WO1983001739A1

WO1983001739A1 - Monoclonal antibodies against brugia malayi

Info

Publication number: WO1983001739A1
Application number: PCT/US1982/001619
Authority: WO
Inventors: And Women's Hospital Brigham; John R. David; Willy F. Piessens
Original assignee: Brigham & Womens Hospital
Priority date: 1981-11-17
Filing date: 1982-11-17
Publication date: 1983-05-26
Also published as: EP0093775A1; IL67294A0

Abstract

Immortal, antibody-producing, hybridomally-produced clones which produce antibodies that react specifically with antigens produced by the nematode parasite Brugia malayi without cross-reacting with other closely related parasites. Also disclosed are methods of producing the clones and antibodies and methods of using the antibodies in diagnostic tests and pharmaceutical preparations.

Description

Monoclonal Antibodies Against Brugua Malayi

Technical Field

The present invention relates to hybridoma cell lines and to monoclonal antibodies produced thereby that are species and stage specific for members of the nematode parasite Brugia malayi.

Background Art

Helminth (worm) infections are the most prevalent diseases of humans and animals When considered on a world-wide scale. Trematodes (flukes), cestodes (tapeworms), and nematodes (roundworms) are the three major parasitic classes of helminths. Of these the nematodes generally have a higher zoological organization, possessing a tough impermeable cuticle, muscle, nerve fibers, and a complete digestive system. Because of their closer zoological relationship to man and animals than bacteria and other parasites, they are difficult to treat. Of the nematodes, filarial forms, which live in tissue as opposed to the intestine or lungs, are particularly inaccessible and difficult to treat. Malayan filoriasis, caused by the parasitic nematode Brugia malayi, is a disease of run-down urban areas in the tropics where it is carried by culicine mosquitoes. The disease pathology is marked by blockage of the lymphatic vessels by adult worms. Adult worms take some 3-15 months to mature and then live or up to 15 years. Filarial worms are viviparous and fertile females produce large numbers of larvae,

Oλ-PI known as icrofilariae. Microfilariae can develop only if ingested by a culicine mosquito. There they develop and reach the infective stage (third stage larva) within 1 or 2 weeks. At this point the bite of a mosquito injects larvae into a human host where the life cycle is completed when the larvae migrate to the lymphatic system and become sexually mature.

The initial symptoms of different types of filarial diseases are easily confused with each other and with the initial symptoms of other nematode-caused diseases. However, different nematode diseases respond to different chemotheraputic agents and cannot all be treated by the same drugs. Accordingly, rational treatment requires initial diagnosis of the genus and species of the infecting nematode.

Disclosure of the Invention

Accordingly, it is an object of this invention to provide a rapid, im unological method of assaying for the presence of Brugia malayi in biological fluids. It is a further object to provide a method of assaying for the presence of Brugia malayi that does not cross-react to a significant extent with other genera of nematodes.

It is a still further object of this invention to provide a method of assaying for specific stages of the life of Brugia malayi.

It is a yet further object of this invention to provide a method of assaying for any of the stages of the life cycle of Brugia malayi using one monoclonal antibody.

These and other objects of the invention as will hereinafter become more readily apparent have been

PI accomplished by providing an immortal antibody- producing, hybrido ally-produced clone and an antibody produced thereby, wherein said antibody is an immunoglobulin specific for Brugia malayi.

Best Mode for Carrying Out the Invention

Cell lines prepared by the procedures described herein are exemplified by a culture now on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. This culutre was deposited on November 13, 1981, and is identified by ATCC # HB8100, ATCC # HB8101, and ATCC # HB8102.

Although it was known prior to the present invention that monoclonal antibodies can be produced against various antigens, it was not known whether monoclonal antibodies produced against an antigen derived from Brugia malayi would be specific for particular stages of the nomatode's life cycle, for all stage of the life cycle, or whether cross-reactions with other closely related nematodes would occur. Applicants have discovered that monoclonal antibodies produced using antigens from the cell membrane of Brugia malayi will react selectively with the same species and even allow identification of specific stages of the life cycle. Such antibodies will prove useful in the area of diagnostic medicine, as they will allow rapid immunological testing of biological fluids for the presence of Brugia malayi. Since specific species and stages can be identified without danger of misidentification, rational chemotherapy can be more rapidly and cheaply initiated than was previously possible using earlier methods of identification.

Monoclonal antibodies of the invention may be produced that are specific for any or all of the stages of the life cycle of Brugia malayi. By specific is meant that antibodies bind to antigens derived from an organism to which the antibody is said to be specific so that at least three times as many antibodies bind to the specific organism as to any other nematode (or stage of the nematodes life cycle) for any given concentrations of antibody and nematode antigen in which the concentration of antibody is the limiting factor. Particularly preferred are antibodies specific for the microfilarial, third stage larva, and adult worm stages of the parasite. Antibodies that are even more specific, for example, against specific stages of the life cycle, are also within the scope of the invention. Antibodies induced initially by antigens derived from a particular stage of Brugia malayi produce antibodies that react with that stage without reacting with other stages of the same species. Such antibodies are especially important for epidemiology studies . Many different techniques of hybridoma formation and monoclonal antibody production are known and may be applied in carrying out the objects of the invention. In general, the process comprises sensitizing an animal with an antigen to induce an immune response, obtaining immune cells from the animal, and fusing the immune cell with a malignant cell line using one of a variety of fusing agents. Resulting hybrids are then grown in a medium which precludes the expansion of the original malignant cell line. After an initial period during which non-hybrid cells die, growing hybrids may be observed microscopically. Each of these colonies is assayed for the immune function sought. Colonies which demonstrate antibody secretion against one or more stage of Brugia malayi are cloned. These clones are then grown in large quantity, for example by stepwise transfer to larger wells, flasks, and bottles. Each of these steps is discussed in somewhat greater detail in the following paragraphs. However, it will be recognized by a practitioner in the area of hybridoma technology that many modifications and additions may be made to the techniques included in this discussion, which is intended to be exemplary and not limiting, while remaining within the scope of the present invention.

The first step of raising monoclonal hybrids is generally immunization of an animal. Because fusion occurs preferentially with proliferating cells, it is preferred to schedule immunization to obtain as many immunoblasts as possible, with harvesting occurring about 3-4 days after the last inoculation being most preferred. In the present application suitable antigens for use in immunization include proteins , glycoproteins, lipoproteins, and other macromolecules present on the surface of or excreted by any stage of the life cycle of Brugia malayi. Antigenic macromolecules may be used in purified form, but suitable results are also obtained using either whole, killed nematodes or membrane prepartions derived from all or part of the nematode. Whole worms are preferrably rendered non-viable by chemical treatment, e.g. formaldehyde or glutaraldehyde fixation, heat or irradiation, in order minimize biohazard problems . Membrane preparations can be obtained by any methods that disrupt the nematode and allow purification of the membrane faction. Suitable methods include grinding or freeze-thawing followed by differential centrification or density gradient purification. For particulate antigens, such as membrane preparations, which are

O PI often strong immunogens, either intraperitoneal, intravenous, subcutaneous, or intr -foot pad injection can be used with success. The immunization schedule may entail two or more injections, at interavls of up to a few weeks, with the last injection being three to four days before the fusion. Adjuvants may be included if desired and are preferred when soluble antigens are used. It is also possible to employ immune cells which have been sensitized naturally, to use cells stimulated by polyclonal activators such as lipopolysaccharides, or to carry out in vitro sensitization of either B or T lymphocytes.

The cell line chosen for hybridization should be capable of rapid growth, be deficient in its metabolism for a component of the growth medium, and have potential for good fusion frequency. The species from which the immortalizing line is derived should be closely related to the species from which the antibody- producing cell is obtained. Intraspecies fusions, particularly between like strains, work better than interspecies fusions. Especially preferred are plasmocytoma-derived cell lines obtained from the same species and strain as the immune cells.

Readily available cell lines, many of which are mutants selected for their inability to secrete immunoglobulin, include the following:

1. P3-NSl-l-Ag4-l, or NSl, a variant of the P₃ (MOPC₂-_j_) mouse myeloma line.

2. MPC_ι;L-X45-6TG, or X45, a Balb/c mouse plasmacytoma. 3. P3-X63-Ag8, or X63, the mouse myeloma cell line originally used by Kohler and Milstein. Recent mutants have been developed (e.g. X63-Ag8.653) which no longer secrete immunoglobulins. 4. sp2/0-Agl4, another BALB/c mouse myeloma line. 5. GD-36-A.Agl lymphoblastoid cell line obtained by injection of SV40 virus into Syrian hamsters.

6. Subclone Y3 - Ag 1.2.3., or Y3, from the rat myeloma mutant 210.RCY3.Agl. With the growth of hybridoma technology, new immortal cell lines suitable for hybrid formation are continually being developed. Such cell lines are also suitable for carrying out the hybridization processes described in connection with the present invention. Once immune cells are obtained and a suitable immortal cell line chosen, the cells are fused to form the hybrid cell line. Various techniques are available for inducing fusion and include virus-induced fusion and polyethyleneglycol-induced fusion. Inactivated Sendai virus is preferred for virus-induced fusion.

Other viruses can induce fusion of somatic cells if the two parental cells are both susceptible to the virus. Among suitable viruses are HVJ and Epstein-Barr virus. Polyethyleneglycol (PEG) can also be used as a fusing agent. PEG itself is toxic for cells at high concentrations and various concentrations should be tested for effects on viability before attempting fusion. PEG having molecular weights varying from 1000 to 6000 may be used. PEG should be diluted with 30-50% saline or serum-free medium. Since PEG is toxic for the cells, the time exposure to PEG should be limited. Exposure to PEG for 1-2 minutes is best for many cell lines. Other conditions that should be controlled for increased fusion efficiency are temperature and cell ratios . Extremes of temperature should be avoided during fusion. Preincubation of each component of the fusion system at about 37° prior. o fusion is

O PI preferred. The ratio between spleen cells and malignant cells should be optimized to avoid self- fusion among spleen cells . Myeloma/spleen-cell ratios ranging from 2:1 to 1:20 are suitable, with 1:8 to 1:10 being preferred.

The mixture of cells obtained after fusion contains hybrids, fused and unfused parental spleen cells, and malignant cells. Spleen cells cannot maintain growth in routine culture medium and will eventually die out. Malignant cells would keep on dividing and soon overgrow the hybrids unless a selective medium is used that will allow only the growth of hybrids. The malignant cell lines must therefore be selected so that they are unable to grow on the chosen culture medium. ^" For example, several available cell lines are hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient and will not grow in aminopterine-containing medium because of their inability to synthesize purines from thymidine and hypoxanthine. Some HGPRT revertants may occur among the malignant cells and these should be periodically purged with 8-azaguanine. The selection medium used to allow only growth of hybrids is composed of hypoxanthine, 1 x 10 M; aminopterine, 4 x 10 M; and thymidine, 1.6 x 10~⁵M, (HAT medium). Other culture media and deficient cell lines may also be used in the practice of the invention.

The fusion mixture can be grown in HAT medium immeditely after fusion or at a later time. The feeding schedules for the fused cells may vary, but obligatory feeding of HAT medium (or another deficient medium) at intervals, for example on days 1, 6, and 11, is required, followed by growth in either regular culture medium or a medium containing hypoxathine and

OMPI -9-

thymidine .

Standard tissue culture medium may be used to support the growth of hybrids. Good results may be obtained with Iscove's medium, Dulbecσo's modified Eagle's medium (DMEM), or HY medium: DMEM enriched with 4.5g glucose/liter, 10% NCTC 109, 20% serum, and 0.15% glutamine. HY medium is preferred.

Serum used in media should be tested for its ability to support the growth of the malignant cell line prior to use. Hybrids may grow in, for example, horse or calf serum, but fetal bovine serum has no immunoglobulin, an important consideration that makes screening for antibody-producing cells much easier, and is therefore preferred. Hybrids may also be grown in serum-free media supplemented with 10-20% of a serum albumin, e.g., bovine serum albumin, and trace elements.

Feeder cells may be used in the initial stages of cell growth to enhance the survivability of the isolated cells. Irradiated thymocytes, spleen cells, myeloma cell lines, and mouse peritoneal macrophages may be used for feeder layers. Preferred feeder cells for use with mouse hybrids are mouse pertinoneal macrophages from the same species as the spleen donor, obtained by washing the peritoneal cavity of a mouse with aqueous sucrose. Macrophages harvested and plated

(about 5 x lθ⁴/ml x 10⁴/^{ml) the dav before} fusion are preferred.

Rapid identification of antibody-producing hybrids is important in order to avoid expenditure of time and resources on cultivation of extraneous cells. Early detection of hybridoma antibodies may be performed using standard im unological assays, for example, where

OMPI the antigen is bound to a solid support and allowed to react with hybridoma supernatants. The presence of antibodies may be detected by sandwich techniques using a variety of indicators, such as rabbit anti-mouse antibodies that are labelled with radioactive isotopes or enzymes. Most of the common methods for detecting immunological activity are sufficiently sensitive for use in detecting antibody-producing cells Several assay systems are discussed in more detail in a later section.

If a solid phase assay system is used, several methods are available to bind the immunogen to a solid phase. For example, many soluble antigens bind by adsorbtion to plastic surfaces. Such plates or wells may be rigorusly washed without affecting antigen binding. When whole cells or particulate fractions comprise the antigen, glutaraldehyde or an underlayer of antibody can be used to fix cells or membranes to a plastic surface. Dessication of cells under vacuum can also be used. This technique also perserves cells for prolonged periods of time if they are used as an antigen.

The reagents used to detect the presence of the antibody/immunogen complex is chosen according to the species involved in the fusion. For example, when mouse .cells are used, anti-mouse immunoglobulins may be used Protein A can also be used because of its ability to bind to the Fc portion of IgG. These reagents may be labeled with radioactive isotopes (radio-immunoassay, RIA) or with enzymes (enzyme-linked immunoassay, ELISA or EIA) . Both RIA and ELISA can be used in automated screening procedures if desired.

Hybrids obtained by fusion are initially heterogenous colonies. In order to obtain a

OMPI homogeneous cell line these colonies must be cloned. By this is meant the process of achieving growth of a cell line from a single parental cell. Cloning of hybrids is preferably performed after 5-16 days of cell growth in selective medium. Later cloning of hybrids usually results in colonies which are slow growers and low yielders of antibody.

Cloning is performed by the limiting dilution method in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilutions, cell suspensions are diluted serially to yield samples which have a statistical probability of having only one cell per well. The agarose technique begins with seeding of cells in a semi-solid upper layer of agarose over a lower layer containing feeder cells. The colonies from the upper layer are picked up and transferred to individual wells. Feeder cells, such as peritoneal macrophages, can be used to improve the cloning efficiency. Antibody secreting hybrids grown in tissue culture flasks generally yield a supernatant with an antibody concentration in the range of 10-100 yg/ml. In order to obtain higher concentrations, hybrids may be transferred into animals with inflammatory ascites. Ascites may be induced, for example, by intraperitoneal injection of mineral oil or 2,6,10,14- tetamethylpentadecane (pristane) 10-30 days in advance of inoculation with hybridoma cells. Under these conditions, antibody-containing ascites can be harvested 8-12 days after intraperitoneal injection of about 10 __ to 10__ cells. The ascites contains a higher concentration of antibodies (1-15 mg/ml) , but includes both monoclonal antibodies and immunoglobulins from the inflammtory ascites. Ascites are generally induced in an animal of the same species as the one from which the

OMP -$ ,^~ WIP immune and immortal cells were derived since growth of hybrids in interspecies animals requires immunosuppression of the host, for example by total body irradiation and administration of antilymphocyte serum prior to hybrid injection, in addition to the procedure normally followed for intraspecies growth of hybrids. Athymic nude mice can be used as an immunodepressed host if desired.

Antibodies may be purified by any of the standard techniques of protein separation such as differential precipitation using, for example, ammonium sulfate? electrophoresis; chromatographic separation based on molecular size, such as Sephadex chromatography; or various techniques based on binding properties of or to the antibodies, much as af inity chromatography.

Complete purification is not required since only the desired i munoglobulin is present and other components do not generally interfere with its immunological action. A particularly preferred sequence for the production of monoclonal antibodies is described in the following paragraphs. BALB/c mice are immunized with a homogenized membrane preparation from any stage of the life cycle, preferably microfrlaria, of Brugia malayi prepared in the following manner. The adult or larval parasite forms are isolated and disrupted by freeze- thawing and homogenization. The membraneous and soluble fractions (100,000 xg supernatant) are used for inoculation after isolatios. The mice are allowed to rest for about three weeks and then immunized again. Their spleens are removed 3-4 days after the second inoculation in order to obtain dividing B cells for fusion.

A preferred myeloma cell line is P3-NSI/-AG 4-1 (NS1), which is an azaguanine resistant, non-secretor myeloma line previously described by Kohler and Milstein, Eur. J. Immunol. _6_:511 (1976). When fused it produces K chains. Sp210-Ag is also a preferred cell line. Thirty percent polyethylene glycol (PEG-1000, Gallard-Schlesinger) is preferred as a fusion promotor. Spleen cells are fused with the NSI cell line at a ratio of 8-10 spleen cells to 1 NSI cell. The NSl/spleen-cell mixture is washed in serum-free MEM medium and suspended in 30% PEG in MEM buffered with 0.02M Hepes, pH 7.2, and centrifuged at 800 rpm for 6 min after a total of 8 min in the PEG-containing medium. The PEG medium is removed, and the cells are plated onto microtiter plates in Dulbecco's MEM with high glucose (4.5 g/l) supplemented with 20% fetal calf serum, 10% NCTC 109 medium, 0.150 mg/ml oxalacetate, 0.050 mg/ml pyruvate, 0.200 units/ml bovine insulin, and 20 mM glutamine and containing 1.6 x 10 M thymidine and 10 M hypoxanthine. An equal volume of the above medium containing 8 x 10 M aminopterin is added 24-48 hr later to make hypoxanthine-aminopterin- thymidine (HAT) selective medium.

Fourteen to twenty-one days later the supernatants

] I O C are ready for an indirect radioimmunoassay. A --^"-"i- rabbit anti-mouse F(ab' )~ with at least 2 x 10 cpm/ μg is used to detect the antibodies produced by the hybridoma that bind to the target parasite cell. The adult or larval parasite forms or eggs are isolated and disrupted by freeze-thawing and homogenization. The membranous and soluble fraction (100,000 x g supernatant) is hydrophobically attached to polyvinylchloride "U" bottom microliter plates or attached to polylysine-coated (M.W. 150,000) "U" bottom microliter plates and used for assay after blocking remaining sites with fetal calf sera. Culture supernatants (25 μl)are incubated overnight at 5°C. The plates are then washed five times with phosphate- buffered saline containing 5% fetal calf serum. Then ¹²⁵I-rabbit anti-mouse F(ab' )₂, 10 /cp in 25 μl of assay buffer, is added to each well, and the plates are again incubated overnight at 5°C. The plates are then washed five times, dried, and the wells cut out and counted. Positive wells are cloned in soft agar over a feeder layer of 3T3 fibroblasts from BALB/c mice. In

3 this procedure, approximately 10 hybrid cells in 4 ml

0.3% soft agar in Dulbecco's MEM containing 20% fetal calf serum are cloned on 60mm Falcon petri dishes that have been plated with 3T3 fibroblasts overlaid with 0- 5% agar in Dulbecco's as above. After 10-14 days, the surviving clones are selected and transferred to microliter wells containing 3T3 fibroblasts and agar as above. As the clones grow, they are transferred to larger wells and finally to Falcon flasks (25 cm ) . The hybrids can be grown in tissue culture or as ascites cells in mice. For the latter, mice are prepared by injecting 0.5 c Pristane (2,6,10,14- tetramethylpentadecane) intraperitoneally 3 to 4 weeks before injecting hybrid cells intraperitoneally.

Karryotype analysis is carried out on the clones, and their products are subjected to isoelectric focussing to verify the presence of monoclonal antibodies.

The hybridomally-produced anti-Brugia malayi antibodies of the present invention can be used in any of the array of available immunoassay techniques which utilize the binding interaction between the antibody and an antigen. The present invention is not limited to any of these techniques in particular. The most common of these is radioimmunoassay (RIA) . RIA is a well- known technique and will not be described in detail here. For particulars, reference is made to Chard, "An Introduction to Radioimmunoassy and Related Techniques", North-Holland Publishing Company, 1978, which is herein incorporated by reference. Any of the many variations of RIA can be used, such as homogenous phase RIA, heterogeneous or solid phase RIA, single antibody methods or double antibody methods, and direct (forward) or reverse sandwich assays. Particularly preferred are solid phase systems wherein the antibody (IgG or IgM) is covalently coupled to an insoluble support so that both the antibody and the bound complex after incubation can be readily separated from the soluble free fraction. A wide variety of solid phase supports have been described, which include particles of dextran, cellulose, continuous surfaces such as polystyrene or polypropylene discs, walls of plastic tubes, glass discs, glass particles, and the like. Particulate solid phases are widely used for a variety of different assays and are included in the present invention. Antibodies are attached to the particles by any of a number of techniques designed to yield a non- reversible covalent or non-covalent link between protein and particle, for example, directly or by cyanogen bromide activation. Other alternatives are the use of antibodies entrapped in the interstices of a polyacrylamide gel or bound to magnetic particles An assay tube is set up containing either sample or standard, along with the tracer and an appropriate amount of solid phase bound antibody, plus a detergent to prevent aggregtion of the particles and non-specific absorption of the tracer. After an incubation period during which the tubes are continuously mixed, the solid phase is sedimented by centrifugation; the

_OMPI supernatant is removed and the solid phase subject to two or more washes with buffer in order to remove free tracer trapped within and between the particles. The counts on the solid phase (bound fraction) are then measured. Immunoradiometric assays, as described in Chards at page 423, can also be used. When a second antibody radioimmunoassay system is used, the second antibody may be IgM or may be IgG.

Another immunoassay technique useful with the antibodies of the present invention is enzyme immunoassay. This technique is also well known to the art and reference -is made to Schuurs and VanWeemen, Clinica Chimica Acta, 81:1-40 (1977), which is herein incoporated by reference. In this technique, enzymes are applied as labels on antigen or antibodies for identification and localization of the immunoreactants. Ariy method in which the extent of binding of enzyme-labeled antigen or enzyme-labeled antibody to its immunoreactant is measured is included in this invention. Enzyme immunoassays can be classified as homogenous or heterogeneous, depending on whether the labeled reagent behaves differently or identically whether or not it is bound to specific counterparts in the immunoreaction, and which therefore may or may not require physical separation of the reactants into two fractions. The variety of enzymes used, methods of linking enzymes to the immunological components, purification of the conjugates, as well as various assay principles and methods are well described in the aforementioned Schuurs and VanWeemen article.

Needless to say, any enzyme immunoassay which has used antibodies in the past can be used with the specific, high-affinity, monoclonal antibodies of the present invention. Another immunoassay method useful in the present invention is the latex agglutination method. In this method, latex particles are coated with antigen derived from Brugia malayi and incubated with hybridomally produced IgM antibodies. Inhibition of agglutination will occur when a sample of physiological fluid containing the same Brugia antigen is incubted with this mixture. The inhibition of agglutination can either be followed with a counter or by recently developed infrared absorption techniques. An alternative is to coat the latex particles with the hybridomally-produced anti-Brugia antibodies. Incubation of these coated particles with physiological fluid comprising Brugia antigen will cause aggutination. Instead of latex particles, animal cells such as red blood cells can of course be used. In this case, the technique becomes a variation of the well known hemagglutination technique used with IgG antibodies and red blood cells. Other useful immunoassay techniques are those using other labeling techniques that result in other types of detectably-tagged antigens such as: fluorescent dyes, Aalbeses, R.C., Clin. Chim. Acta _48:109-111 (1973); electron-dense compounds (such as ferritin) ,

Singer, S.J. et al, J. Biophys. Biochem. Cyto. 9 519- 537 (1961); protein - bacteriophage conjugates, Haimovich, J. et al, Biochim. Biophys. Acta 207: 115-124 (1970); or stable free radicals, Bastiani, R.J. , et al, Am. J. Med. Technol. 39_. 211-216 (1973).

The previously described immunoassay systems are used to assay for the presence of antigens from Brugia malayi in biological fluids, such as serum, urine, or fluid obtained directly from the site of an apparent parasitic infection. It is also possible to use the detectably-tagged monoclonal antibodies in epidemiology studies of the insect vector or other aspects of the life cycle of Brugia malayi. The detectably-tagged antibodies of the invention can be prepared in kit form ready for use in an assay procedure. The kit will contain the antibody in any stable form, for example, lypholized, frozen, or in solution, along with any other necessary reagents and accessories, such as Brugia antigen standards, reaction vessels, such as plastic tubes with or without antigen or antibody coatings, and sample transfering devices, such as pipets. Many such kits are now available for other immunoassays, and the manufacturing of such kits is now standard practice.

The monoclonal antibodies of the invention may also be used in the chemotherapy of filariasis as carriers of drugs having anti-Brugia activity. The antibody is attached chemically, usually by a covalent bond, to a drug which is administered at the normal rate for that drug, usually intravenously. The antibody acts to concentrate the drug at the location of the nematode infection and thereby to increase its effectiveness. Accordingly, in many cases it is possible tύ reduce the amount of drug administered in this manner.

Since the antibody is a protein, any method of attachment to the drug that does not destroy the medicinal properties of the drug or the antigen- antibody binding properties of the antibody is suitable. Suitable methods depend on the structure of the drug and can normally be accomplished by a reaction joining a non-essential functional group of the drug with a non-essential amino acid of the antibody. Since only a fraction of the surface of the antibody is involved in binding with the Brugia antigen, random attachment will generally preserve binding affinity. Suitable drugs include diethylcarbamozene, suramin, mebendazole, and amoscanate and their derivatives.

The antibody with the drug attached may be made up into a pharmaceutical composition ready for use, comprising the drug-antibody combination either alone or in the presence of a pharmaceuticaly acceptble carrier. The pharmaceutical carrier in which the composition is suspended or dissolved may be any solvent or solid that is non-toxic to the inoculated animal and chemcially compatable with the drug-antibody complex. Suitable pharmaceutical carriers include liquid carriers, such as normal saline and other non- toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose. Having now generally described this invention, the same will be better understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless specified.

Example 1.

Female BALB/c mice were injected with membrane rich preparations isolated f om Brugia malayi. The membrane preparations were obtained by disrupting the parasites with freeze-thawing and homogenization, followed by subfractionation by differential centrif gation. The method of fusion was that of Kennett et al, in Current Topics in Microbiology and

OMPI Immunology, Vol. 81, 77, Melchers et al, eds . , Springer-Verlag, New York (1978), which is herein incorporated by reference. Antibody production was evaluated between days 14 to 17 after fusion by an indirect radioactive binding assay utilizing filarial antigen preparations. The adult or larval parasite forms or eggs were isolated and disrupted by freeze- thawing and homogenization. The membranous and soluble fraction (100,000 x g supernatant) were hydrophobically attached to polyvinylchloride "U" bottom microliter plates or attached to polylysine-coated (M.W. 150,000) "U" bottom microliter plates and used for assay after blocking remaining sites with fetal calf sera. Culture supernatants (25 μl)were inocubated overnight at 5°C. The plates were then washed five times with phosphate-buffered saline containing 5% fetal calf serum. Then ³I-rabbit anti-mouse F(ab' )_2* 10 /cpm in 25 μl of assay buffer, was added to each well, and the plates were again incubated overnight at 5°C. The plates were then washed five times, dried, and the wells cut out and counted.

The results of one fusion, in which mice immunized with B. malayi microfilarial served as the spleen cell donors can be summarized as follows. Ey solid phase radioimmunoassay using saline extracts of distinct parasite stages as the source of antigens, 11 of 28 established clones produced antibody that reacts with an antigen present only on microfilariae, 15 with antigens shared by third stage larvae and adult worms, and 2 with antigens shared by infective third stage larvae and microfilarviae. Similar results are shown in the following Table which summarizes the results of a serparate experiment. Cross reactivity was tested against the canine heartwom (Dirofilaria immitis) , Trichinella spiralis (which causes trichinosis), and

_OM -21-

Schistosoma mansoni with negative results

Table

Reaction with Brugia malayi Antigens from

Clone Designation MLcrOfilariae Mult worms Third stage larvae

MF-1 +

MF-19 +

MF-30 +

MF-2 +

MF-3 +

MF-18 +

MF-4 +

MF-11 +

MF-22 + +

MF-5 + +

MF-21 + +

MF-7 TO MF-10 + +

L3II - 1HL1 - H5 + L3II - 1HL1 - Dll + L3II - 1HL1 - A6 + L3II - Ml - Dl + + L3II - 4B12 - BIO + + + L3II - 4BL2 - B9 + +

In this liable + signifies a two-fold increase of bound radioactive counts over background. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

OMPI

Claims

1. An immortal, antibody producing, hybridomally- produced clone, wherein said antibody is specific for the parasite Brugia malayi.

2. The clone of Claim 1, wherein said antibody is specific for one stage of the life cycle of said parasite.

3. The clone of Claim 2, wherein said stage is microfilaria, third stage larva, or adult worm.

4. The clone of Claim 1, wherein said antibody reacts with microfilaria, third stage larva, and adult worm stages of said metazoan.

5. The clone of Claim 1, wherein said clone is obtained from the cell line ATCC # HB8100, ATCC # HB8101, or ATCC # HB8102.

6. A monoclonal antibody against a Brugia malayi nematode, wherein said antibody is a hybridoma-produced monoclonal immunoglobulin specific for the parasite Brugia malayi.

7. The antibody of Claim 6, wherein said antibody is specific for one stage of the life cycle of said parasite.

8. The antibody of Claim 7, wherein said stage is microfilaria, third stage larva, or adult worm.

9. The antibody of Claim 6, wherein said antibody reacts with microfilaria, third stage larva, and adult worm stages of said metazoan.

10. The antibody of Claim 9, wherein said antibody is detectably-tagged.

11. The antibody of Claim 9, wherein said

OMPI antibody is attached to a drug having anti-Brugia activity.

12. A pharmaceutical composition comprising the antibody of Claim 11.

13. The pharmaceuticl composition of claim 12, wherein said composition further comprises a pharmaceutically acceptable carrier

14. The antibody of Claim 6, wherein said antibody is produced by a cell line obtained from the cell line ATCC # HB 8100, ATCC # HB 8101, or ATCC # HB8102.

15. An immunoassay system utilizing an antibody to assay for a Brugia nematode, wherein said antibody is a hybridoma produced monoclonal immunoglobulin specific for the parasite Brugia malayi.

16. The immunoassay system of Claim 15, wherein said antibody is specific for one stage of the life cycle of said parasite.

17. The immunoassay system of Claim 16, wherein said stage is microfilaria, third stage larva, or adult worm.

18. The immoassay syse of Claim 15 or 17, wherein said antibody reacts with microfilaria, third stage larva, and adult worm stages of said metazoan.

19. The immunoassay system of Claim 15, wherein said antibody is produced by a cell line obtained from the cell line ATCC # HB8100, ATCC # HB8101, or ATCC # HB8102..

20. An immunoassay kit comprising the monoclonal antibody of Claim 6.

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