Monoclonal antibodies

Atlantic billfishes (family Istiophoridae) are overexploited and often illegally harvested. To address both of these problems, a rapid means of identifying billfish carcasses is needed. This thesis describes a simple and rapid Nalge Nunc-Immuno(TM) Stick-based direct sandwich assay for sailfish identification that can be performed in the field. A species-specific anti-sailfish monoclonal antibody, covalently bound to the Nalge Nunc-Immuno(TM) Stick's polystyrene surface, was used to capture a biomarker molecule---serum albumin---from sailfish tissue samples. This antibody-antigen interaction was visualized by utilizing peroxidase-conjugated anti-billfish monoclonal antibodies or polyclonal antibodies together with a precipitating substrate. This technique successfully differentiated between sailfish and other fishes within 15 minutes, with a high degree of sensitivity and accuracy. This assay has many potential applications for species identification.

A fusion was conducted using spleen cells from a hyperimmunized Balb/C mouse and a murine myeloma cell line, in an attempt to create monoclonal antibodies specific to parvalbumin from Centropomus undecimalis. The fusion protocol utilized a 24 well culture plate protocol. Initial results indicated the successful generation of antibody producing hybridomas specific to the desired parvalbumin; however, attempts to isolate and clone these cells were unsuccessful. The study results clearly demonstrate some potential drawbacks to using the 24-well fusion protocol versus a 96-well protocol. The 24-well protocol seems to favor rapidly growing non-secreting cells due to the lower dilutions involved. This study was successful in isolating murine polyclonal antiserum reactive in isolating murine polyclonal antiserum reactive with piscine parvalbumin. Further, initial cultures of hybridomas were able to distinguish C. undecimalis from several other species of fishes thus confirming the suitability of parvalbumin as a biomaker protein.

A monoclonal antibody (MAb) produced against sailfish (Istiophorus albicans) serum albumin (SFA)/(Rossi et al. 1992) was used in conjunction with rabbit polyclonal antibodies (PAbs) to formulate a sandwich-style enzyme immunoassay (sEIA). The sEIA specifically identifies sailfish tissues utilizing samples such as heart, kidney, white muscle, red muscle, and blood serum. The assay is sensitive to 20ng of SFA. Of the tissues tested, kidney yields the strongest signal. The entire assay is carried out in a 3cc syringe and, excluding sample preparation, can be executed in 30 minutes. No specialized equipment or training is required. MAbs were also produced against serum albumin purified from white marlin (Tetrapturus albidus) and Atlantic blue marlin (Makaira nigricans). These Mabs were examined for their utility in identification assays for these species.

Monoclonal antibodies (MAbs) were produced against sailfish (Istiophorus platypterus) albumin and investigated for their potential use in species identification. Balb/c mice were immunized with albumin purified from sailfish serum. Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) for reactivity with the purified albumins of sailfish, blue marlin (Makaira nigricans) and white marlin (Tetrapturus albidus). MAbs from 18 different hybridomas exhibited activity against sailfish albumin. Sixteen MAbs showed cross-reactivity with the marlin species. Two MAbs displayed distinct specificity for sailfish albumin. One of these MAbs (M2D1) was conjugated to horseradish peroxidase to construct an ELISA for the identification of sailfish from serum. The assay correctly identified 18 sailfish from 36 billfish samples. The MAb-peroxidase conjugate was highly specific toward sailfish with no detectable reaction against the heterologous species.

This study evaluated two methods for the isolation and purification of anti-DNA antibodies. A two-step affinity purification with streptavidin (SA) biotinylated oligodeoxythymidine (dT) M-280 and protein G Dynabeadsª was compared to a two step method using Melon(TM) Gel and cellulose DNA. Although Melon gel allowed for faster antibody purification and a higher recovery rate it gave a product of less purity than the magnetic bead method. Further characterization of the antibodies was done by PhastGel(TM) non-reducing SDS-PAGE and isoelectric focusing in order to analyze purity and confirm the polyclonal nature of anti-DNA antibodies. Agilent 2100, with a higher resolution then SDS-PAGE, revealed possible subclasses of different MW not detected by SDS-PAGE. ELISA showed that all four IgG antibody subclasses were present, while Western blot confirmed the presence of human IgGs. Ultraviolet spectroscopy, Agilent, and fluorescence based assays were used to demonstrate DNA hydrolytic activity of purified anti-DNA antibody.