Development and Characterization of a Multiplex Assay to Quantify Complement-Fixing Antibodies against Dengue Virus.

Antibodies capable of activating the complement system (CS) when bound with antigen are referred to as "complement-fixing antibodies" and are involved in protection against Flaviviruses. A complement-fixing antibody test has been used in the past to measure the ability of dengue virus (DENV)-specific serum antibodies to activate the CS. As originally developed, the test is time-consuming, cumbersome, and has limited sensitivity for DENV diagnosis. Here, we developed and characterized a novel multiplex anti-DENV complement-fixing assay based on the Luminex platform to quantitate serum antibodies against all four serotypes (DENV1-4) that activate the CS based on their ability to fix the complement component 1q (C1q). The assay demonstrated good reproducibility and showed equivalent performance to a DENV microneutralization assay that has been used to determine DENV serostatus. In non-human primates, antibodies produced in response to primary DENV1-4 infection induced C1q fixation on homologous and heterologous serotypes. Inter-serotype cross-reactivity was associated with homology of the envelope protein. Interestingly, the antibodies produced following vaccination against Zika virus fixed C1q on DENV. The anti-DENV complement fixing antibody assay represents an alternative approach to determine the quality of functional antibodies produced following DENV natural infection or vaccination and a biomarker for dengue serostatus, while providing insights about immunological cross-reactivity among different Flaviviruses.

The limitations of commercial serological assays for detection of chlamydial infections in Australian livestock.

Chlamydia pecorum and Chlamydia abortus are related ruminant pathogens endemic to different global regions. Potential co-infections combined with the lack of species-specific serological assays challenge accurate diagnosis. Serological screening revealed low C. abortus seropositivity with the peptide-based ELISA (1/84; 1.2%) in Australian sheep yet moderate seropositivity in a Swiss flock with history of C. abortus-associated abortions (17/63; 26.9%). By whole cell antigen complement fixation tests (CFT) and ELISA, chlamydial seropositivity was significantly higher in all groups, suggesting cross-reactivity between these two chlamydial species and non-specificity of the tests. However, only C. pecorum DNA could be detected by qPCR in Chlamydia seropositive Australian animals screened, suggesting chlamydial seropositivity was due to cross-reactivity with endemic C. pecorum infections. These results suggest ascribing Chlamydia seropositivity to chlamydial species in livestock using whole-cell antigen CFT or ELISA should be treated with caution; and that peptide-based ELISA and qPCR provide greater chlamydial species-specificity.

Evaluation of three competitive ELISAs and a fluorescence polarisation assay for the diagnosis of bovine brucellosis.

Bovine brucellosis is an infectious disease of worldwide public health and economic importance. The usual tests for the diagnosis of this disease include the Rose-Bengal test (RBT), complement fixation test (CFT), serum agglutination test (SAT) and indirect ELISA. New tests such as competitive ELISAs (C-ELISA) and fluorescence polarisation assay (FPA) have been developed. However, C-ELISA may correspond to different protocols and a wide variation may exist in their diagnostic performance. The aim of this study was to evaluate three commercially available C-ELISA kits (C-ELISA1-3) and FPA for the diagnosis of bovine brucellosis and compare test performance with RBT, CFT, indirect ELISA and FPA. Sera submitted to EU laboratories in 2011 from 5111 adult cattle were tested. Individual test sensitivities (Se) and specificities (Sp) were estimated. Threshold assessment using the receiver operating characteristic method was also performed. The most sensitive tests were FPA (99.0%; 95% confidence interval [CI], 97.9-100%), C-ELISA1 (98.4%; 95% CI, 97.0-99.8%) and RBT (97.7%; 95% CI, 95.9-99.3%). The most specific tests were CFT (99.98%; 95% CI, 99.93-100%), SAT (99.98%; 95% CI, 99.93-100%) and RBT (99.89%; 95% CI, 99.79-99.99%). Among the new tests, none of the three C-ELISA kits studied could be recommended as a single screening test because of their low specificity, especially when used in a herd. C-ELISA3 could not be recommended as confirmatory test on individual animals to determine whether false positive serological test results had occurred.

Combining complement fixation and luminol chemiluminescence for ultrasensitive detection of avian influenza A rH7N9.

The complement fixation test (CFT) is a serological test that can be used to detect the presence of either a specific antibody or antigen to diagnose infections. In a conventional CFT, the assay result is determined by observing the clarity of the reaction solution or the sediment of red cells by the naked eye. Although the assay conditions are thereafter simplified, the sensitivity of the assay would be sacrificed due to the limitation of bulk observation. Inspired by the forensic scientists to examine blood at the scene of the crime, we rationally argued that the luminol chemiluminescence (CL) reaction could be applied in the CFT to sense physiological complement-mediated haemolytic phenomena for sensitive protein detection. The combination of the CFT and the luminol CL system was demonstrated in detection of rH7N9, a recombinant avian influenza virus protein. The testing can be accomplished within 2.5 h and the linear detection range covers 0.25 fg mL(-1) to 25 ng mL(-1). The feasibility of the CL based CFT in assaying a real biopsy was successfully demonstrated by specifically detecting rH7N9 and the carcinoembryonic antigen (CEA) in human serum. This new type of protein detection approach inherits the beauty of complement-mediated assay, such as being fast, and no protein immobilization, blocking and washing. In addition, the participation of luminol CL enables us to quantitatively analyse the intensity of a haemeolysis process, ameliorating the limitation of bulk observation in traditional CFT. It is anticipated that the luminol CL-CFT assay would be particularly suitable for investigation of small molecules, toxins, and short peptides.

Varicella zoster virus antibody detection: A comparison of four commonly used techniques.

BACKGROUND: Antibody tests for the varicella zoster virus (VZV) include neutralization, fluorescent antibody to membrane antigen (FAMA), immune adherence hemagglutination (IAHA), enzyme immunoassay (EIA), glycoprotein-based enzyme-linked immunosorbent assay (gpELISA), and complement fixation (CF) tests. Of these, FAMA is considered the most sensitive. However, in Japan, the EIA method is most frequently employed. OBJECTIVE: The VZV antibody detection rate of the FAMA, EIA, gpELISA, and IAHA methods was compared. METHODS: Four types of antibody tests were conducted with sera collected from 83 college students. The relationships between two antibody tests were examined using Pearson's correlation coefficients. RESULTS: All 83 subjects were observed to be VZV antibody-positive using the FAMA method. The Pearson correlation coefficients of gpELISA, EIA, and IAHA relative to FAMA were 0.808, 0.782, and 0.356, respectively. The positive agreement rate of IAHA relative to FAMA was 88.0% (73/83), whereas those of gpELISA and EIA were both 97.6% (81/83). Furthermore, EIA showed 100% positive agreement with gpELISA and a high correlation coefficient of 0.911, whereas these values for IAHA compared to gpELISA were much lower (90.1% and 0.530). The calculated Pearson correlation coefficient for comparison of the EIA and IAHA methods was 0.498, with a positive agreement rate of 90.1% (73/81). CONCLUSIONS: The EIA method should be employed in Japan based on the similarity of the positivity between EIA and gpELISA, as it is more available and practical than gpELISA.

Serology in chronic Q fever is still surrounded by question marks.

Detection of antibodies using immunofluoresence tests (IFAT) is recommended for diagnosis of chronic Q fever, but other commercial antibody assays are also available. We compared an enzyme-linked immunosorbent assay (ELISA) (Virion/Serion) and a complement fixation test (CFT) (Virion/Serion) for the detection of Coxiella burnetii IgG phase I and IgA phase I in early- and follow-up serum samples from patients with chronic Q fever, diagnosed according to an algorithm that involves IFAT. For this, we tested sera of 49 patients, including 30 proven, 14 probable and five possible chronic Q fever cases. Sensitivity of CFT for diagnosis of chronic Q fever was suboptimal (85 %), as eight patients, including five with chronic Q fever, tested negative at time of diagnosis, whereas IgG phase I antibodies were detected in these five patients by ELISA. Sensitivity of ELISA was higher, although three probable patients were missed. No differences in ELISA IgA phase I detection between proven chronic Q fever and probable were observed; instead possible patients were in majority IgA negative (60 %). Serological examination using ELISA and CFT in follow-up sera from 26 patients on treatment was unsatisfactory. Like IFAT, all kinetic options were possible: decreasing, remaining stable or even increase during time. This study demonstrated that the sensitivity of CFT-based phase I antibody detection is low and therefore not recommended for diagnosis of chronic Q fever. Based on our results, serological follow-up to guide treatment decisions was of limited value.

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep.

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n=118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA). The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.

Effect of incubation temperature on the diagnostic sensitivity of the glanders complement fixation test.

The complement fixation test (CFT) is the only serological test prescribed by the World Organisation for Animal Health (OIE) for the diagnosis of glanders in international trading of equids. However, false-positive reactions have caused financial losses to the animal owners in the past, and false-negative tests have resulted in the introduction of glanders into healthy equine populations in previously glanders-free areas. Both warm (incubation at 37°C for 1 h) and cold (overnight incubation at 4°C) procedures are recommended by the OIE for serodiagnosis of glanders. In a comparison of the sensitivity and specificity of the two techniques, using the United States Department of Agriculture antigen, warm CFT was found to be significantly less sensitive (56.8%; p < 0.0005) than the cold CFT (83.6%). Cold CFT thus increases the detection rate of glanders but a lower diagnostic specificity has to be accepted. The immunoblot was used as the gold standard.

Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment.

The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever.

[A simple method for quantification of modified low-density lipoproteins].

A simple method for quantification of modified low-density lipoproteins (mLDL) in the blood serum containing 8.9% polyvinylpyrrolidone solution 12600 +/- 2700 has been developed. The results show that 10 min incubation of serum in a buffer containing 8.9% PVP leads to complete aggregation mLDL. Atherogenicity of aggregated mLDLs is experimentally confirmed by two independent tests (mLDLs bind and activate the complement system of a guinea pig (pro-inflammatory effect) and cause platelet hyperaggregation (thrombogenic effect)). The proposed method is simple and involves only two steps: mixing the serum with a solution of PVP and recording the turbidity. The method allows to register the presence of mLDL directly in serum without its prior fractionation.

A spectrophotometric method to precisely determine endpoint titers in complement fixation assays.

Since the early 20th century, complement fixation (CF) testing has been used to quantify the humoral response to various pathogens. The qualification of a positive result is based on a subjective determination of 30% lysis of sheep red blood cells, which can lead to variability in the analysis. A spectrophotometric reading of a standard with a known 30% lysis was used to standardize the currently used CF method and tested with controls and patient sera for various fungal assays. By utilizing this method a precise and non-subjective determination of endpoint titers was achieved.

Performance evaluation of two serological tests for contagious bovine pleuropneumonia (CBPP) detection in an enzootic area using a Bayesian framework.

A Bayesian approach, allowing for conditional dependence between two tests was used to estimate without gold standard the sensitivities of complement fixation test (CFT) and competitive enzyme-linked immunosorbent assay test (cELISA) and the serological prevalence of CBPP in a cattle population of the Central Delta of the Niger River in Mali, where CBPP is enzootic and the true prevalence and animals serological state were unknown. A significant difference (P = 0.99) was observed between the sensitivities of the two tests, estimated at 73.7% (95% probability interval [PI], 63.4-82.7) for cELISA and 42.3% (95% PI, 33.3-53.7) for CFT. Individual-level serological prevalence in the study population was estimated at 14.1% (95% PI, 10.8-16.9). Our results indicate that in enzootic areas, cELISA performs better in terms of sensitivity than CFT. However, negative conditional sensitivity dependence between the two tests was detected, implying that to achieve maximum sensitivity, the two tests should be applied in parallel.

Complement-fixing anti-type VII collagen antibodies are induced in Th1-polarized lymph nodes of epidermolysis bullosa acquisita-susceptible mice.

The environment encountered in secondary lymphoid organs (e.g., lymph nodes) influences the outcome of immune responses. Immunization of mice with type VII collagen, an adhesion protein expressed at the cutaneous basement membrane, induces experimental epidermolysis bullosa acquisita (EBA). In this model, clinical disease is associated with the H2s haplotype of the MHC found in SJL/J mice. Most other strains (e.g., BALB/c, C57BL/6, NZM2410/J) are resistant to clinical disease, despite autoantibody production. Comparison of autoantibody response in EBA-resistant and -susceptible mice showed an IgG2-dominated response in the latter. We hypothesized that EBA susceptibility is due to specific cytokine gene expression in draining lymph nodes (dLN). To challenge this hypothesis, EBA-susceptible (SJL/J) and -resistant (BALB/c, C57BL/6) mice were immunized with type VII collagen, followed by analysis of clinical phenotype, subclasses of circulating and tissue-bound autoantibodies, complement activation, and cytokine gene expression in dLN. Disease manifestation was associated with induction of complement-fixing autoantibodies, confirming previous observations. Furthermore, however, IFN-γ/IL-4 ratio in dLN of EBA-susceptible mice was significantly increased compared with EBA-resistant strains, suggesting a Th1 polarization. Immunization of H2s-congenic C57BL/6 mice (B6.SJL-H2s) led to Th1 polarization in dLN and clinical disease. In addition to their cytokine milieu, EBA-susceptible and -resistant mice also differed regarding the expression of FcγR on peripheral leukocytes, in which a higher FcγRIV expression in SJL/J and B6.SJL-H2s mice, compared with C57BL/6, was associated with skin lesions. In summary, blistering in experimental EBA is regulated by both adaptive (divergent class switch recombination due to polarized cytokine expression) and innate (FcγR expression) immune mechanisms.

Comparison of complement fixation test, competitive ELISA and LppQ ELISA with post-mortem findings in the diagnosis of contagious bovine pleuropneumonia (CBPP).

The complement fixation test (CFT), the c-ELISA and an indirect LppQ ELISA were compared to post-mortem (PM) inspection for the diagnosis of contagious bovine pleuropneumonia (CBPP). Sera from 797 cattle in the CBPP affected area of Kazungula, Zambia and 202 sera from Lusaka, Zambia, a CBPP-free area were used. The clinical history of CBPP was recorded and all the cattle from Kazungula were slaughtered and PM inspections conducted. The prevalence of CBPP in Kazungula was 67.5% (95%CI 67.2%, 70.8%), 52.6% (95%CI 49.2%, 56.2%), 59.0% (95%CI 55.5%, 62.4%) and 44.4% (95%CI 41.0%, 47.9%) using PM inspection, CFT, c-ELISA and LppQ ELISA, respectively. Three of the 202 negative control animals tested positive on the c-ELISA although they were from a known CBPP negative zone. In this study, the c-ELISA was more sensitive in detecting cattle with lesions in the chronic stage than any other test whilst the CFT detected more during the onset stage. No single serological test could detect all stages of CBPP infection, therefore the use of more than one test is advised.

Clinical usefulness of a novel C1q assay to detect immunoglobulin G antibodies capable of fixing complement in sensitized pediatric heart transplant patients.

BACKGROUND: Donor-specific antibodies (DSA) against human leukocyte antigens complicate transplantation with the potential for acute antibody-mediated rejection (AMR). Complement-fixing antibodies are required to initiate the complement cascade. Not all DSAs, however, can fix complement. METHODS: A novel C1q assay was developed to detect the sub-set of immunoglobulin G (IgG) antibodies capable of fixing complement. Sera from 18 pediatric heart transplant patients were analyzed for DSAs using a Luminex platform (Luminex Inc, Austin, TX) and commercially available single-antigen bead assay kits. Biopsy specimens were assessed for AMR using histopathologic criteria and immunohistochemical staining. RESULTS: During the study period, 5 patients had AMR; of these, 2 were C1q virtual crossmatch positive (VXM+) and had persistent C1q DSAs after transplant, and 3 were C1q VXM- but antibody developed immediately after transplant. A positive C1q assay in the immediate post-transplant period had a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 100%, with 100% sensitivity and 100% specificity (Fisher exact p = 0.001). Of 11 patients who were IgG VXM+, 5 had AMR; the IgG VXM had a PPV of 45% and NPV of 100%, with 100% sensitivity and 54% specificity (Fisher exact p = 0.101). CONCLUSIONS: The C1q assay can detect a sub-set of antibodies capable of fixing complement and predicts AMR early after transplant. Avoiding only the donor antigens that would be recognized by the C1q assay may accelerate time to transplant by expansion of the donor pool and potentially allows transplantation of previously "incompatible" organs.

Bioactive arabinogalactans from the leaves of Opilia celtidifolia Endl. ex Walp. (Opiliaceae).

The leaves of the tree Opilia celtidifolia have a long tradition for being used in Mali and other West African countries against various ailments such as for wound healing. Previous studies on polysaccharides from these leaves showed the presence of pectic-like polymers with an effect on the human complement system as well as the ability to activate macrophages. The present study shows that bioactive arabinogalactans isolated by water of 50°C could be separated into two acidic fractions, Oc50A1 and Oc50A2. The former could, by gel filtration on Sephacryl S-400, be separated into two fractions, which were further purified on a Superdex 200 column to give the fractions Oc50A1.I.pur and Oc50A1.II.pur. These fractions were subjected to chemical and biological studies. The polysaccharides consisted mainly of heavily branched type II arabinogalactans and minor amounts of rhamnogalacturonan I regions. The isolated polymers had a high human complement-fixating ability, as well as the ability to stimulate rat macrophages and dendritic cells (DCs) and to induce B cell proliferation. These effects were especially pronounced for the higher molecular weight fraction of Oc50A1.I.pur. The fractions Oc50A1.I.pur and Oc501.II.pur stimulated secretion of pro-inflammatory cytokines from purified B cells or DCs. Collectively, these results indicate that the arabinogalactan type II polymers present in the leaves of O. celtidifolia may be used to develop medical devices for regulating inflammatory processes.