Antibody Production – background
In the 1970s-1980s, scientists developed procedures to generate, purify and modify antibodies for use as antigen-specific probes.
In 1988 Harlow and Lane publish a manual (Antibodies: A Laboratory manual) standardizing protocol. These protocols are still in use today. Not much has changed as far as methods are concerned.
Animal immune systems harness the ability to produce antibodies – these antibodies have the ability to bind to specific antigens, therefore creating an opportunity for research and development scientists to manufacture probes for specific molecule detection.
Antibodies’ protein structure is relatively uniform. This uniformity enables antibody purification, labelling, and detection in a predictable and reproducible method.
In this article, we take a look at the processes required during antibody production.
Planning Antibody Production:
Producing antibodies successfully requires careful planning and preparation, with specific reference to the following steps:
- Peptide of hapten (target antigen) synthesis (or purification)
- Immunogenic carrier protein selection
- Create immunogen by conjugating the carrier protein and antigen
- Use the appropriate schedule and adjuvant formula to immunize animals
- Serum screening to determine antibody titer and isotype (characterization)
Antibody Purification
Antibody purification refers to the process of isolating polyclonal antibodies from serum, culture supernatant of a hybridoma cell line (monoclonal antibody), or ascites fluid. Methods used to purify antibodies range from very specific to general and crude:
- Very specific purification methods: purification of specific antibodies in a sample which binds to a specific antigen molecule
- General purification: purifying antibody classes regardless of antigen specificity
- Crude purification: Subset serum protein precipitation including immunoglobulin
How to characterize antibodies:
- Screening – required step during production, identifying the animals and hybridoma clones that produce high levels of antigen-specific antibodies. Use ELISA techniques for this process.
- Measuring antibody concentration or titer. Antibody titer refers to the effective potency of the antibody sample – it is, however, related to concentration.
- Measuring titer establishes the functional dilution of an antibody sample required for a specific assay for example ELISA
- Estimate antibody concentration with these two methods:
- a general protein assay
- a species or immunoglobulin specific method utilizing micro-agglutination assay kits
- Isotyping antibodies (purified monoclonal antibody class and subclass). This is a critical antibody production step. Isotyping is important when selecting the molecule purification and modification method. Use commercial off-the-shelf isotyping kits as it is the easiest way to Isotype antibodies.
Antibody Fragmentation
In general and most often, antibodies are used in the whole-molecule form.
However, in some cases, the performance of experiments and techniques can be improved when using antibodies where non-essential parts of the molecule are removed (parts that are not necessary for the binding antigen).
This process is known as antibody fragmentation. The most frequently IgG fragments created are Fab and F(ab)’2.
Labelling and Immobilization:
Scientists label antibodies in order for the modified antibody to meet the purpose of a particular assay or immunological method.
A variety of reagents for example radioactive isotopes, fluorophores, biotin, and enzymes are regularly used to implement detection signals in biological assays. It is important to understand the functional groups that are available on an antibody, as it is key when choosing a method for modification.
Modification techniques include covalent immobilization, antibody labelling, and cross-linking. The most common labelling strategies use primary amines, sulfhydryl groups, or carbohydrates as a target.
Conclusion:
Antibody production as per the above discussion entails general and specific processes. In a general sense, it refers to the whole process during antibody production including immunogen preparation, immunization, creating hybridoma, collecting, screening, isotyping, purifying, and the labelling antibodies – i.e. all processes in order for the antibody to be used in a particular method or technique.
Specific processes, however, refer to the complete antibody production process leading up to the generation of antibodies, excluding antibody purifying and labelling processes.
Producing monoclonal antibodies is by far more complex than polyclonal antibodies (recovered directly from serum/bleeds).
Monoclonal antibody production entails fusing antibody-secreting spleen cells from immunized mice with immortal myeloma cells. This creates monoclonal hybridoma cell lines, which in return express the specific monoclonal antibody in the supernatant of cell cultures.
Take care when planning and implementing monoclonal or polyclonal antibody production. Successful antibody production determines the success or failure of a good deal of research and or diagnostic applications.
References:
1. Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY. Alberts, B., et al. (1983).
2. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Harlow, E. and Lane, D. (1988).
3. Basic & Clinical Immunology. Lange Medical Publication, Los Altos, CA. Sites, D.P., et al. (1976).
About Marinda Stuiver
Dip, NHDip Microbiology (Vet) TUT (1989)
Marinda Stuiver is a qualified Veterinary Microbiologist with more than 12 years of laboratory experience.
Her experience includes histology, Electron Microscopy, and ELISA testing. Following the sale of the laboratory where she worked, she ventured into the commercial side of the business.
Marinda sold IDEXX ELISA test kits to various laboratories in Southern Africa (Zimbabwe, Botswana, Malawi) and trained laboratory staff to set up and conduct testing.
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