Optimization of oxidized antibody labeling with lucifer yellow CH.

The oxidation of antibody carbohydrate residues is a common approach used for the site-specific immobilization or modification of antibodies. One way of following this oxidation process is to label the resulting aldehyde groups with a dye such as Lucifer yellow CH (LyCH). This study examined the optimum conditions for preparing and purifying antibody-LyCH conjugates. A 250-fold excess of LyCH reacted with antibody at pH 6.5 for two or more hours gave maximum labeling. Nonreacted LyCH could be effectively removed by passing the labeled antibody through a size exclusion column, followed by one or two dialysis cycles. The LyCH antibody conjugates were found to be stable for at least three weeks when stored in pH 7.4 phosphate buffer.

Chromatographic and electrophoretic studies of protein binding to chiral solutes.

Protein interactions are important in determining the transport, metabolism and/or activity of many chiral compounds within the body. This review examines data that have been obtained on these interactions by various chromatographic and electrophoretic methods, especially those based on either high-performance liquid chromatography or capillary electrophoresis. Zonal elution, frontal analysis and vacancy methods are each considered, as are approaches that employ either soluble or immobilized proteins. There are a variety of different items that can be learned about a solute-protein system through these techniques. This includes information on the binding constants and number of binding sites for a solute-protein system, as well as the thermodynamic parameters, rate constants, interaction forces and binding site structure for the protein and solute. Numerous examples are provided throughout this review, as taken from the literature and from work performed within the author's laboratory.

Determination of the diol content of chromatographic supports by capillary electrophoresis.

A capillary electrophoresis (CE) method was developed for determining the diol content of supports used in high-performance affinity or size exclusion chromatography. This method involved oxidizing the diol-bonded support with periodate, followed by the use of CE to separate and quantitate the iodate produced by this reaction. Both the oxidation and separation conditions were considered in optimizing this assay. The final method was performed by reacting a known amount of support with a 20-fold excess of periodate in pH 4.0, 0.5 M acetate buffer, with pyromellitic acid being used as an internal standard. After allowing 5-10 min for oxidation, the mixture was filtered and the filtrate was injected onto a 57 cm x 50 microns I.D. fused-silica capillary operated at 25 kV and containing pH 4.0, 0.5 M acetate as the running buffer. The total separation time was 5 min per run and gave a detection limit of 0.1 mM iodate (or 0.1 mumol diol groups) for a 6-nl injection of a 1-ml reaction mixture. By varying the amount the support that was assayed, this method could be used with either porous or non-porous supports. This technique showed good correlation with an iodometric titration but required much less sample and time to perform.

Effect of mobile phase composition on the binding kinetics of chiral solutes on a protein-based high-performance liquid chromatography column: interactions of D- and L-tryptophan with immobilized human serum albumin.

This work examined the kinetic interactions of chiral solutes on immobilized protein columns, using the binding of D- and L-tryptophan to human serum albumin as a model. Based on band-broadening studies and previous measurements of the association equilibrium constants (Ka) for this system, estimates were obtained for the dissociation and association rate constants (kd and ka) for D- and L-tryptophan under a variety of operating conditions. The relative importance of ka versus kd in creating changes in the overall binding affinity was then considered. For example, an increase in temperature from 4 to 45 degrees C gave a large change in ka for L-tryptophan that was due both to an increase in kd and to a decrease in ka, while ka and kd for D-tryptophan showed a parallel increase that led to a much smaller temperature dependence for Ka. Similar comparisons between ka, kd and Ka were performed over a range of pH values, ionic strengths and solvent polarities. It was also possible from these studies to examine the changes in enthalpy and entropy that accompanied the formation of the activated complex between human serum albumin and each solute. The results from this work were then used to illustrate the importance of kinetics and band-broadening in protein-based chiral separations, and an example was provided showing how this type of kinetic data might be used to help optimize such separations.

Non-linear elution effects in split-peak chromatography. II. Role of ligand heterogeneity in solute binding to columns with adsorption-limited kinetics.

The split-peak effect is a useful phenomenon in studying the kinetic behavior of chromatographic supports. This work examined the combined role of ligand heterogeneity and non-linear elution conditions (i.e., sample load dependence) on the solute free fractions that are measured during split-peak studies. Exact expressions were derived to describe the effects of ligand heterogeneity under linear elution conditions, and simulation models were developed to specifically examine the combined effects of ligand heterogeneity and non-linear elution in systems with adsorption-limited rates for solute binding. The simulations showed that ligand heterogeneity increased the amount of free solute seen at any flow-rate or sample size, with this being most noticeable when using low flow-rates or large samples. One application in which these increases were examined in detail concerned the use of the split-peak effect for association rate constant measurements. It was found that linear extrapolation methods developed for homogeneous systems (as a correction for non-linear elution conditions) could successfully be applied to columns containing heterogeneous ligands. Columns containing immobilized protein A and/or protein G were used as experimental models to test the validity of the simulations; the behavior of these columns showed good quantitative and qualitative agreement with the predicted theoretical results.

Role of binding capacity versus binding strength in the separation of chiral compounds on protein-based high-performance liquid chromatography columns. Interactions of D- and L-tryptophan with human serum albumin.

Frontal analysis was used to examine changes in the association constant (Ka) and moles of binding sites (mL) for D- and L-tryptophan on an immobilized HSA column under various elution conditions. Both enantiomers had single-site interactions under all conditions tested. At pH 7.0 and 25 degrees C, the strength of L-tryptophan/HSA binding was determined mostly by the change in enthalpy of the system, while D-tryptophan/HSA binding was dominated by the change in entropy. The interactions of L-tryptophan with HSA showed a large change when varying the temperature, pH, ionic strength or 1-propanol content of the mobile phase. In each case, changes in Ka accounted for most of the shifts in retention that were seen for L-tryptophan during zonal elution studies. However, mL for this compound was also affected when varying the pH and 1-propanol levels. Changes in Ka were responsible for most of the shifts in D-tryptophan retention that were seen when adjusting the mobile phase pH or ionic strength. In addition, the value of mL for D-tryptophan was affected by pH, temperature and 1-propanol levels. It was concluded that varying such chromatographic conditions can alter either the binding strength or number of binding sites for solutes injected onto immobilized protein columns.

Development of dihydrazide-activated silica supports for high-performance affinity chromatography.

A method of preparing dihydrazide-activated silica was developed for use in high-performance affinity chromatography (HPAC). This support was made by oxidizing diol-bonded silica and reacting it with oxalic or adipic dihydrazide. The steps involved in this synthesis were studied and confirmed by FTIR. Items considered in optimizing the preparation of the support included the amount of dihydrazide added and the reaction time or pH used. Control of dihydrazide bifunctional attachment was obtained by varying the extent of diol-bonded silica oxidation. This support was successfully used in the immobilization of oxidized antibodies, horse radish peroxidase (a glycoenzyme) and transfer RNA. In each case, data indicated that immobilization was through site specific coupling rather than non-specific adsorption. Dihydrazide-activated silica was found to be stable for 2-6 weeks after preparation when stored at 5 to 25 degrees C. The linkage between oxidized biomolecules and this support was stable for at least one month in the presence of various solvents commonly used in HPAC.

Optimization of post-column chemiluminescent detection for low-molecular-mass conjugates of acridinium esters.

In this study, various factors are examined that affect the post-column chemiluminescence detection of low-molecular-mass compounds labelled with acridinium esters, using 9-phenyl acridinium ester (PAE) as the initial model. Reaction conditions examined included the effect of hydroxide or hydrogen peroxide concentration, ionic strength and surfactant content on the degree and length of light production by the acridinium ester label. Based on these results, a system was developed for the post-column detection of acridinium ester conjugates in HPLC. The final post-column reactor had optimum conditions similar to those for a benchtop luminometer but gave light production over slightly longer periods of time and had a broader range of reagent concentrations that gave a maximum response. The detection limit of this system for PAE was 7 x 10(-19) mol per injection, with linear and dynamic ranges that extended up to 3 x 10(-15) and 3 x 10(-13) mol, respectively. Some preliminary work was conducted examining the use of this system in chromatography-based competitive binding immunoassays with a thyroxine-acridinium ester conjugate being used as the label. The estimated limit of detection was 2.5 x 10(-17) mol (or roughly 10(-12) M for a 25 microliters sample) based on the retained fraction of the conjugate.

Characterization of the protein binding of chiral drugs by high-performance affinity chromatography. Interactions of R- and S-ibuprofen with human serum albumin.

Zonal elution and high-performance affinity chromatography were used to study the different binding characteristics of R- and S-ibuprofen with the protein human serum albumin (HSA). This was done by injecting small amounts of R- and S-ibuprofen onto an immobilized HSA column in the presence of a mobile phase that contained a known concentration of R- or S-ibuprofen as a competing agent. These studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column. In addition, S-ibuprofen had at least one other major binding region. The association equilibrium constant for R-ibuprofen with HSA was found to be 5.3 x 10(5) M-1 at pH 6.9 and 25 degrees C. Under the same conditions, the association constants for S-ibuprofen at its two sites were 1.1 x 10(5) M-1 and 1.2 x 10(5) M-1. The S-ibuprofen sites were present in about a 1:1 ratio and appeared to exhibit some allosteric interactions at high S-ibuprofen concentrations. The chromatographic technique used in this work is a general one which can be adapted for use in studying the interactions of other chiral compounds with either HSA or additional proteins.

Antibody immobilization to high-performance liquid chromatography supports. Characterization of maximum loading capacity for intact immunoglobulin G and Fab fragments.

This study examined various factors that affect the maximum amount of intact immunoglobulin G (IgG) or Fab fragments that can be covalently immobilized to silica and other HPLC-grade supports for use in immunoaffinity chromatography or immunoextractions. Factors that were considered included the amount of surface area available for immobilization, the pore size of the support, the type of immobilization method and the nature of the support matrix. The main factor in determining the extent of immobilization was found to be the relationship between the support's surface area and the ability of the IgG or Fab fragments to reach this surface. Access to the support surface was a function of the size of the protein being immobilized and the support porosity, with maximum immobilization being obtained with supports having pore sizes of approximately 300 A for intact IgG and 100 A for Fab fragments. Some differences in the maximum level of immobilization were noted between different coupling methods. Supports like Poros and Emphaze gave similar results to those seen with HPLC-grade silica when a comparison was made between materials with comparable pore sizes. Many of the trends observed in this work for IgG and Fab fragments should apply to other proteins that are to be immobilized to HPLC supports.

Chiral separations in capillary electrophoresis using proteins as stereoselective binding agents.

Proteins are receiving increased attention in capillary electrophoresis (CE) for use as ligands in chiral separations. This review examines the types of proteins that have been employed in CE for the analysis of chiral compounds and provides a summary of applications for such an approach. Several formats for this type of analysis are discussed, including methods that use immobilized proteins (i.e., affinity capillary electrochromatography and capillary gel affinity electrophoresis) or techniques that use proteins as running buffer additives (i.e., affinity capillary electrophoresis). The role of various experimental factors in these separations are considered, and a comparison is made between the general analytical properties of CE systems that use immobilized versus solution-phase proteins.

Affinity chromatography: a review of clinical applications.

Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes.

Survey of recent advances in analytical applications of immunoaffinity chromatography.

Methods that use immunoaffinity chromatography (IAC) for sample preparation or detection are becoming increasingly popular as tools in the analysis of biological and nonbiological compounds. This paper presents an overview of immunoaffinity chromatography and examines some recent developments of this technique in analytical applications. The emphasis is placed on HPLC-based IAC methods or those that combine IAC with other instrumental techniques; however, novel approaches that employ low-performance IAC columns for chemical quantitation are also considered. Particular applications that are examined include (1) the use of IAC in the direct detection of analytes, (2) the extraction of samples by IAC prior to on- or off-line detection by other methods, (3) the use of IAC in chromatographic-based immunoassays, and (4) the development of postcolumn reactors based on IAC for the detection of analytes as they elute from other types of chromatographic columns. The advantages and limitations for each approach are considered. In addition, a summary is provided of reports in the literature that have used IAC for these various formats.

Chiral separations in capillary electrophoresis using human serum albumin as a buffer additive.

This study examined the theory and mechanisms of chiral separations in capillary electrophoresis based on the use of proteins as buffer additives. Human serum albumin (HSA) was used as the model ligand; D,L-tryptophan and (R,S)-warfarin were used as the test analytes to be separated by this protein. Items examined in this work included the amount of HSA adsorbed to the capillary wall and the stability of this adsorbed protein layer. These were investigated by performing frontal analysis on the capillary with HSA and by injecting neutral markers through the capillary at different applied voltages before and after HSA treatment. The role of adsorbed HSA vs HSA in the buffer in determining the stereoselectivity of the CE system was also examined. Adsorbed HSA was the predominant agent involved in the separation of (R,S)-warfarin, while HSA in the buffer had the most significant effect in the resolution of D,L-tryptophan. Two distinct separation mechanisms were proposed to explain these differences. Good agreement was obtained between the results predicted by these mechanisms and the experimental data. Under optimized conditions, both pairs of enantiomers were separated with baseline resolution in less than 12 min.

Automated determination of antibody oxidation using flow injection analysis.

The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10(-8) and 4 x 10(-7) M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10(-5) and 7 x 10(-3) M. The within-run precision was +/- 5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.

Chiral separation mechanisms in protein-based HPLC columns. 1. Thermodynamic studies of (R)- and (S)-warfarin binding to immobilized human serum albumin.

This work characterizes the thermodynamic processes involved in the binding and separation of (R)- and (S)-warfarin on a high-performance human serum albumin (HSA) column. Frontal analysis was used to determine the strength and degree of binding for each enantiomer. (R)- and (S)-warfarin were found to bind at the same region on HSA; however, (R)-warfarin had a larger number of column binding sites. The number of binding sites for both enantiomers showed a slight increase with temperature. The total changes in free energy for (R)- and (S)-warfarin binding were similar at 37 degrees C, but the contribution due to entropy was greater for the R-enantiomer. These results suggested that (R)-warfarin was interacting mainly with the binding site interior, while (S)-warfarin interacted more with the site's outer surface. This model was confirmed by examining the retention of (R)- and (S)-warfarin on the HSA column under various pH, ionic strength, and organic modifier conditions. The different changes in entropy for these solutes made it possible to vary their separation by changing column temperature. Both thermodynamic properties and column binding capacities were found to be important in determining the degree of separation obtained for these compounds.

Characterization of thyroxine-albumin binding using high-performance affinity chromatography. I. Interactions at the warfarin and indole sites of albumin.

A high-performance affinity column containing immobilized human serum albumin (HSA) was used to study the binding of thyroxine at the warfarin and indole sites of HSA. Frontal analysis, using R-warfarin and L-tryptophan as probes for these sites, demonstrated that the immobilized HSA had binding behavior equivalent to that observed for HSA in solution. By injecting R-warfarin or L-tryptophan in the presence of excess thyroxine, it was found that thyroxine was binding directly to both types of site. The warfarin and indole sites had relatively strong binding for thyroxine, with association constants at 37 degrees C of 1.4 x 10(5) and 5.7 x 10(5) M-1, respectively. The value of delta G for these sites ranged from -7 to -8 kcal/mol and had a significant entropy component. The techniques used in this study are not limited to thyroxine-HSA interactions, but should also be valuable in examining the site-specific binding of other drugs and hormones to HSA.

Dual-column determination of albumin and immunoglobulin G in serum by high-performance affinity chromatography.

High-performance affinity chromatography was used for the simultaneous determination of albumin and immunoglobulin G in serum. Two columns in series, the first containing immobilized anti-albumin antibodies and the other containing protein A for binding immunoglobulin G, were eluted separately at pH 3 by means of a column-switching system. This method gave results in good agreement with commercially available methods, while requiring only 2 microliter of serum and 6.0 min per cycle. It was shown that albumin and immunoglobulin G were selectively retained, with little interference from other components, including immunoglobulins A and M.

High-performance immunoaffinity chromatography and chemiluminescent detection in the automation of a parathyroid hormone sandwich immunoassay.

An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.