Pool Of Monoclonal Antibodies Research Articles

Leveraging bioanalytical characterization of fractionated monoclonal antibody pools to identify aggregation-prone and less filterable proteoforms during virus filtration.

Monoclonal antibodies (mAbs) are an essential class of biotherapeutics. A platform process is used for mAb development to ensure clinically safe and stable molecules. Regulatory authorities ensure that mAb production processes include sufficient viral clearance steps to achieve less than one virus particle per million doses of product. Virus filtration is used for size-based removal of enveloped and nonenveloped viruses during downstream processing of mAbs. Process development in mAb purification relies on empirical approaches and often includes adsorptive prefiltration to mitigate virus filter fouling. Opportunities for molecular-level prediction of mAb filterability are needed to plug the existing knowledge gap in downstream processing. A molecular-level approach to understanding the factors influencing mAb filterability may reduce process development time, material loss, and processing costs due to oversized virus filters. In this work, pH step gradient fractionation was applied on polished bulk mAb feed to obtain concentrated pools of fractionated mAb variants. Biophysical properties and quality attributes of fractionated pools, including oligomeric state (size), isoelectric point profile, diffusion interaction parameters, and glycoform profile, were determined using bioanalytical methods. Filterability (loading and throughput) of fractionated pools were evaluated. Statistical methods were used to obtain correlations between quality attributes of mAb fractions and filterability on the Viresolve Pro virus filter.

Countercurrent preferential precipitation of acidic variants from monoclonal antibody pools

The process of countercurrent multistage precipitation (CnMP) was developed for reduction of the acidic variant (av) content in monoclonal antibody (mAb) pools. The process was performed in polyethylene glycol (PEG) solutions of low ionic strength, at which av was the most prone to precipitate. To design the process, a mathematical model was formulated, which consisted of steady-state mass balance equations and underlying thermodynamic dependencies. The model was solved for different combinations of the process variables, including the protein concentration in the feed material and the PEG concentration in subsequent precipitation stages. The model solution was projected onto two-dimensional planes, where the performance indicators, such as the separation yield and the av reduction level, were correlated with the process variables. CnMP overperformed crosscurrent multistage precipitation (CsMP) and ion exchange chromatography (IEX) used in previous studies; in the comparison with CsMP, the 3-stage CnMP process allowed up to 28% increase in yield and up to 25% increase in the av reduction level, and in the comparison with IEX, up to 37% increase in yield and up 12.5% increase in the av reduction level. The yield benefit of CnMP depended on the composition of the feed material and the demand for the av reduction. The concept was experimentally verified; the 2- and 3-stage CnMP processes were performed using the input process variables provided by the model solution. The performance indicators calculated by the model and experimentally measured were in a good agreement, which indicated both feasibility and predictability of CnMP.

Coupling of chromatography and precipitation for adjusting acidic variant content in a monoclonal antibody pool

The acidic charge variants (av) of monoclonal antibodies (mAb) are often reported to have reduced therapeutic potency compared with the main (mv) and basic variants (bv), therefore reduction in the av content in mAb pools is often prioritized over reduction in the bv content.In previous studies we described two different methods for reducing the av content, which were based on either ion exchange chromatography or selective precipitation in polyethylene glycol (PEG) solutions. In this study, we have developed a coupled process, in which advantages of simplicity and ease in realization of PEG-aided precipitation and high separation selectivity of anion exchange chromatography (AEX) were exploited. The design of AEX was supported by the kinetic-dispersive model, which was supplemented with the colloidal particle adsorption isotherm, whereas the precipitation process and its coupling with AEX was quantified by simple mass balance equations and underlying thermodynamic dependencies. The model was used to assess the performance of the coupling of AEX and precipitation under different operating conditions. The advantage of the coupled process over the stand-alone AEX depended on the demand for the av reduction as well as the initial variant composition of the mAb pool, e.g., the improvement in the throughput provided by the optimized sequence of AEX and PREC varied from 70 to 600% for the initial av content changed from 35 to 50% w/w, and the reduction demand changed from 30 to 60%.

Preferential precipitation of acidic variants from monoclonal antibody pools.

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins.In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps.Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

Combined Single-Cell DNA Genotyping and Protein Quantification (DAb-seq) in Acute Myeloid Leukemias Reveals Distinct Immunophenotypic Subsets Among Pathogenic Clones

Genetic and immunophenotypic diversity is a hallmark of hematopoietic malignancies, and clonal evolution has been implicated as a driver of tumor initiation, progression, treatment response, and relapse. Consequently, a deeper understanding of the complex relationship between mutational profiles and disease phenotypes is critical to better understand the underlying disease processes. Probing the question of genotype-immunophenotype interplay at scale has historically proven difficult due to technical limitations permitting capture of only a single datatype from an individual cell. In acute myeloid leukemia (AML), flow cytometry for detection of measurable residual disease (MRD) has become standard of care for monitoring of treatment response and risk for relapse. At the same time, DNA-based molecular profiling of AML provides prognostic information and can guide therapeutic decisions at the time of diagnosis and disease progression. However, it remains a challenge to effectively integrate these assessments into a comprehensive model of disease state that can be used for clinical decision-making. Here we present DNA and Antibody sequencing (DAb-seq), a novel technology for simultaneously performing high-throughput, single-cell measurements of both DNA genotype and protein markers from >5,000 single cells. To our knowledge, DAb-seq is the first technology to directly integrate readouts of DNA genotype and cellular phenotype at the single-cell level. The experimental workflow leverages the single-cell microfluidic technology on the Mission Bio Tapestri platform, a system optimized to conduct multiplex single-cell targeted genomic DNA amplification. In our modified protocol, cells are stained with a pool of monoclonal antibodies conjugated with barcoded oligonucleotides, enabling a sequencing-based readout of single-cell protein signatures. The sequencing data is analyzed with a bioinformatic pipeline that separates antibody signal from targeted genotyping data on a cell-by-cell basis. We apply DAb-seq to the analysis of multiple AML patient samples, using a panel of 50 DNA targets comprising recurrently mutated genes in AML and 23 antibodies commonly used in flow cytometry-based MRD detection. In a single test case of pediatric AML, we identify cells harboring known pathogenic KRAS and FLT3 mutations at diagnosis and relapse. Among these genetically defined cell subsets, we observe differential protein expression of CD56, CD11b, and CD15. Specifically, we find a significant positive correlation between KRAS mutational status and combined expression of CD56 and CD11b. Similarly, FLT3 mutant cells, the dominant clone at relapse, are found to express elevated levels of CD15. We further apply DAb-seq technology to the profiling of mixed phenotype leukemia and monitoring the phenotypic dynamics of genetic clones in response to CD33 targeted therapy. Our data suggest cell populations defined by single variants in reality comprise multiple immunophenotypic subpopulations, underscoring the complex interplay between genotype and protein expression. While the precise mechanism underlying this immunophenotypic diversity remains to be elucidated, our work demonstrates the utility of DAb-seq in uncovering broader patterns of genotypic and phenotypic evolution in large AML patient cohorts. Ultimately, such studies could support further stratification of disease subtypes and increase the precision with which therapies are applied. Figure Disclosures Ruff: Mission Bio Inc.: Employment. Ooi:Mission Bio: Employment, Equity Ownership. Smith:Astellas Pharma: Research Funding; Abbvie: Research Funding; fujiFilm: Research Funding; Revolution Medicines: Research Funding. Abate:Mission Bio, Inc.: Equity Ownership.

Abstract 3527: Combined high-throughput DNA genotyping and protein quantification in single cancer cells

Abstract Clonal genetic diversity is a hallmark of tumor biology. In many cancers, clonal evolution has been implicated as a driver of tumor initiation, progression, and relapse, and can be a prognostic indicator of disease outcome. Cancer cells are also phenotypically heterogeneous, constituting features which can enable intravasation and metastasis, or which can impart immunoevasive capabilities. Clinically, a combination of single-cell genotype and phenotype data from cancer biopsies could provide valuable insights into oncological disease states, such as metastatic risk or drug response. Here we present a multiomic technology for the simultaneous, high-throughput capture of DNA genotype and protein information from single cells, demonstrated through the analysis of acute myeloid leukemia (AML) derived cell lines. The experimental workflow leverages the high-throughput single-cell microfluidic droplet technology on the Mission Bio Tapestri platform, a system optimized to conduct multiplex targeted genomic DNA amplification from >50 AML-associated loci. In our modified workflow, cells are stained with a pool of monoclonal antibodies conjugated with barcoded oligonucleotides, enabling a sequencing-based readout of single-cell protein signatures. The sequencing data is analyzed with a bioinformatic pipeline that separates antibody signal from targeted genotyping data on a cell-by-cell basis. We find that on a mixed population of myelogenous leukemia cells and B-cells, single-cell genotype information effectively distinguishes between cell lines, while the protein data independently recapitulates the biological signal from conventional flow cytometric methods. In conclusion, this technology opens up a new era for discovery using multiomic analysis of the complex relationships between genotype and phenotype in cancer, paving the way for future studies analyzing primary tissue samples. Citation Format: Benjamin Demaree, Cyrille Delley, David Ruff, Sebastian Treusch, Dennis Eastburn, Keith Jones, Adam Abate. Combined high-throughput DNA genotyping and protein quantification in single cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3527.

PS975 HIGH‐RESOLUTION CLONAL ANALYSIS OF TUMORS WITH SIMULTANEOUS SINGLE‐CELL DNA SEQUENCING AND CELL SURFACE PHENOTYPING

Background:Intra‐tumor heterogeneity is a common feature of many blood cancers and is correlated with poor clinical outcomes. Single‐cell DNA sequencing can unambiguously resolve the genetic heterogeneity of tumors missed with traditional bulk next generation sequencing (NGS). Despite the advantages of single‐cell DNA sequencing, previous studies have relied upon laborious, expensive and low‐throughput approaches that are not scalable for routine analysis of tumors. Additionally, to provide maximum insight into disease progression, clonality of mutations within a tumor would also be correlated with cell lineage.Aims:We sought to demonstrate how single‐cell analysis of both cell surface markers as well as variant calling across hundreds of disease relevant loci can provide unprecedented insight into tumor architecture.Methods:To enable high‐resolution profiling of intra‐tumor heterogeneity, we previously developed a unique microfluidic droplet platform that can accurately identify genome variants at hundreds of disease relevant loci across 10,000 individual cells per sample run. This platform has a rapid, robust and easy to use workflow that, for the first time, enables routine analysis of genetic heterogeneity for hematologic malignancies.Results:Here, we report our continued work developing and applying targeted single‐cell sequencing panels for AML and other hematologic malignancies such as CLL. The high‐throughput single‐cell approach we developed has consistently revealed rare tumor cells at diagnosis and remission (MRD) that are critically important with respect to therapy response and disease progression. Additionally, for the first time, we also demonstrate integrated multi‐omic capability on our platform by enabling single‐cell measurement of cell surface protein expression levels together with genomic variant identification in a single NGS readout. In our modified workflow, cell lines, PBMCs or bone marrow aspirates are stained with a pool of monoclonal antibodies (e.g., CD34, CD33, CD19, CD45, CD30 and CD3) that are conjugated with oligonucleotides containing unique protein identifying tags. The sequencing data is analyzed with a bioinformatic pipeline that quantifies antibody levels associated genotyping data from a myeloid specific targeted sequencing panel on a cell‐by‐cell basis. With this approach the need for upstream flow sorting of cells can often be eliminated and the well‐characterized catalog of hematology cell surface markers employed to more directly correlate phenotype and cell‐state information of single cells to mutational burden.Summary/Conclusion:The multi‐omic capability we present, together with other improvements to the platform, make our technology ideally suited to fully resolve clonal architecture of hematologic malignancies. Correlation of mutation status with phenotype using high‐throughput single‐cell multi‐omics is a powerful approach with the potential to better predict disease progression and inform therapy selection.

Evaluating the effect of in-process material on the binding mechanisms of surrogate viral particles to a multi-modal anion exchange resin

Bacteriophage binding mechanisms to multi-modal anion exchange resin may include both anion exchange and hydrophobic interactions, or the mechanism can be dominated by a single moiety. However, previous studies have reported binding mechanisms defined for simple solutions containing only buffer and a surrogate viral spike (i.e. bacteriophage ΦX174, PR772, and PP7). We employed phage spiked in-process monoclonal antibody (mAb) pools to model binding under bioprocessing conditions. These experiments allow the individual contributions of the mAb, in-process impurities, and buffer composition on mechanistic removal of phages to be studied. PP7 and PR772 use synergetic binding by the positively charged quaternary amine and the hydrophobic aromatic phenyl group to bind multi-modal resin. ΦX174′s binding mechanism remains inconclusive due to operating conditions.

A New Approach to CD34+ Hematopoietic Progenitor Cell Counting

In the absence of a gold standard for hematopoietic progenitor counting, the intra-laboratory variation between commonly used strategies for progenitor assessment was compared. We used a pool of FITC-conjugated monoclonal antibodies (CD14, CD11b) and PE-CD34 to facilitate CD34 counting, excluding CD14(+)/CD11b(+) bright cells. We compared this protocol with other common methodologies, such as the single-staining approach, known as the Milan method, and the ISHAGE multiparameter method. We show that the CD14/CD11b protocol is a valid approach to progenitor cell counting. Though different methods give different results for progenitor cell counting, Lin's coefficient shows high concordance among flow cytometry counts but a substantial difference with colony- forming unit counts. Both the ISHAGE and CD14/CD11b protocol give higher counts than the Milan method. Moreover, on revising the ISHAGE analysis, we described a rare population of cells with the CD34(+)CD45(neg) phenotype, which could have an impact in CD34 counting. We have designed a valid alternative approach for hematopoietic progenitor cell counting, and we show that different methods give different results.

Human rhinovirus infection in young African children with acute wheezing

BackgroundInfections caused by human rhinoviruses (HRVs) are important triggers of wheezing in young children. Wheezy illness has increasingly been recognised as an important cause of morbidity in African children, but there is little information on the contribution of HRV to this. The aim of this study was to determine the role of HRV as a cause of acute wheezing in South African children.MethodsTwo hundred and twenty children presenting consecutively at a tertiary children's hospital with a wheezing illness from May 2004 to November 2005 were prospectively enrolled. A nasal swab was taken and reverse transcription PCR used to screen the samples for HRV. The presence of human metapneumovirus, human bocavirus and human coronavirus-NL63 was assessed in all samples using PCR-based assays. A general shell vial culture using a pool of monoclonal antibodies was used to detect other common respiratory viruses on 26% of samples. Phylogenetic analysis to determine circulating HRV species was performed on a portion of HRV-positive samples. Categorical characteristics were analysed using Fisher's Exact test.ResultsHRV was detected in 128 (58.2%) of children, most (72%) of whom were under 2 years of age. Presenting symptoms between the HRV-positive and negative groups were similar. Most illness was managed with ambulatory therapy, but 45 (35%) were hospitalized for treatment and 3 (2%) were admitted to intensive care. There were no in-hospital deaths. All 3 species of HRV were detected with HRV-C being the most common (52%) followed by HRV-A (37%) and HRV-B (11%). Infection with other respiratory viruses occurred in 20/128 (16%) of HRV-positive children and in 26/92 (28%) of HRV-negative samples.ConclusionHRV may be the commonest viral infection in young South African children with acute wheezing. Infection is associated with mild or moderate clinical disease.

Isolation, kinetic analysis, and structural characterization of an antibody targeting the Bacillus anthracis major spore surface protein BclA

One method of laboratory- or field-based testing for anthrax is detection of Bacillus anthracis spores by high-affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re-formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross-reactivity between species. These results were rationalized by determination of the three-dimensional crystallographic structure of the scFv-tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter-spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high-affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure-based antibody design for this difficult target.

A Rapid Microscopic Method for the Confirmation of Rhinoviruses in Cell Culture

Objective: Culture-confirmation of rhinovirus is done using acid lability testing, a laborious and time-consuming method which delays the reporting of patient results by 1–2 days. A fluorescent monoclonal antibody pool (Light Diagnostics Pan-Enterovirus Blend; Millipore Inc., Temecula, Calif., USA) developed to identify various enterovirus isolates in culture was recently reported to also cross-react with rhinoviruses. We evaluated the use of this cross-reacting antibody, used in tandem with non-cross-reacting enterovirus antibodies (D³ IFA; Diagnostic Hybrids Inc., Athens, Ohio, USA) to rapidly identify rhinoviruses in cell culture. Methods: Microscope slides were prepared from cell cultures of 11 rhinovirus clinical isolates and a variety of other respiratory viruses. Slides were stained using both enterovirus antibody pools and examined for fluorescent activity. Results: Positive fluorescence was observed in all the rhinovirus isolates tested using the pan-enterovirus antibody blend, but yielded negative results when stained using the D³ antibodies. Both antibody products produced positive fluorescence for enterovirus isolates and produced negative results when a variety of other respiratory viruses were examined. Conclusion: Staining suspected rhinovirus isolates with each antibody pool affords a rapid means of identifying rhinoviruses and distinguishing them from enteroviruses, without the need for acid lability testing.

Respiratory Viruses in Pediatric Necropsies: An Immunohistochemical Study

Infections of the respiratory system are responsible for the majority of hospitalizations and deaths in pediatric patients in developing countries. We selected 177 necropsies of pediatric patients who died as a result of serious respiratory infections. The histopathological findings and epidemiological data were reviewed, and lung tissue samples were separated for immunohistochemistry testing. Conventional immunohistochemistry techniques were used to detect viral antigens in formalin-fixed, paraffin-embedded (FF-PE) lung tissue samples using a pool of monoclonal antibodies against respiratory viruses (respiratory syncytial virus, influenza A and B, adenovirus, and parainfluenza 1, 2, and 3 viruses) as primary antibodies. The histopathological findings were classified into bronchopneumonia (BCP) and interstitial pneumonitis (IP) patterns. The immunohistochemistry results were compared with histopathological patterns and epidemiological data. Positive results for viruses were found in 34% and 62.5% of the BCP and IP cases, respectively. Males and infants below 1 year of age were more frequent in the group that had positive results for viruses. Acute enteritis was the main cause of hospitalization and sepsis the most frequent cause of death in this group. A clear seasonal distribution was observed, with the majority of cases occurring in the 2nd and 3rd trimesters (autumn and winter) of each year in the period studied. Immunohistochemistry is an affordable and easy-to-perform method for viral-antigen detection in FF-PE tissue samples. Although BCP is a classic histopathological pattern found in bacterial infections, it is possible that children with serious respiratory infections had concomitant viral and bacterial infections, regardless of their previous immunologic state.

Diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the North-East of England

Background Since its discovery in 2001 human metapneumovirus (hMPV) has been shown to be a significant cause of human respiratory disease, responsible for 5–8% of respiratory infections in hospitalised children. Diagnosis hitherto has been largely carried out by reverse tanscriptase polymerase chain reaction (RT-PCR) but immunofluorescence staining of cells from nasopharyngeal secretions (IF) offers advantages for some laboratories and may produce a more rapid result in urgent cases. We have recently demonstrated that IF with a rabbit antiserum gave sensitivity equal to that of RT-PCR. However, monoclonal antibodies offer a more plentiful, uniform IF reagent. Objectives Here we have evaluated a pool of anti-hMPV monoclonal antibodies in the routine diagnosis of respiratory infections in hospitalised infants and children. Study design Eight hundred and fifty-seven routine respiratory specimens were tested by IF with rabbit polyclonal antiserum and monoclonal antibody pool in parallel. A further 1003 specimens were tested with the monoclonal antibody pool alone. All specimens were also tested for a panel of other respiratory viruses by IF. Results Both rabbit polyclonal antiserum and monoclonal antibody pool gave positive results in 56 and negative results in 797 specimens. The rabbit polyclonal antibody detected virus in a further two specimens which were negative when tested with the monoclonal pool giving a concordance of 96.6% and a specificity of 100% for the monoclonal antibody pool. Overall hMPV was detected in 5% of specimens whilst 18.4% were positive for hRSV. Conclusions The monoclonal antibody pool-based IF is a robust assay suitable for routine use with a sensitivity only slightly less than that of the other major diagnostic methodologies available.

A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129 bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments.

The 2β 2–2β 3 loop of anthrax protective antigen contains a dominant neutralizing epitope

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, 312SFFD 315, in the 2β 2–2β 3 loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2β 2–2β 3 loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.

Production of monoclonal antibodies against Rift Valley fever virus: Application for rapid diagnosis tests (virus detection and ELISA) in human sera

This paper describes the production and characterization of RVFV monoclonal antibodies. The characteristics of 32 out of 55 ELISA and/or IFA positive monoclonal antibodies were determined, including the RVFV components against which they are directed. One monoclonal antibody recognized the nucleoprotein, 15 the Gc and 16 the Gn. Among the latter ones, five monoclonal antibodies possess another specificity and recognized both Gn and either the nucleoprotein (four of them) or the NSs protein (one). To validate the use of these monoclonal antibodies for diagnosis tests, a pool of monoclonal antibodies reacting with the structural proteins was prepared and used successfully to detect RVFV from cell culture as well as viral antigen–antibody complex in ELISA.

A simplified plaque assay for respiratory syncytial virus—direct visualization of plaques without immunostaining

Due to their generally small size, detection of respiratory syncytial virus (RSV) plaques commonly relies on immunostaining using either polyclonal antisera or a pool of monoclonal antibodies against the surface fusion glycoprotein. Disadvantages include the costs of the antibodies and substrate, and microscopic examination is often needed for counting of plaques. By optimizing many parameters, greatly improved plaque assays for RSV A2, Long and RSV B 18537 strains using Vero or HEp-2 cells have been developed. Although many groups use Vero cells for plaquing, after optimization plaques were much larger in HEp-2 cells. Both cell types needed to be confluent at infection and agarose was more suitable than carboxymethyl cellulose for the overlay. Overlay medium for HEp-2 was DMEM/F12, 0.3% agarose, and for Veros it was Liebovitz L15, 5% fetal calf serum, 0.3–0.5% agarose. After 5–7 days, monolayers were fixed with 1% formal saline overnight, agarose was removed and monolayers were stained with 0.05% neutral red in water. Plaques were readily visible by eye and plates could be dried and stored permanently. Parainfluenza virus type 3 also formed large plaques in HEp-2 cells under the conditions optimal for RSV.

A comparison of virus isolation, indirect immunofluorescence and nested multiplex polymerase chain reaction for the diagnosis of primary and recurrent herpes simplex type 1 and type 2 infections

134 swabs in viral transport medium were received from 126 patients with suspected clinical HSV-1 and HSV-2 infections. They were tested by (i) nested multiplex polymerase chain reaction NMPCR (strongly positive specimens had visible bands on both rounds of PCR) without prior extraction, (ii) culture in primary rhesus monkey kidney, E6-Vero, RD and HEp-2 cells and (iii) antigen detection by immunofluorescence (IF). Antigen detection employed four novel pools (A–D) of monoclonal antibodies (Mab): A was HSV-1 specific, B was HSV-2 specific while C and D were generic. In comparison to NMPCR the sensitivity and specificity of (i) culture was 59% (22/37) and 100% (134/134), (ii) IF by Pool A was 59% (16/27) and 100% (117/117), (iii) IF by Pool B was 40% (4/10) and 100% (130/130) and (iv) IF by Pools C and D were 60% (18/30) and 100% (96/96). Specimens positive by culture were more likely to be strongly positive by NMPCR ( χ 2 P=0.004). Typing by each method concurred on all occasions. NMPCR was cost effective, easier to perform and was the most sensitive method for HSV detection. It should become the method of choice for HSV diagnosis.