Particulate labeling

Fig. 31 (A) Principle of a sandwich immunoassay using FDA particulate labels. The analyte is first immobilized by the capture antibody preadsorbed on the solid phase (a) and then exposed to antibody-coated microparticle labels (b). Every microparticle contains 108 FDA molecules. High signal amplification is achieved after solubilisation, release, and conversion of the precursor FDA into fluorescein molecules by the addition of DMSO and NaOH (c). (B) Calibration curves of IgG-FDA microcrystal labels with increasing surface coverage of detector antibody (a-d) compared with direct FITC-labeled detector antibody (e). The fluorescence signals increase with increasing IgG concentration. FDA microcrystals with a high IgG surface coverage (c,d) perform better than those with lower surface coverage (a,b). (Reprinted with permission from [189]. Copyright 2002 American Chemical Society)...

Antibody molecules cannot be seen with the light microscope unless they are labeled. A variety of labels have been used including fluorescent compounds and their active enzymes, all with the property of inducing the formation of a colored reaction product from a suitable substrate system to allow visualization. Some of these systems can be employed in electron microscopy by rendering the reaction product electron dense through appropriate treatment. Alternatively, particulate labels such as gold, ferritin, or viral particles can be used (Leong 1993). 

Other particulate labels are most commonly used for electron microscopy. These labels include ferritin, and colloidal gold see Chapters 30, 37-42). More recently, the availability of Q-dot nanocrystals (Invitrogen, Molecular Probes), small, intensely fluorescent, semiconductor crystals, has made it possible to use the same probe for electron microscopy as well as fluorescence microscopy. 

A last method of localizing HRP is by oxidizing ionic silver to give a visible deposition of elemental silver as a label. This method uses buffers and reagents that require a low pH and cause destruction of cellular morphology. Therefore, this method is not recommended except for specific cases where discrete particulate label is needed. 

A third class of labels for antibodies that can be used for bright filed microscopic immunocytochemistry is particulate labels. Antibodies can be labeled with gold... 

Lanthanide chelate-dyed polystyrene particles containing carboxylic acid groups have been covalently coated with antibodies, streptavidin [95], and nucleic acids [96] for different biomolecular-binding assays. Particulate labels can be coated with antibodies and used as such, but the obvious steric and kinetic problems associated with the large molecular size can be particularly avoided by indirect detection of the bound antibodies using, e.g., biotin-streptavidin interaction [97,... 

Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100°C and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the en me reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (l- )-P-D-galactan. The supernatant was analysed for soluble labelled products. 

Process controls include daily testing of water for injection (USP), conformation of fill doses and yields, checking and approving intermediate production tickets, and checking label identity and count. Finished product control includes all the tests necessary to ensure the potency, purity, and identity of the product. Parenteral products require additional tests, which include those for sterility, pyrogens, clarity, and particulate analysis, and for glass-sealed ampoules, leaker testing. 

Iodine-131-labeled Lipiodol, a polyiodinated poppy seed oil which becomes particulate on dispersion in aqueous media, has been used for some time in treatment of hepatocellular carcinoma by arterial injection. This targeting approach has been coupled with the superior radioactivity properties of rhenium radioisotopes by exploiting the lipid solubility of... 

Hall M, Kazakova I, Yao YM (1999) High sensitivity immunoassays using particulate fluorescent labels. Anal Biochem 272 165-170... 

To date the evidence seems to favor the binding of tumor promoter to phospholipid in the cell membrane. Specific binding of [3h]TPA to mouse epidermal particulate matter is susceptible to phospholipases C and A2, less susceptible to protease, and completely resistant to glycosidase (32). Photoaffinity labelling studies with [20-3h]-phorbol 12-p-azidobenzoate 13-benzoate indicates that the irreversible binding of this photolabile phorbol ester to mouse brain membrane is predominantly to the phospholipid (specifically phosphatidylethanolamine and phosphatidylserine) portion rather than to the protein portion (33). 

Figures. Comet assay is shown for human K562 cells exposed to an extract produced from particulate matter released from sample PM 5 filters, to an extract derived from an unloaded filter, or to hydrogen peroxide (100 pM) as a control. Cells with little or no DNA damage are labeled 1 and 2, and those with extensive damage are labeled 3 and 4.

Rao KS, Recknagel RO. 1969. Early incorporation of carbon-labeled carbon tetrachloride into rat liver particulate lipids and proteins. Exp Mol Pathol 10 219-228. 

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