Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains |
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| Authors: | Iain Thompson John McGiven Jason Sawyer Rachel Thirlwall Nicola Commander Judy Stack |
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| Affiliation: | Brucella Research Group, Statutory and Exotic Bacteria, Veterinary Laboratories Agency (VLA), Woodham Lane, New Haw, Surrey KT15 3NB, United Kingdom,1. Technology Transfer Unit, Biotechnology Department, Veterinary Laboratories Agency (VLA), Woodham Lane, New Haw, Surrey KT15 3NB, United Kingdom2. |
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| Abstract: | Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISASPSS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.Species of the genus Brucella cause serious chronic infections, collectively known as brucellosis. Brucellosis is a mammalian disease infecting many economically important animal species as well as humans. With a global distribution, brucellosis causes considerable animal and human health problems as well as huge economic costs. Brucella species are gram-negative, nonmotile, facultative intracellular coccobaccilli belonging to the α-2 subdivision of proteobacteria. The genus consists of six classical species, namely, Brucella abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae, plus more recently discovered strains from marine mammals. Of the Brucella species, B. abortus, B. melitensis, and B. suis are of principal human health and economic importance. These species have smooth lipopolysaccharide (sLPS), which is considered a major virulence factor of disease (23), whereas B. ovis and B. canis have rough LPS (1).The World Organization for Animal Health (OIE) prescribed and alternative serological tests for diagnosis of brucellosis caused by smooth strains rely largely upon the measurement of the host antibody response to the O antigen of the sLPS (8, 22). Classical tests include the Rose Bengal test, the complement fixation test (CFT), and the serum agglutination test (SAT), all of which employ a whole-cell antigen as the key diagnostic reagent. More contemporary techniques, such as indirect enzyme-linked immunosorbent assay (iELISA), competitive ELISA (cELISA), and fluorescent polarization assay (FPA), employ purified LPS or O antigen as the diagnostic reagent. The immunodominance of the LPS O antigen is the basis for the generally excellent sensitivity of these assays (21). However, the use of this antigen can lead to false-positive serological results when animals are infected with bacteria possessing O antigens of similar structure (6), such as Yersinia enterocolitica O:9.ELISAs are readily amenable to high-throughput testing due to the standardized nature of the technology and reagents. This allows for many efficiency savings compared to the classical assays, including the use of effective automation (16). Despite the advantages of ELISA over the classical tests in this regard, ELISAs still require several steps to complete, including separation steps. Although these steps can be automated, they are a vital part of the assay and are a frequent source of imprecision, error, and mechanical breakdown. Assays which have the advantages of ELISA, such as a 96-well format, objective assessment, and good sensitivity and specificity, but which reduce the burden of work and opportunity for error are clearly desirable.The Meso Scale Discovery (MSD) electrochemiluminescence (ECL) platform uses electrochemical stimulation of reporter molecules conjugated to biological components to generate a light signal measured by photodetectors (2, 31), such as a charge-coupled device (CCD) camera. Carbon electrodes are integrated into the bottom of 96-well microtiter plates, to which biological components (for example, LPS) from traditional assays such as ELISA can be passively adsorbed. Biological conjugates, for example, monoclonal antibodies (MAbs), can be labeled with the reporter molecule ruthenium(II) tris-bipyridal [Ru(bpy)32+], which upon electrical stimulation emits light at 620 nm if it is within sufficient proximity to the carbon electrode excitation source. The reaction is enhanced by the addition of read buffer, which contains coreactants, including tripropylamine. Nonspecific signals are minimized as the stimulation mechanism (electricity) is decoupled from the signal (light).MSD ECL assays have the potential for separation-based (“wash”) and non-separation-based (“no-wash”) immunoassays due to the proximity-based nature of signal generation, where only labels near the surfaces of the electrodes are stimulated. The non-separation-based approach allows the addition of read buffer (MSD) directly to the components of the assay; this has an advantage over separation-based assays in that it removes a source of variation and reduces the time and labor required to perform the assay. In addition, MSD ECL assays can be multiplexed by spotting up to 10 different antigens onto discrete areas of the carbon electrode within each well of a 96-well plate; thus, the potential exists for multiplexed no-wash serological assays. The signal from each spot can be detected independently by a CCD camera, providing a quantitative measurement of the amount of light detected. Furthermore, the time taken to read a 96-well plate, approximately 70 s, is comparable to that for ELISAs and compares favorably to those for some other multiplex and ECL systems.We assessed the feasibility and potential of wash and no-wash MSD ECL assays for veterinary serodiagnosis of brucellosis. Biological components (B. melitensis 16 M sLPS antigen and BM40 [10], an anti-M O-chain epitope MAb) from the brucellosis cELISA produced by the Veterinary Laboratories Agency (26) were applied to the MSD platform. As with the parent cELISA, the wash and no-wash ECL assays allow competition between serum antibodies and the MAb BM40 [labeled with Ru(bpy)32+ for the MSD ECL assay] for sLPS bound to the carbon electrode. This affects the quantity of BM40 able to bind to this target. Thus, a positive result yields a low-intensity signal and a negative result yields a high-intensity signal. The new assays were validated against existing serological methods, using sera from Brucella-infected and noninfected ruminants. |
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