Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.     

Research on smoking and lung cancer: a landmark in the history of chronic disease epidemiology

This paper describes the history of the epidemiologic research on lung cancer prior to 1970 and its effect on chronic disease epidemiology. In the 1930s, epidemiology was largely concerned with acute infectious diseases. As the evidence grew that the incidence of lung cancer was increasing among men, however, epidemiologists undertook research into the etiology of the disease. In 1950, Doll and Hill, in England, and Wynder and Graham, in the United States, published substantial case-control studies that implicated the use of tobacco as a major risk factor for the disease. A controversy developed over the credibility of this finding and was increased in 1954 when a cohort study by Doll and Hill and another by Hammond and Horn each gave estimates that the risk of lung cancer was greatly increased among smokers relative to the risk among comparable non-smokers. An account is given of the disputes surrounding these and related studies. The controversy had a stimulating effect in fostering the developing discipline of chronic disease and epidemiology.     

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.     

Stoichiometric binding of apolipoprotein B-specific monoclonal antibodies to low density lipoproteins

The structure of apolipoprotein B and its stoichiometry on plasma lipoproteins has been a major issue and one refractory to a variety of analyses. Immunochemical analyses represent an independent approach. Examinations of apolipoprotein B (apo-B) epitopes on human plasma low density lipoproteins (LDL) using monoclonal antibodies have consistently revealed the existence of extensive apo-B heterogeneity. In the present study, we have addressed the solution of the stoichiometry problem using quantitative analysis of the maximum number of identical antibodies that can be bound per LDL particle in which we take into account this ligand heterogeneity. We have estimated the molecular weight of apo-B by quantifying the number of times a given apo-B epitope is expressed on the surface of LDL. The quantitative binding of eight previously characterized monoclonal antibodies was measured in a fluid phase radioimmunoassay. The results were analyzed by Scatchard analysis and expressed on the basis of independent measurements of the maximum amount of LDL that could be bound by each antibody. Affinity constants for each of the eight antibodies varied between 8.5 X 10(7) and 80 X 10(7) M-1. For these same antibodies, the concentration of maximally bound antibody at a normalized LDL concentration of 1000 ng/ml was estimated to be 0.9-1.8 nM with a mean of 1.23 nM. Adopting a molecular mass from physicochemical analysis for LDL apo-B of 550,000 daltons, the molar ratio between bound antibody and LDL varied between 0.5 and 1.2 (mean 0.75 +/- 0.15). The results supported the hypothesis that apo-B is present as a single large molecular weight polypeptide in LDL.     

Comparison of Direct and Indirect Solid-Phase Microradioimmunoassays for the Detection of Viral Antigens and Antiviral Antibody

Viral antigens were fixed to the surface of microtiter wells, and serial dilutions of antiviral antibody were added. The amount of antiviral antibody bound to viral antigens was determined by measuring the extent to which the antiviral antibody either inhibited the specific binding of (125)I-labeled antiviral immunoglobulin G (IgG) (direct technique) or enhanced the specific binding of (125)I-labeled anti-IgG (indirect technique). Immune complexes composed of viral antigens and antiviral antibody (human) could be detected by the binding of (125)I-labeled rheumatoid factor. Specific binding was influenced by the concentration of protein in the diluents used during the different steps of the procedure. A high concentration of protein in the diluent used with the viral antigens decreased specific binding, whereas a high concentration of protein in the diluent used with (125)I-labeled anti-IgG increased specific binding by decreasing nonspecific attachment of the labeled anti-IgG. Under the conditions employed, the titer of a given antiviral serum was several hundredfold greater by the indirect than by the direct technique.     

Equilibrium theory for the binding of bivalent antibodies to regularly spaced sites on a DNA molecule

In the autoimmune disease, Systemic Lupus Erythematosus, an individual produces antibodies that bind to his or her own DNA. In this paper we consider a single, long DNA-like molecule in a solution containing bivalent antibodies that can bind to the DNA molecule at regularly spaced sites. The antibody can be attached to DNA by either one or two binding sites. We assume that, when an antibody molecule binds through both its sites, it spans a fixed number of free sites that remain accessible to antibody binding. In this model, antibody molecules can interdigitate along the DNA molecule. We allow steric hindrance within such interdigitating clusters of bound antibodies. We derive analytical expressions for the average number of free, monovalently bound and bivalently bound antibodies, and see how this distribution is influenced by steric hindrance and by the relative binding strengths of the singly and doubly bound antibody.     

Do antibody binding techniques identify polysomes synthesizing a specific protein?

Specific binding of antibody directed against MOPC-21 myeloma protein to MOPC-21 polysomes has been demonstrated. It was also shown that this same antibody bound specifically to the ribosomal subunits of these polysomes, suggesting that the antibody is not binding exclusively to the nascent polypeptide chains on the polysomes. The antibody was bound specifically to polysomes of a nonproducing variant XC1 which had been isolated in the presence of MOPC-21 myeloma protein while the antibody was not bound to XC1 polysomes isolated in the absence of myeloma protein. This suggests that the myeloma protein is adsorbed to the polysomes during the isolation procedure.     

碱性磷酸酶标A蛋白加强的PAP技术

本文介绍一种碱性磷酸酶标Ａ蛋白加强的ＰＡＰ技术。采用ＰＡＰ技术、碱性磷酸酶标Ａ蛋白（ＰＡＡＰ）技术ＰＡＡＰ和加强的ＰＡＰ（ＰＡＰ－ＰＡＡＰ）技术显示下丘脑室旁核催产素（ＯＴ）能神经元。结果发现,其中使用ＰＡＰ－ＰＡＡＰ技术免疫反应产物的显色最深。此技术的原理可能是,由于Ａ蛋白分子至少有四个位点能与ＩｇＧ分子的Ｆｃ段高亲合性地结合,故在该技术中,先经过ＰＡＰ程序的三步免疫反应并显色后,每个与一抗结合的二抗分子上和每个与二抗结合的ＰＡＰ复合物分子上各暴露一个能与Ａ蛋白分子结合的Ｆｃ段,在随后经过ＰＡＡＰ技术处理时,部分ＰＡＡＰ复合物分子就结合在这些Ｆｃ段上,经显色后,ＰＡＡＰ技术显示的浅紫兰色与ＰＡＰ技术显示的浅棕褐色重叠,变成更深的反差明显的深棕褐色。     

Antibody microarrays: an evaluation of production parameters

Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home-made slides, commercially available systems were also included in the analysis.     

Improved cholesterol phenotype analysis by a model relating lipoprotein life cycle processes to particle size

Increased plasma cholesterol is a known risk factor for cardiovascular disease. Lipoprotein particles transport both cholesterol and triglycerides through the blood. It is thought that the size distribution of these particles codetermines cardiovascular disease risk. New types of measurements can determine the concentration of many lipoprotein size-classes but exactly how each small class relates to disease risk is difficult to clear up. Because relating physiological process status to disease risk seems promising, we propose investigating how lipoprotein production, lipolysis, and uptake processes depend on particle size. To do this, we introduced a novel model framework (Particle Profiler) and evaluated its feasibility. The framework was tested using existing stable isotope flux data. The model framework implementation we present here reproduced the flux data and derived lipoprotein size pattern changes that corresponded to measured changes. It also sensitively indicated changes in lipoprotein metabolism between patient groups that are biologically plausible. Finally, the model was able to reproduce the cholesterol and triglyceride phenotype of known genetic diseases like familial hypercholesterolemia and familial hyperchylomicronemia. In the future, Particle Profiler can be applied for analyzing detailed lipoprotein size profile data and deriving rates of various lipolysis and uptake processes if an independent production estimate is given.     

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.     

Immunological detection of propolypeptide of von Willebrand factor on platelet surface

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet alpha-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.     

A novel CD44 antibody identifies an epitope that is aberrantly expressed on acute lymphoblastic leukaemia cells

Previous studies have shown that the antibody 7H9D6 identifies CD44, a glycoprotein receptor for hyaluronic acid. 7H9D6 recognizes an epitope of CD44 that is not always present on CD44 molecules. The 7H9D6 antibody bound to the hyaluronic acid binding domain of CD44 and inhibited cell adhesion to immobilized hyaluronic acid. However, the expression of the 7H9D6 epitope was not sufficient for hyaluronic acid binding. Immunofluorescent staining with 7H9D6 revealed a punctate surface staining pattern, suggesting that CD44 molecules recognized by 7H9D6 are located in clusters on the cell surface. In contrast, other CD44 antibodies produced a uniform staining pattern. Early bone marrow B cells were negative for 7H9D6 but reactive with other CD44 monoclonal antibodies. In contrast, leukaemic cells from 65% of patients (28 of 43) with B lineage acute lymphoblastic leukaemia bound 7H9D6. Patients expressing the 7H9D6 epitope on their leukaemic cells had an increased risk of death (HR 3.5 95% CI 1.1-10.9, P = 0.029) and of disease relapse (HR 3.2 95% CI 1.2-8.5, P = 0.017) when corrected for white cell count. This antibody may be useful for the detection of residual disease in B lineage acute lymphoblastic leukaemia and as a prognostic indicator and for the study of CD44 function.     

Bioluminescent immunosorbent for rapid immunoassays

We describe a bioluminescent immunoassay procedure which does not require a separation step to remove excess free label. A luminescent immunosorbent constituted of bacterial luciferase, FMN oxidoreductase, and an antibody coimmobilized on Sepharose is used to determine specifically the label enzyme (glucose-6-phosphate dehydrogenase, coupled to an antigen) bound by a specific antibody. The immunosorbent confines the bioluminescent reaction in a small volume, and the bound label produces NADH, which is directly used by the nearby luciferase FMN oxidoreductase enzyme system. On the contrary NADH produced by dehydrogenases in solution is directly oxidized without emitting light. Dehydrogenases contained in the biological sample do not interfere with the assay, which can be performed directly on 25 microliter of serum. In this paper we describe the general procedure and we analyze the different parameters that must be optimized.     

Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection

Antibodies are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated.     

Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis

A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.     

Quantitative immunochromatographic analysis: theory and application to theophylline immunoassay

The development of an immunochromatographic technique suitable for rapid analysis of biological fluids is described. Quasi-one-dimensional antibody lattices specific for theophylline were constructed by packing Sepharose beads conjugated with specific antibody into specially designed narrow capillary tubes. The design of these capillary columns was such that they would subtract a preset threshold quantity of antigen (label and analyte) from the total amount presented. Labeled antigen, which appeared in the flowthrough, could then be used to precisely quantitate the analyte present. The ideal format would permit very precise subtraction of 100% of the available antigen up to the threshold amount and none of the remainder. The microcolumn described here comes close to this ideal behavior through the attainment of very high ratios of bound/free antigen. The elevated bound/free ratio could be explained by theoretical analysis of the effect on equilibria of the high antibody concentration in this quasi-one-dimensional system. Lattices containing anti-theophylline antibodies were used to develop a competitive enzyme immunoassay for theophylline which demonstrated a dose-response that was closely similar to that predicted by theoretical treatment. The entire assay procedure was performed in less than 30 min and demonstrated a sensitivity limit of approximately 20 ng/ml. Preliminary studies on clinical serum samples suggest that this assay has potential for the routine analysis of biological fluids.     

Partition of free and monoclonal-antibody-bound horseradish peroxidase in a two-phase aqueous polymer system--novel procedure for the determination of the apparent binding constant of monoclonal antibody to horseradish peroxidase.

The principle that the antigen and the antibody prefer different phases in an aqueous two-phase system is the analytical basis of the work presented here. The antigen horseradish peroxidase, which is bound to a monoclonal antibody (mAb), is separated from free Ag in an aqueous phase system (polyethylene glycol (PEG)/dextran) as a function of the concentration of mAb. The plot of the partition coefficient kappa of horseradish peroxidase versus the concentration of mAb yields a sigmoidal curve similar to the curve obtained by enzyme-linked immunosorbent assay (ELISA). Comparing the plots normally used for ELISA in order to determine the apparent binding constant of mAb and the number of epitopes on the Ag we derived a relationship between the difference in partitioning of the free Ag and the bound Ag (delta kappa) and the concentration of mAb. The new linear plot of reciprocal delta kappa versus reciprocal concentration of mAb gives the apparent binding constant of mAb, which is evaluated from the slope. From the intercept at the ordinate the maximum difference of the partition coefficient of the free and bound antigen is derived and the apparent partition coefficient of the free monoclonal antibody can be calculated.     

Immunoassay of bovine and human low-density-lipoprotein receptors using monoclonal antibodies.

Two methods are described for the assay of low-density-lipoprotein (LDL) receptor protein based on the binding of receptor to microtitre plate wells coated with a specific monoclonal antibody or with LDL, followed by detection with radioactive antibody that recognizes a different part of the molecule. The two-antibody procedure detected approx. 2 ng of pure bovine receptor at twice background, and there was a linear relationship on a double-logarithm plot between radioactive antibody bound and the amount of receptor added, up to at least 500 ng of receptor protein per well. The procedure using immobilized LDL was less sensitive and the binding of receptor was inhibited by low concentrations of NaCl, which restricted its usefulness for routine assay of tissue extracts. LDL receptor protein could be readily assayed using the two-antibody procedure in normal human skin fibroblast extracts prepared by bulk-elution from small columns of DEAE-cellulose followed by rapid desalting. No radioactive antibody bound with extracts of cells from a receptor-negative familial hypercholesterolaemic subject. The LDL receptor content of normal fibroblasts preincubated with lipoprotein-deficient serum was estimated, using bovine receptor as standard, to be approx. 60 ng of receptor protein/mg of cell protein.     

Mining Pure,Strict Epistatic Interactions from High-Dimensional Datasets: Ameliorating the Curse of Dimensionality

Background

The interaction between loci to affect phenotype is called epistasis. It is strict epistasis if no proper subset of the interacting loci exhibits a marginal effect. For many diseases, it is likely that unknown epistatic interactions affect disease susceptibility. A difficulty when mining epistatic interactions from high-dimensional datasets concerns the curse of dimensionality. There are too many combinations of SNPs to perform an exhaustive search. A method that could locate strict epistasis without an exhaustive search can be considered the brass ring of methods for analyzing high-dimensional datasets.

Methodology/Findings

A SNP pattern is a Bayesian network representing SNP-disease relationships. The Bayesian score for a SNP pattern is the probability of the data given the pattern, and has been used to learn SNP patterns. We identified a bound for the score of a SNP pattern. The bound provides an upper limit on the Bayesian score of any pattern that could be obtained by expanding a given pattern. We felt that the bound might enable the data to say something about the promise of expanding a 1-SNP pattern even when there are no marginal effects. We tested the bound using simulated datasets and semi-synthetic high-dimensional datasets obtained from GWAS datasets. We found that the bound was able to dramatically reduce the search time for strict epistasis. Using an Alzheimer''s dataset, we showed that it is possible to discover an interaction involving the APOE gene based on its score because of its large marginal effect, but that the bound is most effective at discovering interactions without marginal effects.

Conclusions/Significance

We conclude that the bound appears to ameliorate the curse of dimensionality in high-dimensional datasets. This is a very consequential result and could be pivotal in our efforts to reveal the dark matter of genetic disease risk from high-dimensional datasets.