Antibody antibiotin

The above represent the past and present of the most common enzyme-mediated methods of antigen detection. There are alternate procedures available, involving such methods as antibiotin antibody steps that combine the avidin-biotin systems with a further antibiotin/antienzyme sandwich for still greater sensitivity. Also, there are methods that follow a PAP procedure with a biotinylated antibody to the PAP immunoglobulin followed by ABC detection (15). The obvious problem created with this approach is the tremendous... 

Gently remove the cover slips from the slides following incubation in blocking solution and remount the slides with 60 pL of the working solution of antibiotin antibody (made in mouse). Incubate for 1.5 h at 37°C in a humidified chamber. 

The normal rabbit serum/BSA is a block to nonspecific binding of the primary (antibiotin, made in mouse) and secondary (biotinylated antimouse) antibodies. When removing the cover slips after this incubation, care should be taken to leave a thin film of serum to which the primary antibody is added. 

There are other alterations as well to the basic procedures outlined in this chapter and in Chapters 20 and 21, which involve combinations of the PAP, ABC, and LAB methods. For example, a biotinylated antibody can be recognized by an antibiotin antibody, and a linking reagent can be used to bind the antibiotin antibody to a PAP type of complex, or a secondary biotinylated antibody can be employed following the antibiotin step, which could then result in an ABC means of detection. Alternatively, an antiavidin may be used to amplify a LAB technique further. With each successive step, a greater level of amplification can be achieved. However, the possibility of higher background and poorer resolution from severe steric hindrance is also increased. 

HIAA. A ready-to-use competitive EIA kit has been developed for measuring chemically derivatized 5-HIAA. In this procedure, dichloromethane is used to convert 5-HIAA to its methyl ester during sample preparation. This methylated derivative competes with biotin-labeled 5-HIAA for a limited number of binding sites of an antibody immobilized on microtiter plates. After incubation, the wells are washed to remove unbound biotin-labeled HIAA and then incubated with antibiotin alkaline phosphatase before a p-nitrophenyl phosphate substrate is added. The amount of biotinylated HIAA bound to the antibody is inversely proportional to the 5-HIAA concentration. This EIA method shows excellent correlation with HPLC, and interassay reproducibility coefficient of variation (CV) is about 7% at a 5-HIAA level of 7.5mg/L. 

Mouse antibiotin monoclonal antibody (Sigma Chemicals, St. Louis, MO) 4.6 mg/mL. 

Conjugation of Antibiotin Monoclonal Antibody with Colloidal Selenium... 

Adsorb monoclonal antibody against biotin onto colloidal selenium by mixing 5 pL of goat antibiotin antibody (4.6 mg/mL) with 0.4 mL of phosphate buffered saline and then adding 5 mL of the above selenium colloid. 

Second probe In instances where biotin-lectin conjugates are used, an enzyme conjugate of streptavidin, avidin, or antibiotin antibody is required and should be suitably diluted in a quench buffer. 

PAMAM G4 labeled by ferrocenyl groups was also immobilized on the surface of a gold electrode covered by a monolayer of DTSP [26] or MUA activated by EDAC and pentafluorophenol [27]. This PAMAM G4 ferrocene conjugate mono-layer was biotinylated. The bioelectrocatalytic oxidation of glucose by GOx appeared to be inhibited by addition of avidin or by a monoclonal antibiotin antibody because of surface shielding (Fig. 6.5). 

Inhibition of amperometric detection of glucose in the presence of specific antibiotin antibody or avidin. 

Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides novel nucleic acid affinity probes (nucleotide analog/DNA and RNA polymerase/avidin-sepharose/antibiotin antibody/immunoprecipitation). Proc Nat Acad Sci USA 78 6633-6637... 

Biotin can be detected using fluorescent avidin, modified avidins such as UltraAvidin (Leinco Technologies) or NeutraLite Avidin (Molecular Probes), streptavidin, or antibiotin antibodies. Different secondary reagents may work better in different tissues unmodified avidin tends to result in higher background than the other choices. 

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