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In this paper, we describe procedures for antibody humanization with an emphasis on strategies for designing humanized antibodies with the aid of computer-guided modeling of antibody variable domains, using as an example the humanized anti-CD25 monoclonal antibody, Zenapax.
Laboratories regulated by Good Laboratory Practice (GLP) will have to follow fixed procedures for antibody validation (see http://www.mhra.gov.uk/Howweregulate/Medicines/Inspectionandstandards/GoodLaboratoryPractice/Structure/ and http://www.epa.gov/compliance/monitoring/programs/fifra/glpsops.html).html
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Western blotting was performed according to standard procedures (for antibodies, see the Supplemental Experimental Procedures).
The protocol could be easily adapted to a high-throughput procedure for antibody production.
A completely cell-based procedure for antibody selection is an advantage for many membrane-associated proteins, as these proteins are often difficult to express and purify.
Furthermore, when the standard two-step procedure for antibody purification as performed by the manufacturer (based on ammonium sulfate precipitation and ion exchange chromatography) was extended by a third step of hydrophobic interaction chromatography, the "IFN-like" activity was lost and the neutralizing capacity of the respective antibodies prevailed (Fig. 1A C).
Details of the testing procedure for antibodies against both latent and lytic KSHV antigens are described elsewhere (Newton et al, 2003a, 2003b; Bourboulia et al, 2004).
They do not normally cause severe Arthus reactions, but they can transmit infections and it was many years before effective procedures for rendering antibody preparations virus-free were developed.
Alternatively, one could impose either purification or viral inactivation procedures for the antibodies or await the development of human or humanized monoclonal antibodies before instituting this therapy.
The staining procedures for these antibodies conformed to those previously described [ 5, 6].
Detailed IHC procedures for the antibodies which correlated significantly with miR-182 (FGF2, Hif2α and MMP-7) have been previously published [ 21- 23].
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