Biomedical subjects

E W Voss

Publications and source records attributed to E W Voss.

At least 37 records · Page 2Linked to original sources

PubMed1997

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Animals↗

PubMed1996

Transition-state theory and secondary forces in antigen--antibody complexes.

Secondary forces, defined as those interactions between the antigen (epitope including the surrounding environment) and areas immediately adjacent to the antibody active site, were investigated using monofluorescein-derivatized synthetic peptides of varying electrostatic properties. Secondary forces were quantitated by measuring the unimolecular rate constants at two different temperatures using the high-affinity anti-fluorescein monoclonal antibody 4-4-20 complexed with fluorescein-derivatized synthetic peptides. Unimolecular rate constants were correlated with transition-state theory to explain secondary effects. An acidic peptide produced a large temperature-dependent effect upon binding including a significant enthalpic factor (+33.28 kcal/mol) relative to the binding of fluorescein ligand (+23.96 kcal/mol). Binding of a basic peptide produced both a relatively smaller temperature effect and enthalpy factor than fluorescein ligand. The antibody-ligand binding results were interpreted invoking the concepts of thermally averaged metatypic (liganded) states of the antibody as well as potential biochemical interactions between the antigen and accessible surface regions of the antibody's complementarity determining regions.

Antibodies, Monoclonal↗

PubMed1996

Anti-metatype antibody stabilization of Fv 4-4-20 variable domain dynamics.

Anti-metatype (anti-Met) antibodies are immunoglobulins that specifically recognize and stabilize antibodies in their liganded or metatypic state, but lack specificity for either the hapten or the unliganded antibody. Autologous anti-Met antibodies were previously observed in vivo, suggesting that a metatypic autoantibody response could play a physiological role in the immune network, e.g. controlling the clearance of immune complexes from circulation. The first elicited anti-Met antibodies were against the fluorescein-liganded high affinity murine anti-fluorescein monoclonal antibody 4-4-20. The fluorescein-hapten system has proved to be an invaluable tool for both the recognition and characterization of the metatypic response by utilization of its spectral properties. In this investigation, hydrostatic pressure measurements, in conjunction with fluorescence spectroscopy, were performed on the recombinant Fv derivative (Fv 4-4-20) of the high affinity anti-fluorescein monoclonal antibody 4-4-20 complexed to anti-Met antibodies to study the influence of anti-Met antibodies of Fv 4-4-20 intervariable domain interactions. Anti-Met antibodies bound to liganded Fv 4-4-20 were observed to cause a change in the fluorescence properties of fluorescein that was not observed when anti-Met antibodies were bound to the liganded parent immunoglobulin. The variation of these spectral properties upon addition of anti-Met antibodies was shown to be correlated with dissociation of the variable domains in Fv 4-4-20 in response to its interaction with the anti-Met antibody. The ability to cause variable domain dissociation was dependent on whether monoclonal or polyclonal anti-Met antibodies were bound to the metatype. A model was proposed that elucidated the interaction of anti-Met antibodies, polyclonal and monoclonal, with variable domains of the primary anti-antigen antibody.

Animals↗

PubMed1996

Temperature and pH dependence of fluorescein binding within the monoclonal antibody 9-40 active site as monitored by hydrostatic pressure.

In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 degrees C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 degrees C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212Arg-34L indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten in this active site was solvent accessible. Imperfect packing, which leads to increased conformational dynamics, was determined as a possible cause of the low affinity for mAb 9-40.

Antibodies, Monoclonal↗

PubMed1996

Comparative properties of the single chain antibody and Fv derivatives of mAb 4-4-20. Relationship between interdomain interactions and the high affinity for fluorescein ligand.

Recombinant Fv derivative of the high affinity murine anti-fluorescein monoclonal antibody 4-4-20 was constructed and expressed in high yields, relative to the single chain antibody (SCA) derivative (2 3-fold), in Escherichia coli. Both variable heavy (VH) and variable light (VL) domains, that accumulated as insoluble inclusion bodies, were isolated, denatured, mixed, refolded, and affinity-purified to yield active Fv 4-4-20. Affinity-purified Fv 4-4-20 showed identical ligand binding properties compared with the SCA construct, both were slightly lower than the affinities expressed by Fab or IgG 4-4-20. Proper protein folding was shown to be domain-independent by in vitro mixing of individually refolded variable domains to yield functional Fv protein. In solid phase and solution phase assays, Fv 4-4-20 closely approximated the SCA derivative in terms of both idiotype and metatype, confirming identical active site structures and conformations. The equilibrium dissociation constant (Kd) for the VL/VH association (1.43 x 10(-7) M), which was determined using the change in fluorescein spectral properties upon ligand binding, was relatively low considering the high affinity displayed by the Fv protein for fluorescein (Kd, 2.9 x 10(-10) M). Thus, domain-domain stability in the Fv and SCA 4-4-20 proteins cannot be the sole cause of reduced affinity (2-3-fold) for fluorescein as compared with the Fab or IgG form of 4-4-20. With their identical ligand binding and structural properties, the decreased SCA or Fv affinity for fluorescein must be an ultimate consequence of deletion of the CH1 and CL constant domains. Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors.

Amino Acid Sequence↗

PubMed1996

Perturbation of antibody bound bifluorescent-ligand probe by polyclonal anti-metatype antibodies interacting with epitopes proximal to the liganded antibody active site.

General localization of metatypic determinants recognized by polyclonal anti-metatype antibodies relative to the antibody active site of the high-affinity anti-fluorescein monoclonal antibody 4-4-20 was achieved through use of a unique bifluorescent-ligand probe. The fluorescent probe possessed intrinsic energy-transfer properties with the fluorescein hapten serving as the energy acceptor. The donor group 5-(2-iodoacetyl) aminoethylaminonaphthalene-1-sulfonic acid (IAEDANS) proved environmentally sensitive both to binding of the FITC-cys-AEDANS ligand and to subsequent anti-metatype antibody interactions involving the antibody variable domains of 4-4-20. Spectral changes in ligand-conjugated AEDANS upon specific reactivity of the antibody with FITC suggested secondary interactions between AEDANS and the topological protein surface adjacent to the 4-4-20 active site. Results indicated that some anti-metatype antibodies (Fab fragments) within the polyclonal population bound to sites immediately surrounding the liganded active site and perturbed the interactions of AEDANS with topological sites. The results are discussed in terms of the types of interactions that may occur between the AEDANS moiety and the 4-4-20 antibody protein surface and subsequent perturbation of those interactions by anti-metatype antibodies.

Amino Acid Sequence↗

PubMed1996

Effects of secondary forces on the ligand binding properties and variable domain conformations of a monoclonal anti-fluorescyl antibody.

Biochemical interactions occurring external to the antibody active site or pocket (i.e. secondary forces) that directly effect ligand binding efficiency, and the microenvironment-sensitive spectral properties of bound homologous ligand, residing within the active site of high affinity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previously reported. This study describes the synthesis and characterization of a series of specially designed and chemically distinct mono-fluoresceinated peptides of equal size (13-mer) as well as the changes in the spectral properties and free energy in the binding of each fluorescein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the ligand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluorescein ligand. In addition to the effects on the fluorescence quenching of fluorescein and intrinsic tryptophan residues, and the free energy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with polyclonal antimetatype (conformational dependent anti-liganded state) antibodies. Studies comparing the results of a solid-phase inhibition assay, with the binding of antimetatype antibodies in solution, suggested that variant metatypic states of mAb 4-4-20 resulted from binding of the various fluorescein derivatized peptides. Depiction of the mAb 4-4-20 active site as a series of thermally averaged substates is proposed as a model and framework to interpret further the results. It was concluded that secondary forces can dictate conformer selection from the various substates. thereby modulating the primary antibody ligand interaction.

Antibodies, Monoclonal↗

PubMed1996

Detection of protease activity using a fluorescence-enhancement globular substrate.

Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase resulted in fluorescence enhancement of 4300%. Both alpha-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 x 10(-6) units for I ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 10(-3) units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.

Alkylating Agents↗

PubMed1996

Macrophage mediated processing of an exogenous antigenic fluorescent probe: time-dependent elucidation of the processing pathway.

To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.

Adenosine Triphosphate↗

PubMed1995

Durable elimination of high affinity, T cell-dependent antibodies by low molecular weight antigen arrays in vivo.

Ongoing Ab responses to a T cell-dependent Ag can be suppressed in hyperimmune animals by exogenous, multivalent Ag arrays. The pharmacologic basis for this suppression was studied by varying the molecular mass, ligand valence, and dose of Ag arrays, and then determining their efficacy, pharmacokinetics, and tissue distribution. Arrays ranging in molecular mass from 30 to 500 kDa caused initial clearance of specific serum Abs, but only the smaller arrays caused persistent suppression despite their relatively lower binding avidity and shorter retention in vivo. Suppression by the smaller arrays at lower doses was biphasic, implying two distinct modes of Ab elimination. High affinity IgG was eliminated preferentially, as shown by calibrated variable ligand-density ELISA. Suppressive arrays were localized discretely in the splenic germinal centers of hyperimmune animals. These results indicate that Ag array mass, ligand valence, and dose all play critical roles, and histologic compartmentalization may also be a pertinent parameter, in determining suppressive efficacy in vivo.

Animals↗

PubMed1995

Relative conformational stabilities of single-chain pocket and groove-shaped antibody active sites including HCDR transplant intermediates.

Stability measurements of SCA 04-01/212 (anti-ssDNA) which possesses a groove-shaped active site were performed by Gdn-HCl-induced unfolding, analyzed assuming a simple two-state equilibrium, and expressed as the free energy of unfolding, delta Gn-u. A delta Gn-u of 1.44 +/- 0.13 kcal/mol was determined experimentally for SCA 04-01/212. In addition, the conformational stabilities of HCDR transplants, hybrid antibody molecules resulting from the transplantation of HCDRs from SCA 4-4-20 (anti-fluorescein) into the corresponding regions of 04-01 in all combinations, were determined using the identical protocol applied to SCA 04-01. On the basis of the results of these stability experiments, the HCDR transplants were categorized into three groups, representing low, intermediate, and high stability. Data were discussed in terms of the relationships between structure-function and conformational stability pertaining to the groove-shaped antibody active site of SCA 04-01/212 and the pocket-shaped active site of SCA 4-4-20/212.

Antibodies, Monoclonal↗

PubMed1995

Primary structures of three Armenian hamster monoclonal antibodies specific for idiotopes and metatopes of the monoclonal anti-fluorescein antibody 4-4-20.

This paper reports the complete V gamma, V kappa, C gamma 1 and C kappa nucleotide and deduced amino acid sequences of two hamster monoclonal anti-metatype antibodies, 3A5-1 and 4A6. These antibodies have been previously characterized in terms of their binding and molecular stabilization properties with liganded murine monoclonal and single-chain antibody 4-4-20 active sites. Also reported are the complete V kappa and C kappa nucleotide and deduced amino acid sequence of hamster monoclonal anti-idiotype antibody 1F4, which is specific for the unliganded 4-4-20 active site. Oligonucleotide primers based on the 5' ends of murine variable genes, along with primers specific for murine IgG C gamma 1 and kappa constant region genes, have been used in cDNA and polymerase chain reactions (PCRs) to amplify IgG cDNA from Armenian hamster/mouse hybridomas. The hamster C gamma 1 and C kappa domain sequences are highly homologous to previously reported murine sequences. The anti-idiotype mAb V kappa gene demonstrated strong similarity to the murine V kappa V gene subgroup while the two anti-metatype mAb V kappa genes approximated more closely to the murine V kappa III gene subgroup. The two anti-metatype mAbs utilized highly homologous V gamma genes, with differing HCDR 3 regions, that appeared similar to the murine V gamma I(a) subgroup. These sequence determinations represent the first primary structures reported for antibodies with anti-metatype activity and are additions to the relatively sparse hamster immunoglobulin genetic database. Results are discussed in terms of 4-4-20 active site specificity and anti-metatype activity, as well as immunoglobulin structural diversity in an anti-Ig immune response.

Amino Acid Sequence↗

PubMed1995

Effects of secondary forces on the primary antibody-ligand interaction.

The binding efficiency of high affinity monoclonal antifluorescyl antibody 4.4-20 with the homologous ligand situated in different protein environments has been investigated to quantitate the effect of non-active site secondary factors. To synthesize monofluoresceinated proteins, fluorescein 5-isothiocyanate was reacted with a 100-fold molar excess of ribonuclease, lysozyme, lactalbumin and bovine serum albumin. Absorption and emission spectra, as well as fluorescence life-time measurements which yielded discrete components and proteolytic studies suggested that fluorescein was conjugated to a specific lysine residue consistent with a non-random distribution of lysines within each protein population. The derivatized residue was probably a surface moiety based on accessibility analyses with iodide as a dynamic quencher. Dissociation rate analyses indicated that the relative release time of 4.4-20 with each monofluoresceinated protein was Fl-RNAse > or = Fl-lyso > or = FDS > Fl-lact > or = Fl-BSA which correlated with changes in free energy of binding. Relative fluorescence quenching measurements of the fluorescein moiety indicated that 4.4-20 showed decreasing quenching in the order FDS > Fl-RNAse > Fl-lyso > or = Fl-lact > Fl-BSA. Because spectral data indicated that fluorescein was conjugated to a specific residue or a non-random distribution of residues in each protein population, the results represented the effect of a single distinct environment or a weighted average of different microenvironments. Results have been interpreted within the theoretical framework of a dynamic antibody model involving conformer selection and the relative effects of primary and secondary interactions.

Animals↗

PubMed1995

1.85 A structure of anti-fluorescein 4-4-20 Fab.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

Amino Acid Sequence↗

PubMed1995

Lupus-derived autoantibodies with dual autoactivity: anti-DNA and anti-Fc. I. Comparison of IgG autoreactivities with single-chain Fv derivatives.

Investigations into the intrinsic affinity and reactivity of autoanti-DNA active sites were initiated through the use of purified monoclonal IgG and the synthesis of single-chain Fv derivatives of murine monoclonal anti-DNA autoantibodies BV 04-01 and BV 17-45. Results showed that relative to the respective IgG hybridomas, only the BV 04-01 SCA derivative showed demonstrable reactivity with DNA. The monovalent single-chain derivative of BV 17-45 showed no reactivity with DNA in solution or solid-phase assays, even though the parental IgG had been previously described as high affinity. However, 17-45 displayed reactivity as a bivalent single-chain derivative. In addition, upon concentration, BV 17-45 IgG formed a highly stable, papain-resistant precipitate. Investigations into the nature of the precipitate revealed that BV 17-45 possessed significant, DNA-inhibitable autobinding to its own IgG molecule. BV 04-01 also possessed similar anti-self reactivity. Thus, both monoclonal autoantibodies examined in this study possessed dual binding specificity; anti-DNa and anti-self.

Amino Acid Sequence↗

PubMed1995

Lupus-derived autoantibodies with dual autoactivity: anti-DNA and anti-Fc. II. Fine specificity of anti-self autoreactivity.

The anti-immunoglobulin reactivity of two monoclonal, dual specific, autoantibodies, BV 17-45 and BV 04-01 was examined. The current study further defined the anti-immunoglobulin autoreactivity of these MoAbs to be Fc-specific. Both BV 17-45 and BV 04-01 bound their own Fc domains in addition to Fc regions of other MoAbs of similar isotype with varying levels of activity. The different anti-Fc reactivity patterns of BV 17-45 and BV 04-01 suggested that these MoAbs recognized distinct epitopes. Neither BV 17-45 nor BV 04-01 bound Fab fragments or single-chain antibody derivatives, which confirmed that the anti-immunoglobulin reactivity of these autoantibodies was Fc-specific. In addition, abrogation of anti-Fc reactivity was observed when affinity-labelled MoAbs were used as coating antigens in solid-phase ELISAs. These results implied that active-site ligand binding induced conformational changes which altered the Fc epitope(s) recognized by BV 17-45 and BV 04-01.

Animals↗

PubMed1994

Effect of transplantation of antibody heavy chain complementarity determining regions on ligand binding.

Active site structure-function analyses of anti-fluorescein single chain antibody 4-4-20 and anti-single-stranded DNA single chain antibody 04-01 were conducted studying the ligand binding properties of hybrid antibodies resulting from systematic transplantation of heavy chain complementarity regions (HCDRs) from monoclonal antibody 4-4-20 into 04-01. Two prototype monoclonal antibodies were chosen because the primary structures of their respective light chains were nearly identical but the specificities and shape of their active sites were distinctly different. Based on nearly identical light chains, the diverse active site conformations (i.e. 4-4-20 pocket and 04-01 cleft) and subsequent specificities of the two antibodies were likely dictated by heavy chain properties. As a result, specificities of each HCDR transplant were analyzed in terms of binding reactivity with either fluorescein or (dT)8. Results of binding studies, together with idiotypic and secondary structure analyses, were used to determine the relative contribution of each HCDR to the active site conformations and ligand specificities of monoclonal antibodies 4-4-20 and 04-01. Collectively the various analyses led to the conclusion that the intradomain conformational dynamics and cooperativity necessary for the structural integrity of the low affinity cleft-shaped 04-01 anti-single-stranded DNA active site are probably less stringent than those of a high affinity pocket-shaped anti-fluorescein active site.

Amino Acid Sequence↗

PubMed1994

Inter-active-site distance and solution dynamics of a bivalent-bispecific single-chain antibody molecule.

The solution dynamics of a bivalent bispecific single-chain antibody (BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDNA) were investigated. Fluorescence resonance energy transfer (FRET) studies were performed in order to estimate the average distances, R, between the anti-Fl and the anti-ssDNA active sites. In separate experiments, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride coupled to the 5' end of an oligothymidylate polymer of 6 residues length (2,5-DNS-dT6) served as energy donor to Fl or eosin isothiocyanate coupled to the 5' end of an oligothymidylate polymer of 6 residues length (eosin-dT6) served as energy acceptor from Fl. Labeling of dT6 with 2,5-DNS or eosin did not significantly interfere with recognition by the anti-ssDNA binding site. With the 2,5-DNS/Fl energy transfer pair, the calculated values of R(k2 = 2/3), R(min), and R(max) were 44, 37, and 54 A, respectively. With Fl/eosin (opposite direction of FRET), values of 40, 33, and 51 A, respectively, were obtained. Considering the sizes of the two SCA domains and the length of the interdomain polypeptide linker, an R value of approximately 140 A would be expected for the extended molecule. The fact that measured R distances were on average 3-fold shorter than 140 A indicated that BiSCA was not an extended and rigid molecule. The efficiency of energy transfer increased with increasing temperature in the range of 10-30 degrees C, suggesting that conformational fluctuations of the protein resulted in decreased average distance between BiSCA active sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Bispecific↗