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The authors have withdrawn their manuscript due to becoming aware of methodology issues related to the curation of the training set used to determine cut-off values for Biotyper cluster assignation and lack of replicate measurements on different days for the isolates analysed. It is therefore unclear whether the conclusions of the manuscript are founded and no further work is possible to correct these issues as the instrument is no longer available to the authors. If you have any questions, please contact the corresponding author.

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BACKGROUND: Hospital sinks are environmental reservoirs that harbour healthcare-associated (HCA) pathogens. Selective pressures in sink environments, such as antibiotic residues, nutrient waste and hardness ions, may promote antibiotic resistance gene (ARG) exchange between bacteria. However, cheap and accurate sampling methods to characterize these factors are lacking. AIMS: To validate a workflow to detect antibiotic residues and evaluate water chemistry using dipsticks. Secondarily, to validate boric acid to preserve the taxonomic and ARG ('resistome') composition of sink trap samples for metagenomic sequencing. METHODS: Antibiotic residue dipsticks were validated against serial dilutions of ampicillin, doxycycline, sulfamethoxazole and ciprofloxacin, and water chemistry dipsticks against serial dilutions of chemical calibration standards. Sink trap aspirates were used for a 'real-world' pilot evaluation of dipsticks. To assess boric acid as a preservative of microbial diversity, the impact of incubation with and without boric acid at ∼22 °C on metagenomic sequencing outputs was evaluated at Day 2 and Day 5 compared with baseline (Day 0). FINDINGS: The limits of detection for each antibiotic were: 3 µg/L (ampicillin), 10 µg/L (doxycycline), 20 µg/L (sulfamethoxazole) and 8 µg/L (ciprofloxacin). The best performing water chemistry dipstick correctly characterized 34/40 (85%) standards in a concentration-dependent manner. One trap sample tested positive for the presence of tetracyclines and sulphonamides. Taxonomic and resistome composition were largely maintained after storage with boric acid at ∼22 °C for up to five days. CONCLUSIONS: Dipsticks can be used to detect antibiotic residues and characterize water chemistry in sink trap samples. Boric acid was an effective preservative of trap sample composition, representing a low-cost alternative to cold-chain transport.

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Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.

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Human chorionic gonadotropin (hCG) belongs to the family of glycoprotein hormones. All members of the family are composed of an identical alpha subunit and structurally related beta subunit which confers biological specificity. Specific quantification and functional analysis of hCG require the use of monoclonal antibodies recognizing different epitopes of hCGbeta. This study describes the production and characterization of monoclonal antibodies (MAbs) to hCGbeta with no cross-reactivity to other glycoprotein hormones. Spleen cells from Balb/c mice immunized with hCG were fused with mouse SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of highly purified and recombinant glycoprotein hormones, their subunits and peptides representing the C-terminal end of hCGbeta (hCGbeta-CTP) by ELISA and immunoblotting. The affinity constant (K(aff)) was also determined by ELISA. Three murine hybridomas designated G5M1, B12M2 and F4M3 were obtained that secrete MAbs specific for hCGbeta. The G5M1 MAb reacts only with hCGbeta, hCGbeta-CTP and intact hCG with no detectable cross-reaction with hCGalpha or any of the other glycoprotein hormones. The specificity of B12M2 MAb is very similar to G5M1, but it does not react with hCGbeta-CTP. The F4M3 MAb also has similar specificity to G5M1 and B12M2, but it strongly cross-reacts with hLH. The affinity constant (Kaff) of G5M1, B12M2 and F4M3 was found to be 4.28 x 10(9), 5.2 x 10(8), and 1.97 x 10(9) M(-1), respectively. Our results indicate that G5M1 and B12M2 MAbs are specific for hCG and recognize epitopes restricted to hCGbeta, but F4M3 recognizes a common epitope expressed both on hCGbeta and hLHbeta.

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Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.

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Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.

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Human IgG is comprised of four subclasses (IgG(1), IgG(2), IgG(3), and IgG(4)). Each subclass possesses different biological properties. One of the differential specificities of human IgG subclasses is binding of Fc fragment of IgG(1), 2, and 4 but, not IgG(3) to staphylococcal protein A (SPA). This study was conducted to produce, select and characterize a monoclonal antibody (MAb) recognizing human IgG subclasses with specificity similar to SPA. Splenocytes from Balb/c mice immunized with Fc fraction of a human IgG(1) myeloma protein were fused with Sp2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAb was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. A murine hybridoma designated 6F11E1 was obtained that secretes an MAb specific for the Fc fragment of the immunizing protein. This MAb reacts with isotypic epitope common to IgG(1), 2 and 4 subclasses. An allelic epitope linked to IgG(3) molecules is also recognized by 6F11E1. This pattern of reactivity was found to be highly similar to that of SPA. Our findings imply that similar or overlapping epitopes are recognized by 6F11E1 and SPA.

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