SELECTION AND CLONING PROCEDURE

108 spleen immunized cells and 107 Sp2/O myeloma cells(Sp20-Ag14, mouse myeloma, lymphoblast suspension cellline) were fused between 1/10 ratio in the presence of poly-ethylene glycol (PEG 3700, Sigma Chemical Co.), followingstandard procedures.2 After fusion, cells were distributed in ten96-well tissue culture plates with RPMI supplemented with 100IU mL�1 of penicillin, 100 �g mL�1 of streptomycin, 2 mML-glutamine, and 10% of heat-inactivated fetal calf serum sup-plemented with HAT (100 mM hypoxantine, 0.4 �Maminopterine, and 16 �M thymidine). After 14–21 days, hybridcells showing antibody activity against canine leukocytescreened by cell ELISA, for the detection of antigens of fixedcanine PBMC, were cultured further using a layer of feedercells from a non-immunized Balb/c mouse to assist in the earlystages of growth. The IH1 mAb was subjected to three subse-quent sub-cloning steps by limiting dilution.

SPECIFIC ANTIGEN IDENTIFIED

The specificity of this monoclonal antibody has been deter-mined by ELISA, indirect immunofluorescence, and immuno-histochemistry on canine PBMC and canine frozen tissue sec-tions of lymph node, spleen, liver, and skin as well asimmunofluorescence on peripheral blood monocytes-derivedmacrophages and flow cytometry analyses. The results indi-cated that this monoclonal antibody specific for canine leuko-cytes did not react with epitopes on human and mouse leuko-cytes.

HEAVY AND LIGHT CHAIN OFIMMUNOGLOBULIN

MAb was identified as the IgG class, which were IgG1, usinghybridoma subisotyping kit (Monoclonal Antibody-BasedMouse Ig Isotyping Kit, BD PharMingen, Los Angeles, CA)system.

ANTIGEN USED FOR IMMUNIZATION

Canine blood was collected by venipuncture of the cephalicvein into tubes containing enough heparin to produce a finalconcentration of 10 IU mL�1. The peripheral blood mononu-clear cells (PBMC) were isolated by centrifugation of dilutedblood at 1:2 HBSS (Hank’s balanced salt solution, SigmaChemical Co., St. Louis, MO) layered on Histopaque 1077(Sigma Chemical Co.). Cells were washed twice in HBSS. Mitogen-stimulated PBMC were obtained by incubating thesecells at the concentration of 5 � 106/mL, with 5 �g/mL of con-canavalin A (ConA, Sigma Chemical Co.) in RPMI 1640(Sigma Chemical Co.) supplemented with 100 IU mL�1 of peni-cillin, 100 �g mL�1 of streptomycin, 2 mM L-glutamine, and20% of heat-inactivated fetal calf serum (GIBCO, Gaithersburg,MD) for 48 h in a 100% humidified atmosphere containing 5%CO2 at 37°C. Stimulated or non-stimulated cells were adjustedto a total of 107 cells, sonicated, and emulsified in an equal vol-ume of complete or incomplete Freund’s adjuvant (SigmaChemical Co.).

METHOD OF IMMUNIZATION

Two groups of five 8-week-old BALB/c mice were immunizedwith antigen from ConA-stimulated or with non-stimulated ca-nine PBMC with complete Freund’s adjuvant. The immuniza-tion consisted of one intraperitoneal followed by two subcuta-neous injections (totaling 107 cells per animal in 200 �L). Twoidentical boosters were given 2 and 4 weeks after the first im-munization, using the same amount of cells, emulsified in in-complete Freund’s adjuvant. Two weeks after the secondbooster, the mice were tested for the presence of circulating an-tibody against canine PBMC by indirect enzyme-linked im-munosorbent assay (ELISA).1 The animal with the highest an-tibody titer was injected intravenously with 107 sonicatedcanine PBMC in 200 �L of phophate-buffered saline (PBS),0.15 M, pH 7.2. Three days later, the animal was sacrificed anda single-cell splenocyte suspension was obtained for fusion.

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HYBRIDOMA AND HYBRIDOMICSVolume 23, Number 5, 2004© Mary Ann Liebert, Inc.

Monoclonal Antibody Against CanineMonocytes/Macrophages

Monoclonal Antibodies

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MONOCLONAL ANTIBODIES327

AVAILABILITY

Tissue culture supernatant Yes ✓ NoAscitic fluid Yes ✓ NoHybridoma cells Yes No ✓

ADDRESS CORRESPONDENCE TO:

Dr. Paulo Henrique Palis AguiarLaboratório de Patologia e Bio-Intervenção (LPBI)Gonçalo Moniz Research Center (CPqGM)Oswaldo Cruz Foundation (FIOCRUZ)Av. Valdemar Falcão n. 121Brotas, Salvador, BahiaBrazil 40295-001E-mail: phpa@ufba.br

orDr. Lain Carlos Pontes-de CarvalhoLaboratório de Patologia e Bio-Intervenção (LPBI)

Gonçalo Moniz Research Center (CPqGM)Oswaldo Cruz Foundation (FIOCRUZ)Av. Valdemar Falcão n. 121Brotas, Salvador, BahiaBrazil 40295-001E-mail: lain@cpqgm.fiocruz.br

REFERENCES

1. Baumgarten H: A cell ELISA for the quantitation of leuko-cyte antigens. J Immunol Methods 1989;94:91–98.

2. Harlow E, and Lane D: Antibodies: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor,NY, 1988.

3. Cobbold S, and Metcalf S: Monoclonal antibodies that de-fine canine homologues of human CD antigens. Summaryof the First International Canine Leukocyte Antigen Work-shop (CLAW). Tissue Antigens 1994;43:137–154.