Evaluation of a Novel Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Salmonella, Employing a Stable Coating of Lipopolysaccharide-Derived Antigens Covalently Attached to Polystyrene Microwells

Abstract

Abstract. Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from uninfected pigs. This ELISA proved itself by having a good long-term durability and a high degree of reproducibility, including low dayto-day variations and low interplate variations. Furthermore, the ELISA showed good specificity and sensitivity when data were compared with the optical density levels of a panel of pig sera as determined by a conventional ELISA on the basis of passive coating of the two Salmonella LPS antigens (the mix-ELISA). The covalent anthraquinone mix-ELISA shows promise as a stable and durable alternative to the existing conventional ELISA for serological surveillance of Salmonella infections in pigs.Bacterial lipopolysaccharides (LPSs) are important serotype-defining antigens of gram-negative bacteria, 17 and they are often, therefore, used as antigens in serological tests for the detection of serotype-specific antibodies. 2,10 Lipopolysaccharide consists of the hydrophobic lipid A part and the hydrophilic polysaccharide (PS) part. The latter can be further subdivided into a constant core region roughly similar in all Enterobacteriaceae and the variable O-antigenic PS specific for the bacterial serotype. 4,13 Lipopolysaccharide is anchored to the outer bacterial membrane by lipid A, whereas the highly immunoreactive PS part is exposed and able to interact with the immune system of the infected host. 17 In conventional solid-phase immunoassays (e.g., ELISA) for detection of anti-LPS antibodies, purified LPS is passively adsorbed to the plastic surface by hydrophobic interactions between the lipid A and the plastic surface, leaving the antigenic PS exposed to the solvent and freely accessible to the antibodies. However, coating by passive adsorption is strongly dependent on the overall amphiphilicity of the LPS, being in turn dependent on the specific type of LPS, and because purified LPS is heterogeneous because of the From the Danish Veterinary Laboratory,

Pathogen specific carbohydrate antigen microarrays: a chip for detection of Salmonella O-antigen specific antibodies

“…Currently used ELISA diagnostic tests most often use rather crude capsular polysaccharide (CPS) or LPS antigen preparations for direct coating of the plastic wells. Such preparations are inherently heterogeneous and batch-to-batch variation may be substantial, since chemical characterization is rarely performed [3,6]. Product quality control relies instead primarily on costly, extensive and time-consuming functional tests.…”

“…In addition to verifying disease in the individual human patient (as with the controversial 'Widal-test' [4,5]), serological tests are particularly well suited for large seroepidemiological surveillance studies of livestock in the food industry. The most commonly used serological technique is the enzyme-linked immuno-sorbent assay (ELISA) [6,7]. In ELISA as well as in all serological techniques, the most crucial parameter is the purity and specificity of the selected antigen and the reproducibility of its preparation [8].…”

Synthetic Peptides for the Immunodiagnosis of Human Diseases

“…Usually peptides do not have the same conformation free in solution as when adsorbed or covalently coupled to microtiter plates, the orientation of an immobilized antigen being important for recognition by an antibody. A strategy to overcome the low coating efficiency of highly hydrophilic peptides to microtiter plates and to avoid unpredictable orientation of the peptides on the surface is based on the covalent attachment of the synthetic peptides to the immunoassay surfaces [30][31][32][33][34][35][36]. Covalent binding, unlike simple physical binding, may orientate the immobilized peptides in a defined way on the solid phase.…”

Control Panels of Meat Juice Samples for a Salmonella Enzyme-Linked Immunosorbent Assay

“…For another Salmonella ELISA, it was observed that the SD was dependent on the amount of antibodies present. 10 The same phenomenon seems to be present for this ELISA, because an ordering of the test results from the 39 pools according to CV% resulted in groups with OD% within a certain range (Table 1). It has been reported that pools with a low OD% might have higher CV% than do pools with a higher OD%.…”