Monoclonal antibodies to TEM-1 plasmid-mediated beta-lactamase

Abstract

At least 28 plasmid-mediated j-lactamases have been described in gram-negative bacteria. To assess the relationship among these enzymes, we produced and characterized 28 murine monoclonal antibodies to the TEM-1 plasmid-mediated ,i-lactamase. Radial immunodiffusion identified 3 monoclonal antibodies as immunoglobulin M (IgM), 18 as subclass IgGl, 2 as IgG2a, and 5 as IgG2b. Using a newly described enzyme immunoassay, cross-reactivity of 16 of these monoclonal antibodies was tested against 24 plasmid-determined ,-lactamases. The 16 monoclonal antibodies cross-reacted with TEM-2 and TLE-1 and, to a certain extent, SHV-1. Different levels of cross-reactivity were also observed with OXA-3 (11 of 16), OXA-7 (8 of 16), OXA-1 (2 of 16), OXA-6 (2 of 16), and AER-1 (2 of 16). Six monoclonal antibodies demonstrated partial neutralization of I8-lactamase activity. This study suggests that common epitopes are shared by nine biochemically distinct plasmid-mediated I-lactamases. On the basis of cross-reactivities with these monoclonal antibodies, we identified four epitopes on TEM-1, TEM-2, TLE-1, and SHV-1 P-lactamases.Four mechanisms of bacterial resistance to ,-lactam antibiotics have been described in gram-negative bacteria (13): (i) modification of cell-wall permeability (46), (ii) modification of penicillin-binding proteins (54), (iii) tolerance to the antibiotic (58), and (iv) enzymatic modification of the drug (34). Among these, the most frequent mechanism is the production of P-lactamases (EC 3.5.2.6) which hydrolyze the antibiotic, rendering it inactive (34). These enzymes are produced by almost all bacterial species and differ from each other in their enzymological and physicochemical properties. Although three classifications have been proposed (1, 42, 56), we tend to refer to ,B-lactamases as plasmid-mediated or chromosomal enzymes. At least 28 plasmid-mediated ,-lactamases are known (5,15,19,24,26,30,31,36,38,45,53), including the newly described BRO-1 (11), NPS-1 (25), OHIO-1 (51), and SAR-1 (39). Of all these enzymes, the most widely distributed is the TEM-1 P-lactamase (26,44,52).Three techniques are available to identify plasmidmediated P-lactamases precisely: analytical isoelectric focusing (27), DNA hybridization (23), and immunological assays (40,41). Several studies have reported immunological comparisons between plasmid-mediated P-lactamases using polyclonal antisera directed against TEM

Detection of β-Lactamase Residues in Milk by Sandwich ELISA

“…Several researchers have reported that different β-lactamases may share a certain degree of homology [22,23,24]. Because several types of proteins are present in milk, it is crucial to assess any cross-reactivity in milk using the developed β-lactamase detection method.…”

“…Immunologic methods such as sandwich ELISA are effective for the detection of proteins and bacteria [18,19,20,21,22]. Suitable antibodies for the detection of β-lactamases by ELISA have been developed.…”

“…Morin et al developed and characterized 28 murine monoclonal antibodies (mAbs) against plasmid-mediated TEM-1 β-lactamase. Of the 28 mAbs, 16 monoclonal antibodies had cross-reactivity with TEM-2, TLE-1, and, to some degree, SHV-1 [22]. Even though mAbs have been produced, no analytical method has been developed.…”

Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV β-Lactamases

“…Studies with anti-ROB-1 ␤-lactamase, anti-TEM-1 ␤-lactamase, and anti-PSE-4 ␤-lactamase antibodies have addressed in a qualitative manner the effects of point mutations on steady-state ␤-lactamase levels (10,14,25,27,30). To our knowledge, only two anti-␤-lactamase antibodies have been used in both Western blot and enzyme-linked immunosorbent assay (ELISA) formats to measure TEM-1 and PC1 ␤-lactamases (15,22).Here, we describe the production, purification, and characterization of polyclonal anti-SHV-1 and anti-CMY-2 ␤-lactamase antibodies. We chose to develop these antibodies because SHV-1 is the second most common class A ␤-lactamase found in Escherichia coli and Klebsiella pneumoniae, and CMY-2 ␤-lactamase is the most common plasmid-determined AmpC ␤-lactamase (3, 21).…”