Editing Hybridoma production (section)

=== Representative Results ===

'''Figure 1''' shows an example of the antigen-specific serum titer for one immunization performed in the lab. In this figure, the immune sera A and B were titrated from 1:50 to 1:5,000,000 in comparison to a serum of a naïve mouse. The antigen was coated on the solid phase, and the specific antibodies were detected with a POD-conjugated goat anti-mouse Ig antibody. Both sera of the immunized mice showed a significantly higher antigen-specific titer compared to the naïve mouse. The signal at a dilution of 1:5,000 is sufficient to move on to the next step, the cell fusion.

The growth of hybridomas was visible after 7 to 10 days of HAT selection by the development of cell clones, as shown in '''Figure 2'''. The image was taken from a 96-well plate 10 days after fusion. The cell clones and the aminopterinemono- or polyclonality of the wells were monitored by individual microscopic analyses (magnification: 10X objective lens, 10X eye-piece). Monoclonal wells were labeled with a point on the top of the plate. When the plates were tested in ELISA, antigen-specific signals from monoclonal hybridomas could be easily identified. Positive hybridoma cultures were recloned, expanded, and cryoconserved until the culture was monoclonal, stable, and antigen-specific. The culture supernatant was collected during the time of cultivation in order to purify the produced monoclonal antibody via protein A-mediated affinity chromatography. '''Figure 3 '''shows a standard elution profile of a monoclonal antibody with an IgG1 isotype.

Purified monoclonal antibodies were further characterized by an SDS PAGE, as shown in '''Figure 4'''. At 50 and 25 kDa, the heavy and light chains were clearly visible, indicating the purification of an antibody molecule. Lane 2 showed a control antibody with the same isotype.

[http://cloudflare.jove.com/files/ftp_upload/54832/54832fig1.jpg [1]]<br/>'''Figure 1:''''''Serum Titration after Immunization. '''The sera from two mice were titrated in dilutions from 1:50 till 1:5,000,000 and tested for antigen specificity by an ELISA. As control serum of a non-immunized mouse was used in a dilution of 1:50. The antigen-specific signals were positive for both immunized mice and the splenocytes of mouse A were used for hybridoma generation. Please click here to view a larger version of this figure.

[http://cloudflare.jove.com/files/ftp_upload/54832/54832fig2.jpg [2]]<br/>'''Figure 2:''''''Photographs of Mono- and Polyclonal Hybridomas. '''Mono- and polyclonal hybridomas were microscopically analyzed 10 days after cell fusion. The pictures show the conventional outcome for stable hybridoma cell lines with monoclonal '''(A)'''and polyclonal '''(B) '''properties. A scale bar: 10 µm; B scale bar: 10 µm. Please click here to view a larger version of this figure. [http://cloudflare.jove.com/files/ftp_upload/54832/54832fig3.jpg [3]]'''<br/>Figure 3:''''''Standard Elution Profile for a Monoclonal Antibody with an IgG1 Isotype. '''The generated monoclonal antibody was purified via protein A-mediated affinity chromatography, and the elution profile of an IgG1 antibody is shown in the figure. The first peak indicates the eluted monoclonal antibody molecule at pH 5.0. The following peaks indicate pH 3.5 and pH 2.0. The pH reduction is necessary to completely clean the column of all substances and to regenerate the column matrix for the next purifications. Please click here to view a larger version of this figure.

[http://cloudflare.jove.com/files/ftp_upload/54832/54832fig4.jpg [4]]<br/>'''Figure 4:''''''Standard SDS PAGE for a Monoclonal Antibody with an IgG1 Isotype. '''The purity of the antibody eluent was determined by a Coomassie-stained SDS-PAGE. 5 µg of the antibody solution were applied to L2 and compared to 5 µg of a standard IgG1 antibody in L3 under reduced conditions. Please click here to view a larger version of this figure.

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