Peer Reviewed: Chromatographic Immunoassays

Biointeraction analysis by high-performance affinity chromatography: Kinetic studies of immobilized antibodies

“…It should also be possible to use this approach with a wider variety of support materials and surfaces than can be currently examined by SPR. In addition, this chromatographic approach to kinetic studies can be adapted for use with a variety of detectors (i.e., as has already been demonstrated for immunoaffinity chromatography in general) [7,8,14], making it easier to use than SPR with dilute analytes or those that do not produce a large change in signal as they bind to the surface of an SPR sensor. All of these features make this approach an attractive alternative to SPR for the direct characterization of immunoaffinity materials and for kinetic studies of immobilized biological molecules.…”

“…The ability of an antibody to recognize a specific target and bind this with high affinity gives these methods good selectivity and low limits of detection [7–14]. In addition, the use of immobilized antibody supports in combination with other methods (e.g., reversed-phase chromatography) has seen growing use in environmental and biological applications as a means for extracting and concentrating a given group of analytes from complex samples [8,9,15–23]. …”

Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

“…As shown in Figure 1(a), one of these 18 O labels can be added during cleavage of the peptide's amide bond (i.e., 16 18 O-enriched water. The relative intensities measured in these initial studies will be referred to here by using the terms a 0 , a 1 , a 2, a 3 , a 4 and a 5 for the monoisotopic peak (M+1) through the M+6 peptide peaks in the 16 O-labeled sample and by using b 0 , b 1 , b 2, b 3 , b 4 and b 5 to refer to the same peaks in the 18 O-labeled sample. When these two peptide samples are combined, the intensity of each isotope peak in the mixture (I n ) should be equal to the sum of intensities from the isotope peptide peaks produced in the 16 O-water digest ( ) and the 18 O-water digest ( ), as illustrated in Figure 2.…”

Analysis of drug–protein binding by ultrafast affinity chromatography using immobilized human serum albumin

“…HSA is the most abundant serum protein and is an important binding agent for a wide range of drugs and small solutes in serum [17–19,21–23]. An advantage of using HSA in place of antibodies in affinity microcolumns for the rapid extraction of free drug fractions is the lower association equilibrium constants of drugs for HSA (10 3 –10 5 M −1 ) vs. antibodies (10 6 –10 12 M −1 ) [17,18,38]. This lower binding affinity should make it possible to use isocratic elution with HSA microcolumns during the extraction and measurement of free drug fractions (see Figure 1).…”

“…This lower binding affinity should make it possible to use isocratic elution with HSA microcolumns during the extraction and measurement of free drug fractions (see Figure 1). This feature should, in term, eliminate the time-consuming elution and regeneration steps that are often needed with immobilized antibody columns [38]. A second advantage of using HSA is the ability of this protein to bind to many different drugs [17–19,21–23], thus providing an affinity microcolumn that could be adapted for use with more than one analyte in free fraction measurements.…”