Prof. Montarop Yamabhai

Year	Title	Journal
2021	Determination of Chinese hamster ovary (CHO) cell densities and antibody titers from small volumes of cell culture supernatants using multivariate analysis and partial least squares regression of UV-Vis spectra Jarusintanakorn S., Phechkrajang C., Khongkaew P., Mastrobattista E., Yamabhai M.	Analytical and Bioanalytical Chemistry
Abstract: Antibody titer and viable cell density (VCD) are two important parameters that need to be closely monitored during the process of cell line development and manufacturing of therapeutic antibodies. Typically, determination of each parameter requires 10–100 μL of supernatant sample, which is not suitable for small scale cultivation. In this study, we demonstrated that as low as 2 μL of culture supernatants were sufficient for the analysis using UV-Vis spectrum assisted with partial least squares (PLS) model. The results indicated that the optimal PLS models could be used to predict antibody titer and VCD with the linear relationship between reference values and predicted values at R2 values ranging from 0.8 to > 0.9 in supernatant samples obtained from four different single clones and in polyclones that were cultured in various selection stringencies. Then, the percentage of cell viability and productivity were predicted from a set of samples of polyclones. The results indicated that while all predicted % cell viability were very similar to the actual value at RSEP value of 6.7 and R2 of 0.8908, the predicted productivity from 14 of 18 samples were closed to the reference measurements at RSEP value of 22.4 and R2 of 0.8522. These results indicated that UV-Vis combined with PLS has potential to be used for monitoring antibody titer, VCD, and % cell viability for both online and off-line therapeutic production process. Graphical abstract: [Figure not available: see fulltext.]. © 2021, The Author(s). Keyword: CHO; Multivariate data analysis; Partial least squares regression; Therapeutic antibody titer; UV-Vis spectroscopy; Viable cell density Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85114093523&doi=10.1007%2fs00216-021-03549-4&partnerID=40&md5=3b8fdee12617931653770e91f21962fc DOI: 10.1007/s00216-021-03549-4
2021	Development and characterization of human single chain antibody against Iranian Macrovipera lebetina snake venom Eskafi A.H., Bagheri K.P., Behdani M., Yamabhai M., Shahbazzadeh D., Kazemi-Lomedasht F.	Toxicon
Abstract: Snakebite is an important public health problem in tropical and subtropical regions. Macrovipera lebetina is one of the most dangerous snakes in Iran. Envenoming by this snake can lead to respiratory distress, heart attack, bleeding, and death. The specific treatment available is immunized equine serum, which has several side effects like serum sickness. Nowadays, single-chain fragment variable antibodies (scFvs) are one of the fast growing classes of monoclonal antibodies, which are suggested for treatment of envenoming. This study aimed to achieve a fully human scFv antibody against M. lebetina venom from human non-immune library. In this study, scFvs against M. lebetina venom were isolated by phage display technique. Using three rounds of biopanning, two specific scFvs (C37 and C69) with the highest affinity were selected. The selected scFvs purified by nickel affinity chromatography. The specific binding of purified antibodies were confirmed by enzyme-linked immunosorbent assay. The LD50 as well as HD50 concentration of the crude venom were obtained to be 45 μg and 120 μg/ml, respectively. C69 neutralized 48% of the hemolysis activity of M. lebetina venom and C37 survived 66% of mice after 115 min of envenoming. Taken together, the results indicate the potential of human non-immune libraries for selection of functional antibodies against M. lebetina venom. © 2021 Elsevier Ltd Keyword: Human non-immune library; Macrovipera lebetina; Phage display; Single-chain variable fragment (scFv) Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85105835719&doi=10.1016%2fj.toxicon.2021.04.017&partnerID=40&md5=04c934d2268bd65c32694fb17987db6b DOI: 10.1016/j.toxicon.2021.04.017
2021	Anti-inflammatory activity of soluble chitooligosaccharides (CHOS) on VitD3-induced human THP-1 monocytes Jitprasertwong P., Khamphio M., Petsrichuang P., Eijsink V.G.H., Poolsri W., Muanprasat C., Rangnoi K., Yamabhai M.	PLoS ONE
Abstract: Chito-oligosaccharides (CHOS) are oligomers of D-glucosamine and N-acetyl-glucosamine. Anti-inflammatory activities of a wide variety of CHOS mixtures have previously been reported, mainly based on studies with mouse models and murine macrophages. Since the mouse and human immune systems are quite different, gaining insight into the activity of CHOS on human cell lines, using well-characterized CHOS mixtures, is of considerable interest. Bacillus subtilis chitosanase (BsCsn46A) can efficiently convert chitosan to mixtures of water soluble low molecular weight CHOS. Here, the anti-inflammatory activity of a properly characterized CHOS mixture was studied, using human THP-1 cells that were differentiated to mature monocytes using vitamin D3. Addition of CHOS reduced the production of multiple pro-inflammatory cytokines associated with bacterial lipopolyssacharide (LPS)-stimulated inflammation, in a dose-dependent manner and without affecting cell viability. Interestingly, only minimal effects of CHOS were observed in similar experiments with phorbol 12-myristate 13-acetate- (PMA-) differentiated, macrophage-like, THP-1 cells. Altogether, in addition to showing promising biological effects of well-characterized low molecular weight soluble CHOS in a human system, the present study also points at Vitamin D3- stimulated THP-1 cells as a favorable system for assessing the anti-inflammatory activity of bioactive compounds. © 2021 Public Library of Science. All rights reserved. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85100522592&doi=10.1371%2fjournal.pone.0246381&partnerID=40&md5=1b9377e223009bea8b11f23a2ff022b6 DOI: 10.1371/journal.pone.0246381
2021	Human Hexa-Histidine-Tagged Single-Chain Variable Fragments for Bioimaging of Bacterial Infections Min T.T., Yamabhai M.	ACS Omega
Abstract: The single-chain variable fragment (scFv) of monoclonal antibodies is a promising recombinant nanostructure for various medical applications, including bioimaging and targeted therapy. While numerous scFv antibodies against eukaryotic cell surface proteins (especially cancer biomarkers) have been generated and engineered to suit various purposes, only a few specific scFv against bacterial cell surfaces have been developed, especially those of human origin. Recent incidents of emerging multidrug-resistant pathogenic bacteria and the realization of the importance of a balanced microbiota on the health of the host has led to more interests in the development of recombinant antibacterial antibodies as a detection probe or targeted therapy for bacterial infections. This study reports the generation of two specific human antibacterial scFv using phage display antibody technology. The recombinant scFv fragments of about 30 kDa and a diameter of 5 nm were produced and purified from engineered Escherichia coli that can enhance cytosolic disulfide bond formation. As a proof of principle, Propionibacterium acnes and Pseudomonas aeruginosa were used as model Gram-positive and Gram-negative bacteria, respectively. Specificity at the strain and species level to both planktonic and biofilm forms of these bacteria were demonstrated in various assay formats, namely, ELISA, flow cytometry, western blot, immunofluorescence, and electron microscopy via the hexa-histidine tag. This recombinant scFv generation platform can be applied for other bacteria, and since the scFv obtained has a benefit of being a human origin, it could be conveniently engineered for various therapeutic or theranostic applications with minimized adverse immunoreaction. © 2020 The Authors. Published by American Chemical Society. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85099030562&doi=10.1021%2facsomega.0c05340&partnerID=40&md5=b4f303860e10113cd1d5bbbb6e7bfc5b DOI: 10.1021/acsomega.0c05340
2019	Development of a human scFv antibody targeting the lethal Iranian cobra (Naja oxiana) snake venom Kazemi-Lomedasht F., Yamabhai M., Sabatier J.-M., Behdani M., Zareinejad M.R., Shahbazzadeh D.	Toxicon
Abstract: Snakebite is one of the health concerns worldwide. Naja oxiana is one of the venomous snakes with a high mortality rate. Anti-serum therapy is the only treatment of the victims. However, in some cases, antiserum injection leads to some side effects in host like serum sickness and anaphylactic shock. It is crucial to develop a neutralizing agent with low side effects. The human antibody library (non-immunized library) was used to isolate specific antibodies against N.oxiana venom components. Four rounds of biopanning were performed to enrich scFv-displaying phages against the venom of N. oxiana. Enrichment of scFv-displaying phages against N. oxiana venom was analyzed by polyclonal Enzyme-Linked Immunosorbent Assay (ELISA). Specific antibody fragments against N. oxiana venom were selected through monoclonal ELISA, and were expressed in E. coli bacterial cells. Purification of the selected clones was performed by using nickel affinity chromatography. Neutralization and protective capacity of specific antibody fragments were analyzed in C57BL/6 mice (i.v. injection). Results of biopanning and polyclonal ELISA demonstrate a successful enrichment process. Five specific antibody fragments with the highest signal in monoclonal ELISA were selected, expressed, and purified. The purity of expressed antibody fragments was monitored by SDS-PAGE and Western blot. The selected antibody fragments were able to neutralize two LD50 of N. oxiana venom and protected all mice when injected 15 min post-envenomation. The data indicate that such selected antibodies are promising tools for further studies and in the development of novel protective agents against N. oxiana venom. © 2019 Elsevier Ltd Keyword: Human antibody; Naja oxiana; Phage display; Snake venom Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85073677607&doi=10.1016%2fj.toxicon.2019.10.006&partnerID=40&md5=9ec38415c10b747098b1ea4028ba59be DOI: 10.1016/j.toxicon.2019.10.006
2019	Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology Sompunga P., Pruksametanan N., Rangnoi K., Choowongkomon K., Yamabhai M.	Talanta
Abstract: This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL−1, with a limit of detection (LOD) of 20 and 2 ng mL−1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based. © 2019 Elsevier B.V. Keyword: Alkaline phosphatase fusion; Competitive ELISA; Phage display; Recombinant antibody; scFv; Zearalenone Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85064316025&doi=10.1016%2fj.talanta.2019.04.034&partnerID=40&md5=c478a61b63baff1dea670ab86494504a DOI: 10.1016/j.talanta.2019.04.034
2019	Novel Recombinant Antibody and Protein-based Approaches for Analysis of Food and Food Contaminants with Particular Relevance to Asia Yamabhai M., Rangnoi K., Sompunga P., O'Kennedy R.	Food Chemistry, Function and Analysis
Abstract: An overview of food safety issues in Asia is presented in this chapter. An update on research and innovations related to novel recombinant antibody and protein-based approaches for analysis of food and food contaminants in Asia is reported, with China as the leading country of relevance, followed by South Korea, Japan, and Thailand. The main focus is on mycotoxins, followed by pesticide detection. Different recombinant formats, especially scFv and VHH, have been used. In addition, anti-idiotypic VHH and peptide mimotopes have been used in ELISA-based formats. For foodborne pathogens, recombinant antibodies and peptides identified using phage display technology have been used as affinity reagents for rapid detection. © The Royal Society of Chemistry 2019. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85067363061&doi=10.1039%2f9781788016322-00195&partnerID=40&md5=20566f20a98afdd01ee0a1273484ebed DOI: 10.1039/9781788016322-00195
2019	Correction: CD4+ T cells promote humoral immunity and viral control during Zika virus infection (PLoS Pathog (2019) 15: 1 (e1007474) DOI: 10.1371/journal.ppat.1007474) Ngono A.E., Young M.P., Bunz M., Xu Z., Hattakam S., Vizcarra E., Regla-Nava J.A., Tang W.W., Yamabhai M., Wen J., Shresta S.	PLoS Pathogens
Abstract: In the Funding statement, one of the grant numbers is missing. The correct Funding statement is as follows: This work was funded by NIAID/NIH grants R01 AI116813, R21 NS100477, R21 AI140063, and R01 NS106387 to SS and the Chiba-UCSD Center for Mucosal Immunology, Allergy and Vaccine Development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2019 Elong Ngono et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85067272529&doi=10.1371%2fjournal.ppat.1007821&partnerID=40&md5=6112df718e6608dedf94f639aac3043a DOI: 10.1371/journal.ppat.1007821
2019	CD4+ T cells promote humoral immunity and viral control during Zika virus infection Elong Ngono A., Young M.P., Bunz M., Xu Z., Hattakam S., Vizcarra E., Regla-Nava J.A., Tang W.W., Yamabhai M., Wen J., Shresta S.	PLoS Pathogens
Abstract: Several Zika virus (ZIKV) vaccines designed to elicit protective antibody (Ab) responses are currently under rapid development, but the underlying mechanisms that control the magnitude and quality of the Ab response remain unclear. Here, we investigated the CD4+ T cell response to primary intravenous and intravaginal infection with ZIKV. Using the LysMCre+Ifnar1fl/fl (myeloid type I IFN receptor-deficient) C57BL/6 mouse models, we identified six I-Ab-restricted ZIKV epitopes that stimulated CD4+ T cells with a predominantly cytotoxic Th1 phenotype in mice primed with ZIKV. Intravenous and intravaginal infection with ZIKV effectively induced follicular helper and regulatory CD4+ T cells. Treatment of mice with a CD4+ T cell-depleting Ab reduced the plasma cell, germinal center B cell, and IgG responses to ZIKV without affecting the CD8+ T cell response. CD4+ T cells were required to protect mice from a lethal dose of ZIKV after infection intravaginally, but not intravenously. However, adoptive transfer and peptide immunization experiments showed a role for memory CD4+ T cells in ZIKV clearance in mice challenged intravenously. These results demonstrate that CD4+ T cells are required mainly for the generation of a ZIKV-specific humoral response but not for an efficient CD8+ T cell response. Thus, CD4+ T cells could be important mediators of protection against ZIKV, depending on the infection or vaccination context. © 2019 Elong Ngono et al. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85060542752&doi=10.1371%2fjournal.ppat.1007474&partnerID=40&md5=d46dcd3f7fdfa677f23bb9de2b5936d5 DOI: 10.1371/journal.ppat.1007474
2018	Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling Rangnoi K., Choowongkomon K., O'Kennedy R., Rüker F., Yamabhai M.	Journal of Agricultural and Food Chemistry
Abstract: A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a nalve 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 nalve 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. © 2018 American Chemical Society. Keyword: aflatoxin; chain shuffling; enzyme-linked immunosorbent assay (ELISA); single-chain fragment variable (scFv) Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85047753364&doi=10.1021%2facs.jafc.8b01141&partnerID=40&md5=c4bffcd9a73d36ff4a4f64d97bfab926 DOI: 10.1021/acs.jafc.8b01141