Introduction

I started to make monoclonal antibodies (mAbs) at the University of Alabama in Birmingham (UAB) in the early 1980’s when an Australian Dentist/Immunologist colleague and friend of mine (John F. Kearney) convinced me that I should quit making polyclonal antibodies in rabbits and use a new and exciting method of mAb production where he had personally been involved in its discovery and further development (1). Thus, the key and most important message/information from me in this presentation is that if anyone wants to produce mAbs they should use the draining Lymph Nodes (i.e. not lymphocytes from the Spleen) as their source of activated lymphocytes for cell fusion and subsequent mAb hybridoma production. The reason for using the draining lymph nodes (rather than the Spleen), as the source of antigen-activated lymphocytes, is that many of these cells are capable of producing mAbs that recognise epitopes (both protein and carbohydrate) in biological molecules that have been highly conserved in Nature and therefore their epitopes are common to proteoglycan structural domains from many different animal species. However, if one uses the Spleen as the source of lymphocytes for mAb production, these lymphocytes that they are harvesting are predominantly making antibodies that recognise epitopes that are different from (i.e. not identical to) those in the mouse amino-acid sequences and carbohydrate antigenic domains of the immunogenic molecule, so as to not produce an autoimmune response to these similar molecules that are in the cells and tissues from the immunised mouse; i.e. the process of “self/non-self recognition” in antibody production. Furthermore, this ‘draining lymph node method’ has the additional advantage that the immunisation time is only 5 injections in a 2 week immunisation period and in just 2 months after the first injection one can have prepared purified mAbs that are ready for the many different mAb experimental analyses we basic scientists want to perform (see reference (2) below for more details of this immunisation procedure).

Content

Experimental Methods: The details of how to perform this ‘draining Lymph node lymphocyte method’ for mAb production are described in more detail in one of my earlier papers (2). Consequently, over several years of research, our laboratory has made numerous mAbs that detect epitopes and neo-epitopes (i.e. enzymatic digestion generated ‘new’ epitopes) on numerous glycosaminoglycan and proteoglycan molecules that, in general, give positive results with similar molecules in tissues and organs derived from a wide variety of different animal species.

Results & Discussion: In my presentation I will provide examples of how these mAbs have been used in a variety of different immuno-chemical analyses; i.e. quantification & location of intracellular, cell surface and extracellular matrix proteoglycans derived from several different tissues and animal species including humans. Also, I will present examples of their use in studying connective tissue proteoglycan metabolism in arthritis. More complete details of these studies can be found in the full paper and review publications (2)-(11) cited below.

References

References:
(1)Hammerling, G.J., Hammerling V. & Kearney J.F. Eds (1981). Monoclonal Antibodies & T-cell Hybridomas. New York: Elsevier/North Holland.
(2) Caterson, B., Christner, J.E. & Baker, J.R. (1983). Identification of a monoclonal antibody that specifically recognises Corneal and Skeletal Keratan Sulphate. Journal of Biological Chemistry 258, 8848-8854
(3) Caterson, B., Christner, J.E., Baker, J.R. & Couchman, J.R. (1985). Production and characterisation of monoclonal antibodies directed against connective tissue proteoglycans. Fed. Proc. 44, 386-393
(4) Caterson. B., Griffin, J., Mahmoodian, F. & Sorrell, J.M. (1990). Monoclonal antibodies against chondroitin sulphate isomers: their use as probes for investigating proteoglycan metabolism. Biochem. Soc. Trans. 18, 820-823
(5) Hughes, C.E., Caterson, B., White, R.J., Roughley, P.J. & Mort, J.S. (1992). Monoclonal antibodies recognising protease-generated neo-epitopes from cartilage proteoglycan degradation. Journal of Biological Chemistry 267, 16011-16014
(6) Visco, D.M., Johnstone, B., Hill, M.A., Jolly, G.A. & Caterson, B. (1993). Immunohistochemical analysis of 3-B-3(-) and 7-D-4 epitope expression in canine osteoarthritis. Arthritis & Rheumatism 36, 1718-1725
(7) Johnstone, B., Markopoulos M., Neame, P. & Caterson, B. (1993). Identification and characterisation of glycanated and non-glycanated forms of Biglycan and Decorin in human intervertebral disc. Biochem. J. 292, 661-666
(8) Caterson, B., Hughes, C.E., Roughley, P. & Mort, J.S. (1995). Anabolic and catabolic markers of proteoglycan metabolism in osteoarthritis. Acta Orthop Scand 66, 121-124
(9) Hughes, C.E., Caterson, B., Fosang, A.J., Roughley, P.J. & Mort, J.S. (1995). Monoclonal antibodies that specifically recognise neoepitope sequences generated by ‘aggrecanase’ and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro. Biochem. J. 305, 799-804
(10) Hayes, A.J., Hughes, C.E. & Caterson, B. (2008). Antibodies and immuno-histochemistry in extracellular matrix research. Science Direct: Methods 45, 10-21
(11) Bondeson, J., Wainwright, S., Hughes, C. & Caterson, B. (2008). Clin. Exp. Rheumatology 26, 139-145