Додатки

Рис. Конечный комплекс, образующийся при PAP – методе

А - первичное антитело против антигена содержит метку, например, флюорохорм. Б - первичное антителдо против антигена не имеет метки, после инкубации с первичным антителом добавляют вторичное антитело, которое соединяется с первичным. Метка находится на вторичном антителе.

Первичное антитело и антитело против пероксидазы в PAP - комплексе должны быть получены от животных одного и того же вида. Следующим шагом в развитии систем визуализации стали системы с использованием антител против пероксидазы и щелочной фосфатазы (PAP и APAAP-комплексы). Последовательность процедуры следующая: 1 этап: сначала наносятся первичные антитела; 2 этап: наносятся вторичные антитела против первичных (связующее антитело), при этом один из антигенсвязывающих участков вторичных антител связывается с первичным антителом, а второй остается свободным; 3 этап: вносятся антитела против пероксидазы или щелочной фосфатазы, полученные от животного того же вида, от которого получены первичные антитела, антигенсвязывающие участки которого заняты соответствующим ферментом. Эти антитела связываются со вторым антигенсвязывающим участком вторичных (связующих) антител. Таким образом, формируется комплекс, где с одним антигеном оказываются связанными уже 2 молекулы фермента (рис.), что увеличивает чувствительность метода в 2 раза.

The avidin-biotin system

The avidin–biotin system is a technique for studying the interaction between two biomolecules in an indirect manner, as follows: Biotin is chemically coupled to a binder molecule (e.g., a protein, DNA, hormone, etc.) without disturbing the interaction with its target molecule; avidin is then used to “sandwich” between the biotinylated binder and a reporter molecule or probe. This allows for a variety of tasks, including localization and identification of the binder or target molecule. Consequently, the avidin-biotin system can frequently replace radioactive probes. Together with Ed Bayer, Wilchek established the Avidin-biotin system as a powerful tool in biological sciences. Early in the 1970s, they exploited Avidin as a probe and developed new methods and reagents to biotinylate antibodies and other biomolecules. Today, the system is applied in research and diagnostics as well as medical devices and pharmaceuticals. Examples include western blot, ELISA, ELISPOT and pull-down assays^[11]. More recently, Wilchek participated in structural studies of the avidin–biotin complex, to characterize the unique properties of this strong interaction. The studies have culminated in the determination of the 3D structure of the avidin–biotin complex by X-ray crystallography^[12], which aids in the design of specific artificial recognition sites^[13].

Three-dimensional structures of avidin and the avidin-biotin complex

O Livnah, E A Bayer, M Wilchek, and J L Sussman

Proc. Nat. Acad. Sci. 90: 5076–5080..

AbstractThe crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.

Alternatives to Antigen-Antibody

Reactions

As a defense against host antibodies, some bacteria have evolved the ability to make proteins that bind to the Fc region of IgG molecules with high affinity (Ka ~ 10⁸). One such molecule, known as protein A, is found in the cell walls of

some strains of Staphylococcus aureus, and another, protein G, appears in the walls of group C and G Streptococcus. By cloning the genes for protein A and protein G and generating a hybrid of both, one can make a recombinant protein,

known as protein A/G, that combines some of the best features of both. These molecules are useful because they bind IgG from many different species. Thus they can be labelled with flourochromes, radioactivity, or biotin and used to detect

IgG molecules in the antigen-antibody complexes formed during ELISA, RIA, or such fluorescence-based assays as flow cytometry or fluorescence microscopy. These bacterial IgG-binding proteins can also be used to make affinity columns for the isolation of IgG.