Monoclonal Antibody Production to Neonectria Ditissima

Keane, Gary (2016) Monoclonal Antibody Production to Neonectria Ditissima. Project Report. East Malling Research. (Unpublished)

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Abstract

A culture of Neonectria ditissima (labelled 09/05) was provided by EMR as the antigen source against which they required monoclonal antibodies that recognised mycelial growth.
To this end, mice were immunized with a preparation of mycelial extract as described in the “antigen preparation” below. Tail bleeds were analysed by ELISA (Enzyme-linked Immunosorbent assay) and a fusion carried out based on these results. Monoclonal antibodies were produced and analysed.
From the cell culture medium, the antibody class (IgG, IgA or IgM) and the subclass (IgG1, IgG2a, IgG2b or IgG3) was determined for each cell line. The method chosen in this study was that of the Isostrip mouse monoclonal antibody isotyping kit (cat. No. 11-493-027 001 from Roche Diagnostics). The results were obtained within 5 minutes by the use of the lateral flow devices. As little as 10μl of cell culture medium was required for use with each test.
Information of antibody isotype is important for assay design and to determine the best method for antibody purification. The serum content of tissue culture medium provides contaminants of immunoglobulins which may have an inhibitory effect on subsequent assay development.
Where an IgM antibody class is identified, the isolation and purification process would not prove suitable using a Protein A or Protein G affinity column. Instead, a Hi TrapTM IgM purification HP column (cat. no. 17-5110-01 from GE Healthcare) could be used. If a monoclonal antibody is determined to be of an IgG class, then isolation and purification of the antibody directly from cell culture medium may prove optimal using a Hi TrapTM Protein G HP column (cat. no. 17-0404-03 from GE Healthcare).
Cross reactivity of an antibody with other fungal isolates/species can be an important issue as well. In order to provide some extra information on the antibodies produced, EMR has provided some extra isolates against which the antibodies produced can be checked for cross reactivity (by ELISA). These include two further Neonectria ditissima isolates (labelled TL88 & R28/15) as well as an isolate of Fusarium lateritium, Venturia inequalis, Nectria cinnabarina, Monilinia laxa, Phomopsis diaporthe, Colletrichum acutatum and Botryosphaeria obtusa.

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