Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.     

Analysis of gyrA mutations related to quinolone resistance in Escherichia coli isolates originating from pet, human, vegetable and ice in Bangkok and vicinity

Escherichia coli was used to investigate quinolone resistance and mutations in gyrA gene of E. coli isolated from pet (dog and cat), human (pet's owner), vegetable and edible ice in Bangkok and vicinity. Susceptibility test for nalidixic acid (NA) showed similar percent resistance among the sample sources but a lower ciprofloxacin (CIP) resistance was found particularly in human source. Mutations within quinolone resistance determining region of gyrA gene analyzed using non-radioactive single-strand conformation polymorphism (SSCP) and sequencing showed 10 different SSCP patterns. E. coli isolates from pet, vegetable and ice showed more variety of patterns than strains isolated from human. Four out of 10 SSCP patterns were identified as having mutations in amino acids positions 83 (Ser to Leu) and position 87 (Asp to Asn). These mutations were observed only in NA-resistant strains and combined mutations were observed only in E. coli isolated from humans and pets. As only 24% of NA- and CIP-resistant E coli isolates contained gyrA mutations, other quinolone resistant mechanisms may be involved. Nevertheless, gyrA mutations may be used to monitor nalidixid acid resistance in E. coli.     

Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein

The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test.     

Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel V_HHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-V_HH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the V_HHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.     

Toxic marine puffer fish in Thailand seas and tetrodotoxin they contained

A total of 155 puffers caught from two of Thailand's seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.     

Human ScFv that block sodium ion channel activity of tetrodotoxin

Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C₁₁H₁₇N₃O₈) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.