2014 Lab 4

Immunochemistry

We have looked at microscopy techniques and how to grow and fix cells. Now we will begin to look at analytical techniques in cell biology. 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 or immunocytochemistry.

Objectives

Immunochemistry

• Understand historic background of immunology
• Brief understanding of immune response
• Brief understanding of Polyclonal antibodies
• Brief understanding of Monoclonal antibodies
• Understand major antibody techniques

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.

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 | Nobel Prize César Milstein autobiography | MRC Labs - Cesar Milstein | The Nobel Prize in Physiology or Medicine 1996

Immune Response

Therapeutic antibody applications

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

monoclonal antibody

• Myeloma is a bone marrow tumor that has been adapted to grow permanently in cell culture.
• Fusion of myeloma cells with antibody-producing spleen cells.
• Hybrid cell (hybridoma) can produce and secrete large amounts of a monoclonal antibody.

• MCB Movie: Preparing Monoclonal Antibodies

• See also terms HAT Medium.
• Identifying cell organelles by antibody Abcam Subcellular Poster

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

Historic - Golgi-specific antibodies

Immunofluorescence Labelling

Uses fluorescent labelled antibody or the anti-immunoglobulin antibody used to detect the intracellular location of proteins with a the fluorescence microscope. (see also Microscopy Methods confocal microscopy, TIRF microscopy)

Links: MBoC Figure 9-15. Immunofluorescence

Immunohistochemistry

Uses enzymes linked to antibodies to generate a coloured reaction product deposited at the site of antibody binding in the cell or tissue section.

Links: Biochemistry - Immunoelectron Microscopy

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.

Links: MBoC Figure 8-2. A fluorescence-activated cell sorter

Western Blotting

(immunoblot) - Uses a labelled antibody to specifically detect proteins separated by SDS polyacrylamide gel electrophoresis (SDS PAGE). Can also be modified as a dot-immunobinding technique, spotting a specimen directly onto a nitrocellulose membrane followed by reaction with monoclonal antibody and a biotin-avidin-peroxidase indicator system.

Links: Biochemistry - Figure 4.36. Western Blotting | MBoC Figure 8-18. Western blotting

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.

Links: Metallic Silver Deposit | EBS - Immunogold Labelling in Scanning Electron Microscopy | Immunogold labeling EM level

Enzyme-Linked ImmunoSorbent Assay

(ELISA, enzyme immunoassay or EIA )- Uses a labelled antibody to detect and quantify isolated proteins usually in a 96-well microtiter plate.

Links: Biochemistry - Figure 4.35 Indirect ELISA and Sandwich ELISA |

Antibody Microarray

Uses antibodies bound to a slide or substrate to specifically and quantitatively bind proteins from a cell extract (proteomic profiles), the protein levels in one sample are compared to those in a second sample.

Links: Clontech - Antibody Array

Antibodies in Tissue Culture

Uses antibodies bound to a tissue culture plate

Use of an Immobilized Monoclonal Antibody to Examine Integrin alpha5beta1 Signaling Independent of Cell Spreading. Bao W, Strömblad S. Biol Proced Online. 2002 Nov 11;4:81-87. PMID: 12734565

Alternative Techniques

Antibody Production in Yeast

A yeast platform for the production of single-chain antibody-green fluorescent protein fusions. Huang D, Shusta EV. Appl Environ Microbiol. 2006 Dec;72(12):7748-59. Epub 2006 Oct 6. PMID 17028228 PMC

Antibody Production in Prokaryotes

Production of fluorescent single-chain antibody fragments in Escherichia coli. Schwalbach G, Sibler AP, Choulier L, Deryckère F, Weiss E. Protein Expr Purif. 2000 Mar;18(2):121-32. PMID 10686142

Phage Display

Phage display is a technique which physically links antibody DNA with antibody protein, i.e.e genotype with phenotype.

CysDisplay

CysDisplay is an improved version of phage display allowing specific elution of antibody displaying phage binding to antigens through the disruption of a disulfide bridge between phage and antibody. This ensures that all specific binders are eluted independent of their binding affinity to the antigen. (SeroTec)

SeroTec - (Human Combinatorial Antibody Library

Immunochemistry Method

There are many descriptions of immunochemistry specific methods. The steps below give an example of some typical basic steps.

1. Fixation - Cells/tissue section is fixed and if necessary permeablised.
2. Blocking - with a protein solution to prevent non-specific binding of antibody.
3. Primary Antibody Incubation - diluted to appropriate concentration in buffer or blocking solution. A range of times and temperatures can be used for this step e.g. 1 h at 37C, 2 h at RT, overnight at 4C.
4. Washing - a series of washing steps to remove the excess primary antibody.
5. Secondary Antibody Incubation - diluted to appropriate concentration in buffer or blocking solution. A range of times and temperatures can be used for this step e.g. 1 h at 37C, 2 h at RT, overnight at 4C.
6. Washing - a series of washing steps to remove the excess secondary antibody.
7. Mounting - there are a number of lab-made and commercial mountants that preserve either flourescence or colour precipitate.

Web Links

• The Nobel Prize The Nobel Prize in Physiology or Medicine 1984

• Oklahoma State University Department of Biochemistry and Molecular BiologyOSU Biochemistry and Molecular Genetics of Antibodies

• Nature Protocols immunological techniques

• Commercial Resources Millipore - Antibodies Tutorial | Invitrogen - Molecular Probes | SeroTec - Human Combinatorial Antibody Library | Abnova - Protocol Movies

References

Journals

• Journal of Histochemistry and Cytochemistry
• Histochemistry and Cell Biology
• Biotechnic and HistochemistryEuropean Journal of Histochemistry
• Journal of Molecular Histology

Textbooks

Essential Cell Biology

• Antibodies Panel 5-3 pp158-159.

Immunobiology

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

• Figure A.14. The production of monoclonal antibodies
• A-12. Monoclonal antibodies

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

• Figure 24-10. Primary and secondary antibody responses
• Figure 24-8. The clonal selection theory
• MBoC Figure 9-15. Immunofluorescence
• MBoC Figure 8-2. A fluorescence-activated cell sorter
• MBoC Figure 8-18. Western blotting

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

• MCB Figure 6-10. Procedure for producing a monoclonal antibody to protein X
• MCB Movie: Preparing Monoclonal Antibodies

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

Biochemistry

• Biochemistry - Immunoelectron Microscopy
• Biochemistry - Figure 4.35 Indirect ELISA and Sandwich ELISA
• Biochemistry - Figure 4.36. Western Blotting

Molecular Imaging and Contrast Agent Database

• Optical

Search Online Textbooks

• "polyclonal antibody" Bookshelf
• "monoclonal antibody" Bookshelf
• "immunochemistry" Bookshelf

PubMed

• PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed

• PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC

• Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM

• Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

• "immunochemistry" Entrez all databases
• "polyclonal antibody" Entrez all databases
• "monoclonal antibody" Entrez all databases

Reviews

• Wingren C, Borrebaeck CA. High-throughput proteomics using antibody microarrays. Expert Rev Proteomics. 2004 Oct;1(3):355-64. Review. PMID: 1596683 PMID: 1596683

Articles

• Chaga GS. Antibody arrays for determination of relative protein abundances. Methods Mol Biol. 2008;441:129-51. PMID: 18370316

• Schwalbach G, Sibler AP, Choulier L, Deryckère F, Weiss E. Production of fluorescent single-chain antibody fragments in Escherichia coli. Protein Expr Purif. 2000 Mar;18(2):121-32. PMID: 10686142

• Huang D, Shusta EV. A yeast platform for the production of single-chain antibody-green fluorescent protein fusions. Appl Environ Microbiol. 2006 Dec;72(12):7748-59. Epub 2006 Oct 6. PMID: 17028228 PMC

• Pope ME, Soste MV, Eyford BA, Anderson NL, Pearson TW. Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. J Immunol Methods. 2009 Feb 28;341(1-2):86-96. Epub 2008 Nov 28. PMID: 19041872

• Patel JD, Joseph JM, Falkler WA Jr. Direct detection of Chlamydia trachomatis in clinical specimens by a dot-immunobinding technique using monoclonal antibody. J Immunol Methods. 1988 Apr 6;108(1-2):279-87. PMID: 3280688

Terms

adjuvant substance that increases the antigenic immune response and is used to increase production of antibody.

antibody (Ab) the protein secreted by mature B lymphocytes, plasma cells, in response to an antigen.

antigen any substance that induces the formation of antibodies.

ascites the accumulation of serum in the peritoneal cavity of the abdomen. Used in animals to generate a high concentration of antibody solution.

Freund's Complete Adjuvant (FCA) the adjuvant that produces very high antibody levels by stimulating a hypersensitive, painful inflammation at the injection site. A water in oil emulsion with inactivated and dried mycobacteria, usually Mycobacterium tuberculosis. Named after Jules T. Freund (1890-1960) a Hungarian-born American immunologist.

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.

hybridoma the cell produced by the fusion of a myeloma cell and an antibody-producing cell, the resulting hybrid is both long-lived and produces a continuous supply of antibody.

lymphocyte a particular type of white blood corpuscle; B lymphocytes arise in bone marrow; lymphocytes in the thymus develop into T-cells.

monoclonal arising from a single cell, in the immune system these cells produce a single antibody specific for a particular antigen and derived from hybridoma cells.

myeloma a tumor of B lymphocyte cells arising in the bone marrow.

Related Topics

organelle labels, aldehyde cross-linking, artefatcs of fixation, immunochemistry, monoclonal antibodies

Lab 4 Individual Assessment

1. Identify an antibody against an adhesion junction protein that is commercially available.
2. Add a link to the original data sheet page and identify the type of adhesion junction.
3. Include the following information: type of antibody (polyclonal, monoclonal), species raised in, species reacts against, types of application uses, and if available any reference using that antibody.

2014 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2013 Revision | Development | 2014 Revision

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture | Microarray Visit | Stem Cells Lab

2014 Projects: Group 1 | Group 2 | Group 3 | Group 4

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G