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Current diagnosis of acute myocardial infarction involves quantification of circulating cTn levels. This work endeavoured to generate and enhance recombinant antibody fragments targeting various epitopes on the N- and C-terminals of the cTnI molecule, thereby facilitating highly sensitive detection of the troponin molecule. From this approach, two anti-cTnI scFv antibodies were successfully selected using either pHage display or structural reformatting of full length anti-cTnI IgG. Their antibody binding affinity was further optimised via chain shuffling and/or site directed mutagenesis, resulting in scFv with heightened sensitivity when compared to the wild-type scFv. If used in conjunction with existing anti-mid fragment cTnI antibodies, these N- and C- terminal-targeting scFvs show high potential for the enhancement of current cTnI detection assays by limiting the effects from cTnI degradation or troponin complex formation.

The daily clinical practice frequently force physicians to make adecision of whether a patient is coursing an infectious disease at theacute or the chronic stage, since treatment may be different dependingon it. This is the case of toxoplasmosis, a worldwide disease caused bythe intracellular parasite Toxoplasma gondii. When a pregnant woman isinfected for the first time by T. gondii and is not treated, the parasite cancross the placenta and severely affect the fetus. In case of primary infection,the treatment protocol includes using antiparasitic drugs toprevent serious damages, including death of the unborn baby. However,antiparasitic chemicals have side effects that should be avoided, unlesstreatment is mandatory. That is why discrimination between long-termand primary T. gondii infection turns out to be critical in infectedpregnant women (Montoya and Remington, 2008).In this work we have used rP22a, an acute-phase recombinant protein werecently described (Costa et al., 2017), to assess anti-T. gondii IgG AI,calculated by two new easier approaches, one based on the area underthe avidity curve (AUAC) and the other one based on the E-P titrationmethod with minor variations. We compare our results with those obtainedwhen calculating the AI by the conventional methods abovementioned, and the performance to render a proper sample classificationas compared to that obtained having used a commercial kit asstandard. Fil: Garcia, Valeria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Peverengo, Luz María. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Peretti, Leandro Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: González, Verónica Doris Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Dalla Fontana, Maria L.. Laboratorio Central de la Provincia de Santa Fe; Argentina Fil: Marcipar, Iván Sergio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lagier, Claudia Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina

The aim of this study was to evaluate the performance of conventional serology (Q-Preven™ and ELFAVIDAS™) and flow cytometry-based serologic tools for early serologic diagnosis of congenital toxoplasmosis. The study groups included prospectively confirmed cases of congenital toxoplasmosis (TOXO=88) and age-matching non-infected controls (NI=15).The results demonstrated that all samples tested positive/indeterminate for anti-T. gondii IgM screening at birth using air-dried whole blood samples. Serum samples collected at 30-45days after birth tested positive for ELFAVIDAS™ IgG in both groups. While all NI tested negative for ELFAVIDAS™ IgM and IgA, only 78% and 36% of TOXO tested positive for IgM and IgA, respectively. Flow cytometry-based anti-T. gondii IgM, IgA and IgG reactivity displayed moderate performance with low sensitivity (47.6%, 72.6% and 75.0%, respectively). Regardless the remarkable specificity of IgG1, IgG2 and IgG3 subclasses for early diagnosis, weak or moderate specificity was observed (Se=73.9%, 60.2% and 83.0%, respectively). The analysis of IgG avidity indices (AI) demonstrated the highest performance among the flow cytometry-based methods (Se=96.6%; Sp=93.3%), underscoring the low avidity index (AI60%) within TOXO (97.0%) in contrast with the high avidity index (AI60%) in NI (93%). Analysis of anti-T. gondii IgG and IgG3 reactivity for mother:infant paired samples may represent a relevant complementary tests for early diagnosis. In conclusion, a feasible high-standard algorithm (Accuracy=97.1%) was proposed consisting of Q-Preven™ IgM screening at birth, followed by ELFAVIDAS™ IgM and flow cytometric IgG avidity analysis at 30-45days after birth as a high performance tool for early serological diagnosis of congenital toxoplasmosis.

Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies.

Shiga toxins (Stxs) are major causative agents for bloody diarrhea and hemolytic uremic syndrome, a life-threatening disease in humans. No effective treatment is available. Early detection of Stxs in clinical samples is critical for disease management. As bacteria evolve, new Stxs are produced; therefore, methods used to identify them need to be improved as well. In this study, new monoclonal antibodies (mAbs) against Stx1d and 1e were developed and used to improve a commercial Stx1 kit. Incorporation of the new mAbs into the Abraxis Stx1 kit not only increased the assay sensitivity to Stx1d, but the assay was conferred the ability to detect Stx1e, a newly identified subtype of Stx1 produced by an atypical Stx-producing bacterial strain, Enterobacter cloacae M12X01451, isolated from a clinical specimen. This toxin was not detectable using existing commercial kits. The signal to noise ratio (s/n) of the new assay was increased 3-fold for Stx1d, and 44-fold for Stx1e at toxin concentration of 10ng/mL. The limit of detection (LOD) was 10pg/mL for Stx1a, and 100pg/mL for Stx1c, 1d and 1e. When used for bacterial strains, the sensitivity of the new assay was improved 2.5- to 60-fold depending on subtypes produced. In summary, high affinity mAbs against Stx1d and 1e were developed and incorporation of these mAbs into the Stx1 kit significantly enhanced the assay sensitivity and broadened the subtype-specificity. This improvement should be useful for reducing product recalls and disease mistreatment due to failures of detecting less common but clinically important subtypes of Stxs.

Limited data exists regarding antibody avidity for human papillomavirus (HPV). We describe development of a multiplex electrochemiluminescent avidity ELISA for four HPV types (HPV 6, 11, 16, 18) by adding a dissociating step to our established multiplex HPV VLP ELISA. Initial experiments exploring ammonium thiocyanate, sodium thiocyanate and guanidine hydrochloride (GuHCl) as dissociating agents identified GuHCl as most promising. Dissociation conditions with GuHCl were varied (concentration, incubation time, temperature) to select conditions with minimal impact on VLP integrity as measured with monoclonal antibodies to conformational epitopes. Avidity index (AI) was calculated based on a standard curve as ratio of bound IgG in GuHCl treated versus untreated sample. To evaluate our assay we determined AI in sera with known HPV titers. We selected 32 residual anonymized sera from individuals with a wide range of titers for HPV6, 11, 16, and 18. AIs were similar across multiple dilutions of serum within the assay's dynamic range and were reproducible with two plate lots. This assay will aid in understanding HPV antibody avidity and maturation in response to natural infection and varying vaccine schedules. This is the first report of a VLP-based multiplexed avidity ELISA that evaluates assay parameters for all nine HPV vaccine types.

Monoclonal antibodies have become essential tools for diagnostic and therapeutic purposes. Antibody affinity is one of the critical factors influencing the therapeutic success of tumor targeting antibodies. Therefore, developing an accurate and reliable method for determining antibody affinity is crucial. In this study, we describe a fluorescent cell-based immunosorbent assay that can accurately measure antibody affinity (KD) in the nanomolar range. This method involves the addition of fluorescently labelled antibodies to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The KD values obtained by this method were comparable with values obtained by the flow cytometry and radioactive (I125) scatchard assay. Our results demonstrate that this modified cell-based fluorescent method allows for a convenient and efficient identification of therapeutically relevant leads.

Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein. A subset of the monoclonal antibodies was immobilized to characterize antigen capture in solution. It can be advantageous to utilize antibodies that recognize distinct epitopes when choosing reagents for detection and diagnostic assays. We determined the uniqueness of the epitope recognized by the anti-VP40 antibodies using a checkerboard format that exploits the 6×6 array of interactions monitored by the Bio-Rad ProteOn XPR36 SPR instrument. The results demonstrate the utility of surface plasmon resonance to characterize monoclonal and recombinant antibodies. Additionally, the analysis presented here enabled the identification of pairs of anti-VP40 antibodies which could potentially be utilized in sandwich type immunoassays for the detection of Ebola virus.

Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection. However, antibody titer provides only a partial measure of antibody function, which is dependent on both the variable region (Fv) to bind the antigen target, and the constant region (Fc) to elicit an effector response from the innate arm of the immune system. In the case of some diseases, such as HIV, for which an effective vaccine has proven elusive, antibody effector function has been shown to be an important driver of monoclonal antibody therapy outcomes, of viral control in infected patients, and of vaccine-mediated protection in preclinical and clinical studies. We sought to establish a platform for the evaluation of the Fc domain characteristics of antigen-specific antibodies present in polyclonal samples in order to better develop insights into Fc receptor-mediated antibody effector activity, more fully understand how antibody responses may differ in association with disease progression and between subject groups, and differentiate protective from non-protective responses. To this end we have developed a high throughput biophysical platform capable of simultaneously evaluating many dimensions of the antibody effector response., Highlights • High-throughput array-based characterization platform for polyclonal antibodies. • Development of a biophysical proxy for antibody effector function. • Antigen and Fc receptor recognition characteristics are captured. • Enables systematic serologic studies of NHP and human antibody samples.

Monitoring recency of infection helps to identify current transmission in vulnerable populations for effective disease control. We have established an in-house avidity based hepatitis C virus (HCV) recency assay based on the Monolisa Anti-HCV PLUS Version 3 ELISA kit for use of dried serum/plasma spots (DS/PS) in order to distinguish recent and long-term infections. A first panel of DS/PS (n = 218; genotype 1 n = 170 and non-genotype 1 n = 48) consisting of primary and at least one follow up sample was used to analyze the temporal changes of the Avidity Index (AI) over time. Sub-panels of longitudinal DS/PS (n = 66) and acute cases (26 weeks; n = 34) were taken to calculate the Mean Duration of Recent Infection (MDRI) and the False Long-term Rate (FLTR), respectively. A second panel of DS/PS104 weeks (n = 132) and a third panel of DS/PS prepared from resolved infections (≥180 days since last positive; n = 32) were used to calculate the False Recent Rate (FRR). For all genotypes, the optimal AI cut-off was determined to be 40% resulting in an MDRI of 364 days (95% CI: 223-485). FLTR was 5.9% (95% CI: 0.7-19.7), 8.3% (95% CI: 1-27), and 0% (-) and FRR was 13.6% (95% CI: 8.3-20.7), 11.7% (95% CI: 6.6-19), and 30.6% (95% CI: 9.1-61.4) for all genotypes, genotype 1, and non-genotype 1 infections, respectively. For resolved infections, the FRR was 53.1% (95% CI: 35.8-70.4). Thus, this assay performs particularly well for genotype 1 reaching a high rate of correct discriminations between infections acquired less than a year before diagnosis and those acquired earlier by applying an AI cut-off of 40%. Due to a rapid decline in avidity post resolution of an HCV infection this assay is not recommended to be used in HCV RNA negative patients.

The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection in enzyme-linked immunosorbent, Western blot, flow cytometry, and immunofluorescence staining analyses in Escherichia coli, mammalian cell lines, yeast, and plant. In addition, our RA-tag/47RA mAb combination showed high sensitivity and reliable affinity (KD = 5.90 × 10−8 M) when compared with conventional tags. Overall, our results suggest that the RA-tag system could facilitate the development of a broadly applicable tag system for biological research.

Cytokeratin 18 (CK18), the main scaffold protein of keratinocyte, is distributed in epithelial cells. This structural protein maintains the integrity and continuity of epithelial tissue. Cytokeratin is also frequently used as an immunohistochemical marker of tumor growth. In recent years, immune repertoire (IR) evaluation using next-generation sequencing (NGS) have become increasingly efficient. Here we deep sequenced the mouse IR of the immunoglobulin heavy chain (IGH) after CK18 immunization. We comprehensively analyzed the IR based on complementarity determining region 3 (CDR3) abundance, germline gene usage polarization, clone diversity, and lineage. We found many convergence characteristics after CK18 immunization. Convergence represents a phenomenon that antigen stimulation or pathogen exposure induces the antigen specific clone expansion and enrichment. The convergence could be used for the immune evaluation and antibody screen. After immunization, the IGHV5 gene clusters became preponderant. The abundance and length of the most frequent CDR3 both increased, nevertheless the IR diversity level decreased. From the convergent IGH repertoires, we selected and expressed six antibodies with the most frequent CDR3s and IGH V-J combinations. The ELISA results suggested all screened six antibodies bound CK18 specifically. The most potential antibody had 9.424E-10M M affinity for the interaction with the CK18. Therefore, this is the NGS platform has been first used for anti-CK18 monoclonal antibodies (MAbs) discovery. These analyses methods could also be used for vaccine evaluation.

A panel of 9 fully human monoclonal antibodies against human erythropoietin (EPO) with defined characteristics (non-neutralizing, neutralizing, various isotypes, affinities) representative of those evident in antibody-mediated pure red cell aplasia (PRCA) and non-PRCA patients were formulated and lyophilized. The panel was evaluated in a multi-centre international collaborative study comprising eighteen different laboratories using different assay platforms including those in routine use. These included binding assays, some based on use of novel technologies and neutralization assays predominantly employing EPO responsive cell-lines. Results showed that detection and titre varied depending on antibody characteristics and the method used. Only selective assay platforms were capable of detecting the diverse repertoire of EPO antibodies in the panel indicating that some clinically relevant antibodies are likely to be missed in some assays. Importantly, the clinical samples from PRCA patients were distinguished as antibody-positive and the healthy donor serum as antibody negative across all different platforms tested. For neutralization, data was generally consistent across the assays for the different samples regardless of the cell-line and the assay conditions. The heterogeneity in data from the study clearly indicated the need for reference standards for consistency in detecting and measuring EPO antibodies across different assay platforms for monitoring the safety and efficacy of erythropoiesis stimulating agents. Therefore, the WHO ECBS at its meeting in October'15 established the EPO antibody panel, available from NIBSC, to facilitate decision-making on assay selection for testing antibodies against human EPO, for evaluating assay performance of antibody assays for clinical use, for assay validation and for standardization.

The presence of (1 → 3)-β-D-glucan in human plasma is a marker for fungal infections. Currently, the Limulus amebocyte lysate (LAL)-based assay is widely used for the quantification of plasma (1 → 3)-β-D-glucan. However, it has limitations in clinical use, such as an unstable supply of natural resources, complicated manufacturing process, and low-throughput of the reagents. Alternative assays exploiting specific antibodies against (1 → 3)-β-D-glucan have been developed to overcome these challenges. However, these methods are associated with low sensitivity and poorly correlate with the data obtained by the LAL-based assay. The aim of this study is to develop a novel enzyme immunoassay that is as sensitive and accurate in determining plasma (1 → 3)-β-D-glucan levels as compared to that obtained with the LAL-based assay. We generated specific monoclonal antibodies against (1 → 3)-β-D-glucan that recognizes four-unit glucose oligomers with (1 → 3)-β-D-linkages, and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) using these antibodies. The newly developed ELISA showed proportional increase in absorbance with the volume of (1 → 3)-β-D-glucan added. The limit of detection of the assay was 4 pg/ml of plasma (1 → 3)-β-D-glucan that was equivalent to the LAL-based assay and the working range was 4–500 pg/ml. The intra-assay coefficient of variation was 2.2–5.4% using three different concentrations of plasma samples. We observed strong correlation (R = 0.941, slope = 0.986) between the measurements obtained by our ELISA and Fungitec G test ES Nissui, a commonly used LAL-based assay, using 26 types of plasma samples. This could be attributed to the epitopes of the antibodies. Both antibodies could inhibit the LAL-based assay, suggesting that the antibodies recognize the identical regions in β-D-glucan, thereby inactivating factor G, an initiation zymogen for coagulation cascade, in the LAL-based assay. Thus, the ELISA developed in this study can detect fungal infections in clinical settings with similar efficiency as the LAL-based assay. This assay is characterized by good performance, stable supply of materials, and simple manufacturing process and is more suitable for the high-throughput diagnosis of fungal infections.

Monoclonal antibodies (mAb) are unique tools in therapeutics and immunodiagnostics applications but many of these applications rely on conjugated mAbs. Whether conjugating drugs or tracers, the conjugation process, frequently taking advantage of primary amines on lysine residues, may affect the binding activity of the antibodies. Furthermore, due to the sticky nature of many mAbs, unfavorable interactions may become eminent, with the result of high background signals. The workload associated with producing mAbs, able to withstand conjugation, preserving stability and affinity and avoiding off-target interactions, is comprehensive and related with only incidental success. We designed a method, where uncloned hybridomas were pre-selected for secretion of mAbs with the above characteristics. Using human collectin K1 (CL-K1, alias CL-11, Colec11) as a model antigen, mAbs present in culture supernatant from uncloned hybridomas were immobilized on Protein A beads, followed by solid phase biotinylation and subsequent elution. ELISA was employed to compare the binding activity of conjugated vs. unconjugated mAbs, and furthermore for their application in combination with other antibodies. From a group of 96 uncloned hybridomas we accomplished in obtaining five suitable mAbs, among which, two mAbs were superior. The successful conjugation of the selected mAbs with fluorophores and subsequent applications in microscopy and flow cytometry were further demonstrated. In conclusion, pre-selection of uncloned hybridomas, by testing of their mAbs' ability to withstand conjugation with tracers or drugs, is a successful strategy to avoid a huge workload of cloning numerous hybridomas, in order to obtain conjugatable mAbs.

Covalent immobilization of antibodies to protein G beads is a basic molecular biology method, although the beads present poor recovery results. Our aim was to reuse the immobilized antibody-protein G complex on a very small scale, therefore we optimized the crosslinking procedure to be used on the wells of a standard 96-well microplate. The method used involves the affinity binding of the antibody to the protein G surface, followed by the immobilization step using different crosslinking reagents, DMP and BS3, quenching the crosslinking reaction, and binding the antibody-specific antigen. By scaling down the procedure, we were able to reuse the anti-EGFR crosslinked wells more than 20 times. This method can be used to perform assays on a wide range of solid supports containing the protein G in an immobilized form, including functionalized nanosensors, for immunoprecipitation, protein and cell lysate purification, target protein enrichment.

Background Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity and variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resulted in homogeneous dense binding to mouse melanoma cells and significant tumor growth inhibition. Methods Surface-related tumor-specific mutations on B16-F10 cells were identified. Peptides containing the single amino acid mutation were synthesized for 9 different neoantigens. Rabbits were vaccinated with each of these peptides and high affinity polyclonal antibodies to each peptide were obtained. The 9 antibodies were combined as a cocktail and mice with implanted B16-F10 cells were treated with and without PD1 inhibitor. Results Even a single dose of the antibody cocktail inhibited tumor growth and prolonged survival. PD1 inhibitor alone had little effect on tumor growth. The antibody cocktail plus PD1 inhibition increased tumor response and 4 doses of the cocktail completely prevented tumor growth in 50% of the mice. Complete responses were durable. The complete responders were highly resistant to tumor re-challenge at 6 months. No adverse events were identified in the antibody treated mice. Conclusions Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple doses were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free.

Antibody-based diagnostics and therapeutics have huge commercial value. However, applications of antibodies are often limited by instability, particularly for recombinant antibody formats. This paper describes the conversion of a single-chain variable fragment (scFv) antibody to a single-chain antibody fragment (scAb) with notably improved stability characteristics. This scAb retains antigen-binding activity (i) at high temperature (up to 60 °C), (ii) in guanidine hydrochloride (GdnHCl, up to 1 M), and (iii) when stored at 37 °C for 6 months. However, limited improvement was observed when the original scFv was converted to a larger fragment antigen-binding (Fab) format. Certain Cys-to-Ala mutations in the third complementarity determining region of the antibody heavy chain (CDR H3) also led to stability improvements. Our findings indicate that the stability of an antibody derivative depends on its format and on the positions of cysteines in the CDRs.

Infectious bursal disease (IBD) is considered a vital viral disease that threatens the poultry industry worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VP2/IBDV through a bacteria display technology, resulting in the enrichment of scFvs. Three scFv clones with different fluorescence intensity were obtained by colony pick up at random. The obtained scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis (FACS). The potential use of the isolated IBDV-specific scFv antibodies was demonstrated by the successful application of these antibodies in Western blotting and ELISA assay. What's more, in vitro neutralization measurement showed that one of the three isolated antibodies possessed the neutralization function against IBDV. This study provides new strategies for screening of antibody library, and scFv antibodies isolated in this study may be utilized as lead candidates for further development of diagnostic or therapeutic antibodies for detection and treatment of IBDV infection.

We developed a method to detect and isolate plasma cells that produce antigen-specific antibodies. An affinity matrix of hapten was constructed on a cell surface, and subsequent incubation allowed cells to secrete antibodies. Anti-hapten antibodies preferentially bound to the affinity matrix on the cells from which they were secreted. We showed that the combination of surface biotinylation and streptavidin which was conjugated with a high valence of hapten was suitable for sensitive detection of antibody binding. Using this protocol, anti-hapten plasma cells from immunized mouse spleen were detected and enriched by flow cytometry. This method allows for isolation of intact plasma cells according to the antibody specificity and may be useful for highly efficient and precise analysis of an antibody repertoire.

Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), is increasingly recognized as a modulator of insulin-like growth factor (IGF) signaling; it cleaves IGF binding proteins causing the release of bioactive IGF. Accumulating evidence supports an important role of PAPP-A in both normal physiology and under different pathological conditions. However, antibodies for the detection of PAPP-A in non-human tissues have been lacking, although needed for use with several animal models which are currently being developed. To develop a monoclonal antibody suitable for the immunohistochemical detection of PAPP-A, we therefore selected a phage-derived scFv antibody, PAC1, specifically recognizing an epitope of PAPP-A, which is highly conserved between multiple animal species. We first converted this antibody into bivalent IgG, and verified its ability to recognize PAPP-A in sections of formalin-fixed and paraffin-embedded tissue. For increased sensitivity, affinity maturation to sub-nanomolar affinity was then carried out. The resulting recombinant antibody, PAC1-D8-mIgG2a, detects PAPP-A specifically and sensitively in human tissue. In addition, this antibody allows detection of PAPP-A in non-human species. We demonstrate its usefulness for the visualization of PAPP-A in murine and porcine tissues.

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

We have developed a highly sensitive immunoassay-called digital ELISA-that is based on the detection of single enzyme-linked immunocomplexes on beads that are sealed in arrays of femtoliter wells. Digital ELISA was designed to be highly efficient in the capturing of target proteins, labeling of these proteins, and their detection in single molecule arrays (SiMoA); in essence, the goal of the assay is to "capture every molecule, detect every molecule". Here we provide the theoretical basis for the design of this assay derived from simple equations based on bimolecular interactions. Using these equations and knowledge of the concentrations of reagents, the times of interactions, and the on- and off-rates of the molecular interactions for each step of the assay, it is possible to predict the number of immunocomplexes that are formed and detected by SiMoA. The unique ability of SiMoA to count single immunocomplexes and determine an average number of enzymes per bead (AEB), makes it possible to directly compare the number of molecules detected experimentally to those predicted by theory. These predictions compare favorably to experimental data generated for a digital ELISA for prostate specific antigen (PSA). The digital ELISA process is efficient across a range of antibody affinities (K(D)~10(-11) -10(-9) M), and antibodies with high on-rates (k(on)>10(5) M(-1) s(-1)) are predicted to perform best. The high efficiency of digital ELISA and sensitivity of SiMoA to enzyme label also makes it possible to reduce the concentration of labeling reagent, reduce backgrounds, and increasing the specificity of the approach. Strategies for dealing with the dissociation of antibody complexes over time that can affect the signals in an assay are also described.

Monoclonal antibodies are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, the use of anticarbohydrate antibodies to target glycans for these purposes has been less successful. Glycans contain less hydrophobic functionality than do proteins or nucleic acids, thus individual glycan-antibody interactions are relatively weak. Information encoded by glycans often involves subtle variations of branched oligosaccharides that cannot be detected with conventional antibodies. Here we describe a new phage display selection strategy for identification of high-affinity and specific glycan antibodies. We designed and characterized a phage clone that functionally displays the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120. The two heavy chain variable domains of 2G12 exchange positions to create an extended recognition surface containing four oligomannose binding sites per IgG molecule. We designed and characterized a phage clone in which this domain exchange architecture was recapitulated as an antigen binding fragment dimer [(Fab)(2)]on the phage surface by protein engineering. The functional display of the 2G12 (Fab)(2) fragment was validated by Western blot and phage enzyme-linked immunosorbent assay. Furthermore, we demonstrate that this 2G12 (Fab)(2) display system is amenable to selection of functional clones using a mock selection. These results provide proof-of-concept that the privileged 2G12 domain-exchanged scaffold can be used for design of novel antibody libraries that are biased toward glycan recognition.

Carcinoembryonic antigen related cell adhesion molecule (CEACAM) 6 is over-expressed in different types of cancer cells. In addition, it has also been implicated in some infectious diseases. Targeting this molecule by an antibody might have applications in diverse tumor models. Single domain antibody (sdAb) is becoming very useful format in antibody engineering as potential tools for treating acute and chronic disease conditions such as cancer for both diagnostic as well as therapeutic application. Generally, sdAbs with good affinity are isolated from an immune library. Discovery of a new target antigen would require a new immunization with purified antigen which is not always easy. In this study, we have isolated, by phage display, an sdAb against CEACAM6 with an affinity of 5 nM from a llama immunized with cancer cells. The antibody has good biophysical properties, and it binds to the cells expressing the target antigen. Furthermore, it reduces cancer cells proliferation in vitro and shows an excellent tumor targeting in vivo. This sdAb could be useful in diagnosis as well as therapy of CEACAM6 expressing tumors. Finally, we envisage it would be feasible to isolate good sdAbs against other interesting tumor associated antigens from this library. Therefore, this immunization method could be a general strategy for isolating sdAbs against any surface antigen without immunizing the animal with the antigen of interest each time.

The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217 ng/ml, i.e., 11 nM, comparable to the 7 nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent., Highlights ►MAp19 and MASP-2 were both mainly expressed in hepatocytes. ►The MAp19 concentration in serum was 217 ng/ml, 11 nM, comparable to that of MASP-2. ►In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. ►High levels of MAp19 were found in urine, where MASP-2 was absent. ►MAp19 cannot compete with MASP-2 for binding to MBL, nor inhibit complement activation.

Biopanning of two linear (6- and 15-mer) and two constrained (10- and 17-mer) phage-displayed peptide libraries with two anti-DNP monoclonal antibodies (mAbs) selected seven unique peptide sequences using only the low affinity anti-DNP monoclonal antibody. The selected peptides contained two of 6, one of 10, two of 15 and two of 17 amino acids in length. They were all rich in hydrophobic residues. Both 15-mer peptides had antigenic regions of eight amino acids as revealed by a spot scan assay. Two of the 17-mer and one of the 10-mer peptides displayed on phage competed with free DNP for the low affinity anti-DNP mAb. These findings highlight (i) the selective power of phage displayed peptide libraries to identify peptides that mimic the shape of a small hapten molecule such as DNP, (ii) the possible preferential bias of phage libraries towards low affinity antibodies, (iii) the importance of using a panel of phage libraries for selecting peptide mimics.

Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.

Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)(2)s), were constructed from the monovalent anti-keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)(2)s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the (125)I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)(2)s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)(2)s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)(2)s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.

A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 minutes were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.

We describe the facile generation of a stable recombinant antibody with intrinsic red fluorescent properties for qualitative and potentially quantitative immunofluorescence analysis. The REDantibody based on the X-ray crystallographic structures of the anti-sialyl-Tn antibody B72.3 and 3D model of the monomeric red fluorescent protein was designed to retain optimal spatial geometry between the C- and N-termini of the V(H) and V(L) chains respectively to mimic the domains interface pairing in antibody Fab fragments and to incorporate the red fluorescent protein as a bridging scaffold. The model was further validated by assembling a REDantibody based on CA19.9 the anti-sialylated Lewis (Le)(a) blood group antigen and 4D5-8 the anti-p185(HER2) antibodies. The chimeric heavy and light chains containing red fluorescent protein as a bridge were correctly processed and secreted into Escherichia coli periplasm for assembly and disulphide bond formation, further analysis revealed the molecules to be exclusively monomers. Purified anti-glycan proteins were used for an immunofluorescent analysis of Trypanosoma cruzi epimastigotes, and the anti-p185(HER2) used to determine the binding properties. The REDantibody platform facilitates rapid generation of scFv chimeras that could be used for screening antibodies against cell surface markers. Furthermore, such modular assembly should permit the interchange of binding sites and of fluorophores to create robust panels of coloured antibodies.

Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins.

Phage display technology has emerged as a leading approach to select proteins with improved properties for many different types of applications. The selection typically selects not only for improved binding properties but also for other factors such as efficiency of protein production and folding in Escherichia coli, the host in which the proteins and the phage are produced. Furthermore, the selection methodology is likely to influence the character of retrieved variants. We have now defined the extent whereby the charge of the displayed proteins influence the selection process, resulting in an increased average positive charge among selected proteins in comparison to the proteins that are harbored in the library before selection. Implications of and possible routes to minimize this effect are discussed.

The aim of this study was to compare the affinity values obtained for a monoclonal antibody/antigen complex using two different techniques, surface plasmon resonance (SPR) and an enzyme linked immunosorbent assay (ELISA) approach recently described by Bobrovnik S.A. and by Stevens F.J. These two techniques can be used in particular to determine the equilibrium dissociation constant, K(D), of the complex in solution or on a surface. Bobrovnik's method gives two K(D) values that differ by a factor of 100, demonstrating that two populations of complexes are present in solution. In an initial step, one protein binds relatively weakly to the other (high K(D)) and this is followed by a conformational change in the most flexible portion of the antigen, which increases the affinity (low K(D)). Only the higher of the two K(D) values can be detected when complex formation in solution is investigated using SPR, because the interaction measured concerns the fibronectin/antibody complexes of lowest affinity. In contrast, when measuring association at the sensor surface, SPR gives an average result between the two K(D) values because complexes corresponding to both affinities can form in this situation. The constants that characterise the kinetics of the fibronectin-antibody interaction obtained by SPR and ELISA are therefore different, because the methods do not allow the same phenomena to be observed. However they are consistent and complementary.