Allowance for antibody bivalence in the determination of association rate constants by kinetic exclusion assay

This investigation completes the amendment of theoretical expressions for the characterization of antigen–antibody interactions by kinetic exclusion assay—an endeavor that has been marred by inadequate allowance for the consequences of antibody bivalence in its uptake by the affinity matrix (immobilized antigen) that is used to ascertain the fraction of free antibody sites in a solution with defined total concentrations of antigen and antibody. A simple illustration of reacted site probability considerations in action confirms that the square root of the fluorescence response ratio, R_Ag/R_o, needs to be taken in order to determine the fraction of unoccupied antibody sites, which is the parameter employed to describe the kinetics of antigen uptake in the mixture of antigen and antibody with defined initial composition. The approximately 2-fold underestimation of the association rate constant (k_a) that emanates from the usual practice of omitting the square root factor gives rise to a corresponding overestimate of the equilibrium dissociation constant (K_d)—a situation that is also encountered in the thermodynamic characterization of antigen–antibody interactions by kinetic exclusion assay.     

Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay

Prior observations that questioned the validity of kinetic exclusion assays were based on the mistaken assumption that the assays quantified the fraction of those antibody molecules that had unoccupied binding sites. Instead, the standard KinExA assay quantifies the fraction of total antibody binding sites that are unoccupied, regardless of the number of unoccupied sites on each antibody molecule. Although the standard KinExA analysis assumes that there is only a small probability of antibody-site capture by the affinity matrix, the results of numerical simulations demonstrate the reliability of dissociation constants obtained by the standard KinExA analysis for capture probabilities as high as 30%. This finding further strengthens the potential of kinetic exclusion assays as the procedure of choice for the rapid and accurate characterization of immunochemical reactions that forms part of screening processes in the search for therapeutic antibodies.     

Homogeneous time-resolved G protein-coupled receptor–ligand binding assay based on fluorescence cross-correlation spectroscopy

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor–ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.     

Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases

Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI–MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis–Menten constant (K_m), and catalytic turnover rate (V_max/[E]_t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V_max/[E]_t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s⁻¹, respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8 s⁻¹). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K_m. Kinetic parameters obtained by the ESI–MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP–HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI–MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.     

A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E2 using enhanced luminol as substrate

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E₂ (PGE₂) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE₂ antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE₂ conjugate. Then, after preincubating, the anti-PGE₂ antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE₂ conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.     

Detection and comparison of protein-DNA interactions using DNA-BIND plate and horseradish peroxidase-based colorimetric assay

We describe a procedure for detection and comparison of protein–DNA interactions using DNA–BIND plate and horseradish peroxidase (HRP)-based colorimetric assay. Amino-modified oligonucleotide was covalently immobilized on the surface of DNA–BIND plate. After the complementary oligonucleotide was annealed, the plate was incubated with protein to allow sequence-specific DNA binding. Primary antibody and HRP-labeled secondary antibody were then employed, and colorimetric assay was conducted before the absorbance was read. This is a sensitive, specific, and high-throughput method that has been applied not only in the detection of protein–DNA interaction but also in the quantitative comparison of DNA-binding capabilities among wild-type and mutant proteins.     

Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (K_d, ∼10⁻¹¹ M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R² = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R² = 0.834). These results suggest that the BV–KinExA method described here provides a suitably accurate K_d evaluation of mAbs against proteins on the cell surface.     

Competitive immunoassay for analysis of vitamin B(12)

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B₁₂ by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B₁₂ and purified using affinity chromatography. Checkerboard assay was performed with vitamin B₁₂ antibody and vitamin B₁₂–alkaline phosphatase conjugate followed by its conjugate characterization using ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (K_a) of the vitamin B₁₂ antibody was found to be 4.23 × 10⁸ L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B₁₂ that showed 12% to 35%. The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2% and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity, specificity, precision, and recovery of vitamin B₁₂ with spiked multivitamin injections, tablets, capsules, and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000 to 10,000 ng/ml. After extraction of vitamin B₁₂ using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.     

Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcoreTM binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab′

The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti-idiotype antibody 3-2G12 has been used as a model system to demonstrate experimentally the influence of non-ideal binding effects on BIAcore^TM binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5×10⁵ M ⁻¹ s⁻¹), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised. In addition, the application of site-specific immobilisation procedures was found to improve considerably the correlation of experimental binding data to the ideal 1:1 kinetic model such that the discrepancy between experimental and fitted curves was within the noise range of the instrument. Experiments performed to measure the equilibrium constant (K_D) in solution resulted in a value of similar magnitude to those obtained from the ratio of the kinetic rate constants, even those measured with a heterogeneous ligand or with a significant mass transfer component. For this system, the experimental complexities introduced by covalent immobilisation did not lead to large errors in the K_D values obtained using the BIAcore © 1997 John Wiley & Sons, Ltd.     

Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)–V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K_D = 0.93 nM to K_D = 348 nM). That decrease in the affinity of drug–ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug–ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.     

High throughput solution-based measurement of antibody-antigen affinity and epitope binning

Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k_a) rate constant measurements suggest that this is mainly caused by inaccurate k_a measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods.     

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200‐μm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His₆‐tagged enzymes via Ni‐NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop‐flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK‐catalysed synthesis of L ‐erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis–Menten model. Results show that the TK kinetic parameters in the IEMR (V_max(app) = 0.1 ± 0.02 mmol min^–1, K_m(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the k_cat for the microreactor of 4.1 × 10⁵ s^?1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His₆‐immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell‐based systems for TK bioprocess characterization. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

Insulin receptor‐insulin interaction kinetics using multiplex surface plasmon resonance

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor‐insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real‐time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR‐A). We used a scaffold approach in which anti‐insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR‐A capture. The multiplex SPR system (ProteOn XPR36?, Bio‐Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two‐site binding pattern, consistent with the literature, with a high‐affinity site (dissociation constant K_D1 = 38.1 ± 0.9 nM) and a low‐affinity site (K_D2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K_D1 = 73.2 ± 1.8 nM and K_D2 = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high‐affinity site was converted to the low‐affinity site. The new SPR assay enables insulin‐eIR‐A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H₄PteGlu_n has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD–serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD–serine is quantified by comparison with standards. This method was used to determine the K_m and k_cat values for 5,10-CH₂–H₄PteGlu₅ of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH₂–H₄PteGlu₅ for an SHMT from any organism.     

Generation and characterization of biotinylated recombinant Fab antibody fragment against cortisol

This paper describes the in vivo generation method of biotinylated recombinant Fab antibody fragments. The original molecular vectors for Escherichia coli Fab fragments expression were designed. In vivo biotinylated recombinant antibody Fab fragment against cortisol was generated. The kinetic parameters of interaction of these antibody fragments with cortisol-BSA complex were measured via the biolayer interferometry method. An equilibrium dissociation constant of this interaction K_D is 5.48 × 10^–10 M. The interaction is reversible and it could be competitively inhibited by free cortisol addition. These results could be used in generating of immunoassays for quantitative cortisol determinations. The in vivo biotinylation system under review is universal and suitable for expression of any biotinylated Fab fragments in the E. coli system.     

A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate

Phytase (EC 3.1.3.–) hydrolyzes phytate (IP₆) present in cereals and grains to release inorganic phosphate (P_i), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P_i liberated from IP₆. This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP₆–lysozyme as a substrate. The assay is based on the principle that IP₆ forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP₆ in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P_i from IP₆. This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl₂, and phosphate, showed a concentration-dependent interference.     

Contributions of cation-π interactions to the collagen triple helix stability

Cation–π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X–Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation–π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host–guest peptide system: (Pro-Hyp-Gly)₃-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)₃, where X is an aromatic residue and Y is a cationic residue, to study the cation–π interaction in the collagen triple helix. Circular dichroism (CD) measurements and T_m data analysis show that the cation–π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation–π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation–π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π–electron density and the cation–π interaction in collagen. Our data demonstrate that the cation–π interaction could play an important role in stabilizing the collagen triple helix.     

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody,M2590, to GM3-ganglioside

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside G_M3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of G_M3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of G_M3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.