14 research outputs found
Mannan biotechnology: from biofuels to health
<div><p></p><p>Mannans of different structure and composition are renewable bioresources that can be widely found as components of lignocellulosic biomass in softwood and agricultural wastes, as non-starch reserve polysaccharides in endosperms and vacuoles of a wide variety of plants, as well as a major component of yeast cell walls. Enzymatic hydrolysis of mannans using mannanases is essential in the pre-treatment step during the production of second-generation biofuels and for the production of potentially health-promoting manno-oligosaccharides (MOS). In addition, mannan-degrading enzymes can be employed in various biotechnological applications, such as cleansing and food industries. In this review, fundamental knowledge of mannan structures, sources and functions will be summarized. An update on various aspects of mannan-degrading enzymes as well as the current status of their production, and a critical analysis of the potential application of MOS in food and feed industries will be given. Finally, emerging areas of research on mannan biotechnology will be highlighted.</p></div
Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody–Ligand Interaction Using Chain Shuffling
A human antiaflatoxin B1 (AFB1) scFv
antibody (yAFB1-c3), selected
from a naı̈ve
human phage-displayed scFv library, was used as a template for improving
and analysis of antibody–ligand interactions using the chain-shuffling
technique. The variable-heavy and variable-light (VH/VL)-shuffled
library was constructed from the VH of 25 preselected clones recombined
with the VL of yAFB1-c3 and vice versa. Affinity selection from these
libraries demonstrated that the VH domain played an important role
in the binding of scFv to free AFB1. Therefore, in the next step,
VH-shuffled scFv library was constructed from variable-heavy (VH)
chain repertoires, amplified from the naı̈ve library,
recombined with the variable-light (VL) chain of the clone yAFB1-c3.
This library was then used to select a specific scFv antibody against
soluble AFB1 by a standard biopanning method. Three clones that showed
improved binding properties were isolated. Amino acid sequence analysis
indicated that the improved clones have amino acid mutations in framework
1 (FR1) and the complementarity determining region (CDR1) of the VH
chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement
in sensitivity over the original scFv clone and was selected for molecular
binding studies with AFB1. Homology modeling and molecular docking
were used to compare the binding of this and the original clones.
The results confirmed that VH is more important than VL for AFB1 binding
Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications
For many decades,
feline infectious disease has been among the
most common health problems and a leading cause of death in cats.
These diseases include toxoplasmosis, feline leukemia virus (FeLV),
and particularly feline immunodeficiency virus (FIV) disease. Early
diagnosis is essential to increase the chance of successful treatment.
Generally, measurement of the IgG level is considered to be indicative
of an individual’s immune status for a particular pathogen.
The antibodies specific to feline IgG are crucial components for the
development of a detection kit. In this study, feline IgG-bound scFv
was selected using phage display technology. Three rounds of biopanning
were conducted against purified feline IgG. Through an indirect enzyme-linked
immunosorbent assay (ELISA), two scFv clones demonstrating the best
binding ability to feline IgG were chosen for biochemical characterization.
In addition, the selected scFv (N14) was expressed and purified in
a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
revealed that the size of the purified N14 was 29 kDa. A sandwich
ELISA was used to evaluate the binding capacity of the purified scFv
to feline IgG. As expected, the purified N14 had the capacity to bind
feline IgG. Furthermore, N14 was modified to create a scFv-alkaline
phosphatase (scFv-AP) fusion platform. The surface plasmon resonance
(SPR) results revealed that N14-AP bound to feline IgG with an affinity
binding value of 0.3 ± 0.496 μM. Additionally, the direct
ELISA demonstrated the binding capacity of N14-AP to feline IgG in
both cell lysate and purified protein. Moreover, N14-AP could be applied
to detect feline IgG based on electrosensing with a detection limit
of 10.42 nM. Overall, this study successfully selected a feline IgG-bound
scFv and developed a scFv-AP platform that could be further engineered
and applied in a feline infectious disease detection kit
Antibody characterization via dilution series.
<p>(<b>A</b>) A checkerboard titration experiment of phage-displayed scFv clone bDOA9rb8. Various amount of phage particles were prepared by 10-fold dilution series starting from 10<sup>13</sup>, 10<sup>12</sup>, 10<sup>11</sup>, 10<sup>10</sup>, and 10<sup>9</sup> pfu/well. Each dilution of phage antibody was used to detect different amount of boiled DOA9 antigen at the protein concentration of 10, 5.0, 2.5, 1.3, 0.6 and 0.3 μg/well of total protein, determined by Bradford assay. Each reaction was done in triplicate. The phages were purified by PEG precipitation and re-suspended in PBS buffer at a concentration of 10<sup>11</sup> pfu/μl. Bound phage was detected by anti-M13 HRP, using ABTS as color reagent. The Y-axis indicated the OD value and the SD measured from triplicate wells. (<b>B</b>) Detection limit of phage displayed scFv clone bDOA9rb8 against boiled DOA9. The 5-fold dilution series of boiled DOA9 protein antigen were prepared in sodium carbonate buffer starting from the protein concentration of 10, 2, 0.4, 0.08, 0.02, 0.003 and 0.00 μg/well. The ELISA was performed by using the optimum concentration of phage antibody (10<sup>12</sup> pfu/well), determined from panel A. The average OD<sub>405</sub> nm values and standard errors from triplicate wells are shown.</p
Schematic outline of the procedure for the construction of the immunized rabbit scFv library.
<p>Once the antibody titer of immunized rabbit, as determined by agglutination method, was higher than 1:1600, the total RNA was extracted from spleen (<b>A</b>). Then, cDNA was synthesized by using a mix of random hexamers and oligo-dT<sub>18</sub> (<b>B</b>). The locations of all PCR primers on the two variable regions genes are indicated (<b>C</b>). The list of all primers used for the construction of the library is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179983#pone.0179983.t001" target="_blank">Table 1</a>. The first PCR step comprised 29 PCR reactions for amplification of V<sub>H</sub> and V<sub>L</sub> gene repertoire. Then, equal amounts of V<sub>H</sub> and V<sub>L</sub>κ, and V<sub>H</sub> and V<sub>L</sub>λ were assembled together via a (G<sub>4</sub>S)<sub>3</sub> linker sequence by overlap extension, followed by pull-through PCR steps (<b>D</b>). The amplified full-length scFv repertoire fragments were then cloned into a pMOD1.0 vector (see full-size map in Supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179983#pone.0179983.s002" target="_blank">S2 Fig</a>), between <i>Sfi</i> I and <i>Not</i> I sites, before being transformed into <i>E</i>. <i>coli</i> TG1 (<b>E</b>). The agarose gel (inset) illustrates an example of DNA fragments from various steps; lane 1: 100 bp DNA ladder (NEB, USA); lane 2: V<sub>L</sub>λ; lane 3: V<sub>L</sub>κ; lane 4: V<sub>H</sub>; and lane 5: assembled scFv fragments (<b>F</b>). Please note that even the PCR products of V<sub>H</sub> obtained from the PCR reactions were quite low as shown as a faint band in the lane 4 of the gel, we were able to use them to generate assemble products of scFv genes by pull-through PCR as shown in lane 5.</p
Amino acid sequence and 3D structure of isolated anti-DOA9 rabbit scFv antibody.
<p>The primary amino acid sequence of the clone bDOA9rb8 is shown at the bottom. The flexible linker (G<sub>4</sub>S)<sub>3</sub> that joins V<sub>H</sub> and V<sub>L</sub> segments is indicated. The three complementarity-determining regions (CDRs) are underlined. The 3D structure was done by Phyre<sup>2</sup> software, using the structure of an scFv antibody in complex with an analogue of the main immunogenic region of the acetylcholine receptor (PDB code: 1F3R) as templates. The 3D structures in both space-filling and ribbon models with CDRs of V<sub>H</sub> and V<sub>L</sub> domains are indicated.</p
SDS-PAGE and Westernblot analysis of purified soluble scFv antibody.
<p>(<b>A</b>) The soluble scFv antibody against <i>Bradyrhizobium</i> sp. strain DOA9 clone bDOA9rb8 was purified from culture supernatant by IMAC. Lane M: protein molecular weight marker; lane S: culture supernatant input; lane FT: flow-through fraction; lanes W1, W2, and W3 indicate the three wash fractions; lanes E1, E2, and E3 are the three elution fractions. The soluble scFv antibody of approx. 30 kDa can be found in elution fractions 1 and 2. This scFv antibody was used to study the binding specificity in the next step. (<b>B</b>) SDS-PAGE and Western blot analysis of free scFv clone bDOA9rb8 obtained from periplasmic extracts.</p
Specific binding of selected phage-displayed scFv clones.
<p>Phage ELISA results of the binding of scFv antibodies against DOA9 and other antigens in pure culture (<b>A</b>) and plant nodules (<b>B</b>) are shown. The two clones of phage-displayed rabbit scFv, i.e., RB9 and RG8, could bind specifically to only strain DOA9 but not to other <i>Bradyrhizobium</i> strains and <i>Bacillus subtilis</i> 168. Phage-displayed human 3C1 scFv antibody was used as a negative control in this assay. The average OD<sub>405</sub> nm values and standard errors from triplicate wells are shown. Note that in panel B, there was no bacillus nodule because it can’t form nodule in plant roots.</p
Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of <i>Bradyrhizobium</i> sp. DOA9 in both free-living and bacteroid forms
<div><p>A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. <i>Bradyrhizobium</i> sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of <i>Aeschynomene americana</i> in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future.</p></div
Selective enrichment of scFv antibodies during the biopanning process.
<p>Selective enrichment of scFv antibodies during the biopanning process.</p