Total binding assay procedure

The saturation binding assays of Lu-D OTATATE were performed using the following concentrations 0.05,0.15,4.5,13.5,40.5,120.5 and 361.5nM for total and non-specific binding assays. The experimental results were analysed using the PRISM-2 program. The procedure detailed in the previous subsection was used. 

The microbiological method for total folate determination is still the most widely used and accepted procedure, and competitive-binding assays are most typically used for blood folate analyses. Separation, identification, and quantification of natural folates using chromatographic procedures, particularly HPLC, can be achieved but is not straightforward and requires careful selection of chromatographic conditions for the required separations (7). 

Fig. 1. Definition of the efficiency of a separation procedure in a radioimmunoassy. In a perfect assay all the labeled antigen would distribute between the free and bound fractions. In practice, some of the free is classified as bound (assay blank, nonspecific binding), and some of the free will be nonimmunoreactive. The greater the total amount of tracer ligand that reacts in the system, the better the assay. From Chard. ...

About 10% of serum proteins are immunoglobulins (Igs). After immunization, the specific antibodies produced are about 1"C5% of this fraction, so the required Ig (in ELISA) may be from 0.1 to 2.5% of the total protein in a serum. Some assays are favored by the relatively crude fractionation of serum to obtain Igs, e.g., for use in binding to plates in trapping (sandwich assays) to avoid competition for plastic binding sites by other serum proteins. Several methods for separation of Igs are available for use in ELISA. These procedures are suitable for polyclonal antibodies but not necessarily for mAbs. The isolation of total Igs as compared to the purification of specific Igs, is relatively simple. 

The activity of serum alkaline phosphatase (ALP) isoenzymes (total, intestinal, liver, and bone-specific) was measured as previously described (11,12). Serum intact osteocalcin was measured by an immunoradiometric assay using a commercial kit (Immutopics, Inc., San Clemente, CA). Serum IGF-I concentration was measured by RIA after the binding proteins were removed by a high-performance liquid chromatography (HPLC) procedure (8,13). Serum leplin concentration was measured by RIA using a conunercial kit (Linco Research, St. Charles, MO). 

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