Kinetic analysis of unpurified native antigens available in very low quantities and concentrations

Affinity measurements of antigen–antibody interactions are generally performed using known concentrations of purified or recombinant materials. In addition, many technologies that measure affinity require the interacting components to be present in at least microgram quantities. Specifically, if the antigen is either available only in low quantities or unable to be purified, or if the quantity is unknown, then the measurement of affinity can be very difficult. Using the Kinetic Exclusion Assay (KinExA) technology, here we describe a method that overcomes the requirement for large amounts of purified and known quantities of antigen. We used this method to precisely measure the affinity of fully human anti-human interleukin 13 (IL13) monoclonal antibodies to IL13 produced in native form from primary T cells derived from a variety of species, including human. These antigens were available only in the limited quantities present in the conditioned cell culture medium, and the affinity was measured directly without further purification.     

Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8

The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen.     

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.     

Chemiluminescence ELISA for the detection of antibodies to bovine leukaemia virus antigens

A chemiluminescence enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine leukaemia virus antigens (BLV) has been developed. The possibility of using an enhanced chemiluminescence reaction for the determination of adsorbed immunoperoxidase conjugates was studied in this work. The intensity of chemiluminescence depends on both the concentration of reagents and experimental conditions used. The efficiency of the assay is determined by the formation of an immobilized antigen monolayer. A relationship between the quantity of the protein added and adsorbed has been shown. The optimal time and temperature for the antigen–antibody incubation steps have been estimated for each system (3h at 37°C was chosen as a standard incubation time). A linear dependence of the chemiluminescence intensity and optical density on the concentration of antibodies to the BLV antigens was observed. The detection limit of antibodies in the chemiluminescence ELISA is 2–3 times lower than that in the spectrophotometric one. The results obtained indicate the possibility of using both methods.     

Unique single‐domain antigen binding fragments derived from naturally occurring camel heavy‐chain antibodies

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy‐chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy‐chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy‐chain antibodies. The presence of the heavy‐chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen‐binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen‐binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti‐idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site. Copyright © 1999 John Wiley & Sons, Ltd.     

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site, proved to be the preferential one, being 6.50 x l0(5), 1.94 x 10(6) and 8.94 x 10(5), respectively. In addition, it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy.     

Species specificity of hybridoma antibodies to surface antigens of guinea pig sperm

The species specificity of hybridoma antibodies to sperm surface antigens was studied. A collection of over 50 hybridoma antibodies that bind to the guinea pig sperm surface was tested for binding to mouse, rat, hamster, and human sperm by indirect immunofluorescence. None of the antibodies bind to mouse sperm. rat sperm, or human sperm. All but three of the antibodies also fail to bind to hamster sperm. AH-30, AH-31, and AH-1032, the three antibodies that crossreact with hamster sperm, show a different topographical localization on hamster sperm from that seen on guinea pig sperm. The three antibodies do not precipitate a ¹²⁵I surface-labeled antigen from hamster sperm extracts. However, from guinea pig sperm extracts, all three antibodies precipitate ¹²⁵I surface-labeled polypeptides with molecular weights (M_r) of 62,000, 52,000, and 38,000. This result suggests that the crossreacting antibodies may be recognizing different antigens on hamster and guinea pig sperm.     

Chromatographic analysis of acetohexamide binding to glycated human serum albumin

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K_a) for the high affinity interactions was in the range of 1.2–2.0 × 10⁵ M⁻¹ and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K_a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K_a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.     

High-affinity binding of seminal plasma PSP94 to human immunoglobulin is through the Fab domain

Prostate secretory protein of 94 amino acids (PSP94) is one of the major proteins present in human seminal plasma. We had earlier reported that PSP94 has the ability to bind to human IgG. The aims of the present study were to further delineate the PSP94–IgG interaction and to understand whether this could have any significance in sperm function. Direct binding of IgG fragments to PSP94 showed maximal binding with F(ab′)₂ followed by Fab, while Fc displayed least binding in ELISA. Binding kinetics of PSP94–IgG interaction using surface plasmon resonance (SPR) revealed high-affinity binding of IgG to PSP94 with a dissociation constant (K_D) of 8.8 × 10⁻¹¹ M. PSP94–IgG interaction was found to be through the Fab domains of IgG. Real-time interaction kinetics revealed association constants for binding of IgG, Fab, and F(ab′)₂ towards PSP94 to be of the same order but with altered dissociation constants. IgG and its F(ab′)₂ fragment once complexed to PSP94 demonstrated negligible dissociation, while dissociation rate of Fab fragment was 6.6 × 10⁻⁴. In silico molecular modeling of PSP94–IgG complex identified N- and C-terminal β-strands of PSP94 to be the most plausible region involved in IgG interaction. Immunofluorescence studies revealed that IgG bound to human spermatozoa predominantly in the tail region, which could be prevented when IgG was preincubated with PSP94. This study reports for the first time that IgG forms a high-affinity complex with PSP94 through its F(ab′)₂ domain and reveals the ability of PSP94 to prevent binding of IgG to spermatozoa.     

Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K _ass) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K _ass of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k _diss) for anti-HRP shoulder of Babs was 21 times higher than k _diss for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody–enzyme conjugates in the case of binding of bivalent Mabs.     

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of¹²⁵I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of¹²⁵I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.     

Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, O9) of Salmonella typhi

A panel of monoclonal antibodies were generated against the surface polysaccharide antigens of the cell envelope of Salmonella typhi. Four clones (IgM) were specific for the capsular Vi polysaccharide, and one clone (IgG3) reacted selectively with the S. typhi lipopolysaccharide in enzyme immunoassay. On the basis of their reactivity pattern and binding affinity, MATy-V7 (IgM) and MATy-O9 (IgG3) antibodies were selected for further characterization of their antigenic specificity. In an inhibition enzyme immunoassay with rabbit factor-specific anti-Salmonella antibodies as the competing agents, the reactivity of MATy-V7 and MATy-O9 were significantly inhibited by the anti-Vi and anti-O9 antisera, respectively. Moreover, both the Vi- and O9-specific monoclonal antibodies were shown to be useful serotyping agents by correct identification in slide agglutination tests of 32 clinical isolates of all the S. typhi and other serogroup D salmonellae among a total of 140 bacterial isolates representing eight different Enterobacteriaceae genera tested.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements

Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K(ds)) to double-stranded DNA to determine K(ds) as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K(ss), the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD.     

Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)

Antibodies to a low molecular weight folate binding protein isolated from human milk were raised in rabbits and used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for immunological characterization of human folate binding proteins (FBPs). The high and low molecular weight FBPs from human milk were immunologically indistinguishable. Furthermore, the FBPs in human urine and cerebrospinal fluid showed a cross-reactivity of 70% and 30%, respectively. No cross-reactivity of the FBP from cow's milk was observed.     

Chromatographic studies of changes in binding of sulfonylurea drugs to human serum albumin due to glycation and fatty acids

This report examines the use of high-performance affinity chromatography as a screening tool for studying the change in binding by sulfonylurea drugs to the protein human serum albumin (HSA) during diabetes. The effects of both the non-enzymatic glycation of HSA and the presence of fatty acids on these interactions were considered using a zonal elution format. It was found that there was a significant increase (i.e., 2.7- to 3.6-fold) in the relative retention of several sulfonylurea drugs (i.e., acetohexamide, tolbutamide, glybenclamide and gliclazide) on columns containing normal versus glycated HSA. The addition of various long chain fatty acids to the mobile phase gave the same trend in retention for the tested drugs on both the HSA and glycated HSA columns, generally leading to lower binding. Most of the fatty acids examined produced similar or moderately different relative shifts in retention; however, palmitic acid was found to produce a much larger change in retention on columns containing glycated HSA versus normal HSA under the conditions used in this study.     

Combinatorial screening of polymer nanoparticles for their ability to recognize epitopes of AAV‐neutralizing antibodies

A library of 17 nanoparticles made of acrylate and methacrylate copolymers is prepared, characterized, and screened against six epitopes of adeno‐associated viruses (AAV)‐neutralizing antibodies to assess their affinity and specificity. Peptide epitopes are immobilized onto the surface of glass beads, packed in filtration microplates, and incubated with fluorescein‐labelled nanoparticles. Following intense washing, the affinity of nanoparticles to immobilized epitopes is assessed by measuring the fluorescence of captured nanoparticles. The results show that polar monomers, acrylic acid in particular, have a positive impact on polymer affinity towards all peptides used in this study. The presence of hydrophobic monomers, on other hand, has a negative impact on polymer binding. The composition of peptides used in this study has no noticeable impact on the affinity of synthesized nanoparticles. The affinity of nanoparticles with the highest affinity to peptide targets does not exceed millimolar level. Overall, it is found that the synthesized library showed modest affinity but lacked specificity, which should be further “tuned,” for example, by using molecular imprinting to achieve an acceptable level of affinity and specificity for practical application.