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High-throughput malaria serosurveillance using a one-step multiplex bead assay
Malaria Journal, 2019
Background: Serological data indicating the presence and level of antibodies against infectious disease antigens provides indicators of exposure and transmission patterns in a population. Laboratory testing for large-scale serosur-veys is often hindered by time-consuming immunoassays that employ multiple tandem steps. Some nations have recently begun using malaria serosurveillance data to make inferences about the malaria exposure in their populations , and serosurveys have grown increasingly larger as more accurate estimates are desired. Presented here is a novel approach of antibody detection using bead-based immunoassay that involves incubating all assay reagents concurrently overnight. Results: A serosurvey in was performed in Haiti in early 2017 with both sera (n = 712) and dried blood spots (DBS, n = 796) collected for the same participants. The Luminex ® multiplex bead-based assay (MBA) was used to detect total IgG against 8 malaria antigens: PfMSP1, PvMSP1, PmMSP1, PfCSP, PfAMA1, PfLSA1, PfGLURP-R0, PfHRP2. All sera and DBS samples were assayed by MBA using a standard immunoassay protocol with multiple steps, as well a protocol where sample and all reagents were incubated together overnight-termed here the OneStep assay. When compared to a standard multi-step assay, this OneStep assay amplified the assay signal for IgG detection for all 8 malaria antigens. The greatest increases in assay signal were seen at the low-and mid-range IgG titers and were indicative of an enhancement in the analyte detection, not simply an increase in the background signal of the assay. Seropreva-lence estimates were generally similar for this sample Haitian population for all antigens regardless of serum or DBS sample type or assay protocol used. Conclusions: When using the MBA for IgG detection, overnight incubation for the test sample and all assay reagents greatly minimized hands-on time for laboratory staff. Enhanced IgG signal was observed with the OneStep assay for all 8 malaria antigens employed in this study, and seroprevalence estimates for this sample population were similar regardless of assay protocol used. This overnight incubation protocol has the potential to be deployed for large-scale malaria serosurveys for the high-throughput and timely collection of antibody data, particularly for malaria seropreva-lence estimates.
Clinical and Vaccine Immunology, 2006
ABSTRACTAntibodies toPlasmodium falciparumare classically measured using the enzyme-linked immunosorbent assay (ELISA). Although highly sensitive, this technique is labor-intensive when large numbers of samples must be screened against multiple antigens. The suspension array technology (SAT) might be an alterative to ELISA, as it allows measurement of antibodies against multiple antigens simultaneously with a small volume of sample. This study sought to adapt the new SAT multiplex system for measuring antibodies against nine malarial vaccine candidate antigens, including recombinant proteins from two variants of merozoite surface protein 1, two variants of apical merozoite antigen 1, erythrocyte binding antigen 175, merozoite surface protein 3, and peptides from the circumsporozoite protein, ring erythrocyte surface antigen, and liver-stage antigen 1. Various concentrations of the antigens were coupled to microspheres with different spectral addresses, and plasma samples from Camero...
2008
IX. Immunoglobulin-or serum protein-binding to infected erythrocytes A. Stripping erythrocytes of bound serum proteins and reformation of rosettes ...... B. Detection of serum proteins on the surface of Plasmodium falciparum-infected
Malaria Journal, 2015
Background: Epidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life. Methods: An assay based on 14 Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia. Results: A significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p < 0.05). For the majority of antigens, antigen-coupled beads were stable for at least 2 months. The assay was very reproducible with limited intercoupling, interplate and intraplate variability (mean RSD <15 %). Estimating seroconversion and seroreversion per antigen using reversible catalytic models and models allowing two seroconversion rates showed higher seroconversion rates in adults. Conclusion: The multiplex bead-based immunoassay was successfully implemented and analysis of field blood samples shows that changes detected in force of malaria infection vary according to the serological markers used. Multivariate analysis of the antibody responses and insights into the half-life of antibodies are crucial for improving the interpretation of these results and for identifying the most useful serological markers of past and recent malaria infection.
Malaria journal, 2015
Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead-based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast. Recombinant proteins or peptides derived from liver or blood stage antigens of Plasmodium falciparum (CSP, LSA141, LSA3, SALSA, PF13-DBL1α1, GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour-coded microsphere (Luminex™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed. Hig...
Tropical Medicine & International Health, 2016
objective To generate monoclonal antibodies (MAbs) for developing a rapid malaria diagnostic urine-based assay (RUBDA), using Plasmodium-infected human urinary antigens. methods Plasmodium-infected human urinary (PAgHU) and cultured parasite (CPfAg) antigens were used to generate mouse MAbs. The reactivity and accuracy of the MAbs produced were then evaluated using microplate ELISA, SDS-PAGE, Western blotting assay, microscopy and immunochromatographic tests. results Ninety-six MAb clones were generated, of which 68.8% reacted to both PAgHU and CPfAg, 31.3% reacted to PAgHU only, and none reacted to CPfAg only. One promising MAb (UCP4W7) reacted in WBA, to both PAgHU and CPfAg, but not to Plasmodium-negative human urine and blood, Schistosoma haematobium and S. mansoni antigens nor measles and poliomyelitis vaccines. conclusion MAb UCP4W7 seems promising for diagnosing Plasmodium infection. Urine is a reliable biomarker source for developing non-invasive malaria diagnostic tests. SDS-PAGE and MAbbased WBA appear explorable in assays for detecting different levels of Plasmodium parasitaemia.
Antimicrobial Agents and Chemotherapy, 2007
Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ϳ0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [ 3 H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r 2 > 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.
Malaria Journal, 2010
Background: The absence of antibodies specific for the 19 kDa C-terminal domain of merozoite surface protein 1 (MSP1 19 ) has been associated with high-density malaria parasitaemia in African populations. The hypothesis that a high prevalence and/or level of anti-MSP1 19 antibodies that may inhibit erythrocyte invasion would be present in apparently healthy individuals who harbour a sub-microscopic malaria infection was tested in this study. Methods: Plasma samples were collected from residents in a region in Nigeria hyperendemic for malaria, who had no detectable parasitaemia by microscopy. Using a competition-based enzyme-linked-immunosorbent assay with two invasion-inhibitory monoclonal antibodies (mAbs) 12.10 and 12.8, the levels and prevalence of specific antibodies were measured. The minimum multiplicity of infection was determined using PCR. The prevalence of anaemia was also measured. Results: Plasma samples from 85% of individuals contained antibodies that bound to MSP1 19 . The inhibition of mAb 12.10 binding was strongly correlated with the prevalence (Spearman correlation test, p < 0.0001) and mean titre of anti-MSP1 19 antibodies (Spearman correlation test, p < 0.001) in the samples. Comparing samples from individuals with multiple infection (group M) and single infection (Group S), group M contained a higher (p = 0.04) prevalence of anti-MSP1 19 antibodies that competed with mAb 12.10. Using a logistic regression model, it was found that the presence of antibodies competitive with mAb 12.10 was affected negatively by anaemia (p = 0.0016) and positively by the carriage of multiple parasite genotypes (p = 0.04). Conclusions: In the search for correlates of protection against malaria, which will be essential to evaluate clinical trials of malaria vaccines based on MSP1, this study examines some potential assays and the factors that need to taken into account during their evaluation, using samples from individuals naturally exposed to malaria infection.
PLoS ONE, 2013
Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA). The assay measures the transmissionblocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds) to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra-and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability). We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in preclinical and early clinical development of transmission-blocking vaccines, and this study strongly supports its further development and application.