Biotin-streptavidin method

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

Avidin-biotin complex (ABC) is based on the high affinity that streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) have for biotin. Biotin is a naturally occurring vitamin. One mole avidin will bind four moles biotin. ABC method affords a several-fold higher antigen detectability than those achieved in the standard indirect method. 

S. Busse, E. Scheumarm, B. Menges, and S. Mittler, "Sensitivity Studies for Specific Binding Reactions using the Biotin/Streptavidin System by Evanescent Optical Methods " Biosensors Bioelectronics 17, 704 - 710 (2002). 

All detection systems can be employed following HER and noticeable improvement of most antigen detection will be observed. However, we recommend using sensitive detection systems, such as streptavidin-biotin-peroxidase methods when antigen density is low. 

One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin—avidin or biotin—streptavidin complexes. The avidin— biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 13) (Savage et al., 1992). 

The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) method are more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemical techniques. The strong noncovalent attraction between biotin and avidin or streptavidin is exploited in many histochemical, immunohistochemical, and in situ hybridization... 

An immunohistochemical examination of PSA using polyclonal antibodies by the peroxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkaline phosphatase has been successfully carried out (Zaviacic et al., 1994). Immunoelectron microscopy in conjunction with the protein A-gold complex can also be used for localizing PSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence localization of PSA is given below. 

Isolation of mRNA by Biotin-Streptavidin Affinity Method... 

Figure 7.4. Isolation of poly-A+ RNA by biotin-streptavidin affinity matrix. Poly-A+ RNA is captured as a hybrid between poly-A+ RNA and biotinylated oligo(dT) by streptavidin matrix. Most mRNAs carry poly-A+ stretch at their 3 end, and hence poly A-containing RNA can be enriched substantially by this affinity capture method. Poly-A+ RNA can be eluted from the beads by low salt or water. The eluted RNA can be ethanol precipitated.

Figure 26. Schematic of the post hybridization detection method. A DNA target solution labeled with biotin is first incubated with the DNA probe functionalized chip. Targets diffuse passively from the solution to the surface where they hybridized with the probes if complementary. A solution containing streptavidin-functionalized magnetic labels is then incubated with the chip. Labels bind through the biotin-streptavidin interaction to where hybridization occurred. DNA hybridization is detected with spintronic transducers.

In a similar method the labeled streptavidin-biotin (LSAB) method also utilizes a biotinylated secondary antibody that links primary antibodies to a streptavidin-peroxidase conjugate (6). In both methods a single primary antibody is subsequently associated with multiple peroxidase molecules, and because of the large enzyme-to-antibody ratio, a considerable increase in sensitivity is achieved compared to direct peroxidase-conjugate methods. 

Figure 3. Labeled Streptavidin-Biotin (LSAB) Method.

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

Figure 10.13. Comparison of immunoperoxidase (Lane A), peroxidase anti-peroxidase (Lane B) and biotin-streptavidin (Lane C) immunoblotting detection methods.15 [Reprinted, with permission, from K. E. Johansson, in Handbook of Immunoblotting of Proteins , O. J. Bjerrum and N. H. H. Heegaard, Eds., CRC Press, Boca Raton, FL, 1988. 1988 by CRC Press, Inc.]...

Method II Indirect binding using biotin-streptavidin interaction. Conjugation of Ru(bpy)32+-COOH to streptavidin. Ru(bpy)32+-COOH was dissolved in DMF to obtain 70 pL of a solution with a concentration of 7.1 10 3 mol/L. 1.5 equivalents of DCC and NHS were added and the reaction solution was gently mixed for 4 h at RT. 630 pL of a 2.0x10 5 mol/L streptavidin solution in 0.1 mol/L borate buffer (pH 9.4) were then added (activated Ru(bpy)32+-COOH streptavidin 40 1 molar ratio). The solution was incubated overnight and the labeled protein was subsequently purified with dialysis against 5 L of PBS. 

The current immunofluorescence method is limited to the detection of adduct levels around 1/10 nucleotides. Coaq>uter-assisted video microscopy systems or the use of biotin-streptavidin staining should further increase sensitivity. It may then be possible to utilize these methods for adduct detection in human samples from occupational or environmental exposures. Since adducts can in theory be visualized in single cells, the small amount of material obtained at biopsy could be utilized. 

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