Improvements in steroid receptor assays including rapid computer analysis of data

Improvements on the steroid receptor assay have been developed which eliminate tedious procedures and give faster and more accurate results. A protein assay (Waddell method) is discussed which directly measures peptide bonds in cytosolic material. It is more rapid and more sensitive than the Lowry method. Programs for the analysis of data obtained by either the dextran-coated charcoal method or the sucrose density gradient have been written. These are superior to previously published programs and can be incorporated into any laboratory with a minimum of cost. The program for the dextran-coated charcoal method converts the counts per minute from both total and nonspecific ³H-steroid binding activity into information needed for a Scatchard analysis. These data are plotted and a linear regression and determination coefficient for best fit of the data are performed. In the sucrose density gradient method program, the counts per minute from both total and nonspecific ³H-steroid binding activity is converted to disintegrations per minute and plotted versus the corresponding fraction number. The specific binding activity in femtomoles/milligram of protein in the various molecular forms can then be obtained through set programmed steps.     

Quantitative evaluation of solution equilibrium binding interactions by affinity partitioning: application to specific and nonspecific protein-heparin interactions

A variation of the quantitative affinity chromatography (QAC) method of Winzor, Chaiken, and co-workers for the analysis of protein-ligand interactions has been developed and used to characterize sequence-specific and nonspecific protein-heparin interactions relevant to blood coagulation. The method allows quantitation of the binding of two components, A and B, from the competitive effect of one component, B, on the partitioning of the other component, A, between an immobilized acceptor phase and solution phase at equilibrium. Under the conditions employed, the differences in total A concentrations yielding an equivalent degree of saturation of the immobilized acceptor in the absence and presence of B defines the concentration of A bound to B in solution, thereby enabling conventional Scatchard or nonlinear least-squares analysis of the A-B equilibrium interaction. Like the QAC method, quantitation of the competitor interaction does not depend on the nature of the affinity matrix interaction, which need only be described empirically. The additional advantage of the difference method is that only the total rather than the free competitor ligand concentration need be known. The method requires that the partitioning component A be univalent, but allows for multivalency in the competitor, B, and can in principle be used to study binding interactions involving nonidentical, interacting, or nonspecific overlapping sites. Both the binding constant and the stoichiometry for the specific antithrombin-heparin interaction as well as the apparent binding constant for the nonspecific thrombin-heparin interaction at low thrombin binding densities obtained using this technique were in excellent agreement with values determined using spectroscopic probes.     

A nonlinear regression program for small computers

A BASIC computer program for performing weighted nonlinear regression is described and a listing of the program is given. The program, which is small and simple to use, has been designed to be run by users with little knowledge of mathematics or computers. Robust methods of analysis are described which may be applied to data in which experimental errors are not normally distributed, and the program incorporates one such method. It is shown that the program is useful for the analysis of data conforming to the Michaelis-Menten equation, a single exponential, and to binding equations, and other applications are discussed.     

Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immunoagglutination assay

A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay. For this purpose a theoretical model of the initial agglutination kinetics, based on particle diffusion combined with a steric factor determined by the level of specific and nonspecific agglutination, was developed. The theoretical model combined with the experimental data can be used to optimize an agglutination-based assay with regard to sensitivity and dynamic range and to estimate the affinity, receptor surface density, molecular and binding site sizes, and level of nonspecific binding that is present in the assay. The experimental results are in good agreement with the theoretical model, indicating the usefulness of the model for immunoagglutination assay optimization.     

Improved gel-filtration method for analysis of estrogen receptor binding

A method has been described whereby 100- to 300-mg amounts of breast tumor tissue can be analyzed for cytoplasmic estrogen receptor binding capacity. This procedure differentiates specific from nonspecific estrogen binding. In addition, competitive adsorption of estrogen to Sephadex gel has been measured and true receptor binding capacities evaluated. By submitting the corrected receptor binding data to Scatchard plot analysis, the dissociation constant of the estrogen receptor complex was determined with greater accuracy.     

Dynamic Strategies for Target-Site Search by DNA-Binding Proteins

Gene regulatory proteins find their target sites on DNA remarkably quickly; the experimental binding rate for lac repressor is orders-of-magnitude higher than predicted by free diffusion alone. It has been proposed that nonspecific binding aids the search by allowing proteins to slide and hop along DNA. We develop a reaction-diffusion theory of protein translocation that accounts for transport both on and off the strand and incorporates the physical conformation of DNA. For linear DNA modeled as a wormlike chain, the distribution of hops available to a protein exhibits long, power-law tails that make the long-time displacement along the strand superdiffusive. Our analysis predicts effective superdiffusion coefficients for given nonspecific binding and unbinding rate parameters. Translocation rate exhibits a maximum at intermediate values of the binding rate constant, while search efficiency is optimized at larger binding rate constant values. Thus, our theory predicts a region of values of the nonspecific binding and unbinding rate parameters that balance the protein translocation rate and the efficiency of the search. Published data for several proteins falls within this predicted region of parameter values.     

Comparison of filtration and equilibrium dialysis methods for [3H]imipramine binding to human platelets

Receptor binding of imipramine in human platelets was assessed by filtration through glassfiber filters and by equilibrium dialysis. Both methods yield drug-receptor dissociation constants of similar magnitude (10^?9m) to literature values. However, the density of binding sites (B_max) was fivefold lower by filtration (473 ± 92 fmol/mg protein) compared to equilibrium dialysis (2652 ± 765 fmol/mg protein). Dialysis allows direct assessment of free imipramine and avoids drug loss during the separation step of the filtration assay. Additional advantages were found for computer nonlinear regression analysis of untransformed data to eliminate errors owing to linear transformation in the Scatchard analysis and for simultaneous quantitation of nonspecific and total drug binding.     

Characterization of human follicle-stimulating hormone binding to human granulosa cells by an immunoenzymological method.

An original, nonradiometric method has been developed for studying the binding parameters of native follicle-stimulating hormone (FSH) to its specific receptors in human ovarian granulosa cells. After binding and washing of the cells, hFSH was desorbed from its receptors and quantitatively measured by a specific enzyme immunoassay (EIA) in which nonspecific binding was estimated in the presence of an excess of equine chorionic gonadotropin (eCG/PMSG), which binds to human FSH receptors but does not interfere in the hFSH EIA. This method makes use of native nonmodified hFSH molecules (in contrast to radiometric methods) and permits direct estimation of the binding parameters (Kd and total number of sites). The Kd of hFSH for its human granulosa receptors measured by this technique (4.8 +/- 0.3 x 10(-10) M) is close to that determined by other methods. However, we found a total number of specific FSH receptors per granulosa cell (1 to 6 x 10(4) higher than that reported by others by Scatchard analysis of competition dose-response curves in radioreceptor assays. The method is also sensitive enough to measure the in vivo occupancy of receptors by endogenous hFSH, which was found to be less than 6% in women undergoing hormonal treatment for in vitro fertilization.     

Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H' DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H' DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.     

Estimation of activity related energy expenditure and resting metabolic rate in freely moving mice from indirect calorimetry data

Physical activity (PA) is a main determinant of total energy expenditure (TEE) and has been suggested to play a key role in body weight regulation. However, thus far it has been challenging to determine what part of the expended energy is due to activity in freely moving subjects. We developed a computational method to estimate activity related energy expenditure (AEE) and resting metabolic rate (RMR) in mice from activity and indirect calorimetry data. The method is based on penalised spline regression and takes the time dependency of the RMR into account. In addition, estimates of AEE and RMR are corrected for the regression dilution bias that results from inaccurate PA measurements. We evaluated the performance of our method based on 500 simulated metabolic chamber datasets and compared it to that of conventional methods. It was found that for a sample time of 10 minutes the penalised spline model estimated the time-dependent RMR with 1.7 times higher accuracy than the Kalman filter and with 2.7 times higher accuracy than linear regression. We assessed the applicability of our method on experimental data in a case study involving high fat diet fed male and female C57Bl/6J mice. We found that TEE in male mice was higher due to a difference in RMR while AEE levels were similar in both groups, even though female mice were more active. Interestingly, the higher activity did not result in a difference in AEE because female mice had a lower caloric cost of activity, which was likely due to their lower body weight. In conclusion, TEE decomposition by means of penalised spline regression provides robust estimates of the time-dependent AEE and RMR and can be applied to data generated with generic metabolic chamber and indirect calorimetry set-ups.     

Development and validation of a kinetic assay for analysis of anti-human interleukin-5 monoclonal antibody (SCH 55700) and human interleukin-5 interactions using surface plasmon resonance

A method to assess the kinetic interactions of a humanized anti-human interleukin-5 (IL-5) monoclonal antibody (SCH 55700) with native human IL-5 using surface plasmon resonance (SPR) has been developed and validated. Since there are no clearly defined validation requirements for a SPR-based binding kinetic assay, the validation strategy was based on the guidelines stipulated by the International Conference on Harmonization for Analytical Method Validation. Due to the uniqueness of the method, however, proper interpretation of the guidance was critical for establishing a validation plan. Validation was designed to assess repeatability, intermediate precision, specificity, linearity, and robustness which included analysis of baseline stability and reproducibility of ligand immobilization. Additionally, system suitability criteria were established to assure that the assay consistently performs as it was intended. The experimental artifacts that can complicate kinetic analysis using biosensor technology, such as heterogeneity of the ligand, mass transport, and nonspecific binding, were considered during the development of this assay. For each run, replicate concentrations of SCH 55700 were injected randomly over the immobilized surfaces to acquire association- and dissociation-phase data. The data were transformed and double referenced to remove systematic deviations seen in the binding responses. Association and dissociation rates were determined using a bivalent analyte model for curve fitting.     

An automated Western blot analysis using the phastsystem

We have developed a rapid, automated method for preparing Western blots of very small amounts of proteins, utilizing a commercially available electrophoresis system (Phastsystem, Pharmacia). This system has been adapted to transfer to nitrocellulose experimental samples that were initially separated in the same system by gradient-sodium dodecyl sulfate gel electrophoresis. The developing unit of the Phastsystem has permitted automation of all the necessary steps including incubation with antibodies, saturation of nonspecific binding sites, and washing. The total elapsed time for protein separation, transfer, and staining is about 6 to 7 h.     

Rapid affinity measurement of protein-protein interactions in a microfluidic platform

A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to μM range.     

Analysis of p53 "latency" and "activation" by fluorescence correlation spectroscopy. Evidence for different modes of high affinity DNA binding

The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the "activation" phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of "latent" and "activated" p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions.     

Differences among techniques for high-abundant protein depletion

The need to identify protein or peptide biomarkers via readily available biological samples like serum, plasma, or cerebrospinal fluid is often hindered by a few particular proteins present at relatively high concentrations. The ability to remove these proteins specifically, reproducibly, and with high selectivity is increasingly important in proteomic studies, and success in this procedure is leading to an ever-increasing list of lower abundant proteins being identified in these biological fluids. The current work addresses some of the potential problems in depleting proteins in typical biomarker studies, including nonspecific binding during depletion procedures and whether low molecular weight (LMW) species bind to the column in a so-called "sponge" effect caused by the ability of albumin or other high-abundant proteins to bind peptides or protein fragments. LC-MS/MS methods were applied to the comparative analysis of an IgG-based immunodepletion method and a Cibacron blue (CB)-dye-based method, for specificity of removing targeted proteins (binding fraction), as well as for assessing efficiency of target removal. This analysis was extended to examine the effects of repeated use of materials (cycles of binding and elution), in order to assess potential for carryover of one sample to the next. Capacity studies and efficiency of protein removal from the serum samples were followed for the IgG-based system using both immunochemical assays (ELISA) as well as LC-MS/MS methods. Additionally, the IgG-based system was further characterized for the removal of LMW polypeptides by nonspecific binding. We conclude that the IgG-based system provided effective removal of targeted proteins, with minimal carryover, high longevity, and minimal nonspecific binding. Significant differences are noted between the depletion techniques employed, and this should be considered based on the expectations set during experimental design.     

Protein patterning on silicon-based surface using background hydrophobic thin film

A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOP). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 microg/ml of total protein concentration, the CYTOP hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin-biotin system.