Biomedical research advances over the past two decades in bioseparation science and engineering have led to the development of new adsorbent systems called monoliths, mostly as stationary supports for liquid chromatography (LC) applications. They are acknowledged to offer better mass transfer hydrodynamics than their particulate counterparts. Also, their architectural and morphological traits can be tailored in situ to meet the hydrodynamic size of molecules which include proteins, pDNA, cells and viral targets. This has enabled their development for a plethora of enhanced bioscreening applications including biosensing, biomolecular purification, concentration and separation, achieved through the introduction of specific functional moieties or ligands (such as triethylamine, N,N-dimethyl-N-dodecylamine, antibodies, enzymes and aptamers) into the molecular architecture of monoliths. Notwithstanding, the application of monoliths presents major material and bioprocess challenges. The relationship between in-process polymerisation characteristics and the physicochemical properties of monolith is critical to optimise chromatographic performance. There is also a need to develop theoretical models for non-invasive analyses and predictions. This review article therefore discusses in-process analytical conditions, functionalisation chemistries and ligands relevant to establish the characteristics of monoliths in order to facilitate a wide range of enhanced bioscreening applications. It gives emphasis to the development of functional polymethacrylate monoliths for microfluidic and preparative scale bio-applications.
The identification of the Hepatitis C virus using molecular cloning techniques, besides making the term Non-A Non-B Hepatitis obsolete, enables the development of specific assays for the detection of antibodies in HCV-infected individuals, thus making it possible to obtain sero-epidemiological data of the disease. The carriage of Hepatitis C antibody varies worldwide. The disease is most prevalent in intravenous drug abusers or haemophiliacs. Parenteral transmission is the most important route of transmission. Sexual, intra-familial and perinatal transmissions are uncommon. About 40% could be community-acquired (sporadic). Diagnostic tests include enzyme-linked immunosorbant (ELISA) anti-HCV assay, recombinant immunoblot assay, HCV-RNA by polymerase chain reaction and HCV-Ag. More than 50% of acute cases becomes chronic and runs a benign and indolent course. About 20% progress to cirrhosis and some of these develop hepatocellular carcinoma. Several published trials have consistently shown that treatment with interferon in some patients is useful. There is however a relapse rate of 50%. Further trials with interferon and other anti-viral agents like ribavirin are awaited for more effective treatment.
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.
The chemical, technological and allergy properties of goat's milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10-3 μM compared to 9.046 × 10-3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.
Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.
Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.