Antibody Isotyping and Characterization Methods

Antibody screening, titering and isotyping are important in-process and final antibody-testing steps in any custom antibody production project. These tests provide the information needed to decide which immunized animals or cell lines to select and continue, what dilutions and secondary reagents should be used in specific applications, and how the antibody can be effectively purified.

• Screening—identifying antibody samples having antigen-binding specificity
• Titering—measuring antibody concentration and functional assay potency
• Isotyping—determining a monoclonal antibody class and subclass identity

This article reviews these methods of antibody characterization.

Explore Antibodies  Introduction to Immunoglobulins

• Antibody screening
• Antibody titering (and quantitation)
• Antibody isotyping

An important component in every antibody production procedure is development of a screening method to assay for hapten-specific antibody. Screening is first performed on periodic test-bleeds (sera) during an immunization schedule to identify which immunized animals are producing a high level of antigen-specific polyclonal antibody. After mice are selected and processed for monoclonal antibody development (fusion of harvested spleen cells to produce hybridomas), the resulting hundreds-to-thousands of resulting monoclonal cell lines must be tested to determine which ones are producers of the desired specific antibody.

Screening for polyclonal antibodies with serum samples (test-bleeds) or for monoclonal antibodies with cell culture supernatants is usually accomplished using an ELISA method, even if this is not the intended final application for the antibody being produced. At later stages of antibody development (e.g., immediately before selecting mice for fusion to make monoclonal antibodies), positive samples identified by screening can be tested more thoroughly for performance in specific assays.

Typical ELISA strategy for antibody screening:

1. Coat purified antigen in an immunoassay (polystyrene) microplate using overnight incubation in pH 9.4 carbonate-bicarbonate buffer. (See important comment below.)
2. Wash and block the plate wells with appropriate reagents.
3. Add one to several dilutions of immune serum to test wells and normal (pre-immune) serum to other wells; cover and incubate the plate for 1 hour to allow antibodies to bind.
4. Wash the plate wells. Add an appropriate enzyme-conjugated detecting secondary antibody, and incubate the plate for 1 hour to allow binding to occur. Usually the goal is to select for IgG, in which case the appropriate secondary antibody for mouse serum samples would be an anti-mouse IgG.
5. Finally, wash the plate and detect active conjugated enzyme by adding the appropriate substrate, such as TMB for an HRP-conjugated secondary.

If the antigen (hapten) is a small peptide, it may not bind effectively in microplate wells using the standard coating condition for passive adsorption. Peptide antigens that were conjugated to carrier proteins for immunization will nearly always bind to microplates. However, anti-serum from the immunized animal will contain antibodies against both hapten and carrier. Therefore, to discriminate between hapten-specific and carrier-specific antibodies, couple the hapten to an irrelevant carrier protein by the same coupling chemistry (e.g., maleimide) used to prepare the immunogen conjugate. For example, bovine serum albumin (BSA) and ovalbumin (OVA) are often used as irrelevant carrier proteins to assess anti-peptide antibody titers when mcKLH has been used as the immunogen. Alternatively, use an activated microplate for covalent immobilization of peptides via amine or sulfhydryl groups.

Learn more: Overview of ELISA
Explore: Antibody Isotyping and Characterization

Before one can successfully and reproducibly labeling antibodies or use them in immunoassays, a researcher must accurately determine the concentration and functional titer of purified antibodies. "Antibody concentration" and "antibody titer" have different meanings:

• Concentration—the total amount of antibody (protein) in solution without regard to function.
• Titer—the functional concentration or dilution-factor of a stock solution of antibody for a given immunoassay.

Depending upon the methods of purification that were used for a sample, only a certain percentage of measured total protein concentration or of total antibody concentration will comprise intact, active, antigen-binding antibody. Antibody concentration can be estimated using either a general protein assay or a immunoglobulin-specific method (see microagglutination assays below).

Antibody titer is related to concentration but refers more specifically to the effective potency of a given antibody sample. Measuring titer usually means determining the functional dilution of an antibody sample necessary to achieve the desired detection range in a given assay, such as ELISA. Screening by ELISA (discussed above) inherently provides some titer information (especially if several dilutions of samples were tested). However, the specific structure and format of the screening assay generally does not correspond to the final intended application for which the antibody is being produced.

Knowledge of both concentration and titer are helpful for subsequent assay development and use of a newly produced antibody.

The concentration of pure antibodies can be estimated from the measured absorbance at 280nm, assuming a value of 13 to 14 for the absorbance of a 1% (10mg/mL) solution in saline or PBS.

The concentration of pure antibodies also can be estimated using any commercial protein assay, such as our BCA Protein Assay Kit or Coomassie Plus Protein Assay Kit. With any protein assay, it is best to use an immunoglobulin reference standard, such as our Bovine Gamma Globulin Standard (BGG), instead of the usual bovine serum albumin (BSA). This is because BSA, while providing an accurate "mean" standard for protein mixtures, responds differently from immunoglobulins in all protein assay methods.

Learn more: Overview of Protein Assays
Explore: Protein Assay Kits

Often, antibodies are produced, obtained, or intentionally stored in a semi-purified state. Antibody solutions often contain stabilizing protein additives (e.g., BSA or gelatin) or retain co-purified serum proteins (e.g., transferrin, albumin) and other immunoglobulin classes. Total protein assays do not distinguish between the antibody of interest and these other proteins.

Our Easy-Titer IgG Assay Kits are simple, mix-and-ready assays that allow accurate determination of the concentration of specific species and classes of intact antibodies. The kits detect and measure specific target antibodies using agglutination of microspheres that are coated ("sensitized") with the specific anti-IgG or IgM polyclonal antibodies. In the appropriate aqueous buffer (supplied in kit), the monodispersed antibody-coated microspheres (> 1µm diameter) have highest absorptivity (l-max) to incident light having a wavelength (340nm) that is equal to approximately half their diameter. When sample is added, two or more microspheres bind to each antibody target via their coated specific polyclonal antibodies, and this agglutination results in a proportional decrease in absorptivity (lower absorbance).

Learn more: Antibody Purification
Explore: Antibody Isotyping and Characterization

As stated above, titer is defined as the functional dilution (or working concentration) of an antibody sample that is necessary to achieve a minimum level of specific detection in a given assay method. The exact minimum acceptable value is established by the researcher, but it is usually defined by reference to a statistically significant signal-to-noise ratio.

Antibody titer is an assay-specific measurement. One immunization protocol, for example, may produce an antibody titer of 1:10000 dilution, while a second protocol may produce an antibody titer of only 1:5000. Alternatively, one bleed of antiserum may have a high titer compared to a second bleed of antiserum tested in the same assay. The higher the dilution factor, the stronger the polyclonal immune response (amount or specificity of antibody).

For any given antibody (clone), titer correlates strongly to concentration. Thus, if two different production and purification batches of a given monoclonal antibody are found to have different titers, it usually means that one batch is more concentrated than the other. The other possibility is that the antibody in one batch became inactivated (denatured, degraded) more than in the other batch.

For different antibodies (different clones), titer differences may have very little correlation to concentration differences. One antibody may simply be much more specific and have a higher affinity to antigen than the other antibody.

Learn more: ELISA Development and Optimization

Isotyping involves determining the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG2a) of a monoclonal antibody. This is a critical step in antibody production, as it is necessary for choosing an appropriate purification and modification method for the antibody. Isotyping is most easily accomplished with commercial, ready-to-use antibody isotyping kits. Most isotyping kits provide determination of the following antibody classes and subclasses of a given species (e.g., mouse): IgG, IgG2a, IgG2b or IgG3, IgA or IgM. and as having kappa or lambda light chains.

Determining the class and subclass identity of an antibody is especially important for choosing the method by which it should be purified and used in immunoassays. For example, if an antibody is determined to be IgM, it cannot be purified effectively with Protein A or G, and it will most likely require fragmentation for use in immunohistochemical procedures. If a monoclonal antibody is determined to be IgG1 composed of kappa light chains (VL-kappa), there is a good possibility that immobilized Protein L can be used to purify it from culture supernatant without contamination of bovine immunoglobulins from the serum supplement.

Isotyping requires the use of specific anti-immunoglobulin antibodies that are capable of detecting the different classes and subclasses of monoclonal antibodies encountered in antibody production. This is the reason that isotyping is most easily done using commercial kits; except in large-scale antibody production operations, it is simply not practical to separately acquire the multiple antibodies required.

Most isotyping kits provide determination of the following antibody classes and subclasses of a given species (e.g., mouse):

• IgG, IgG2a, IgG2b or IgG3, IgA or IgM
• Light chain identity as either VL-kappa or VL-lambda

Isotyping kits are available in two basic formats: ELISA kits or membrane (strip or cassette) kits. Typically, these kits include pre-coated plates or membranes designed to directly capture and react with antibody from the test sample. ELISA kits capture the test antibody to the particular microplate wells that contain the corresponding pre-coated isotype-specific antibody; a generic anti-immunoglobulin antibody provides the final detection of wells to which the test antibody was captured. Cassette kits produce colored bands on the membrane where the dotted, isotype-specific antibodies react with the test antibody. Cassettes are faster for testing one sample at a time; ELISA kits provide higher throughput for testing multiple samples simultaneously.

The most convenient and rapid isotyping kits are antigen-independent assays designed for use with essentially pure, monoclonal antibodies. If antibodies of multiple classes or subclasses are present in a sample at measurable levels, they will be detected more-or-less equally by these assay kits regardless of their antigen specificity.

Although it is rarely necessary, antigen-dependent isotyping can be accomplished in an ELISA format if the researcher possesses the required isotype-specific antibodies. First, antigen is coated in a microplate, then the antibody test-samples are added to all wells to allow antigen-specific binding. Next, washing removes all sample components except the antigen-specific. Finally, the isotype-specific detection antibodies are added to different wells and then subsequently detected with appropriate secondary antibodies. It is important to use isotype-specific antibodies that are derived from a different host species than the test samples.