International Journal of Biological Macromolecules
Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs ... more Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually variable and frequently mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of specific agent in clinical sample is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
Abstract Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing t... more Abstract Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing to tuberculosis (TB). One of the major challenges of TB management is the identification of patients, making TB diagnosis critical for disease management. This study focuses on the 16 kDa heat shock protein (HSP16.3; a potential biomarker for latent TB infection) that is expressed during the latent phase of Mtb growth. In order to explore the dynamics and interactions of HSP16.3, the 3-D structure of HSP16.3 was built via comparative modelling. The predicted structure shows a predominantly beta-sheet dodecamer with alpha-helical folds at its N-terminal. A known protein-hydrophobic probe (1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic acid; bisANS) was docked to the HSP16.3 model. Interacting residues predicted from docking and MD simulations are in good accordance with experimental data reported in the literature. MMPBSA calculation from MD simulation also showed favourable binding free energy of −29.90 kcal/mol, driven mainly by van der waals and non-polar solvation energies. The statistical evaluation and results from the computational study on HSP16.3 indicate the reliability of the built model, which is potentially useful for further structural studies of HSP16.3 for latent TB diagnostics.
Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typ... more Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction...
Biotechnology and applied biochemistry, Jan 21, 2017
Phage display has been applied successfully as a tool for the generation of monoclonal antibodies... more Phage display has been applied successfully as a tool for the generation of monoclonal antibodies. Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional monoclonal antibody generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against BmSXP, a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10(9) . The antibody library was used to screen for monoclonal antibodies against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analysed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The monoclonal antibodies obtained were als...
Toxicon : official journal of the International Society on Toxinology, Jan 14, 2016
Antibody phage display is a useful tool for the isolation and identification of monoclonal antibo... more Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.
Antibody phage display panning involves the enrichment of antibodies against specific targets by ... more Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.
The use of human variable heavy (VH) domain antibodies has been on the rise due to their small sc... more The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline....
Development of vaccines for infectious diseases has come a long way with recent advancements in a... more Development of vaccines for infectious diseases has come a long way with recent advancements in adjuvant developments and discovery of new antigens that are capable of eliciting strong immunological responses for sterile eradication of disease. Tuberculosis (TB) that kills nearly 2 million of the population every year is also one of the highlights of the recent developments. The availability or not of diagnostic methods for infection has implications for the control of the disease by the health systems but is not related to the immune surveillance, a phenomenon derived from the interaction between the bacteria and their host. Here, we will review the immunology of TB and current vaccine candidates for TB. Current strategies of developing new vaccines against TB will also be reviewed in order to further discuss new insights into immunotherapeutic approaches involving adjuvant and antigens combinations that might be of potential for the control of TB.
Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorg... more Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; an...
Current methadone maintenance therapy (MMT) is yet to ensure 100% successful treatment as the opt... more Current methadone maintenance therapy (MMT) is yet to ensure 100% successful treatment as the optimum dosage has yet to be determined. Overdose leads to death while lower dose causes the opioid withdrawal effect. Single-nucleotide polymorphisms (SNP) in cytochrome P450s (CYPs), the methadone metabolizers, have been showen to be the main factor for the interindividual variability of methadone clinical effects. In this study, we investigated the effect of SNPs in three major methadone metabolizers (CYP2B6, CYP2D6, and CYP3A4) on methadone binding affinity. Results showed thatCYP2B6*11,CYP2B6*12,CYP2B6*18, andCYP3A4*12have significantly higher binding affinity toR-methadone compared to wild type.S-methadone has higher binding affinity inCYP3A4*3,CYP3A4*11, andCYP3A4*12compared to wild type.R-methadone was shown to be the active form of methadone; thus individuals with CYP alleles that binds better toR-methadone will have higher methadone metabolism rate. Therefore, a higher dosage of m...
Abstract Introduction Epitopes are categorized into two groups: T-cell and B-cell, and further se... more Abstract Introduction Epitopes are categorized into two groups: T-cell and B-cell, and further separated into linear and conformational epitope. Although conformational epitope prediction is more accurate compare to sequence based epitope prediction, not as much of protein secondary structures were solved. Mapping the epitopes by experimental way is time-consuming, reversible and expensive. Even though lots of epitope predictions are available but performance is yet to be satisfied. Thus, a better sequence based epitope prediction is needed with improvement of the previous methods. Objective To improve the sequence-based B-cell epitope prediction by support vector machine (SVM). Methods This method is an integration of SVM and 32 physiochemical properties. The data obtained from Immune Epitope Database, IEDB, was separated into five categories: bacteria, virus, unicellular, multicellular except vertebrates, and fungus. All modules were developed by using MATLAB programming language. The accuracy of both predictors must be more than 50% (random guessing) in order to make the prediction useful. Results & Discussion The accuracy obtained for both predictors are higher than 50%. The benchmarking results show that the B-cell epitope prediction module has a comparable prediction results to other available predictors. Conclusion The improved sequence based B-cell epitope prediction module could be further developed as a web-based server to benefit potential users.
Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombina... more Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.
International Journal of Biological Macromolecules
Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs ... more Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually variable and frequently mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of specific agent in clinical sample is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
Abstract Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing t... more Abstract Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing to tuberculosis (TB). One of the major challenges of TB management is the identification of patients, making TB diagnosis critical for disease management. This study focuses on the 16 kDa heat shock protein (HSP16.3; a potential biomarker for latent TB infection) that is expressed during the latent phase of Mtb growth. In order to explore the dynamics and interactions of HSP16.3, the 3-D structure of HSP16.3 was built via comparative modelling. The predicted structure shows a predominantly beta-sheet dodecamer with alpha-helical folds at its N-terminal. A known protein-hydrophobic probe (1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic acid; bisANS) was docked to the HSP16.3 model. Interacting residues predicted from docking and MD simulations are in good accordance with experimental data reported in the literature. MMPBSA calculation from MD simulation also showed favourable binding free energy of −29.90 kcal/mol, driven mainly by van der waals and non-polar solvation energies. The statistical evaluation and results from the computational study on HSP16.3 indicate the reliability of the built model, which is potentially useful for further structural studies of HSP16.3 for latent TB diagnostics.
Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typ... more Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction...
Biotechnology and applied biochemistry, Jan 21, 2017
Phage display has been applied successfully as a tool for the generation of monoclonal antibodies... more Phage display has been applied successfully as a tool for the generation of monoclonal antibodies. Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional monoclonal antibody generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against BmSXP, a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10(9) . The antibody library was used to screen for monoclonal antibodies against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analysed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The monoclonal antibodies obtained were als...
Toxicon : official journal of the International Society on Toxinology, Jan 14, 2016
Antibody phage display is a useful tool for the isolation and identification of monoclonal antibo... more Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.
Antibody phage display panning involves the enrichment of antibodies against specific targets by ... more Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.
The use of human variable heavy (VH) domain antibodies has been on the rise due to their small sc... more The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline....
Development of vaccines for infectious diseases has come a long way with recent advancements in a... more Development of vaccines for infectious diseases has come a long way with recent advancements in adjuvant developments and discovery of new antigens that are capable of eliciting strong immunological responses for sterile eradication of disease. Tuberculosis (TB) that kills nearly 2 million of the population every year is also one of the highlights of the recent developments. The availability or not of diagnostic methods for infection has implications for the control of the disease by the health systems but is not related to the immune surveillance, a phenomenon derived from the interaction between the bacteria and their host. Here, we will review the immunology of TB and current vaccine candidates for TB. Current strategies of developing new vaccines against TB will also be reviewed in order to further discuss new insights into immunotherapeutic approaches involving adjuvant and antigens combinations that might be of potential for the control of TB.
Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorg... more Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; an...
Current methadone maintenance therapy (MMT) is yet to ensure 100% successful treatment as the opt... more Current methadone maintenance therapy (MMT) is yet to ensure 100% successful treatment as the optimum dosage has yet to be determined. Overdose leads to death while lower dose causes the opioid withdrawal effect. Single-nucleotide polymorphisms (SNP) in cytochrome P450s (CYPs), the methadone metabolizers, have been showen to be the main factor for the interindividual variability of methadone clinical effects. In this study, we investigated the effect of SNPs in three major methadone metabolizers (CYP2B6, CYP2D6, and CYP3A4) on methadone binding affinity. Results showed thatCYP2B6*11,CYP2B6*12,CYP2B6*18, andCYP3A4*12have significantly higher binding affinity toR-methadone compared to wild type.S-methadone has higher binding affinity inCYP3A4*3,CYP3A4*11, andCYP3A4*12compared to wild type.R-methadone was shown to be the active form of methadone; thus individuals with CYP alleles that binds better toR-methadone will have higher methadone metabolism rate. Therefore, a higher dosage of m...
Abstract Introduction Epitopes are categorized into two groups: T-cell and B-cell, and further se... more Abstract Introduction Epitopes are categorized into two groups: T-cell and B-cell, and further separated into linear and conformational epitope. Although conformational epitope prediction is more accurate compare to sequence based epitope prediction, not as much of protein secondary structures were solved. Mapping the epitopes by experimental way is time-consuming, reversible and expensive. Even though lots of epitope predictions are available but performance is yet to be satisfied. Thus, a better sequence based epitope prediction is needed with improvement of the previous methods. Objective To improve the sequence-based B-cell epitope prediction by support vector machine (SVM). Methods This method is an integration of SVM and 32 physiochemical properties. The data obtained from Immune Epitope Database, IEDB, was separated into five categories: bacteria, virus, unicellular, multicellular except vertebrates, and fungus. All modules were developed by using MATLAB programming language. The accuracy of both predictors must be more than 50% (random guessing) in order to make the prediction useful. Results & Discussion The accuracy obtained for both predictors are higher than 50%. The benchmarking results show that the B-cell epitope prediction module has a comparable prediction results to other available predictors. Conclusion The improved sequence based B-cell epitope prediction module could be further developed as a web-based server to benefit potential users.
Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombina... more Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.
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