Diagnostic Potential of an IgE-ELISA in Detecting Strongyloidiasis

Strongyloides stercoralisinfection is prevalent worldwide and can cause lifelong infection in immunocompetent individuals, and potentially death in immunosuppressed patients. The diagnosis is hindered by the low sensitivity of microscopic examination, thus making serology an important complementary test to improve the detection rate.

However, there were reports that some Strongyloides-infected individuals were negative with specific IgG and IgG4 assays, and other helminth infections were positive with commercial StrongyloidesIgG-ELISAs. Thus, there is a need to develop better serodiagnostic methods for strongyloidiasis. We investigated the diagnostic potential of IgE-ELISAs using Strongyloides larval lysate.
Sera from two groups infected with Strongyloides served as the positive reference, that is, 1) positive by commercial IgG-ELISAs and IgG4 rapid test, and stool samples positive by microscopy and/or PCR (group IA; n = 20); and 2) negative by IgG-ELISAs and IgG4 rapid test, but stool samples were PCR positive (group IB sera; n = 11).

Sera from another two groups served as negative reference (controls), that is, 1) infected with other parasites (group II; n= 73) and 2) healthy donors (group III; n = 22). Results showed a 100% diagnostic sensitivity in detecting sera from groups IA and IB. The latter group of individuals probably had early infection because their IgG and IgG4 assays were negative.

The optical density values of group IB sera were also significantly lower than those of group IA (P< 0.003). The IgE-ELISA was 100% specific when tested against sera from groups II and III. This study highlights the diagnostic potential of IgE-ELISA using larval lysate to detect strongyloidiasis, especially those with probable early infection.

Development of a double-recombinant antibody sandwich ELISA for quantitative detection of epsilon toxoid concentration in inactivated Clostridium perfringens vaccines

Background: Epsilon toxin (ETX) causes a commonly fatal enterotoxemia in domestic animals. Also, ETX causes serious economic losses to animal husbandry. In this study, we selected several clones against ETX using repertoires displayed on filamentous phage. Anti-ETX specific clones were enriched by binding to immobilized antigen, followed by elution and re-propagation of phage. After multiple rounds of binding selection, ELISA analysis showed that most isolated clones had high affinity and specificity for ETX.

Results: Two recombinant monoclonal antibodies against ETX were isolated by phage display technology. B₁ phage VH antibody isolated from DAb library and G₂ soluble scFv antibody isolated from Tomlinson I + J libraries have been applied as the capture and detection antibodies for developing an ETX sandwich ELISA test, respectively.

Conclusions: Designed ETX sandwich ELISA could be a valuable tool for quantitative detection of ETX in inactivated commercial vaccines against enterotoxemia.

A quantitative enzyme-linked immunoassay (ELISA) to approximate complement-fixing antibody titers in serum from patients with coccidioidomycosis

Coccidioidomycosis is most frequently diagnosed serologically, and the quantitative test for complement-fixing antibodies is considered prognostically useful. Because complement-fixing antibody testing is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. In this report, we restrict the complement-fixing, antibody-binding domain to a 200-amino-acid recombinant peptide of the known antigen.

Over-lapping truncations of this peptide do not bind complement-fixing antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal biotin-mimic peptide tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by 1-2 logs in different sera. The newly developed ELISA shows a significant quantitative correlation with complement-fixing antibody titers. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-α antigen were selected and characterized.

For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were determined and a standard curve was established using recombinant chIFN-α.

The sandwich ELISA effectively detected an increased IFN-α production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C), and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize its activity were identified. The newly developed antigen sandwich ELISA developed in this study will be a useful tool to monitor IFN-α production in chickens.

Performance of commercial Mycoplasma hyopneumoniae serum ELISAs under experimental and field conditions

Mycoplasma hyopneumoniae (MHP) is an economically significant pathogen of swine. MHP serum antibody detection via commercial ELISAs is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance.

In Study One, 6 commercial MHP ELISAs were compared using serum samples from 8-week-old CDCD pigs allocated to 5 inoculation groups of 10 pigs each: (1) negative control (2) M. flocculare (strain 27399) (3) M. hyorhinis (strain 38983) (4) M. hyosynoviae (strain 34428), and (5) MHP (strain 232).

Weekly serum and daily oral fluid samples were collected through 56 days post-inoculation (DPI). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period.

Analysis of ELISA performance at various cutoffs found that the manufacturers’ recommended cut-offs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives.

In Study Two, the 3 best performing ELISAs from Study One were compared using serum samples generated under field conditions. Ten8-week-old pigs were intratracheally inoculated with MHP Matched serum and tracheal samples (to establish the true pig MHP status) were collected at 7- to 14-day intervals through DPI 98.

Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current MHP ELISAs and understanding of their use in surveillance.