generation-of-murine-monoclonal-antibodies-by-hybridoma-technology

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

University of Potsdam

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells.

Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein.

Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.

Multiplex localization of sequential peptide epitopes by use of a planar microbead chip.

Antibody use is ubiquitous in the biomedical sciences. However, determining best research practices has not been trivial. Many commercially available antibodies and antibody-conjugates are poorly characterized and lack proper validation. Uncritical application of such useless tools has contributed to the reproducibility crisis in biomedical research. Despite early initiatives such as MIAPAR or PSI-PAR, a best practice guideline for antibody characterization is still not in prospect. Here, we analyze 24 antibody-related databases and compare their content with regard to validation aspects and coverage. We also provide a flowchart for end-users with all necessary steps to facilitate finding and choosing specific and sensitive antibodies for their experiments. Based on a growing demand for better and standardized validation procedures and characterization guidelines for antibody molecules we have summarized our findings in a five-point plan. We intend to keep the discussion alive and hope that properly used antibodies will remain as central to biomedicine as they are today.

How to avoid pitfalls in antibody use.

Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)).

Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers.

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

Antibodies and Selection of Monoclonal Antibodies.

Glycoprotein 2 (GP2), the pancreatic major zymogen granule membrane glycoprotein, was reported to be elevated in acute pancreatitis in animal models.

Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2.

Leukotriene B4 as an inflammatory mediator is an important biomarker for different respiratory diseases like asthma, chronic obstructive pulmonary disease or cystic lung fibrosis. Therefore the detection of LTB4 is helpful in the diagnosis of these pulmonary diseases. However, until now its determination in exhaled breath condensates suffers from problems of accuracy. Reasons for that could be improper sample collection and preparation methods of condensates and the lack of consistently assay specificity and reproducibility of the used immunoassay detection system. In this study we describe the development and the characterization of a specific monoclonal antibody (S27BC6) against LTB4, its use as molecular recognition element for the development of an enzyme-linked immunoassay to detect LTB4 and discuss possible future diagnostic applications.

Article	Year
Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein. Journal of microbiological methods\| PubMed ID: 25451457	2015
Multiplex localization of sequential peptide epitopes by use of a planar microbead chip. Analytica chimica acta\| PubMed ID: 26826697	2016
How to avoid pitfalls in antibody use. F1000Research\| PubMed ID: 26834988	2015
Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers. Arthritis research & therapy\| PubMed ID: 27209064	2016
Antibodies and Selection of Monoclonal Antibodies. Advances in experimental medicine and biology\| PubMed ID: 27236550	2016
Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2. Clinical chemistry and laboratory medicine\| PubMed ID: 27837595	2016
A novel monoclonal antibody suitable for the detection of leukotriene B4. Biochemical and biophysical research communications\| PubMed ID: 27913298	2016

Katja Hanack

Biography

JoVE Journal Publications

Other Publications