Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

Hybridoma Screening by Antigen Capture: Reverse Dot Blot

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then “captured” on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts

In this protocol, hybridization is first performed in conventional aqueous solvents at a temperature well below the melting temperature, and the hybrids are then washed at higher stringency in buffers containing quaternary alkylammonium salts. TMACl is used with probes that are 14–50 nt in length, whereas TEACl is used with oligonucleotides that are 50–200 nt in length.