WO1991002250A1 - Hybridomas and monoclonal antibodies therefrom having specific reactivity toward heavy chain immunoglobulin from catfish - Google Patents

Abstract

A hybridoma cell line which produces a monoclonal antibody with reactivity directed specifically to catfish immunoglobulin heavy chain having a molecular weight of 70,000 daltons was discovered. This hybridoma monoclonal antibody is specific for immunoglobulin of catfish species but not for other fresh-water fish species. Purification and quantitation of immunoglobulin from body fluids and cells of catfish may be accomplished with this monoclonal antibody. The hybridoma cell line and monoclonal antibody produced is also useful for identification and purification of immunogens from economically important agents of catfish.

Description

HYBRIDOMAS AND MONOCLONAL ANTIBODIES THEREFROM HAVING SPECIFIC REACTIVITY TOWARD HEAVY CHAIN IMMUNOGLOBULIN FROM CATFISH

BACKGROUND OF THE INVENTION This invention related to hybridoma cell lines which produce monoclonal antibodies (Mabs) having react¬ ivity specifically toward catfish immunoglobulin (Ig) heavy chain of a molecular weight of about 70,000 daltons. The monoclonal antibodies are useful for purification and quantitation of Ig in body fluids and cells of catfish. These Mabs will be important reagents for development of anti-idiotype vaccines.

Monoclonal antibody to catfish Ig that is specific for 70, 000-dalton heavy chain and useful for purification and quantitation of catfish antibody has not been previously reported. Recently, Miller, et al. [Devel. Co p. Immunol. 11: 739-747 (1987)] reported the use of monoclonal anti¬ bodies to identify and separate functionally distinct subpopulations of channel catfish lymphocytes. The monoclonal antibodies were reported to be reactive with surface lymphocyte immunoglobulin and were believed to be against surface heavy chain immunoglobulin. No evidence was presented for their specificity or reactivity for serum catfish antibody. No reports of Ig concentrations in serum of the channel catfish have been found in the literature. Serum Ig is reported to be a covalent-linked tetramer of about 700 kd [Lobb and Clem, Mol. Immunol. 20: 811-818 (1983); Lobb, Mol. Immunol. 22: 993-999 (1985)]. The Ig is composed of eight heavy (H) and light (L) chains. The molecular weight of H-chain is of about 70 kd, whereas that of the L-chain is about 22-26 kd [Lobb, et al., J. Immunol. 132: 1917-1923 (1984)].

SUMMARY OF THE INVENTION It is an object of this invention to provide urine hybridoma cell lines which produce monoclonal antibodies specific for catfish Ig heavy chain having a molecular weight of about 70,000 daltons.

It is a further object of this invention to provide monoclonal antibodies as immunochemical reagents for the purification of immunogenic protein and as reagents for the development of vaccines.

Other objects and advantages of this invention will become readily apparent from the ensuing description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In one aspect, the present invention relates to hybridoma cell lines which produce monoclonal antibodies (Mabs) that bind ^"selectively to heavy chain catfish immuno¬ globulin (Ig) having a molecular weight of about 70,000 daltons. Four hybridomas designated 1-4, 1-5, E-8, and II- 15 were isolated and shown to produce IgG₁ antibodies. The Mabs bind to catfish Ig as demonstrated by im unoblotting. The purified hybridoma cell line designated E-8 has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 on June 30, 1989 and has been assigned the ATCC Designation HB 10179.

The antibodies produced by the novel hybridoma cell lines are useful as immunochemical reagents for the purification of immunogic proteins and for specific probes of protein structure. I munoaffinity chromatography utilizing these Mabs will be useful in preparation of catfish heavy chain Ig for the formulation of vaccines.

All monoclonal antibodies which bind to catfish heavy chain Ig are encompassed by this invention. In addition to monoclonal antibodies, the invention also encompasses polyclonal antibodies which are produced by immunization of an animal with εe ipurified catfish Ig.

These monoclonal antibodies are produced by hybrid cells which are constructed using conventional techniques [see Kohler and Milstein, Nature 256: 495-497

(1975)]. The antibodies are preferably of the IgG type and bind selectively to catfish Ig. As is well known in the field of monoclonal antibodies, each independent cell line that produces a monoclonal antibody specific for the same antigen is nonetheless different from all the others, as is each of the monoclonal antibodies so produced.

While repetition of the procedures described herein will result in production of hybrid cell lines specific for catfish Ig, it is unlikely that it will yield cell lines that produce monoclonal antibodies which are chemically exact copies of the monoclonal antibodies described herein.

BALB/c mice were immunized by subcutaneous injection at several sites with catfish Ig obtained from a Sephacryl S-300 chromatography of ammonium sulfate precipi- tate of catfish Ig. A second injection with the same Ig- adjuvant mixture was given 6 days later. A final injection of Ig was given by intravenous injection 10-14 days later. Spleen cells were prepared 3-5 days after the final immuni¬ zation and fusion with the myeloma line P3X63-Aq8.563, according to the procedure described by Kohler and Milstein [supra] .

Hybrids were selected in a medium which contained hypoxanthine/aminopterine/thymidine as described ' by Littlefield [Science, 145: 709-710 (1964)], and fused cells were plated in wells of tissue culture plates. Approximately 14-15 days after fusion, culture supernatant was collected from actively growing hybridoma colonies and screened for antibody production by enzyme-linked immuno- absorbent assay (ELISA) using semipurified Ig as antigen. Hybridoma cell lines expressing positive screening react¬ ions were cloned by limiting dilution to obtained single cells. Wells were monitored for the appearance of cell colonies, and culture supernatants from wells with only one colony were again screened by ELISA as above. Positive reacting hybridomas were tested from monoclonality by Ouchterlony analysis [Ouchterlony, Prog. Allergy 5: 1-406 (1958)]. Hybridoma cell lines exhibiting the IgG₁ isotype were selected for expansion and further characterization by immunoblotting to identify heavy chains having molecular weights of about 70,000 daltons. From these hybridomas, cell lines designated as 1-4, 1-5, 11-15, and E-8 were selected which produced Mab having specific reactivity with Ig of catfish but not Ig of other species of fresh-water fish.

The perfused hybridoma E-8 was injected into pristine primed mice and resulting aεcites fluid was collected. The E-8 Mab was purified from the ascites by Protein A affinity chromatography, a known procedure.

The Mab- may be used to purify catfish Ig by preparing an immunoaffinity column or a solid immuno- absorbent purification produced a single precipitation reaction when the purified Ig electrophoresis is reacted with antibody to catfish Ig. The E-8 Mab may be used in immunoaffinity procedures to purify catfish Ig from the serum of catfish immunized to Edwardsiella ictulari, a pathogen of catfish. Thus the invention allows purifi- cation of antigens which cause antibody responses that may be protective. In addition, the purification of specific antibody allows the development of anti-idiotype vaccines by known procedures.

The Mab E-8 quantitates Ig from fluids of cat- fish. The quantitative assay of catfish Ig may be accomp¬ lished by direct, indirect, or sandwich ELISA techniques. Further, this invention allowed the development of a radial immunodiffusion technique (RID) for quantitation of catfish Ig. Results of analysis of catfish Ig at different water temperatures and different fish sizes are shown in Tables I and II, respectively. TABLE I

Relationship of Immunoglobulin (Ig) Concentrations to Water Temperature

Temperature Number Tested Mean of Serum (C) Ig in mg/dl

10 30 398^a

30 15 367,

Numbers followed by different superscript letters differ significantly (P<0.05).

TABLE II

Relationship of Immunoglobulin (Ig) Concentrations to Catfish Size

Size Number Tested

(Inches)

3-6 24 7-10 57 15-18 45

Numbers followed by different superscript letters differ significantly (P<0.05) .

The following Examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. Example 1

FISH Channel catfish were collected from ponds at the Department of Fisheries and Allied Aquacultures, Alabama Agricultural Experiment Station, Auburn University. The fish were bled from the caudal vein and the serum was collected from clotted blood. The catfish were 3-6, 7-10, and 15-18 inches long and were taken from water either -at 10°C or 30°C. Serum from white catfish, blue x channel catfish, carp, tilapia, and largemouth bass was obtained by Dr. John Plumb, Department of Fisheries and Allied Aquacul- tures, Auburn University.

Example 2 PREPARATION OF SEMIPURIFIED IG

Pooled catfish serum was precipitated by ammonium sulfate at 45% saturation. The precipitates collected by centrifugation at 12,100 x G for 10 ins. were dissolved in and dialyzed against 0.05 M Tris-HCL, pH 8.5, containing 0.9 M NaCl, 0.01% EDTA, and 0.22% NaN³ for 3 to 4 days with twice daily changes of buffer. The material was chromato- graphed on a Sephacryl S-300 (Pharmacia, Piscataway, NJ) 1.7 x 65 cm column equilibrated with the Tris-HCL buffer. Fractions of 8 ml were collected at a flow rate of 1 ml/ in. The leading fractions (16 ml) of the first 280 nm peak was collected at a flow rate of 1 ml/min. The leading fractions (16 ml) of the first 280 n peak was collected and dialyzed extensively against 0.5M phosphate buffered saline (PBS), pH 7.2. Example 3

MONOCLONAL ANTIBODY PRODUCTION Male Balb/c mice (6 to 8 weeks old) were sub- cutaneously injected at several sites with 230 μg of semi- purified Ig emulsified 1:1 in Freund's Complete Adjuvant (Gibco, Grand Island, NY) . A subcutaneous injection with the same antigen-adjuvant was given 6 days later. After 10 to 14 days, a final injection of 600 μg in 0.25 ml PSB was given by intravenous injection. Spleens were removed from the mice 3 days later and fused with P3/X63/Ag8.653 mouse myelo e cells. The resulting hybridoma cells produced by polyethylene glycoi 4000 fusion were grown in hypoxanthine, a inopterine, thymidine (HAT) - selective RPMI-1640 supple¬ mented with 15% fetal calf serum in 24-well culture plates [Littlefield, supra] . Supernatants were screened for specific antibodies by indirect ELISA, using horseradish peroxidase labeled anti-mouse immunoglobulin antisera (Miles Scientific, Naperville, IL) and semipurified Ig as antigen. Hybridomas producing antibodies to catfish Ig were cloned by limiting dilution and subclassed by im- munodiffusion using isotype specific anti-mouse antisera (Miles Scientific, Naperville, IL) . IgG, secreting hybri- doma cell lines were selected for expansion. Culture supernatants from serially passaged hybridomas were stored at -70° C until used.

Example 4 ASCITES The hybridomas were grown as ascites in the peritoneal cavity of BALB/c mice primed with Pristane (Sigma Chemical Co., St. Louis, MO) 12 to 14 days prior to injection of two million hybridoma cells. Ascites fluid taken from the intraperitoneal cavity was made cell-free by centrifugation at 800 X G for 15 mins. The Protein A technique recommended by the manufacturer (BioRad Laborat¬ ories, Richmond, CA) was used to isolate IgG, from ascites.

Example 5 ELISA Culture supernatants and ascites were tested for specific ^' monoclonal antibody reactivity by Biodot assay following the procedure recommended by BioRad Laboratories (Richmond, CA) . Nitrocellulose paper (pore size, 0.2 μm) was soaked in 0.1 M PBS, pH 7.4, for 10 mins. and pos- itioned in a dot blot block. A solution of 200 μl con¬ taining 5 μg of protein per ml of semipurified Ig was allowed to filter by gravity for 30 mins., and than a vacuum was applied. Dots were washed three times with PBS- 0.05% Tween 20 (PBS-T) . PBS containing 0.2% immun- oglobulin-free horse serum (blocking buffer) was added (0.1 ml/dot) , and the blot was incubated for 30 mins. before PBS-T washing (3X) . Dots were reacted with a dilution of horseradish peroxidase (HRP) -labeled anti-mouse IgG (Miles Scientific, Naperville, IL) for 20-30 mins. Positive reactions were visualized by color development with 30% hydrogen peroxide and 2,2-azino-di-(3-ethyl-benzthiazoline sulfonic acid-6) diammoniu salt (20 mg/100 ml) of 0.1 M citric acid buffer.

Example 6 IMMUNOAFFINITY PURIFICATION OF IG Monoclonal antibodies of the IgG, isotype were isolated from ascites by a Protein A technique (BioRad Laboratories, Richmond, CA) . The monoclonal antibodies were coupled onto CnBr-activated Sepharose 4B beads (Pharmacia, Piscataway, NJ) according to the procedure recommended by the manufac- turer. Pooled catfish serum was added to the immunoaffinity column containing the immobilized monoclonal antibody, and the unbound material was washed off the column with PBS. The bound Ig was eluted with 0.2 M glycine, pH 2.5, into 1.0 M Tris buffer, pH 7.5. The affinity-purified Ig was dialyzed and concentrated using an Amicon ultra-filtration apparatus with 43 micron filter (Amicon, Danver, MA) .

Coomassie Blue staining of SDS-PAGE gels showed that the Ig had very similar values to those of human IgM. The catfish Ig molecular weight was about 900,000 kda.

Example 7 POLYCLONAL ANTI-CATFISH IG ANTISERUM Adult goats were immunized with 0.5 g of the affinity-purified Ig combined 1:1 in complete Freund's Adjuvant by subcutaneous injections. Antigen was given at 14, 28, and 42 days. An intravenous injection with 0.5 mg was given 7 days later, and the goats were bled weekly for a period of one month. The serum was pooled and IgG was isolated by .33% ammonium sulfate precipitation. The protein concentration of this antibody was determined after extensive dialysis against PBS.

The anti-Ig antiserum reacted exclusively with serum Ig of the channel catfish, white catfish, and blue x channel catfish. Precipitin lines showed identity with each other. Tilapia, carp, or largemouth bass sera showed no reactivity with the catfish anti-Ig antiserum. Example 8

SODIUM DODECYL SULFATE POLYACRYLAMIDE

GEL ELECTROPHORESIS (SDS-PAGE)

Gradient gels of 10-20% SDS-PAGE (Pharmacia, Piscataway, NJ) or 0.75 mm SDS-PAGE discontinuous vertical slab gel (12%) with a 3% stacking polyacrylamide gel [Haverstein, et al., Devel. Comp. Immunol. 12: 773-785 (1988) ] were used. Samples containing the reduced and alkylated H and L chains were prepared according to the method described by Lobb and Clem [supra] . Samples con¬ taining Ig were prepared in sample buffer at concentrations of 0.5 to 1.0 g/ml, heated in boiling water for 4 mins., and held at 39°C for 2 to 3 hrs. Electrophoresis was per¬ formed at a constant 100 mA at 25°C . Gels not used for western blotting were stained by Commassie Brilliant Blue R250 stain. Molecular weight estimations were made by incorporation of human IgG, IgM, and IgA or low molecular weight markers (Sigma Chemicals, St. Louis, MO) into electrophoresis runs. Example 9

WESTERN BLOTTING After SDS-PAGE, proteins were transferred to nitrocellulose [Lobb and Clem, supra] using BioRad Trans- blot apparatus (BioRad Laboratories, Richmond, CA) . Electrophoretic transfer was done at a constant 50 volts for 2 to 4 hrs. The nitrocellulose strips were blocked with a solution of 1% powdered milk or 1% heat-inactivated (56°C) goat serum. The block strips were incubated in monoclonal antibody solutions for 1 hr. , washed with 0.005 M Tris buffered saline (TBS) , and then incubated for 1 hr. with HPR labeled anti-mouse IgG for 1 hr. The strips were washed with TBS and the color was developed with 0.05% 4- chloro 1-naphthol and 0.015% H₂0₂.

Monoclonal antibodies 1-4, 1-5, E-8, and 11-15 all show recognition of the same 70 kda H-chain from catfish Ig in the western blot. The E-8 hybridoma secretes IgG, subclass monoclonal antibody, and it was selected for use in immunoaffinity purification of catfish Ig.

Example 10 RADIAL IMMUNODIFFUSION The levels of serum Ig were quantitated by the

RID method of Mancini, et al. [Immunochemistry 2: 235-254 (1965)]. Briefly, 1% agarose solution containing 6 g/ml of the precipitating polyspecific antiserum and 0.1% sodium azide was poured as 1 mm thick layer in calibrated immuno- diffusion plates (Miles Scientific, Naperville, IL) . The polyspecific antiserum was prepared as ammonium sulfate precipitated Ig which was diluted in 0.05 M phosphate buffered saline (PBS) for use in RID. Serum sample for Ig determinations were diluted 1:10, and 15 μl of each dilu- tion was carefully plated in duplicate or triplicate wells. The plates were incubated in plastic sealed bags for 48 hrs. at room temperature. Precipitin ring diameters were determined directly from each calibrated plate to the nearest 0.1 mm. The serum Ig level was quantitated by reading the mean precipitin ring diameter against a stand¬ ard curve constructed using known concentrations of immuno¬ affinity purified Ig. An immunoaffinity Ig reference of 100 mg/dl was included with each test to ensure reproduci- bility of the RID assay. If the ring diameter of this standard was -within 0.2 mm of the original standard ring diameter, the catfish serum values were read from the standard curve.

Example 11 IMMUNODIFFUSION Double diffusion in agarose was done according to the method of Ocuchterlony [supra] .

Example 12 IMMUNOELECTROPHORESIS The electrophoresis was done using agarose plates prepared and used according to procedures recommended by Corning (Corning, NY) . - li ¬ lt is understood that the foregoing detailed description is given by way of illustration and that modification and variation may be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS :

1. A hybridoma cell line capable of producing in the medium of its growth a monoclonal antibody capable of binding with catfish heavy chain immunoglobulin having a molecular weight of about 70 KD.

2. A hybridoma cell line as described in Claim 1 wherein said hybridoma cell line has the identifying characteristics of ATCC Designation HB 10179.

3. A monoclonal antibody capable of binding with catfish heavy chain immunoglobulin having a molecular weight of about 70 KD.

4. A monoclonal antibody as described in Claim 3 wherein said monoclonal antibody is produced by a hybri¬ doma cell line having the identifying characteristics of ATCC Designation HB 10179.

5. A monoclonal antibody capable of binding exclusively with catfish heavy chain immunoglobulin.