Reactivity Of mAbs Research Articles

Mixed origins: HIV vaccine induced Env-specific antibodies arise from naive and cross-reactive memory B cells

Abstract The most potent and broad HIV envelope (Env)-specific antibodies (Abs) often when reverted to their inferred germline versions representing the naïve B cell receptor, usually fail to bind Env. This may suggest that the primary B cell population that responds to the initial Env exposure is not exclusively comprised of a naïve population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated 66 gp120 reactive mAbs from participants from HVTN105, a phase I trial testing AIDSVAX B/E (gp120 protein) combined with a DNA immunogen. In order to determine the origin of these mAb producing B cells, we used Miseq-based VH lineage tracking approach and identified several of these mAb lineages in peripheral blood prior to vaccination (pre-immune). Further, several of these pre-immune lineages also persist in the bone marrow ~8 months after the final vaccination, including within in the CD138+ long-lived plasma cell compartment. When tested by ELISA, pre-immune mAbs bound to HIV Env, with relatively lower ability than their post-vaccination forms, but showed higher reactivity than their germline form. The majority of the pre-immune lineage members were IgM, however IgG and IgA members were predominant prior to vaccination and the majority of the pre-immune lineage members exhibited somatic hypermutation. These results may indicate that the vaccine-induced Env-specific antibody lineages originated from a mixed ancestor population of naïve and cross-reactive memory cells.

A Computationally Optimized Broadly Reactive Antigen Subtype-Specific Influenza Vaccine Strategy Elicits Unique Potent Broadly Neutralizing Antibodies against Hemagglutinin.

Computationally optimized broadly reactive Ags (COBRA) targeting H1 elicit a broad cross-reactive and cross-neutralizing Ab response against multiple H1N1 viral strains. To assess B cell breadth, Mus musculus (BALB/c) Ab-secreting cells elicited by a candidate COBRA hemagglutinin (HA) (termed P1) were compared with Ab-secreting cells elicited by historical H1N1 vaccine strains. In addition, to evaluate the Ab response elicited by P1 HA at increased resolution, a panel of P1 HA-specific B cell hybridomas was generated following immunization of mice with COBRA P1 and the corresponding purified mAbs were characterized for Ag specificity and neutralization activity. Both head- and stem-directed mAbs were elicited by the P1 HA Ag, with some mAbs endowed with Ab-dependent cell-mediated cytotoxicity activity. P1 HA-elicited mAbs exhibited a wide breadth of HA recognition, ranging from narrowly reactive to broadly reactive mAbs. Interestingly, we identified a P1 HA-elicited mAb (1F8) exhibiting broad hemagglutination inhibition activity against both seasonal and pandemic H1N1 influenza strains. Furthermore, mAb 1F8 recognized an overlapping, but distinct, epitope compared with other narrowly hemagglutination inhibition-positive mAbs elicited by the P1 or wild-type HA Ags. Finally, P1 HA-elicited mAbs were encoded by distinct H chain variable and L chain variable gene segment rearrangements and possessed unique CDR3 sequences. Collectively, the functional characterization of P1 HA-elicited mAbs sheds further insights into the underlying mechanism(s) of expanded Ab breadth elicited by a COBRA HA-based immunogen and advances efforts toward design and implementation of a more broadly protective influenza vaccine.

Serum low replacement medium versus serum rich replacement medium for production of Anti-Schistosoma Monoclonal antibody: A comparative study

Objectiveto assess the production of Anti-Schistosoma Monoclonal Antibodies by growing IgM producing hybridoma cell line in serum low medium and to evaluate their diagnostic potential to replace the old conventional method in animal ascetic fluid. MethodsHighly reactive and specific IgM MAb secreting hybridoma cell line to S. mansoni SEA was cultured in three different media; serum rich replacement medium (conventional), serum low replacement medium (SLRM) and serum free replacement medium (SFRM). ResultsCell count and viability were better in conventional medium and SLRM than SFRM. The lower detection limit of the assay Mab of SLRM culture was 1.1 ng/ml of S. mansoni SEA. SFRM was excluded because of the low concentration and reactivity. MAb was tested against 34 human samples. Sensitivity and specificity of the assay for each were 92% and specificity 90% for SLRM respectively. The diagnostic efficacy of the assay was 94%. Conclusionsthe production and diagnostic efficacy of antischistosomal MAbs was comparable on usage of either in vitro cell culture supernate in SLRM or conventional media.

Precise Immunodetection of PTEN Protein in Human Neoplasia.

PTEN is a major tumor-suppressor protein whose expression and biological activity are frequently diminished in sporadic or inherited cancers. PTEN gene deletion or loss-of-function mutations favor tumor cell growth and are commonly found in clinical practice. In addition, diminished PTEN protein expression is also frequently observed in tumor samples from cancer patients in the absence of PTEN gene alterations. This makes PTEN protein levels a potential biomarker parameter in clinical oncology, which can guide therapeutic decisions. The specific detection of PTEN protein can be achieved by using highly defined anti-PTEN monoclonal antibodies (mAbs), characterized with precision in terms of sensitivity for the detection technique, specificity for PTEN binding, and constraints of epitope recognition. This is especially relevant taking into consideration that PTEN is highly targeted by mutations and posttranslational modifications, and different PTEN protein isoforms exist. The precise characterization of anti-PTEN mAb reactivity is an important step in the validation of these reagents as diagnostic and prognostic tools in clinical oncology, including their routine use in analytical immunohistochemistry (IHC). Here, we review the current status on the use of well-defined anti-PTEN mAbs for PTEN immunodetection in the clinical context and discuss their potential usefulness and limitations for a more precise cancer diagnosis and patient benefit.

New Monoclonal Antibodies for a Selective Detection of Membrane-Associated and Soluble Forms of Carbonic Anhydrase IX in Human Cell Lines and Biological Samples.

Monoclonal antibodies (MAbs) selectively targeting tumor-associated antigens such as carbonic anhydrase IX (CA IX) can significantly contribute to research, diagnostics, and treatment of CA IX-related cancers. CA IX is overexpressed in numerous hypoxic cancers where it promotes tumor progression. Therefore, it is considered as a promising tumor biomarker. A novel collection of MAbs against recombinant CA IX was developed and evaluated in different immunoassays for studying CA IX expression. The reactivity of MAbs with native cell surface protein was confirmed by flow cytometry and the presence of hypoxia-inducible CA IX was investigated in several human cancer cell lines. In addition, the applicability of MAbs for visualization of CA IX-positive tumor cells by immunofluorescence microscopy was demonstrated. MAb H7 was identified as the most promising MAb for different immunoassays. It recognized a linear epitope covering CA IX sequence of 12 amino acid residues 55-GEDDPLGEEDLP-66 within the proteoglycan domain. The MAb H7 was the only one of the collection to immunoprecipitate CA IX protein from cell lysates and detect the denatured CA IX with near-infrared fluorescence Western blot. It was also employed in sandwich enzyme-linked immunosorbent assay to detect a soluble form of CA IX in growth medium of tumor cells and blood plasma samples. The diagnostic potential of the MAb H7 was confirmed on formalin-fixed and paraffin-embedded tissue specimen of cervical carcinoma in situ by immunohistochemistry. The generated MAbs, in particularly clone H7, have great potential in diagnostics and research of CA IX-related cancers.

Abstract 2983: LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral cancer

Abstract Background Podoplanin (PDPN) is a specific ligand of C-type lectin-like recepotor-2 (CLEC-2), and is involved in cancer metastasis. The physiological PDPN function has been discussed in many normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. Herein, we aimed to produce cancer-specific anti-PDPN monoclonal antibodies (mAbs). Furthermore, we investigated whether the reactivity of cancer-specific anti-PDPN mAbs might be a prognostic marker of oral cancer. Materials and methods We immunized mice with LN229/PDPN cells, and screened mAbs, which react with PDPN-expressing cancer cell lines, such as glioblastoma cell lines and lung squamous cell carcinoma cell lines and do not react with PDPN-expressing normal cells, including primary lymphatic endothelial cells and renal epithelial cell lines in flow cytometry. Furthermore, we investigated the reactivity of anti-PDPN mAbs by immunohistochemistry against oral cancers. Anti-tumor activities by mouse-human chimeric LpMab-23 (chLpMab-23) were examined using mouse xenograft models of oral cancer cells. Finally, we analyzed the association between the reactivity of anti-PDPN mAbs and clinical/pathological features of oral cancers. Results We established LpMab-23, a cancer-specific anti-PDPN mAb. LpMab-23 reacted with PDPN-expressing cancer cell lines in flow cytometry. In contrast, LpMab-23 recognized PDPN-expressing normal cells very weakly. LpMab-23 reacted only with PDPN-expressing cancer cells, not with lymphatic endothelial cells in oral cancer tissues by immunohistochemistry. The epitope mapping of anti-PDPN mAbs revealed that LpMab-23 recognized a cancer-specific glycopeptide including Thr55/Ser56. chLpMab-23 revealed high ADCC and anti-tumor activities against oral cancers. The Kaplan-Meier curves of the five-year new metastasis-free survival rate (nMFS) were significantly lower in LpMab-23-positive patients than in the patients with LpMab-23-negative ones. Conclusions A cancer-specific anti-PDPN mAb (LpMab-23) and its mouse-human chimeric mAb (chLpMab-23) were successfully established. LpMab-23 is advantageous in anti-tumor activities. LpMab-23-positive cases could be a useful predictor of poor prognosis for oral cancer. Citation Format: Takashi Miwa, Shinji Yamada, Mika K. Kaneko, Yoshikazu Furusawa, Masato Fukui, Yukinari Kato. LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral cancer [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 2983.

Development of Atroposelective Antibodies by Immunization with a Racemic Mixture of Binaphthyl Derivatives

Abstract The design and creation of chiral recognition elements are important for the synthesis, separation, and detection of chiral molecules. We prepare monoclonal antibodies (mAbs), which are chemically homogeneous antibodies, as tailored chiral recognition elements by immunizing mice with a racemic mixture of a binaphthyl derivative (BN (rac)) conjugated to keyhole limpet hemocyanin (KLH). Immunization with BN (rac) induces an immunoresponse that is as strong as that with enantiomerically pure antigens and yields mAbs for each enantiomer of BN, simultaneously. Notably, one of the mAbs prepared by immunization with BN (rac) recognizes the axial chirality of the BN enantiomer with a 14000-fold difference in affinity. These findings provide a strategy to obtain atroposelective antibodies for each enantiomer of BN with a single immunization. mAbs also recognize the axial chirality of 1,1′-bi-2-naphthol (BINOL) and 1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNPA), which are an important chiral building block and a chiral organic catalyst, respectively. The cross reactivity of mAbs for the partial structure of BN is significantly low. Therefore, mAbs recognize the axial chirality of BN, BINOL, and BNPA by binding their binaphthyl moiety.

Enhancement of binding avidity by bivalent binding enables PrPSc-specific detection by anti-PrP monoclonal antibody 132.

Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119–127, enables us to reliably detect abnormal isoform prion protein (PrPSc) in cells or frozen tissue sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrPSc-specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrPC) are close enough to allow bivalent binding, mAb 132 binds to PrPC. These results indicate that weak monovalent binding to monomeric PrPC diminishes PrPC signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrPSc in a bivalent manner.

A Computationally Optimized Broadly Reactive Antigen (COBRA) Elicits Broadly Neutralizing Antibodies Against a Conserved Influenza Virus Hemagglutinin B-Cell Epitope

Abstract The development of broadly protective influenza vaccines will represent the main countermeasure to overcome influenza virus spread and improve the coverage offered by the current standard of care. A computationally optimized broadly reactive influenza virus hemagglutinin (HA) antigen (COBRA), named P1, elicits a broadly reactive antibody (Ab) response against multiple H1N1 strains. In order to understand the mechanism of P1-conferred breadth of response, we first characterized the breadth of the Ab response at the B-cell level. Specifically, the reactivity of secreted Abs from mice immunized with P1 or seasonal/pandemic vaccine strains was assessed against a panel of H1N1 recombinant HA. Interestingly, while Abs from seasonal/pandemic immunized mice showed a predominant homologous response, those from P1- immunized animals exhibited a broader recognition. In order to dissect the antibody response, a panel of P1-specific B-cell hybridomas was generated. Subsequently, the corresponding purified monoclonal Abs (mAbs) were assessed for the breadth of HA binding and hemagglutination inhibition (HAI) activity against different H1N1 viruses. Collectively, P1-specific mAbs exhibited a broad spectrum of binding and functional activities, spanning from strain-specific to broadly reactive and neutralizing mAbs, while seasonal/pandemic-specific mAbs displayed a narrower spectrum of activity. Epitope mapping studies revealed that P1-elicited broadly reactive and neutralizing Abs recognize unique conserved epitopes within the HA receptor binding site. Collectively, these studies shed light on the mechanism of breadth conferred by P1 and leverage its optimization for the development of a more effective influenza vaccine.

Anti-tumor effects of mAb against L-type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes.

l‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.

Identification of two novel fowl adenovirus C-specific B cell epitopes using monoclonal antibodies against the capsid hexon protein.

The diseases associated with fowl adenovirus (FAdV) infection, such as inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HPS), and gizzard erosion (GE), were first reported in Pakistan in 1987, and subsequent outbreaks have been reported worldwide, especially in China, where severe outbreaks of HPS with high mortality from 30 to 100% were recently reported and resulted in significant economic losses to the poultry industry. The diagnosis methods of FAdVs were mostly limited to the nucleotide sequence of hexon by PCR and DNA sequencing. The aim of this study was to generate B cell epitope maps of the species- and serotype-specific hexon L1 using monoclonal antibodies (mAbs) and bioinformatics tools for the development of novel diagnostic methods. In this study, the hexon L1 (230 amino acids) was expressed and used to generate 10 mAb-expressing hybridoma cell lines against the relative protein peptide. Subsequently, we defined the linear peptide epitopes recognized by these mAbs using a series of partially overlapping peptides derived from the FAdV-C hexon protein amino acid sequence to map mAbs reactivity. Finally, a common B cell epitope (31PLAPKESMFN40) for all species FAdVs and two FAdV-C-specific epitopes (79KISGVFPNP87 and 181DYDDYNIGTT190) were identified. These mAbs and their defined epitopes may support the development of the universal or species-specific differential diagnostic methods of FAdVs.

Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer.

MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.

Variation at position 350 in the Chikungunya virus 6K-E1 protein determines the sensitivity of detection in a rapid E1-antigen test

Chikungunya virus (CHIKV), a mosquito-borne pathogen, consists of three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. Although a current rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity to ECSA-genotype viruses, it showed poor performance against the Asian-genotype virus that is spreading in the American continents. To understand the basis for the low performance of this IC test against Asian-genotype virus, we re-examined the anti-CHIKV monoclonal antibodies (mAbs) used in the assay for their interaction with E1-antigen of the three CHIKV genotypes. We found that the reactivity of one mAb for Asian-genotype virus was lower than that for ECSA virus. Comparison of E1 amino acid sequences revealed that the ECSA virus used to generate these mAbs possesses glutamic acid (E) at position 350, in contrast to WA and Asian, which possess aspartic acid (D) at this position. Site-directed mutagenesis confirmed that the mutation altered mAb reactivity, since E-to-D substitution at position 350 in ECSA reduced recognition by the mAb, while D-to-E substitution at this position in Asian and WA increased affinity for the mAb. Taken together, these results indicate that residue 350 of the CHIKV 6K-E1 is a key element affecting the performance of this IC assay.

Alanine scanning mutagenesis of SP70 epitope in characterizing species‑specific antibodies induced by enterovirus 71‑based antigens

The enterovirus 71 (EV71) SP70 epitope, derived from amino acids 208–222 of VP1, is a neutralizing epitope. The present study aimed to assess the inter-species differences of the antibodies induced by EV71-based antigens in responses to SP70 mutant peptides. BALB/c mice and Lou/C rats were immunized with EV71 SP70. Monoclonal antibodies (Mabs) were produced by hybridoma clones. Serum polyclonal antibodies (Pabs) were produced from BALB/c mice and New Zealand white rabbits immunized with recombinant EV71 VP1 (rEV71-VP1) protein or inactivated EV71. Micro-neutralization and immunofluorescence assays were used to evaluate the capacity of the antibodies to bind to EV71. Reactivity of Mabs and Pabs to mutated SP70 were determined by alanine scanning mutagenesis. Furthermore, serums from EV71-infected patients were collected to examine the affinity of SP70 antibody in the serum to mutated SP70, using competitive ELISA. The binding affinity of mouse Mabs to the SP70 epitope was increased by alanine substitution at sites of 210, 212, 213, 214, and 221. The binding affinity of rat Mabs to the SP70 epitope was increased by alanine substitution at sites 210, 217, 219, and 221. Mouse serum Pabs elicited by inactivated EV71 bound wild-type SP70, but lost affinity for mutated peptides. Conversely, rabbit serum Pabs elicited by inactivated EV71 robustly recognized SP70 mutants. Mouse serum Pabs elicited by rEV71-VP1 presented the same trend as mouse Mabs. Mutations at sites 214, 215, and 217 led to loss of recognition by rabbit Pabs elicited by rEV71-VP1, while most mutations did not influence antibody binding. Compared with the wild-type, mutations at the sites 209, 219 and 221 of SP70 lead to increased affinity with the serum antibodies produced by the EV71-infected patients. Antibody responses triggered by inactivated EV71, rEV71-VP1 and EV71 SP70 differed among species in neutralizing capacity and affinity for SP70 mutant peptides.

Immunocapture isotope dilution mass spectrometry in response to a pandemic influenza threat.

As a result of recent advances in mass spectrometry-based protein quantitation methods, these techniques are now poised to play a critical role in rapid formulation of pandemic influenza vaccines. Analytical techniques that have been developed and validated on seasonal influenza strains can be used to increase the quality and decrease the time required to deliver protective pandemic vaccines to the global population. The emergence of a potentially pandemic avian influenza A (H7N9) virus in March of 2013, prompted the US public health authorities and the vaccine industry to initiate production of a pre-pandemic vaccine for preparedness purposes. To this end, we evaluated the feasibility of using immunocapture isotope dilution mass spectrometry (IC-IDMS) to evaluate the suitability of the underlying monoclonal and polyclonal antibodies (mAbs and pAbs) for their capacity to isolate the H7 hemagglutinin (HA) in this new vaccine for quantification by IDMS. A broad range of H7 capture efficiencies was observed among mAbs tested by IC-IDMS with FR-545, 46/6, and G3 A533 exhibiting the highest cross-reactivity capabilities to H7 of A/Shanghai/2/2013. MAb FR-545 was selected for continued assessment, evaluated by IC-IDMS for mAb reactivity against H7 in the H7N9 candidate vaccine virus and compared with/to reactivity to the reference polyclonal antiserum in allantoic fluid, purified whole virus, lyophilized whole virus and final detergent-split monovalent vaccine preparations for vaccine development. IC-IDMS assessment of FR-545 alongside IC-IDMS using the reference polyclonal antiserum to A/Shanghai/2/2013 and with the regulatory SRID method showed strong correlation and mAb IC-IDMS could have played an important role in the event a potential surrogate potency test was required to be rapidly implemented.

ANTI-N-TERMINAL ABETA MAB 3A1 PREFERENTIALLY RECOGNIZES ABETA AGGREGATES AND DOES NOT CROSS-REACT WITH APP

The murine anti-N-terminal Abeta monoclonal antibody (mAb) 3A1 was generated against dityrosine cross-linked Abeta1–40 protein species (CAPS). 3A1's epitope comprises of Abeta's first 15 amino acids, DAEFRHDSGYEVHHQ. The mAb has demonstrated activity in vivo by decreasing plaque burden and increasing plasma Abeta in an APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease [Frost et al (2015) Neurobiol Aging. 36(12): 3187]. To better understand the mAb's specificity for Abeta we established in vitro the antibody's ability to recognize Abeta1–40 conformers (monomers, dimers, protofibrils), APP, and rodent Abeta. 3A1's avidity for Abeta conformers, APP and rodent Abeta were determined using ELISA, and Western blot assays. Anti-N-terminal Abeta mAb, 6E10 [Kim et al. (1988) Neurosci. Res. Comm. 7:113], was used as positive control. Abeta conformers were generated as previously described [Welzel et al (2012) PLoS One. 7(11):e50317]. MAb 3A1 had similar avidity for plate-immobilized Abeta1–40 conformers as mAb 6E10, with EC50 values in the sub nM range. Both antibodies had essentially no reactivity with rodent Abeta1–40. However, in contrast to 6E10, mAb 3A1 showed a strong preference for aggregated compared with monomeric Abeta1–40, and did not detect APP protein. The N-terminal Abeta reactive MAb 3A1 demonstrated novel specificity by preferentially targeting the aggregated peptide without cross-reacting with APP. This unique activity may be utilized in immunoassay(s) to advance current poor understanding on the role of Abeta species in Alzheimer's disease.

Monoclonal antibodies against recombinant GAPDH of Edwardsiella tarda reveal the conserved nature of the protein

ABSTRACTThe outer membrane protein, encoded by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, of Edwardsiella tarda is a highly conserved immunogenic protein. The GAPDH was cloned and expressed in Escherichia coli. The purified protein was used to produce mouse monoclonal antibodies (MAbs). Four stable hybridomas producing MAbs (3G12, 4E9, 5A11 and 9G1) against rGAPDH were obtained. The heavy chains of antibodies produced by the hybridomas were of the isotypes IgG1 and IgM. Cross reactivity of MAbs (3G12 and 9G1) was observed with GAPDH of Aeromonas hydrophila and Micrococcus luteus. MAbs 3G12 and 4E9 reacted with Vibrio cholerae, Salmonella enterica and Penaeus monodon tissues but not with vertebrate GAPDH. None of the MAbs reacted with Staphylococcus aureus. The results indicate that the level of conservation of GAPDH is high among evolutionarily close species. The MAbs developed will be a useful tool to study the evolutionarily conserved and functionally diverse GAPDH.

Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules

Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases.

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab')2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.

New insights into the phenotype of human dendritic cell populations.

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin-derived DCs, tonsil leukocytes, monocyte-derived DCs, CD34-derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell-surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte-surface molecules.