Monoclonal antibodies with biotin

Warnke, R., and Levy, R. 1980. Detection of T and B cell antigens with hybridoma monoclonal antibodies. A biotin-avidin horseradish peroxidase method. J. Histochem. Cytochem. 8 771-776. 

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. 

This chapter provides protocols for the most convenient methods of labelling mouse IgG monoclonal antibodies with enzymes (alkaline phosphatase and peroxidase), a fluorescent molecule (fluorescein), biotin, and DIG. A general protocol for the evaluation of the labelled monoclonal antibody is also given. 

A wide spectrum of hepatic lesions has been reported in AIDS (H4), but it is not known whether the changes are related to the presence of HIV-1. Therefore, sections from livers of autopsied patients with AIDS were examined for the presence of HIV-1 antigen p 24 (core) and gp 41 (envelope) by the avidin-biotin-peroxidase complex methods using monoclonal antibodies. The most common histologic abnormalities were steatosis, portal inflammation, Kupffer cell hyperplasia, and focal hepatocellular and bile duct damage. Immunoreactivity for HIV-1 antigens was demonstrated in 80% of cases. 

CNTs can be functionalized with protein via non-covalent bond (Li et al., 2005 Kim et al., 2003 Mitchell et al., 2002). For example, (beta-lactamase I, that can be immobilized inside or outside CNTs, doesn t change enzyme s activity (Vinuesa and Goodnow, 2002). Taq enzyme can attach to the outside of CNT, and doesn t change its activity (Cui et al., 2004). Peptide with Histidine and Tryptophan can have selective affinity for CNT(Guo et al., 1998). Monoclonal antibody can attach to SWNTs. Protein-modified CNTs can be used to improve its biocompatibility and biomolecular recognition capabilities (Um et al., 2006). For example, CNTs functionalized with PEG and Triton X-100 can prevent nonspecific binding of protein and CNTs. Biotin moiety is attached to the PEG chains Streptavidin can bind specifically with biotin-CNT (Shim et al., 2002). 

If the antigen of interest is abundant and additional sensitivity is not required, then a two-step direct method, rather than a three-step indirect method, can be anployed. With the two-step method, the primary antibody is a biotin-labeled mouse monoclonal antibody, which is followed directly by the ABC incubation step. Because of the abundance of human immunoglobulins in tissue sections, methods for staining immunoglobulin often employ biotin-labeled mouse antihuman immunoglobulin reagents. 

Recently, monoclonal antibodies were attached to gas-filled microbubbles using this spacer coupHng technology. Testing in vitro, in a cell culture system, demonstrated selective accumulation of decafluorobutane-based lipid shell MP1950 microbubbles with covalently attached anti-lCAM-1 antibodies, onto the surface of activated endotheUum [8]. Anti-P-selectin antibodies attached to such microbubbles via an avidin-biotin scheme showed selective accumulation in the areas of inflammation and ischemia/reperfusion injury in an animal model [93]. In the latter example, biotin was also connected to the microbubble surface via a flexible polymer spacer arm. 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

The sections are incubated for 1 hr with the primary monoclonal antibody, mouse antihuman mast cell tryptase antibody (DAKO), diluted 1 200 with 1% BSA/PBS. They are washed for 10 min in PBS using magnetic stirring, incubated with biotinylated antimouse antibody for 15 min, and washed in PBS. This is followed by adding avidin-biotin-horse-radish peroxidase for 15 min. A Vector DAB Substrate kit is applied to develop the reaction product by using nickel-DAB (5 min developing time) according to the manufacturer s instructions. This step yields a black reaction product at sites of mast cell tryptase. 

In a protein-binding assay with fluorescence detection a microarray of biotin, HPYPP-peptide and WSHHPQFEK-peptide was screened against streptavidin-Cy3 and avidin-Cy5. By following the same principle an anti-human insulin monoclonal antibody was also screened against a set of different peptides. 

For smaller laboratories, the work involved in validation is often difficult, but there are two alternatives. Users can choose a system with an existing standardized and validated protocol and validated interpretation system. Commercially available kits generally provide these, and when utilized exactly as described in the kit insert, are guaranteed to provide diagnostically useful results. A second option would be to use one of the more common standard systems of fixatives with known antibodies, in which publication data has provided some evidence of functionality. As an example, a laboratory could use a 10% neutral buffered formalin fixation with a standard protocol, followed by a biotin-streptavidin HRP system, using a monoclonal antibody combination called AE1/AE3. This has been proven to be a reliable measure of cytokeratin in tissue sections. 

Fung K, Messing A, Ixe VM-Y,Trojanowski JQ. A novel modification of the avidin-biotin-complex method for immuniohistochemical studies of transgenic mice with murine monoclonal antibodies../ Ilistochem. Cytochem 1992 40 1319-28. 

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