An indirect enzyme immunoassay for the measurement of total 17alpha-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17alpha-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.
Arboreal pit vipers of the Trimeresurus complex group are medically important species in Indonesia (west of Wallace's line), but there is no specific antivenom produced in the country for treating related envenomation. Instead, the exiting trivalent Indonesian antivenom, Biosave® Serum Anti Bisa Ular (SABU, indicated for envenoming by Malayan pit viper, Javan spitting cobra and banded krait) is often misused to treat Trimeresus envenoming resulting in poor therapeutic outcome. Here, we investigated the cross-reactivity and neutralization capability of Thai Green Pit Viper Antivenom (GPVAV) against the venoms of four Indonesian Trimeresurus species. Consistently, the venoms of Trimeresurus (Trimeresurus) insularis, Trimeresurus (Trimeresurus) purpureomaculatus, Trimeresurus (Parias) hageni and Trimeresurus (Craspedocephalus) puniceus of Indonesia showed stronger immunoreactivity on ELISA to GPVAV than to Biosave®. The findings correlated with in vivo neutralization results, whereby GPVAV was far more effective than Biosave® in cross-neutralizing the lethality of the venoms by a potency of at least 13 to 80 times higher. The efficacy of GPVAV is partly attributable to its cross-neutralization of the procoagulant effect of the venoms, thereby mitigating the progression of venom-induced consumptive coagulopathy. The paraspecific effectiveness of GPVAV against Trimeresurus species envenoming in Indonesia await further clinical investigation.
The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 microg in mice) but was not lethal to mice at a dose of 1 microg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.
An immunofluorescent assay (IFAT) using whole cell antigen derived from Burkholderia thailandensis used for detection of total antibodies to Burkholderia pseudomallei, was found to compare favorably with a previous published report on a B. pseudomallei IFAT assay. At a 1:20 cut-off titer, the assay had high sensitivity (98.9%) and satisfactory specificity (92.3%), when tested against sera from 94 patients suspected of melioidosis. Sera from 12 patients with culture proven melioidosis gave absolute concordance with the 2 test antigens. No sera from 50 blood donors had a titer of > or =20. Cross-reactivity with patients' sera positive for Chlamydia, Mycoplasma, Legionella and typhoid was not observed, except for 3 sera from typhus patients and one from a patient with leptospirosis. The major advantage of this assay is that the cultivation and preparation of B. thailandensis as antigen can be carried out in any laboratory with basic microbiological set-up. The serodiagnosis of melioidosis can be made safe for medical laboratory personnel, particularly in B. pseudomallei endemic regions.
Candida parapsilosis has emerged as one of the most common causes of bloodstream infection worldwide. The diagnosis of invasive candidiasis etiological agents to the species level remains a laboratory and clinical challenge. Thus, specific monoclonal antibodies to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry would be very useful. Inbred Balb/c mice were immunized with C. parapsilosis antigens, and blood was checked for the presence of reactive antibodies using ELISA. Fusion was performed using the harvested spleen cells and NS1 myeloma cells, and the clones were screened for the presence of antibody producing hybrid cells by dot-blot. The 1B11 clone secreted IgG2a monoclonal antibody that was reactive with the C. parapsilosis antigen at MW of 59 kDa and cross-reacted with C. tropicalis but not with other fungal and bacterial antigens tested. Another 3D1 clone secreted IgG1 monoclonal antibody that was reactive with C. parapsilosis antigen at MW of 30 kDa. The 3D1 monoclonal antibody was found to be species specific. Experimental systemic candidiasis in rats was induced through intravenous injection of C. parapsilosis, and all the vital organs were collected for immunohistochemistry study. These monoclonal antibodies were reactive against surface epitopes on the yeast cells, pseudohyphae, and immune complexes in tissue sections. Sandwich ELISAs using these antibodies were developed and were able to detect circulating antigens in experimental candidiasis in rats at 0.2 μg/μL. These monoclonal antibodies may have potential as primary capture antibodies for the development of rapid diagnostic test for human systemic fungal infection.
Crab is an important source of food allergen. Tropomyosin represents the main crab allergen and is responsible for IgE cross-reactivity between various species of crustaceans. Recently, other new crab allergens including arginine kinase have been identified. However, information on allergens of the local Portunidcrab is not available. Thus, the aim of this study was to identify the major allergens of Portunus pelagicus (blue swimming crab) using the allergenomics approach. Raw and cooked extracts of the crab were prepared from the crab meat. Protein profile and IgE binding pattern were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from 30 patients with crab allergy. The major allergens of the crab were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry analysis of the peptide digests. The SDS-PAGE of raw extract revealed approximately 20 protein fractions over a wide molecular weight range, while cooked extract demonstrated fewer protein bands. The raw extract also demonstrated a higher number of IgE reactive bands than the cooked extract. A heat-resistant protein of 36 kDa has been identified as the major allergen in both raw and cooked extracts. In addition, a heat-sensitive protein of 41 kDa was also recognized as a major allergen in raw crab. The 2-DE gel profile of the raw extract demonstrated about >100 distinct proteins spots and immunoblotting of the 2-DE profile demonstrated at least 12 different major IgE reactive spots with molecular masses between 13 to 250 kDa and isoelectric point (pI) values ranging from 4.0 to 7.0. The 36 and 41 kDa proteins were identified as the crab tropomyosin and arginine kinase, respectively by mass spectrometry. Therefore, this study confirmed that tropomyosin and arginine kinase are the major allergens of the local Portunid crab, P. pelagicus.
Coeliac disease (CD) is an inflammatory disorder of the small intestine. It includes aberrant adaptive immunity with presentation of CD toxic gluten peptides by HLA-DQ2 or DQ8 molecules to gluten-sensitive T cells. A ω-gliadin/C-hordein peptide (QPFPQPEQPFPW) and a rye-derived secalin peptide (QPFPQPQQPIPQ) were proposed to be toxic in CD, as they yielded positive responses when assessed with peripheral blood T-cell clones derived from individuals with CD. We sought to assess the immunogenicity of the candidate peptides using gluten-sensitive T-cell lines obtained from CD small intestinal biopsies. We also sought to investigate the potential cross-reactivity of wheat gluten-sensitive T-cell lines with peptic-tryptic digested barley hordein (PTH) and rye secalin (PTS). Synthesised candidate peptides were deamidated with tissue transglutaminase (tTG). Gluten-sensitive T-cell lines were generated by culturing small intestinal biopsies from CD patients with peptic-tryptic gluten (PTG), PTH or PTS, along with autologous PBMCs for antigen presentation. The stimulation indices were determined by measuring the relative cellular proliferation via incorporation of (3) H-thymidine. The majority of T-cell lines reacted to the peptides studied. There was also cross-reactivity between wheat gluten-sensitive T-cell lines and the hordein, gliadin and secalin peptides. PTH, PTS, barley hordein and rye secalin-derived CD antigen-sensitive T-cell lines showed positive stimulation with PTG. ω-gliadin/C-hordein peptide and rye-derived peptide are immunogenic to gluten-sensitive T-cell lines and potentially present in wheat, rye and barley. Additional CD toxic peptides may be shared.
Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.
Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems.
Dengue is the most prevalent mosquito-borne disease in Southeast Asia, where the incidence of systemic lupus erythematosus (SLE) is approximately 30 to 53 per 100,000. Severe dengue, however, is rarely reported among individuals with SLE. Here, whether sera of patients with SLE cross-neutralize dengue virus (DENV) was investigated. Serum samples were obtained from individuals with SLE who were dengue IgG and IgM serology negative. Neutralization assays were performed against the three major DENV serotypes. Of the dengue serology negative sera of individuals with SLE, 60%, 61% and 52% of the sera at 1/320 dilution showed more than 50% inhibition against dengue type-1 virus (DENV-1), DENV-2 and DENV-3, respectively. The neutralizing capacity of the sera was significantly greater against DENV-1 (P cross-neutralize DENV, which may explain the rarity of severe dengue in individuals with SLE. Further studies, are needed to further substantiate this finding and to elucidate the specific neutralizing epitopes recognized by the sera of individuals with SLE.
The sharp increase in incidence of dengue infection has necessitated the development of methods for the rapid diagnosis of this deadly disease. Here we report the design and development of a reliable, sensitive, and specific optical immunosensor for the detection of the dengue nonstructural protein 1 (NS1) biomarker in clinical samples obtained during early stages of infection. The present optical NS1 immunosensor comprises a biosensing surface consisting of specific monoclonal NS1 antibody for immunofluorescence-based NS1 antigen determination using fluorescein isothiocyanate (FITC) conjugated to IgG antibody. The linear range of the optical immunosensor was from 15-500ngmL-1, with coefficient of determination (R2) of 0.92, high reproducibility (the relative standard deviation obtained was 2%), good stability for 21days at 4°C, and low detection limit (LOD) at 15ngmL-1. Furthermore, the optical immunosensor was capable of detecting NS1 analytes in plasma specimens from patients infected with the dengue virus, with low cross-reaction with plasma specimens containing the Japanese encephalitis virus (JEV) and Zika virus. No studies have been performed on the reproducibility and cross-reactivity regarding NS1 specificity, which is thus a limitation for optical NS1 immunosensors. In contrast, the present study addressed these limitations carefully where these two important experiments were conducted to showcase the robustness of our newly developed optical-based fluorescence immunosensor, which can be practically used for direct NS1 determination in any untreated clinical sample.