Reagents antibodies

Prepare the labeling reagent stock solution. Biotin, biocytin, and desthiobiotin hydrazide derivatives are typically used at a labeling reagent antibody MR of 20-25. Biotin and desthiobiotin hydrazide stock solutions should be prepared at 10-20 mg/mL in DMSO or DMF while biocytin hydrazide can be dissolved in the reaction... 

The assay for transgene expression involves the transduction of tissue culture cells or animals, and testing for the presence of the transgene protein product. Reagents (antibodies, enzymatic substrates, nucleic acid probes) specific for the transgene product should be made in large quantities, so the assay can be repeated several times. 

Random tissue/tumor arrays These arrays contain tissues from multiple sites and contain tumor and or non-tumor tissues. Small arrays of this kind can be used for quality control measures such as monitoring of existing reagents/antibodies as well as work-up of novel reagents. In addition they can also be used as discovery tools. For example a survey of CD10 in tumors arising in multiple tissue sites lead to the discovery of its utility in diagnosing uterine stromal tumors (4). 

Steric hindrance due to high antigen level and possible prozone effect. Re-optimize concentration of the primary antibody and ancillary reagents. Antibody concentration of the primary antibody may be too high. 57-60... 

In assessing the efficacy of in vitro versus in vivo production of reagent antibody, consideration must be given both to the relative concentrations of antibody and to the volumes obtainable. Subcutaneous inoculation of cells in a mouse yields approximately 1 ml of serum 2 weeks later the yield of ascitic fluid harvested 7-14 days after intraperitoneal injection is between 5 and 15 ml. As the concentration of antibody produced in this way is at least 100-fold greater than in tissue culture, 10 ml of ascitic fluid from one mouse would be equivalent to at least 1 liter of tissue culture fluid. In this context, the recent description of hybridomas produced by fusion of a rat myeloma line with rat spleen cells is likely to be of considerable practical significance because of the larger volume of serum obtainable following inoculation of hybridomas in these rodents. ... 

In addition to the ability to work with very small amounts of reagent antibody, the indirect assay principle has the advantage that one labeled antibody may be utilized for a wide variety of assays provided only that the reagent antibody for all the different assays is raised in the same species. The materials used for the production of the labeled antibody are easy to prepare in large quantities (e.g., rabbit IgG and, say, a sheep antiserum to this) and can be carefully characterized at each stage before committing scarce materials to the assay itself. If the two-site assay procedure is used, this also avoids the use of an immunoadsorbent separation step with consequent economy in the use of the primary antigen. 

The indirect two-site assay provides a great economy in the use of reagent antibody at the expense of an additional reaction step. It also allows for the production of one or two iodinated antibodies that will cope with virtually any assay. It seems likely that, if the procedure can be speeded by the judicious use of high temperatures and better geometry of the reaction container, it will prove to be a very satisfactory assay procedure. 

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