UNSW Cell Biology

ANAT3231 Laboratory 05

Introduction

This laboratory is an introduction to immunological methods for analysis of cells and tissues in cell biology.

Previous labs have looked at how to look at cells (microscopy), how to grow cells in vitro (tissue culture) and how to preserve cells (fixation). We will now begin to look at techniques used in analysing cells using antibody techniques (immunochemistry). In cell biology there are many different techniques that use antibodies, only a few examples will be given in this current class.

In order to understand this technique you need a basic understanding of the immune response and antibody-antigen interaction. The laboratory handout and textbook alone contain enough information as an introduction to the subject for this level of study. If you are interested in further reading, I have also included below links to more detailed textbooks and websites with further information and images. Please note this additional information is not necessarily examinable, but may be useful if you have not previously studied immunology.

Note - immunochemistry can also be called immunohistochemistry.

Objectives

Understand historic background of immunology

Brief understanding of immune response

Brief understanding of Polyclonal antibodies

Brief understanding of Monoclonal antibodies

Understand major antibody techniques

Page Links: Before you Start | Start Here | NLM Online Textbooks | History | Immune Response | Polyclonal Antibodies | Monoclonal Antibodies | Techniques |References | Terms | Related Topics | Commercial Resources

Before you Start

The simplest form of analysis involves looking at the cells following fixation, histological staining and analysis of microscopic images of cells.

This analysis includes quantification of: cell size, shape, specialized processes and number of cells.

cell size - can inform about cellular growth.

cell shape - can inform about cell differentiation, cell motility and cell death.

specialized processes - can inform about cell differentiation.

number of cells - can inform about proliferation, cell cycle and cell death.

Start Here

Immunology is an entire subject to itself, so this lab is only an introduction to the key concepts you will require to understand the practical side of its application to cell biology. There are also a huge number of different techniques that use the core of immunology (the interaction/binding between an antigen and and antibody).

History

1938 - Antigen-Antibody binding hypothesis by John Marrack

1948 - antibody production in plasma B cells

1957 - Clonal Selection theory by Frank Macfarlane Burnet (Nobel Prize 1960)

1972 - Structure of the antibody molecule

1973-75 - Specificity of the cell mediated immune defence by Peter Doherty and Rolf Zinkernag (Nobel Prize 1996)

1975 - monoclonal antibodies produced by Georges J.F. Köhler and César Milstein (Nobel Prize 1984)

Links: The Nobel Prize in Physiology or Medicine 1960 | Frank Macfarlane Burnet Guide to Records | The Nobel Prize in Physiology or Medicine 1984 | MRC Labs - César Milstein | The Nobel Prize in Physiology or Medicine 1996 |

Immune Response

Links: MBOC Figure 24-10. Primary and secondary antibody responses | Figure 24-8. The clonal selection theory

Polyclonal Antibodies

Links: Biochemistry Figure 4.32. Polyclonal and Monoclonal Antibodies |

Monoclonal Antibodies

MCB Movie: Preparing Monoclonal Antibodies See also terms HAT Medium.

Links: Immunobiology - Figure A.14. The production of monoclonal antibodies | A-12. Monoclonal antibodies | MCB Figure 6-10. Procedure for producing a monoclonal antibody to protein X MCB Movie: Preparing Monoclonal Antibodies | The Nobel Prize in Physiology or Medicine 1984 |

Antibody Techniques

Immunofluorescence Labelling - Uses fluorescent labelled antibody or the anti-immunoglobulin antibody used to detect the intracellular location of proteins with a the flurescence microscope.

Fluorescence Activated Cell Sorting (FACS) - Uses fluorescent labelled antibody bound to the surface of living cells to identify and sort using a laser to detect the fluorescence.

Western Blotting (immunoblot) - Uses a labelled antibody to specifically detect proteins separated by SDS polyacrylamide gel electrophoresis (SDS PAGE).

Immunoelectron Microscopy - Uses antibodies to detect the intracellular location of proteins at high resolution by electron microscopy. Antibodies are labeled with gold particles and then applied to ultrathin sections, which are then examined in the transmission electron microscope (TEM). Gold particles of different diameters can be used to visualise two or more proteins simultaneously.

Enzyme-Linked ImmunoSorbent Assay (ELISA) - Uses a labelled antibody to detect and quantify isolated proteins usually in a 96-well microtiter plate.

Antibody Microarray -

Links: MBoC Figure 9-15. Immunofluorescence | MBoC Figure 8-2. A fluorescence-activated cell sorter Biochemistry - Figure 4.36. Western Blotting | MBoC Figure 8-18. Western blotting | Biochemistry - Immunoelectron Microscopy | Biochemistry - Figure 4.35 Indirect ELISA and Sandwich ELISA |

NLM Online Textbooks

Molecular Biology of the Cell 4

NCBI MBoC | Publisher (Garland) MBoC | Search MBoC - monoclonal antibody |

Molecular Cell Biology

NCBI MCB | Publisher (Freeman) MCB

The Cell- A Molecular Approach

NCBI The Cell | Publisher (Sinauer) The Cell | Search The Cell - monoclonal antibody

Immunobiology

Janeway, Charles A.; Travers, Paul; Walport, Mark; Shlomchik, Mark New York and London: Garland Science ; c2001

NCBI - Immunobiology | monoclonal antibody

| polyclonal antibody

Web Links

External

Oklahoma State University Department of Biochemistry and Molecular Biology
OSU Biochemistry and Molecular Genetics of Antibodies

Commercial Resources

Chemicon - Introduction to Antibodies
A downloadable online Introduction to Antibodies PDF (27 pages, 2.5 Mb) introducing antibodies and techniques.

References

Journals

Journal of Histochemistry and Cytochemistry
Histochemistry and Cell Biology
Biotechnic and Histochemistry
European Journal of Histochemistry
Journal of Molecular Histology

Terms

HAT Medium (Hypoxanthine Aminopterin Thymidine medium) - selection medium for generating monclonal hybridoma cell lines. Media contains: Hypoxanthine, Aminopterin and Thymidine. Only cell lines expressing both hypoxanthine phosphoribosyl transferase (HPRT+) and thymidine kinase (TK+) can survive in this medium. Aminopterin inhibits de novo synthesis of nucleosides, while HPRT and TK supply them from hypoxanthine and thymidine.