Spin-labeled haptens

Fig. 10. Effect of antibody binding on the paramagnetic resonance spectrum of spin-label hapten (X) in phospholipid vesicles.

Fig. 11. Evidence that a membrane-associated immunochemical reaction (complement fixation) depends on the mobility of the target hapten (IX) in the plane of a model membrane. The extent of the immunochemical reaction, complement fixation, is measured by A Absorbance at 413 nm. Temperature is always 32°C, which is above the chainmelting temperature (23°C) of dimyristoylphosphatidylcholine used for the data given in A and below the chain-melting transition temperature (42°C) of dipalmitoylphosphatidyl-choline used for the data in B. Thus A refers to a fluid membrane and B refers to a solid membrane. The numbers by each curve are equal to c, the mole % of spin-label hapten IX in the plane of the lipid membrane. It will be seen that complement fixation, as measured by A Absorbance at 413 nm is far more effective in the fluid membrane than in the solid membrane at low hapten concentrations (i.e., c 0.3 mo e%). In C the lipid membrane host is a 50 50 mole ratio mixture of cholesterol and dipalmitoylphosphatidylcholine. The immunochemical data suggest that this membrane is in a state of intermediate fluidity. Specific affinity-purified IgG molecules were used in these experiments. (For further details, see Ref. 5.)... 

Kaivarainen, A.I., Rozhkov, S.P., Franek, F., Olshovska, Z. (1983) Intramolecular mobility in antiDNP antibodies and their Fab fragments. ESR spectra of the complexes with a spin-labelled hapten in H20-D20 mixtures at various temperatures, Folia biologica 29, 209-220. 

Fig. 15. Stereomodels of the predicted MOPC 315 site. (A) Constructed from its sequence and inserting the CDR sequences into the framework of McPC 603 (Padlan et al., 1976a). (B) Revised model with incorporation of data from nuclear magnetic reso-nence and spin-labeled haptens (Dwek et al., 1977).

The method was extended to rabbit anti-p-azobenzoate and anti-p-azophenyltrimethyammonium antibodies by Piette et al. (94). Both antibodies were shown to immobilize their corresponding spin-labeled haptens. Variations in the degree of inunobilization of bound hapten were indicated by the amount of separation of the first and third spin-resonance peaks. In the case of anti-p-azobenzoate antibodies, an-... 

We assume that the affinities of these specific IgG molecules for spin-labeled lipid haptens such as (V), (IX), or (X) are of the same order of magnitude. Figure 10 illustrates the effect of antibody binding on the paramagnetic resonance spectra of (X) incorporated into lipid membrane vesicles. 

A series of hapten-labelled (e.g., adamantane, dansyl) phosphoramidites has been prepared and incorporated into ODNs suitable for use in immunodetection assays.EDTA has been conjugated to dU for the determination of protein binding by paramagnetic relaxation enhancement. A spin label has... 

The measuring principle is based on the fact that hundreds of hapten molecules are incorporated into the cell membrane of liposomes, e.g.. by linkage of phosphatidylethanolamine (a functional part of the multilayer membrane). In the presence of an antibody for the formation of an immune complex, destabilization of the entire vesicle occurs. The contents of the liposome, e.g., fluorophores dyes, spin-labeled molecules, or... 

Using the Dnp derivative illustrated above, Stryer and Griffith showed that the Kq value for its interaction with anti-Dnp antibody is similar to that of e-Dnp-t-lysine. By measurements of electron spin resonance they found an inverse stoichiometric relationship between the amount of antibody present and the percentage of hapten which exhibited the characteristic spectrum of unbound spin-labeled material. 

An experiment to estimate the depth of the antibody combining site was carried out by Hsia and Piette (93). They synthesized compounds with the general structure H —d—SL where H represents the 2,4-di-nitrophenyl hapten, d is a spacer which can be varied in size, and SL is the spin-labeled group. 

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