Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Redefining Pleiotropic Drug Resistance in a Pathogenic Yeast: Pdr1 Functions as a Sensor of Cellular Stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 ( CYB5 , SSK1 , SSK2 , HOG1 , TRP1 ). A bZIP transcription repressor of mitochondrial function ( CIN5 ) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata . Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. Importance: Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1 - a key determinant of fluconazole resistance - is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Cell wall protein variation, break-induced replication, and subtelomere dynamics in Candida glabrata.

Candida glabrata is an opportunistic pathogen of humans, responsible for up to 30% of disseminated candidiasis. Adherence of C. glabrata to host cells is mediated by adhesin-like proteins (ALPs), about half of which are encoded in the subtelomeres. We performed a de novo assembly of two C. glabrata strains, BG2 and BG3993, using long single-molecule real-time (SMRT) reads, and constructed high-quality telomere-to-telomere assemblies of all 13 chromosomes to assess differences between C. glabrata strains. We documented variation between strains, and in agreement with earlier studies, found high (~0.5%-1%) frequencies of SNVs across the genome, including within subtelomeric regions. We documented changes in ALP gene structure and complement: there are large length differences in ALP genes in different strains, resulting from copy number variation in tandem repeats. We compared strains to characterize chromosome rearrangement events including within the poorly characterized subtelomeric regions. We show that rearrangements within the subtelomere regions all affect ALP-encoding genes, and 14/16 involve just the most terminal ALP gene. We present evidence that these rearrangements are mediated by break-induced replication. This study highlights the constrained nature of subtelomeric changes impacting ALP gene complement and subtelomere structure.

De novo genome assembly of Candida glabrata reveals cell wall protein complement and structure of dispersed tandem repeat arrays.

Candida glabratais an opportunistic pathogen in humans, responsible for approximately 20% of disseminated candidiasis. Candida glabrata's ability to adhere to host tissue is mediated by GPI-anchored cell wall proteins (GPI-CWPs); the corresponding genes contain long tandem repeat regions. These repeat regions resulted in assembly errors in the reference genome. Here, we performed a de novo assembly of the C. glabrata type strain CBS138 using long single-molecule real-time reads, with short read sequences (Illumina) for refinement, and constructed telomere-to-telomere assemblies of all 13 chromosomes. Our assembly has excellent agreement overall with the current reference genome, but we made substantial corrections within tandem repeat regions. Specifically, we removed 62 genes of which 45 were scrambled due to misassembly in the reference. We annotated 31 novel ORFs of which 24 ORFs are GPI-CWPs. In addition, we corrected the tandem repeat structure of an additional 21 genes. Our corrections to the genome were substantial, with the length of new genes and tandem repeat corrections amounting to approximately 3.8% of the ORFeome length. As most corrections were within the coding regions of GPI-CWP genes, our genome assembly establishes a high-quality reference set of genes and repeat structures for the functional analysis of these cell surface proteins.

CD226 ligation protects against EAE by promoting IL-10 expression via regulation of CD4+ T cell differentiation.

Treatment targeting CD226 can ameliorate experimental autoimmune encephalomyelitis (EAE), the widely accepted model of MS. However, the mechanisms still need to be elucidated. Here we showed that CD226 blockage by anti-CD226 blocking mAb LeoA1 efficiently promoted IL-10 production in human peripheral blood monocytes (PBMC) or in mixed lymphocyte culture (MLC) system, significantly induced the CD4+IL-10+ T cell differentiation while suppressing the generation of Th1 and Th17. Furthermore, CD226 pAb administration in vivo reduced the onset of EAE in mice by promoting IL-10 production and regulating T cell differentiation. Concomitantly, the onset and severity of EAE were reduced and the serum IL-10 expression levels were increased in CD226 knockout mice than that in control mice when both received EAE induction. These novel findings confirmed that CD226 played a pivotal role in mediating autoimmune diseases such as EAE. Furthermore, to our knowledge, we show for the first time that IL-10 is an important contributor in the inhibitory effects of CD226 ligation on EAE.

RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.

Hantaan virus infection induces CXCL10 expression through TLR3, RIG-I, and MDA-5 pathways correlated with the disease severity.

Hantaan virus (HTNV) is a major agent causing hemorrhagic fever with renal syndrome (HFRS). Although the pathogenesis of HFRS is unclear, some reports have suggested that the abundant production of proinflammatory cytokines and uncontrolled inflammatory responses may contribute to the development of HFRS. CXCL10 is one of these cytokines and is found to be involved in the pathogenesis of many virus infectious diseases. However, the role of CXCL10 in the pathogenesis of HFRS and the molecular regulation mechanism of CXCL10 in HTNV infection remain unknown. In this study, we report that CXCL10 expresses highly in the HFRS patients' sera and the elevated CXCL10 is positively correlated with the severity of HFRS. We find that HTNV, a single-strand RNA virus, can act as a double-strand RNA to activate the TLR3, RIG-I, and MDA-5 signaling pathways. Through the downstream transcription factors of these pathways, NF-κB and IRF7, which bind directly to the CXCL10's promoter, the expression of CXCL10 is increased. Our results may help to better understand the role of CXCL10 in the development of HFRS and may provide some novel insights into the immune response of HTNV infection.

Elevated plasma soluble Sema4D/CD100 levels are associated with disease severity in patients of hemorrhagic fever with renal syndrome.

BACKGROUND: Hantaan virus (HTNV) could cause a severe lethal hemorrhagic fever with renal syndrome (HFRS) in humans. Despite a limited understanding of the pathogenesis of HFRS, the importance of host-related immune responses in the pathogenesis of HFRS has been widely recognized. CD100/Sema4D has been demonstrated to play an important role in physiological and pathological immune responses, but the functional role of CD100 in infectious diseases has only been inadequately reported. The aim of this study was to investigate the pathological significance of CD100 in patients after HTNV infection. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples were collected from 99 hospitalized patients in Tangdu Hospital and 27 health controls. The level of soluble CD100 (sCD100) in plasma were quantified by ELISA and the relationship between sCD100 and the disease course or severity were analyzed. The expressions of membrane CD100 on various subpopulations of peripheral blood mononuclear cell (PBMC) were analyzed by flow cytometry. The results showed that sCD100 level in acute phase of HFRS was significantly higher in patients than that in healthy controls (P<0.0001) and the sCD100 level declined in convalescent phase. Multivariate model analysis showed that platelet count, white blood cell count, serum creatinine level and blood urea nitrogen level were associated with sCD100 levels and contributed independently to the elevated sCD100 levels. The expression of membrane CD100 on PBMCs decreased in the acute phase of HFRS patients compared with that of the normal controls and recovered in the convalescent phase. CONCLUSIONS: We reported the elevated level of plasma sCD100 in HFRS patients and the elevated level might be a result from the shedding of membrane CD100 on PBMC. The elevated level of sCD100 was associated with disease severity, suggesting that sCD100 might be a cause or a consequence of progression of HFRS. The underlying mechanisms should be explored further.

High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices.

In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL(-1) to 1600 pg mL(-1), and the measured low limit of detection (LOD) was 3.2 pg mL(-1). No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future.

Down regulation of TRAIL and FasL on NK cells by Cyclosporin A in renal transplantation patients.

TNF-related apoptosis-inducing ligand (TRAIL) and FasL can participate in cell mediated cytotoxicity via their death domain-mediated apoptotic signaling in the host-versus-graft disease occurred after renal transplantation. However, the effect of Cyclosporin A (CsA) commonly used as a drug to prevent and to treat renal transplant rejection, on these molecules have not been fully determined. In the present study, we found that with CsA administration, the expression of TRAIL and FasL predominantly on NK cells from renal transplantation patients was increased at day 5 after operation and went down to normal level on day 13. While, the levels of soluble TRAIL (sTRAIL) and sFasL in the serum increased within 25 days and went down to normal level three month later. In addition, we showed that a remarkable increase of TRAIL and FasL expression both on the surface of activated lymphocytes especially on NK cells and in the supernatants generated from mixed lymphocytes culture (MLC). Furthermore, the enhancement of these two molecules was greatly decreased by adding 500 ng/mL CsA at the beginning of MLC. We conclude that CsA may inhibit the transplant rejection partially by down-regulating the expression of TRAIL and FasL on NK cells.

HLA-A2 and B35 restricted hantaan virus nucleoprotein CD8+ T-cell epitope-specific immune response correlates with milder disease in hemorrhagic fever with renal syndrome.

BACKGROUND: Hantaan virus (HTNV) infection in humans is a serious public health concern in Asia. A potent T cell activation peptide vaccine from HTNV structure protein represents a promising immunotherapy for disease control. However, the T cell epitopes of the HTNV restricted by the HLA alleles and the role of epitope-specific T cell response after HTNV infection remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Five well-conserved novel CD8(+) T-cell epitopes of the HTNV nucleoprotein restricted by the most popular HLA alleles in Chinese Han population were defined with interferon-γ enzyme-linked immunospot assay in 37 patients infected with HTNV during hospitalization. Two epitopes aa129-aa137 and aa131-aa139 restricted by HLA-A2 and B35, respectively, were selected to evaluate the epitope-specific CD8(+) T-cell response. HLA-peptide pentamer complex staining showed that the frequency of single epitope-specific CD8(+) T cell could be detected in patients (95% confidence interval for aa129-aa137: 0.080%-0.208%; for aa131-aa139: 0.030%-0.094%). The frequency of epitope-specific pentamer(+) CD8(+) T-cell response was much higher in mild/moderate patients than in severe/critical ones at the acute stage of the disease. Moreover, the frequency of epitope-specific CD8(+) T cells at acute stage was inversely associated with the peak level of serum creatinine and was positively associated with the nadir platelet counts during the hospitalization. The intracellular cytokine staining and the proliferation assay showed that the effective epitope-specific CD8(+) T cells were characterized with the production of interferon-γ, expression of CD69 and the strong capacity of proliferation. CONCLUSION/SIGNIFICANCE: The novel HLA class I restricted HTNV nucleoprotein epitopes-specific CD8(+) T-cell responses would be closely related with the progression and the severity of the disease, which could provide the first step toward effective peptide vaccine development against HTNV infection in humans.

Hantaan virus RNA load in patients having hemorrhagic fever with renal syndrome: correlation with disease severity.

To investigate the role of viral load in the pathogenesis of hemorrhagic fever with renal syndrome, the Hantaan virus RNA load in plasma from 101 patients was quantified, and the relationships between viral load and disease course, severity, and level of specific humoral immunity were analyzed. The viral load, detectable in 79 patients, ranged from 3.43 to 7.33 log10 copies/mL of plasma. In the early stage of disease, patients in severe/critical group were found to have higher viral loads than those in the mild/moderate group (5.90 vs 5.03 log10 copies/mL; P = .001), suggesting an association between Hantaan virus load and disease severity.

Sustained high level of serum VEGF at convalescent stage contributes to the renal recovery after HTNV infection in patients with hemorrhagic fever with renal syndrome.

To investigate the role of vascular endothelial growth factor (VEGF) in the increased permeability of vascular endothelial cells after Hantaan virus (HTNV) infection in humans, the concentration of VEGF in serum from HTNV infected patients was quantified with sandwich ELISA. Generally, the level of serum VEGF in patients was elevated to 607.0 (542.2-671.9) pg/mL, which was dramatically higher compared with healthy controls (P < 0.001). There was a rapid increase of the serum VEGF level in all patients from the fever onset to oliguric stage, at which the serum creatinine reached the peak level of the disease, indicating that VEGF may be involved in the pathogenesis of renal hyper-permeability. Moreover, the serum VEGF level at convalescent stage was positively correlated with the degree of the disease severity. The sustained high level of serum VEGF at convalescence was observed in critical HFRS patients, suggesting that VEGF would probably contribute to the renal recovery after the virus clearance. Taken together, our results suggested that the VEGF would be involved in the pathogenesis of renal dysfunction at the oliguric stage after HTNV infection, but may function as a recovery factor during the convalescence to help the body self-repair of the renal injury.

The genetic polymorphisms of HLA are strongly correlated with the disease severity after Hantaan virus infection in the Chinese Han population.

The polymorphism of human leukocyte antigen (HLA), which is a genetic factor that influences the progression of hemorrhagic fever with renal syndrome (HFRS) after Hantaan virus (HTNV) infection, was incompletely understood. In this case-control study, 76 HFRS patients and 370 healthy controls of the Chinese Han population were typed for the HLA-A, -B, and -DRB1 loci. The general variation at the HLA-DRB1 locus was associated with the onset of HFRS (P < 0.05). The increasing frequencies of HLA-DRB1∗09 and HLA-B*46-DRB1*09 in HFRS patients were observed as reproducing a previous study. Moreover, the HLA-B*51-DRB1*09 was susceptible to HFRS (P = 0.037; OR = 3.62; 95% CI: 1.00-13.18). The increasing frequencies of HLA-B*46, HLA-B*46-DRB1*09, and HLA-B*51-DRB1*09 were observed almost in severe/critical HFRS patients. The mean level of maximum serum creatinine was higher in HLA-B∗46-DRB1*09 (P = 0.011), HLA-B*51-DRB1*09 (P = 0.041), or HLA-B*46 (P = 0.011) positive patients than that in the negative patients. These findings suggest that the allele HLA-B*46 and haplotypes HLA-B*46-DRB1*09 and HLA-B*51-DRB1*09 in patients could contribute to a more severe degree of HFRS and more serious kidney injury, which improve our understanding of the HLA polymorphism for a different outcome of HTNV infection.

Identification of a novel B-cell epitope of Hantaan virus glycoprotein recognized by neutralizing 3D8 monoclonal antibody.

Hantaan virus (HTNV), a member of the family Bunyaviridae, is a major agent causing haemorrhagic fever with renal syndrome, a high-mortality-rate disease threatening approximately 150 000 people around the world yearly. The 3D8 mAb displays a neutralizing activity to HTNV infection. In this study, the B-cell epitopes of HTNV glycoproteins (GPs) were finely mapped by peptide scanning. A new B-cell epitope (882)GFLCPEFPGSFRKKC(896) of HTNV, which locates on Gc, has been screened out from a set of 15-mer synthesized peptides covering the full-length of HTNV-GPs. It has been shown by the alanine-scanning technique that (885)C, (893)R, (894)K, (895)K and (896)C are the key amino acids of the binding sites of the GPs. The implications of identifying a novel B-cell epitope for hantavirus immunology and vaccinology are discussed.

High sensitivity ELISA for detection of botulinum neurotoxin serotype F.

Botulinum neurotoxins (BoNTs) are classified as category A biological threat agents by the Centers for Disease Control and Prevention (CDC) in the United States for its hazardous and potential bioterrorist threat to the public. About 1% naturally occurring botulisms are caused by Botulinum neurotoxin serotype F (BoNT/F). Most of the immunoassays for detecting BoNTs focus on the serotypes A and B, but few methods have been established for the detection of BoNT/F. Recently, the recombinant Hc subunit of botulinum neurotoxin type F (rFHc) was expressed as an effective vaccine against BoNT/F, indicating that this rFHc could be an effective immunogen to raise monoclonal antibodies (MAbs) for the detection and neutralization of BoNT/F. Here we present a novel sandwich enzyme-linked immunosorbent assay (ELISA) based on two MAbs against rFHc, which were FMMU-BTF-8 and FMMU-BTF-29 as capture antibody and detection antibody, respectively. The limit of detection (LOD) of this ELISA reached 12.09 pg/mL, much less than that of the other reported immunoassays. A simple, sensitive ELISA for detecting and quantifying BoNT/F was established, which can be used as a valuable method to detect and quantify BoNT/F.

Establishment of reverse direct ELISA and its application in screening high-affinity monoclonal antibodies.

Traditionally monoclonal antibody (MAb) titer is determined by indirect enzyme-linked immunosorbent assay (ELISA), which is primarily used to evaluate the quality of MAbs. In this study, the titer and affinity of a group of MAbs against ovalbumin (OVA) were tested by indirect ELISA and the ELISA method reported previously. Data showed that there may be great differences between the indirect ELISA antibody titer and affinity value of MAbs. For the first time, a simple and effective reverse direct ELISA (RD-ELISA) was established for the detection of high-affinity MAbs. Among the group of MAbs to OVA, a certain proportion of antibodies with high affinity but low indirect ELISA titer do exist and can be clearly and efficiently detected by RD-ELISA. This study demonstrates that RD-ELISA is an effective method for high-affinity MAb screening.