Recombinant antibody expression vectors enabling double and triple immunostaining of tissue culture cells using monoclonal antibodies.
Next to the already available mouse monoclonal and laboratory animal-derived polyclonal antibodies, recombinant antibodies offer an additional and virtually unlimited arsenal of new immunohistochemical research tools. The major advantages of recombinant antibodies are their rapid and easy generation against virtually any target. The avidity of antibody fragments can be increased by partial dimerisation. This can be achieved by fusion of CL domains derived of different species to recombinant antibody domains. The VL-linker-VH-CL constructs result in significantly lower dimerisation levels compared to the VH-linker-VL-CL antibody constructs. The most efficient dimerisation occurs with the Jun-tagged scFvs. The very large and rapidly expanding collection of recombinant antibodies already available combined with the ease of introducing various tag sequences allows for an almost unrestricted number of easily adjustable research tools. To our best knowledge we report for the first time that using CL domains derived from different species, in combination with readily available commercial secondary antibodies specific for these CL domains, provides an easy method for the application of recombinant monoclonal antibodies of various origins in immunohistochemical analyses eliminating the problem of co-staining with multiple mono- or polyclonal antibodies. Both double and triple labelling experiments can be performed successfully.
Raats
Jos M H
JM
Department of Biochemistry, 161, Radboud University Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. J.Raats@ncmls.ru.nl
Hof
Danielle
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D045744:Cell Line, Tumor; D002645:Chickens; D019281:Dimerization; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region; D051379:Mice; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D000911:Antibodies, Monoclonal; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins