Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (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 × 109 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed 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 mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.
The leaves of the Mitragynine speciosia tree (also known as Kratom) have long been chewed, smoked, or brewed into a tea by people in Southeastern Asian countries, such as Malaysia and Thailand. Just this past year, the plant Kratom gained popularity in the United States as a "legal opioid" and scheduling it as a drug of abuse is currently pending. The primary alkaloid found in Kratom is a μ-opioid receptor agonist, mitragynine, whose structure contains a promising scaffold for immunopharmacological use. Although Kratom is regarded as a safe opioid alternative, here we report the LD50 values determined for its two main psychoactive alkaloids, mitragynine and 7-hydroxymitragynine, as comparable to heroin in mice when administered intravenously. Given Kratom's recent emergence in the U.S., there is currently no diagnostic test available for law enforcement or health professionals, so we sought to design such an assay. Mitragynine was used as a starting point for hapten design, resulting in a hapten with an ether linker extending from the C9 position of the alkaloid. Bacterial flagellin (FliC) was chosen as a carrier protein for active immunization in mice, yielding 32 potential monoclonal antibodies (mAbs) for assay development. Antimitragynine mAbs in the range of micro- to nanomolar affinities were uncovered and their utility in producing a convenient lateral flow detection assay of human fluid samples was examined. Antibodies were screened for binding to mitragynine, 7-hydroxymitragynine, and performance in lateral flow assays. Two monoclonal antibodies were subcloned and further purified with 93 and 362 nM affinity to mitragynine. Test strip assays were optimized with a detection cut off of 0.5 μg/mL for mitragynine in buffer and urine (reflecting projected clinically relevant levels of drug in urine), which could be beneficial to law enforcement agencies and health professionals as the opioid epidemic in America continues to evolve.
Hepatocellular carcinoma (HCC) is the most common primary liver cancer and its diagnosis on routine stains is usually straightforward, except in some cases where there may be difficulty in distinguishing HCCs from metastatic carcinomas (MC) and cholangiocarcinomas (CC). Hepatocyte Paraffin 1 antibody (Hep Par 1) is a new monoclonal antibody which reacts with normal and neoplastic hepatocytes, and this study aims to determine its specificity and sensitivity in distinguishing hepatocellular carcinoma (HCC) from cholangiocarcinoma (CC) and metastatic carcinomas (MC). Hep Par 1 antibody was applied to 28 cases of HCC, 22 cases of MC from varying sites and 8 CCs, and produced a strong, diffuse, granular, cytoplasmic staining of all benign hepatocytes. 23 out of 28 cases of HCC showed heterogeneously positive staining for Hep Par 1 irrespective of their degree of differentiation, while 2 out of 8 cases of cholangiocarcinoma were positive for Hep Par 1, and all 22 cases of metastatic carcinoma were negative. The sensitivity and specificity of Hep Par 1 for HCC was 82.1% and 93.3% respectively; whereby the antibody was noted to show occasional false positivity in cases of cholangiocarcinoma and non-neoplastic bowel mucosa, while its variable staining in HCC produced false negative results in some small biopsies. Thus, Hep Par 1 should be used in a panel with other antibodies to obtain useful information in distinguishing HCC from CC and MC.
Surface plasmon resonance (SPR) sensing is recently emerging as a valuable technique for measuring the binding constants, association and dissociation rate constants, and stoichimetry for a binding interaction kinetics in a number of emerging biological areas. This technique can be applied to the study of immune system diseases in order to contribute to improved understanding and evaluation of binding parameters for a variety of interactions between antigens and antibodies biochemically and clinically. Since the binding constants determination of an anti-protein dengue antibody (Ab) to a protein dengue antigen (Ag) is mostly complicated, the SPR technique aids a determination of binding parameters directly for a variety of particular dengue Ag_Ab interactions in the real-time. The study highlights the doctrine of real-time dengue Ag_Ab interaction kinetics as well as to determine the binding parameters that is performed with SPR technique. In addition, this article presents a precise prediction as a reference curve for determination of dengue sample concentration.