Generating Monoclonal Antibodies.

Antibodies that are produced by hybridomas are known as monoclonal antibodies. Here we introduce methods for generating and screening monoclonal antibodies, including developing the screening procedure and producing hybridomas.

Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Anti-Immunoglobulin Antibody-Coated Plates.

By definition, a monoclonal antibody should only be of a single class or subclass. Each class of antibody is associated with specific functions, and it can be useful to know the class/subclass of the monoclonal antibody produced by a specific hybridoma. In this protocol, class/subclass-specific antibodies are used to capture the monoclonal antibody from hybridoma supernatant. If the antibody is clonal, it will only be bound by one anti-heavy-chain capture antibody and one anti-light-chain antibody. No antigen is required.

Immunizing Animals.

The traditional method for generating polyclonal and monoclonal antibodies requires the immunization of an animal. Selecting the best species of animal and getting that animal's immune system to respond to a target antigen with an antibody response are essential to obtaining good-quality antibodies and hybridomas. There are only a limited number of opportunities for a researcher to intervene to manipulate and tailor the response to a particular antigen. Here we present advice and methods for designing the way in which the antigen is presented to the immune system (i.e., the immunization protocol), including the choice of animal, the antigen dose, the use of adjuvants, the route and number of injections, and the period between injections.

Determining the Class and Subclass of a Monoclonal Antibody by Ouchterlony Double-Diffusion Assays.

Originally, the Ouchterlony double-diffusion assays were the most common method for determining the class and subclass of a monoclonal antibody, and they still are useful, particularly when only a few assays will be performed. A sample of hybridoma tissue culture supernatant is placed in a well in a bed of agar, and class- and subclass-specific antisera are placed in other wells in a ring surrounding the test antibody. As the antibodies diffuse into the agar, they meet and multimeric immune complexes precipitate to form a visible "precipitin line."

Hybridoma Screening by Antibody Capture: Enzyme-Linked Detection (Indirect ELISA) in Polyvinyl Chloride Wells.

In this antibody capture assay for hybridoma screening, the antigen is immobilized on a solid substrate (the surface of the wells in a polyvinyl chloride [PVC] microtiter plate), and antibodies in the hybridoma tissue culture supernatant are incubated with the antigen. Unbound antibodies are removed by washing, and antibody-antigen complexes are detected by secondary antibody conjugated to alkaline phosphatase (AP), which catalyzes the conversion of a chromogenic substrate to a blue/green product. Alternative secondary antibodies are necessary for experiments in which immunoglobulin-fusion proteins have been used as immunogens or screening proteins.

Hybridoma Screening by Antibody Capture: Immunohistochemistry.

To determine the subcellular location of an antigen, hybridoma tissue culture supernatants can be screened using immunohistochemistry. For antibodies to have access to antigens in fixed and embedded tissue sections, the paraffin must be removed and the tissue must be rehydrated and digested before immunohistochemical staining.

Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Antigen-Coated Plates.

The class or subclass of an antibody is defined by its heavy chain. There are five main classes of antibodies: M, G, A, E, and D. By definition, a monoclonal antibody should only be of a single class or subclass. Each class of antibody is associated with specific functions. The method of antibody purification will differ based on the class. In this protocol, antigen is used to capture antibodies reactive to it. Specific class and/or subclass secondary antibodies are then used to discern which class/subclass has been captured.

Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Permeabilized Cells to Detect Intracellular Binding.

Flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to internal cellular proteins, but the cells must be permeabilized before the hybridoma supernatants are applied. In using this technique, useful controls are positive and negative cell lines with primary and secondary antibodies as well as positive and negative cell lines with secondary antibody alone.

Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Whole Cells to Detect Cell-Surface Binding.

If the antigen of interest is a cell-surface protein, flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to these proteins. Two alternative protocols are presented here-staining in individual tubes and staining in 96-well plates.

Hybridoma Screening by Antibody Capture: Detection of Cell-Surface Binding by Immunofluorescence.

If the antigen of interest is a cell-surface protein, immunofluorescence can be used to identify hybridomas secreting monoclonal antibodies to these proteins. They can be stained on microscope chamber slides, flat-bottomed cell culture plates (96, 48, or 24 well), or imaging plates (96 well). This protocol uses 96-well imaging plates.

Hybridoma Screening by Antibody Capture: Detection of Intracellular Binding by Immunofluorescence.

Hybridoma screening by immunofluorescence stainings of whole cells can be adapted to screen for antibodies to internal proteins by permeabilizing the cells before applying the hybridoma supernatants. In this protocol, cells are attached to a solid support (glass slides), which makes them easy to manipulate and transfer between different reagent solutions.

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).

Hybridoma Screening by Antigen Capture: Immunoprecipitation.

Immunoprecipitation is rarely used for screening hybridoma fusions because the assays are tedious and time-consuming. However, it can be useful when working with complex antigens because the precipitated antigen is normally detected after sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis and thus it is simple to discriminate between true and false positives. Furthermore, the assay provides information regarding the molecular weight of the antigen.

Adoptive Transfer Immunization of Mice.

This procedure is designed to enrich and expand antibody-forming cells for use in generating monoclonal antibodies. Gamma-irradiation is used to wipe out the immune system in a recipient animal, after which spleen cells that have reverted to memory cells are obtained from syngeneic donor animals and transferred to the irradiated animal, allowing the implanted immune cells to take over. This method can produce an 80-fold enrichment of antibody-producing cells over that obtained in the original immunized animal.

Selecting the Antigen.

The classical method for generating polyclonal or monoclonal antibodies relies on the in vivo humoral response of animals. Here we describe the factors that antigens can have that might influence the strength and quality of an antibody response. This introduction is divided into three sections: (1) an overview of immunogenicity, (2) choosing the best form for the immunogen, and (3) methods for modifying antigens to make them more immunogenic.

Hybridoma Screening by Antibody Capture: Dot Blot.

A dot blot is an appropriate hybridoma screening procedure when the antigen is a protein that is available in purified form. The antigen is bound directly to a nitrocellulose sheet and incubated with hybridoma tissue culture supernatant. A dot blot is widely used to determine the productivity of a given hybridoma, and this is described here. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones.

Hybridoma Screening by Antibody Capture: A High-Throughput Western Blot Assay.

Antibody capture assays are often the easiest and most convenient of the hybridoma screening methods. In this procedure, proteins in solution or in a cell lysate are separated according to size by gel electrophoresis and then transferred by blotting to a nitrocellulose sheet. Antigen bound to the solid substrate is incubated with the primary antibody, and the resultant antibody-antigen complexes are detected by a horseradish peroxidase (HRP)-conjugated secondary antibody and a chemiluminescent substrate for HRP.

Induction of Ascites in Mice.

Smaller animals such as rats, mice, hamsters, and guinea pigs are usually poor choices for polyclonal antibody production because only small volumes of serum can be obtained. This problem can be reduced by inducing the formation of ascites in mice, which can provide up to 10 mL of ascites fluid from a single animal. Antibody titers in ascites fluids are almost as high as serum titers.

Inducing, Collecting, and Storing Ascites.

Ascitic fluid (also called ascites) is an intraperitoneal fluid extracted from mice that have developed a peritoneal tumor. For antibody production, the tumor is induced by injecting hybridoma cells into the peritoneum, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities and continue to secrete the antibody of interest, thus creating a high-titered solution of antibodies for collection. A single mouse may yield as much as 10 mL of ascitic fluid or as little as 1 mL per batch. Antibody concentrations will typically be between 1 and 10 mg/mL. The most common problem encountered in storing ascites is contamination of these solutions with bacteria or fungi. This can be prevented by the addition of sodium azide.