Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18 ng mL(-1)), sulfonamides (0.17 ng mL(-1)) and melamine (7.5 ng mL(-1)), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants.     

Directed evolution of an anti-human red blood cell antibody

We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis. A combinatorial library of 10⁷ clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay. Selected V_L and V_H sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab. Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.Key words: phage display, antibody maturation, Fab, antibody folding, scFv, agglutination     

Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency.

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.     

Identification and characterization of single chain anti-cocaine catalytic antibodies

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.     

Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5beta- or 5alpha-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5beta- or 5alpha-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.     

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.     

Docking Substrates to Metalloenzymes

Abstract

Carbonic Anhydrase (CA) is a metalloenzyme that reversibly catalyzes the interconversion between carbon dioxide and bicarbonate anion. A class of sulfa drugs, sulfonamides, are known to inhibit CA. One approach to identifying important binding and specificity interactions between sulfonamides and CA is to analyze the results from docking studies. Previous docking studies have mainly focused on the encounters of substrates with non-metalloenzymes. Here we report the application of MOE-Dock to the CA II – sulfonamide system. After developing a standard docking protocol for the CA II – sulfonamide system we then used the protocol to determine other CA II – sulfonamide complexes.     

Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library

Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest. In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology. After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated. After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography. We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution. This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer. The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure-activity requirements for CV-N's antiviral properties.     

Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies

Monoclonal antibodies (MAbs) with affinities for molecules on the cell surface of the procaryote Myxococcus xanthus were used in a screening strategy for the isolation of mutants lacking particular cell surface molecules. From a large library of independent mutants created by Tn5 transposon mutagenesis, mutants were isolated which lacked reactivities with MAb 1604 (a MAb specific for a cell surface protein) and MAbs 2600, 1733, 1514, 1412, and 783 (MAbs specific for carbohydrate epitopes on the O antigen of lipopolysaccharide [LPS]). The defect in antibody recognition was shown by genetic crosses and DNA hybridization experiments to be caused by the Tn5 transposon acting as a mutation at a single locus. Quantitative enzyme-linked immunosorbent assays showed that particular mutant strains had no detectable affinity for the specific MAb probe. LPS mutants were resistant to myxophage Mx8, and this provided a selection method for isolating a large number of new LPS mutants. A class of Mx8-resistant mutants lacked reactivity with MAb 1514 and therefore was defective in the O antigen of LPS. A class of Mx1-resistant mutants lacked reactivity with MAb 2254, a MAb specific for a carbohydrate epitope on the core of LPS. A comparison of MAb binding to different mutant strains revealed a principle for mapping epitopes and showed that MAbs 1514 and 2254 recognize side-chain carbohydrates rather than backbone carbohydrates within the LPS molecule.     

Rigid-body docking with mutant constraints of influenza hemagglutinin with antibody HC19

An automatic docking algorithm has been applied to the modeling of the complex between hemagglutinin from influenza virus and the Fab fragment of a monoclonal antibody raised against this antigen. We have introduced here the use of biochemical information provided by mutants of hemagglutinin. The docking procedure finds a small number of candidate solutions where three sites of escape mutations are buried and form hydrogen bonds in the interface. The localization of the epitope is improved by additional biochemical data about mutants that do not affect antibody binding. Five candidate solutions with low energy, reasonably well-packed interfaces, and six to ten hydrogen bonds are compatible with mutant information. One of the five stands out as generally better than the others from these points of views. © 1994 John Wiley & Sons, Inc.     

Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.     

Infectivity studies of influenza virus hemagglutinin receptor binding site mutants in mice

The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.     

Screening of stable cutinase from Fusarium solani pisi using plasmid display system

Although enzymes are potential candidates for industrial catalysts, their industrial applications have been limited because they are easily deactivated under harsh operational conditions. In this study, a plasmid display system was used for the screening of stable cutinase in organic solvent (20% acetonitrile) and at high temperature. The fusion proteins were expressed and bound to specific DNA sequences on the encoding plasmids. Proteolysis resistance was used as a selection tool, where well-folded proteins are more resistant to the protease digestion than poorly-folded proteins. Stable mutants, identified to be I183T, I183F, and A56V, were screened in the organic solvent and at high temperature. The I183T and I183F mutants were more stable than the A56V mutant in 20% acetonitrile, while the A56V mutant was superior to the I183T and I183F mutants at high temperature. Molecular modeling was performed in order to investigate the residual characteristics of the stable mutants; secondary structure, residual solvation energy, residual ??-carbon flexibility, number of hydrogen bonds, number of neighboring amino acids, ratio of exposed/buried residue, and surface area. This analysis provided some guidelines for increased stability.     

Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG_1–4), and recognize hIgG_1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.     

Engineered human IgG antibodies with longer serum half-lives in primates

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.     

A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins.     

In silico site-directed mutagenesis of neutralizing mAb 4C4 and analysis of its interaction with G-H loop of VP1 to explore its therapeutic applications against FMD

Investigating the behaviour of bio-molecules through computational mutagenesis is gaining interest to facilitate the development of new therapeutic solutions for infectious diseases. The antigenetically variant genotypes of foot and mouth disease virus (FMDV) and their subsequent infections are challenging to tackle with traditional vaccination. In such scenario, neutralizing antibodies might provide an alternate solution to manage the FMDV infection. Thus, we have analysed the interaction of the mAb 4C4 with a synthetic G-H loop of FMDV-VP1 through in silico mutagenesis and molecular modelling. Initially, a set of 25,434 mutants were designed and the mutants having better energetic stability than 4C4 were clustered based on sequence identity. The best mutant representing each cluster was selected and evaluated for its binding affinity with the antigen in terms of docking scores, interaction energy and binding energy. Six mutants have confirmed better binding affinities towards the antigen than 4C4. Further, interaction of these mutants with the natural G-H loop that is bound to mAb SD6 was also evaluated. One 4C4 variant having mutations at the positions 2034(N→L), 2096(N→C), 2098(D→Y), 2532(T→K) and 2599(A→G) has revealed better binding affinities towards both the synthetic and natural G-H loops than 4C4 and SD6, respectively. A molecular dynamic simulation for 50?ns was conducted for mutant and wild-type antibody structures which supported the pre-simulation results. Therefore, these mutations on mAb 4C4 are believed to provide a better antibody-based therapeutic option for FMD.

Communicated by Ramaswamy H. Sarma     

Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.

Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.     

Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display

In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.