Evaluation of inexpensive blocking agents for ELISA in the detection of antibody in leprosy

Jt, Douglas; Qx, Wu; Gp, Agustin; Mg, Madarang

doi:10.5935/0305-7518.19880007

Cited by 6 publications

(3 citation statements)

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“…11 It is also used in Southern blots and ELISA as a blocking reagent. 12 Casein, the third blocking agent, is the major protein of milk and makes up about eighty percent of the weight of milk protein. It is responsible for most of the binding site saturation effect, ie., it covers much of the phage and limits the binding of bacteria to the phage.…”

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confidence: 99%

Blocking Agent Optimization for Nonspecific Binding on Phage Based Magnetoelastic Biosensors

Shen

Park

et al. 2012

J. Electrochem. Soc.

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Magnetoelastic biosensors have been studied for the past few years for use in detecting bacteria for food safety. These magnetoelastic biosensors are composed of a bio-molecular recognition element and transducer platform (magnetoelastic resonator). Phages (biomolecular recognition element) have been developed to provide very specific binding with Salmonella typhimurium bacteria. However, there is some binding (non-specific binding) of bacteria to the gold areas not covered with phage on both the measurement and control sensors. In those areas, a blocking agent is necessary to reduce the non-specific binding. The purpose of this study is to compare three of the often used blocking agents, BSA, milk and casein in different concentrations to determine the agent and the optimum concentration for blocking. To accomplish this, a set of measurement and control sensors were prepared using identical procedures except the control sensors were not immobilized with phage. The measurement and control sensors were then coated with a blocking agent of concentrations ranging from 0.1 to 5 mg/mL. SEM observations show that coating the magnetoelastic biosensors with BSA at a concentration of 1mg/mL yields the largest amount of specific binding of bacteria cells while also maintaining the largest ratio of specific to non-specific binding. This binding characteristic is a result of the physical and electrical structure of the blocking proteins. To verify that the blocking solutions made good contact with the sensor's surface, contact angle measurements were used to evaluate the wettability of the surface. Changes in the resonant frequency of the biosensors were found to agree with the results from SEM observations.

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confidence: 99%

Blocking Agent Optimization for Nonspecific Binding on Phage Based Magnetoelastic Biosensors

Shen

Park

et al. 2012

J. Electrochem. Soc.

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“…NFDM exhibited a similar blocking effect to CN due to its molecular diversity and amphipathic characteristics of milk proteins [49]. A 10% NFDM offered a better blocking effect than 10% BSA and 20% egg albumin [50]. The superiority of NFDM in ELISA blocking was also demonstrated by Akerstedt [51] and Huber et al [52].…”

Section: Effect Of Protein and Non-protein Blockersmentioning

confidence: 75%

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection

Jiang

Albo

et al. 2021

Foods

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Different types of enzyme-linked immunosorbent assays (ELISA) have been widely used to control food safety and quality. To develop an accurate and reproducible ELISA, false immunodetection results caused by non-specific binding (NSB) and cross-reaction must be prevented. During the case study of sandwich ELISA development for the detection of porcine hemoglobin (PHb), several critical factors leading to NSB and cross-reaction were found. First, to reduce the NSB of the target analyte, the selection of microplate and blocker was discussed. Second, cross-reactions between enzyme-labeled secondary antibodies and sample proteins were demonstrated. In addition, the function of (3-aminopropyl)triethoxysilane (APTES) was evaluated. Overall, this study highlights the essence of both antibody and assay validation to minimize any false-positive/negative immunodetection results.

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“…The assay was performed as previously described. 18 Plates coated with the specified antigen were first blocked with 75 microlitres of 10% skimmed milk in phosphate buffered saline with 0• 1 % Tween-20 (PBS), pH 7-4, and incubated for I hour at 37°C. The sera were diluted I: 500 in 10% skimmed milk in PBS, pH 7•4.…”

Section: Elisamentioning

confidence: 99%

Evaluation of four semi-syntheticMycobacterium lepraeantigens with sera from healthy populations in endemic and non-endemic areas

et al. 1992

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In order to determine the frequency of occurrence of antibodies to semisynthetic antigens of My cobacterium leprae in clinically healthy nonpatient populations and to establish a 'baseline' for comparison with antibody frequen cies in both patients with a history of leprosy and their contacts, ELISAs were conducted using representative sera from two areas: a leprosy endemic area, Cebu City, Philippines and a nonendemic area for leprosy Chicago, Illinois, USA. These sera were tested, by an indirect IgM ELISA, for the presence of antibodies reacting with fo ur semisynthetic antigens based on the phenolic glycolipid I antigen of M. leprae: ND-O-BSA (natural disaccharide with octyl linkage to bovine serum albumin), NT-O-BSA (natural trisaccharide with octyl linkage to BSA), ND-P-BSA (natural disaccharide with phenolic ring linkage to BSA) and NT-P-BSA (natural trisaccharide with phenolic ring linkage to BSA). Using an OD reading ;;. 0•16 as positive, the antigen with the lowest background seroreactivity was ND-O-BSA, which reacted with 5/398 (1'3%) sera from Cebu, and 3/426 (0,7%) sera from Chicago. A total of 10 (2'5%) of 398 sera from the endemic area reacted with at least one antigen and 5 (1'3%) sera reacted with all four semisynthetic antigens. Of the 426 sera fr om Chicago, 12 (2,8%) were reactive with at least one antigen and 3 (0'7%) were reactive with all four semisynthetic antigens. Mean ELISA values for the 22 positive sera for each antigen ranged from 0• 17 to O• 3 OD units, while the mean values for all sera in

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Evaluation of inexpensive blocking agents for ELISA in the detection of antibody in leprosy

Cited by 6 publications

References 0 publications

Blocking Agent Optimization for Nonspecific Binding on Phage Based Magnetoelastic Biosensors

Blocking Agent Optimization for Nonspecific Binding on Phage Based Magnetoelastic Biosensors

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection

Evaluation of four semi-syntheticMycobacterium lepraeantigens with sera from healthy populations in endemic and non-endemic areas

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