Preparation of Hybridomas Producing Monoclonal Antibodies against Aflatoxin B1 as a Tool to Control Hepatocellular Carcinoma

Abstract

Hybridomas that secreted antibodies against aflatoxin B1 for multiple uses were prepared using a unique immunization schedule. Aflatoxin B1-BSA conjugate was used for immunization of Balb/c mice. Spleen cells were harvested from the hyper immunized mice to be fused with myeloma cell line (P3NS1) using polyethylene glycol 3000, 50% concentration as a fusogenic agent. The produced hybridomas were selected using HAT selective medium that was replaced by complete HT medium. From the 10th day after fusion, wells that contain colonies of hybridomas covering 30% or greater of the wells surface were screened for production of monoclonal antibodies against aflatoxin B1 using ELISA. 21 hybridomas were found to be reactive to aflatoxin B1. All were found to belong to IgG2a isotype except one was found to belong to IgM isotype. The prepared monoclonal antibodies and their application to immunoassays represents a useful and rapid quantitative measurement with high affinity and low detection limits in order to purify environmentally occurring levels of this carcinogen specially in areas at high risk for liver cancer.

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“…14 The rush to incorporate those fresh discoveries into cancer therapy had begun once more. [15][16][17] With this unique strategy, clinicians and researchers had only little success, occasionally establishing clinical remissions in metastatic renal cell carcinoma with high-dose interleukin 2 (il-2) 18 and questionable responses in stages iii and iv melanoma with interferon. 19,20 These modest successes were frequently offset by serious negative incidents.…”

Generation of monoclonal antibodies against PBP2a to improve diagnosis and potential treatment of MRSA infections

“…Generation of monoclonal antibodies. 13 Ten BALB/c mice were given the S. aureus immunogen vaccine before being exposed to S. aureus ATCC 29213 as previously mentioned. Mice were given 5ug of vancomycin per g of body weight on day 10 following infection (Sigma-Aldrich, Germany).…”

“…Prior to the development of hybridomas, mice were additionally given three consecutive days of s.c. administration of 10ug of immunogen without adjuvant. Spleens were removed, and, using Milstein and Köhler's procedure with slight modifications, 13 splenocytes were fused to Sp2/0-Ag 14 myeloma cells. Briefly, 1.5x10 7 Sp2/0-Ag14 myeloma cells (DSMZ, Germany) and 5x10 7 spleen cells (Sigma-Aldrich, Germany) were combined, pelleted, and resuspended in 1.5ml of polyethylene glycol-solution before being placed in 20ml of Gibco RPMI medium (Thermo Fisher Scientific, Germany).…”

“…96-well plates containing visible colonies were then used to conduct an ELISA test to check for the development of antigen-specific antibodies. 14,15 Each monoclonal antibody's IgG-subclass was determined 13 in accordance with the manufacturer's instructions using the isotyping kit IsoStrip (Roche, Germany).…”