Attachment antibodies

Fig. 13 Diagrammatic representation of two types of antibody-targeted systems. Drug is either covalently linked directly to the antibody or is contained in liposomes that are targeted by attached antibodies.

Gitman, A. G., Kahane, L., and Loyter, A. (1985a) Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai vims envelopes and neuraminidase-treated cells. Biochemistry 24,2762—2768. 

A variety of reagents have been used to covalently attach antibodies onto a solid phase, upon derivatization of the solid support and/or the antibody ... 

PEG-ylated immunoliposomes for CNS drug delivery. The drug is entrapped within a hposome vector to which is attached antibodies on poly(ethylene glycol) linkers... 

Moreover, Voinea et al. attached antibodies against vascular cell adhesion molecule-1 (VCAM-1) overexpressed on activated human endothelial cells on liposomes with the intention of using them as drug carriers [162], A-gluraryl-PE was used as membrane anchor for the antibody coupling via its free amino groups after its activation with carbodiimide. There is no necessity of antibody modification before the coupling reaction. 

An avidin-biotin system has been used to attach antibodies in the bilayer of DDSs. Xiao et al. developed a three-step strategy to improve the tumor-to-tissue ratio of anticancer agents [184], Two antibodies specific for the CA-125 antigen that is highly expressed on NIH OVCAR-3 cells were used. These cells were prelabeled with biotinylated anti-CA-125 antibody and fluoroscein isothiocyanate (FITC)-labeled streptavidin (SAv) prior to administration of biotinylated liposomes. Both antibodies were specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells, which do not express the specific antibody. Antibody biotinylation did not affect its immunoreactivity. 

Lougrey, H. L., Bally, M. B., and Cullis, P. R. (1987) A novel-covalent method of attaching antibodies to liposomes. Biochim. Biophys. Acta 901,157-160. 

This chapter deals with the detection of antigens that are accessible on the surface of isolated living cells using fluorochrome labels. By incubating live cells at 4°C, to prevent endocytosis of bound molecules, the attached antibody can remain on the cell surface, and either be observed in the live state or subsequently fixed. This method yields the greatest sensitivity and best morphologic preservation for detection of surface molecules using antibodies for microscopy. 

In an interesting variation of the technology. Law et al, (L2) described a liposome-based TSH assay in which the liposomal membrane surface contained covalently attached antibodies, and a modified membrane-impermeable acridinium ester derivative was encapsulated inside the vesicles. A normal sandwich assay was conducted using magnetic particles as the solid phase and antibody-sensitized liposomes as the label. The total assay time was 5 min, compared with the normal 2.5 hr, and showed superior sensitivity. 

During all rinses, as stated previously, the key is to leave solution on the sample, which will prevent drying of the sample and thus prevent drying of the section. After each rinse, leave 10-20% of the liquid on the sample and do not remove all of the solution. Drying of the sample will dislodge cells on the coverslip and permanently attach antibodies to the cells which increases background. A minimum of six 5-10 min rinses are recommended. Many labs use as much as 10 rinses to remove all of the antibody from the previous step... 

After U antibody incubation, all of the antibody must be diluted and washed away. Each rinse will be in 500 p,l of PBS plus rinse buffer and a total of six rinses, each done for 10 min with agitation. For cultures, allowing a sample to drying will tear cells off the coverslip and drying will permanently attach antibodies to the remaining cells increasing background. Rinse a minimum of five times for 5-10 min each. 

Bioassay-based or enzyme-linked immunosorbent assay (ELISA) methods have been applied in epitope mapping [18, 19]. In this approach, different antigens are coated separately on the surface in different weUs of the analytical plate followed by incubation with a specific antibody that is linked to an enzyme. When the enzyme s substrate is added to the solution, a subsequent reaction can produce a detectable signal. This approach, however, is mostly limited to the characterization of linear epitopes. ELISA can also be applied to multisite binding analysis, where one antibody is attached to the surface, the antigen is bound, and the ability of the second antibody to bind to the attached antibody-antigen... 

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