Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 μg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes

Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 μg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes

Deep Sequencing for the Detection of Virus-Like Sequences in the Brains of Patients with Multiple Sclerosis: Detection of GBV-C in Human Brain

<div><p>Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1–2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a “hit”. Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4–10 million sequences (“reads”) each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.</p> </div

Virus-like sequence short read assembly.

1<p>students t-test (minimum value) corrected for multiple comparisons by the Bonferroni method.</p>2<p>maximum length of all the assembled reads that aligned to the indicated virus.</p>3<p>number of MS brain specimens that had reads with significant homology (MegaBlast Expect ≤0.1) to the indicated viral sequences.</p>4<p>The assembly of short reads did not improve alignment with the specified viral sequences.</p>5<p>Assembly revealed homology to human mitochondrial and host integration sites.</p

Representational Analysis.

<p>Virus-like sequences overrepresented in MS brain specimens compared with controls are displayed. Bonferroni corrected p-values beginning with 0.05 are displayed. Each row (labeled, N = 64) represents an overrepresented viral taxon. Each column (N = 17) represents a different MS brain specimen. The shaded yellow boxes represent significant hits. The GBV-C sequences confirmed by PCR in one of the specimens is circled in red.</p

Virus-like sequence short read assembly.

1<p>students t-test (minimum value) corrected for multiple comparisons by the Bonferroni method.</p>2<p>maximum length of all the assembled reads that aligned to the indicated virus.</p>3<p>number of MS brain specimens that had reads with significant homology (MegaBlast Expect ≤0.1) to the indicated viral sequences.</p>4<p>The assembly of short reads did not improve alignment with the specified viral sequences.</p>5<p>Assembly revealed homology to human mitochondrial and host integration sites.</p

Assembly of GBV-C sequences from the deep sequencing reads.

<p>Sanger sequencing results from brain specimen MS-6 are shown in green. Deep sequencing reads from subject MS-6 that aligned perfectly (36/36 bp matches) to the GBV-C genome are displayed in orange.</p

Amplification of GBV-C RNA from the brain of MS-afflicted subject MS-6.

<p>Following the failure of published primers to amplify sequences in this specimen, specific primers were constructed based on the sequences of specific reads obtained from the Illumina GAII sequencing of this specimen. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031886#pone.0031886-Souza1" target="_blank">[27]</a> A. Four regions of the GBV-C genome were selected for amplification including 2 sites in the 5′ non-translated region, the E2 (envelope protein) gene, and NS3 (non-structural). B. Amplicons were derived from each of these primer sets, including + (genome) and − (replication intermediate) strands. Sequencing of 3 of these amplicons spanning 906 bp revealed identity with >100 published GBV-C isolates, indicating that this subject had a novel strain of GBV-C in her brain tissue which appeared to be replicating at the time of her death.</p

Deep sequencing results for viruses previously implicated in MS pathogenesis.

1<p>Viral taxa are defined as sequences in GenBank below the level of family. This includes sequences identified as belonging to genus or species or subspecies (strain). It also includes sequences not assigned to a specific species or genus.</p>2<p>The MS Specimen Hit Rate is defined as the number of hits for a given taxon or species divided by the number of total non-ribosomal reads. This method is used to normalize these values between specimens. The range of hit rates among the 17 sequenced MS brain specimens is displayed.</p>3<p>The control specimen hit rates for each viral taxon are expressed as the mean ± SEM among the 8 control specimens.</p>4<p>Null hypothesis = the tested MS specimen falls within the expected distribution of control samples. Values provided are two-tailed. The minimum p-value among all 17 tested MS samples is shown.</p>*<p>The p-value could not be calculated because the control hit rates are not normally distributed.</p