• hybridoma Ca-3 is hybridoma Ca-3, said hybridoma being deposited pursuant to Budapest Treaty Requirements with the American Type Culture Collection (ATCC) , Rockville, MD 20852 U.S.A. on May 10, 1989 and given the ATCC designation HB 10139.
• ATCC American Type Culture Collection
• hybridoma Ca-3 can be used, as is well known in the art, to produce other stable cell lines that produce a subject monoclonal antibody, and thus production of a subject monoclonal antibody is not dependent on the culturing of hybridoma Ca-3 per se. See Eurporean Patient Office Publication WO 89/00999.
• anti-116 kD antibody in its various grammatical forms refers to a composition containing immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules that are immunospecific for an epitope expressed on a 116 kD P. carinii antigen reduced or antigenically crossreactive fragments of this antigen.
• the term "immunospecific” in its various grammatical forms refers to an antibody molecule that immunologically binds a particular epitope or a substantial portion of a particular epitope expressed by a P. carinii 116 D antigen and does not immunologically bind other unrelated epitopes.
• epitope in its various grammatical forms refers to that portion of an antigen that is specifically recognized by an antibody combining site. It is also referred to as the determinant or antigenic determinant.
• reduced refers to a protein that has had any intra-peptide and inter-peptide disulfide bonds broken or reduced by the addition of a reducing agent such as 2- mercaptoethanol to the protein and boiling in the presence of sodium dedecylsulfate (SDS) .
• a reducing agent such as 2- mercaptoethanol
• apparent molecular weight refers to the size of a protein in kilodaltons (kD) determined by comparing the protein's mobility in a sizing system, e.g. SDS- PAGE as described in Gel Electrophoresis of Proteins. Hanes and Rickwood, eds. IRL Press, Washington, D.C., 1981, to the mobility of other proteins of known molecular size in kilodaltons.
• a sizing system e.g. SDS- PAGE as described in Gel Electrophoresis of Proteins. Hanes and Rickwood, eds. IRL Press, Washington, D.C., 1981, to the mobility of other proteins of known molecular size in kilodaltons.
• a diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, an anti-116 kD antibody, as a separately packaged immunochemical reagent. Instructions for use of a packaged immunochemical reagent are also typically included.
• a package refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits a polyclonal antibody or monoclonal antibody of the present invention.
• a package can be a glass vial used to contain milligram quantities of a contemplated antibody or it can be a microtiter plate well to which microgram quantities of a contemplated antibody have been operatively affixed, i.e., linked so as to be capable of immunologically binding an antigen.
• Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
• a diagnostic system of the present invention further includes a detection means capable of signaling the formation of a complex containing the 116 kD P. carinii antigen and/or a anti-116 kD antibody molecule.
• detection means refers to any method of detecting the presence of a complex containing the 116 kD P. carinii antigen without the need for any label. Such detection means are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins, methods and systems. Exemplary detection means include methods known as biosensors and include biosensing methods based on detecting changes in the reflectivity of a surface, changes in the absorption of an evanescent wave by optical fibers or changes in the propagation of surface acoustical waves. In preferred embodiments, a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of a complex containing the P.
• complex refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction.
• exemplary complexes are immunoreaction products.
• label and
• indicating means in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
• the label can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochro e (dye) that is a useful immunofluorescent tracer.
• Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC) , tetramethylrhodamine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
• fluorescein isocyanate FAC
• FITC fluorescein isothiocyante
• DANSC 5-dimethylamine-l- naphthalenesulfonyl chloride
• TRITC tetramethylrhodamine is
• the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, alkaline phosphatase or the like.
• HRP horseradish peroxidase
• additional reagents are required to visualize the fact that a antibody-antigen complex (immunoreactant) has formed.
• additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
• An additional reagent useful with HRP is 2,2'- azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) .
• Radioactive elements are also useful labeling agents and are used illustratively herein.
• An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124 I, 125 I, 128 I, 132 I and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 25 I.
• Another group of useful labeling means are those elements such as 11 C, 18 F, 15 0 and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such 111 indium of 3 H.
• labeling of, polypeptides and proteins is well known in the art.
• antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as -a component in the culture medium.
• radioisotope-containing amino acids provided as -a component in the culture medium.
• the techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol.. Vol. 8 Suppl. 7:7-23 (1978),
• the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
• a "specific binding agent” is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself an antibody molecule composition of the present invention.
• Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like.
• the specific binding agent binds the reagent species when that species is present as part of a complex.
• the specific binding agent is labeled.
• the agent is typically used as an amplifying means or reagent.
• the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
• the diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of 116 kD P. carinii antigen in blood, serum, or plasma.
• ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample.
• a description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al., published by Lange Medical Publications of Los Altos, CA in 1982 and in U.S. Patents No. 3,654,090; No. 3,850,752; and No. 4,016,043, Which are all incorporated herein by reference.
• an anti- 116 kD antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
• a reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and antigen are well known to those skilled in the art can be used.
• Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine
• reagent species labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
• the enzyme's substrate can also be provided in a separate package of a system.
• a solid support such as the before-described microliter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
• the packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such materials include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like.
• P. carinii cyst antigen P. carinii cyst antigen.
• P. carinii organisms were purified from infected human lung tissue using the method of Gigliotti et al., Journ. Infect. Pis.. 154:315-322, 1986, with the only modification being the use of a Stomacher (Tech Marr Products, Cinn. , Ohio) to homogenize the tissue. Briefly, the infected lungs were homogenized in a Stomacher. The homogenate was passed through cotton gauze to remove any large clumps of tissue, and then passed through a 12 micron filter 2(Nuclepore, Pleasanton, CA) . The filtered homogenate was centrifuged at 400 g for 30 minutes at room temperature.
• PBS phosphate buffered saline
• penicillin/mL 200 Mg of streptomycin/mL
• amphotericin B/mL 4 mg of amphotericin B/mL to inhibit any bacterial and fungal growth.
• the number of P. carinii organisms present was determined according to Pifer et al., Pediatr. Res. 11:305- 316, 1977. The isolated P. carinii organisms were stored in small aliquotes at -70'C.
• mice were mixed with 2 volumes of complete Freund's adjuvant and injected into female Balb/c mice according to Gigliotti, et al., Journ. Infect. Pis.. 154:315-322, 1986. After the initial challenge the mice were further challenged with P. carinii infected human lung homogenate mixed with 2 volumes of incomplete Freund's adjuvant. The immunized spleenocytes were fused with murine myeloma P3-X63A68, ATCC No. TIB-9 according to the methods of Pe St. Groth et al., Immun. Methods 1-2, 1980 and Hoffman et al., IX International Congress for Human Mycology, P3-1, 1985.
• the resulting hybrids were screened for antibody production using an ELISA assay.
• the antibody producing hybrids were further screened for production of P carinii specific monoclonal antibodies using the Western blotting technique.
• the hybrids producing P. carinii specific monoclonal antibodies were subcloned twice by the limiting dilution method to produce clonal cell lines.
• the isotype of the antibody produced by the cell lines was determined using standard methodologies.
• the cell lines were injected in Balb/c mice to produce an antibody rich ascities fluid.
• the antibody was purified from the ascities fluid using ammonium sulphate fractionation and then conjugated to alkaline phosphatase according to Immunochemical Techniques. Van Vunakis and Langone, eds., in Meth.
• P. carinii organisms prepared according to Example 1 were electrophoresed through a 10% polyacrylamide gel containing sodium dodecylsulfate (SDS-PAGE) described in Gel Electrophoresis of Proteins . Hanes and Rickwood, eds., IRL Press, Washington, D.C., 1981.
• the electrophoresed proteins were then transferred (affixed) to a sheet of modified nitrocellulose, (Nitro Plus 2000, Micro Separations Inc.) using the standard Western blotting techniques described in Salinovich et al.. Anal. Bioche .. 156:341-347, 1986.
• the modified nitrocellulose sheet containing the transferred proteins was processed as described in Example 2 to form and detect an immunoreaction product.
• the monoclonal antibody Ca-3 immunoreacted with a single protein species having an apparent molecular weight of about 116 kD.
• a blood sample was collected from a patient having P. carinii pneumonitis.
• the serum fraction of the blood sample was separated by means well known in the art.
• the serum sample 4 microliters, was applied to a 0.5% agarose gel (Beckman, Brea, CA) .
• the gel containing the serum sample was maintained at room temperature for five minutes and then placed in an electrophoresis system (Beckman, Brea, CA) containing barbital buffer consisting of 10 mM 5,5-diethylbarbituric acid and 50 mM 5,5-diethylbarbituric acid, sodium salt at pH. 8.6.
• the gel containing the serum sample was electrophoresed at 100 volts for 25 minutes.
• the gel was then removed from the electrophoresis system and placed on a sheet of 0.45 mM pore size modified nitrocellulose, Nitro Plus 2000 (Micro Separations Inc. , Westboro, MA) , that was presoaked in transfer buffer (0.4 M Tris, 0.35 M magnesium chloride, and 0.052 M Glycine in a 27% solution of'methanol at a pH of 8.0).
• transfer buffer 0.4 M Tris, 0.35 M magnesium chloride, and 0.052 M Glycine in a 27% solution of'methanol at a pH of 8.0.
• the gel and modified nitrocellulose sheet were then sandwiched between two sheets of Whatman 3MM filter paper (Bio Rad Laboratories, Richmond, CA) that had been presoaked in transfer buffer, thus forming a transfer pack.
• the transfer pack was maintained at room temperature about 0.5 hours to about 18 hours or until all of the proteins are transferred out of the gel onto the modified nitrocellulose sheet, i.e., were affixed to, the nitrocellulose.
• the transfer pack was then disassembled and the modified nitrocellulose sheet placed in a solution of containing 10 mM sodium phosphate, 0.35 M sodium chloride, 0.05% Tween-20 and 0.025% casein according to the method described by Berry et al. , Journ. Imm. Methods . 16 : 293 , 1985.
• the modified nitrocellulose sheet was maintained in this solution for 15 minutes at room temperature.
• the modified nitrocellulose sheet was rinsed twice with a solution containing 10 mm sodium phosphate and 0.35 M sodium chloride at a pH of 7.4. The modified nitrocellulose sheet was then placed in a solution containing 10 mm sodium phosphate, at a pH of 7.4, 0.35 M sodium chloride and a 1 to 1000 dilution of anti-116 kD monoclonal antibody, MAB Ca-3, conjugated to alkaline phosphatase (AP) , (Bio-Rad Laboratories, Richmond, CA) according to the methods described in Immunochemical Techniques. Van Vunakis and Langone, eds. , in Meth. Enzymol.. 70:1-525, 1980.
• AP alkaline phosphatase
• the modified nitrocellulose sheet was maintained in the solution containing the AP- conjugated monoclonal antibody for 2 hours at room temperature. During this time period, the P. carinii 116 kD antigen immobilized on the modified nitrocellulose sheet immunoreacted with the-AP-conjugated monoclonal antibody to form a labeled immunoreaction product on the modified nitrocellulose sheet.
• the immunoreaction product was visualized by maintaining the modified nitrocellulose sheet in a solution containing 0.05 M Tris at pH 9.2, 1.02% 2-amino-2-methyl 1, 3-propanediol, 0.1% magnesium chloride and 161 mg/ml 5-bromo-4-chloro-3-indolyl phosphate for about 30 minutes to greater than 18 hours. During this time an insoluble colored reaction product formed on the modified nitrocellulose sheet at the location of the AP- conjugated monoclonal antibody. The modified nitrocellulose sheet was rinsed twice in tap water to remove any unreacted substrate and allowed to dry.
• the pelleted material was resuspended in SDS loading buffer containing the standard amount of 2ME (about 0.14 M)according to the procedures described in Gel Electrophoresis of Proteins. Hanes and Rickwood, eds., IRL Press, Washington, D.C., 1981.
• the sample was electrophoresed in a standard sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) containing 10% polyacrylamide.
• SDS-PAGE sodium dodecylsulfate polyacrylamide gel
• the electrophoresed proteins were then transferred to a sheet of modified nitrocellulose, Nitro Plus 2000, (Micro Separations Inc.) using the standard Western blotting techniques described in Salinovich et al., Anal. Bioche .. 156:341-347, 1986.
• the modified nitrocellulose sheet containing the transferred proteins was processed as described in Example 2 to form and detect an immunoreaction product.
• the MAB Ca-3 immunoreacted with two reduction products having an apparent molecular weight of 23 kD, and also immunoreacted with a reduction product having an apparent molecular weight of about 56 kD.

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• 1990
  • 1990-05-10 PT PT9400290A patent/PT94002A/pt not_active Application Discontinuation
  • 1990-05-10 CA CA 2016524 patent/CA2016524A1/en not_active Abandoned
  • 1990-05-11 AU AU56443/90A patent/AU5644390A/en not_active Abandoned
  • 1990-05-11 JP JP50760990A patent/JPH04505253A/ja active Pending
  • 1990-05-11 EP EP19900907860 patent/EP0472599A4/en not_active Withdrawn
  • 1990-05-11 GR GR900100355A patent/GR1001087B/el unknown
  • 1990-05-11 WO PCT/US1990/002632 patent/WO1990013670A1/en not_active Application Discontinuation
  • 1990-05-11 IE IE171990A patent/IE901719L/xx unknown

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