Production and Characterization of Chimeric Monoclonal Antibodies against Burkholderia pseudomallei and B. mallei Using the DHFR Expression System

Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection

Production and Characterization of Chimeric Monoclonal Antibodies against Burkholderia pseudomallei and B. mallei Using the DHFR Expression System

Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection

Medulloblastoma and Brucellosis - Molecular Evidence of Brucella sp in Association with Central Nervous System Cancer

Neurobrucellosis has been reported to cause lesions in a number of different locations in the central nervous system. Histologically or radiologically, these lesions were consistent with an infection. In response to parents who believed their child's brain tumor, histologically typical of medulloblastoma, was in reality neurobrucellosis, formalin-fixed paraffin-embedded tumor tissue from the medulloblastoma was sectioned, DNA extracted, and tested by polymerase chain reaction (PCR). Specific primer/probe sets, designed in our laboratory to target Brucella species, B. melitensis, B. abortus and B. suis, and designated OMP31, B-m, B-a and B-s, respectively, were used in TaqMan real-time PCR to amplify those gene targets in two separate blocks of the child's tumor. Sections from two blocks were positive only for Brucella species. Although the patient grew up in a European country known to harbor brucella in foods, such as unpasturized milk and cheese, the patient was seronegative for B. mellitensis, B. suis, and B. abortus. In an effort to test whether a relationship existed between the presence of brucella and medulloblastoma, 20 medulloblastomas were retrieved from the tissue repository of the AFIP. The above four primer/probe sets were again used to amplify brucella DNA. Five of 20 tumors (25%) contained Brucella species DNA by the OMP31 primer/probe set. None of the 20 medulloblastomas had specific sequences for B. mellitensis, B. suis, or B. abortus. Is chronic brucellosis similar to other infectious agents such as helicobacter that is associated with tumor formation?</p