Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Abstract

Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outer membrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/c mice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM. MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cell antigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and nonpathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted with non-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.

LipL32 Is a Subsurface Lipoprotein of Leptospira interrogans: Presentation of New Data and Reevaluation of Previous Studies

“…6) [19], while only one cell per microscopic field was shown in the other study [38]. LipL32 monoclonal antibodies [24], [25] have also been utilized in IFA, however the interpretation of the data is impossible given the lack of controls for the integrity of the outer membrane [24]. In fact, when we assessed LipL32 surface exposure using these same monoclonal antibodies, we found that the antibodies recognized the protein only after the OM have been disrupted (Fig.…”

“…LipL32 is the most abundant protein in pathogenic Leptospira [17], [20] and arguably the most widely studied protein in leptospirosis research [17], [24], [25], [33]–[36]. The lipoprotein nature of LipL32 and its presence in outer-membrane fraction was previously reported [17].…”

“…In addition to anti-LipL32 rabbit serum [10] and monoclonal antibodies for LipL32 [24], [25] raised against whole protein, LipL32-specific antibodies from human clinical leptospirosis sera were obtained by affinity purification (Fig. 2).…”

“…LipL32 monoclonal antibody 1D9 [24], [25] was a kind gift from Dr. José Antonio Guimarães Aleixo (Universidade Federal De Pelotas, Pelotas, Brazil). Patient sera from leptospirosis outbreaks in 1996 and 1997 in Salvador, Brazil, were kindly provided by Dr. Albert I. Ko (Yale University School of Public Health, New Haven, CT).…”

Evaluation of the Anti‐LipL32 Monoclonal Antibodies Potential for Use in Leptospirosis Immunodiagnostic Tests

“…Procedures for production of MAbs were as described previously. [12] Sixweek-old female BALB/c mice were injected intraperitoneally on day 1 with 75 mg of rLipL32 prepared 1:1 with sterile PBS and Freund's complete adjuvant. Injections were repeated on days 14, 21, and 28 using Freunds incomplete adjuvant.…”