Artifacts and limitations of enzyme immunoassay

Aj, Pesce; Michael, J.Gabriel

doi:10.1016/0022-1759(92)90070-a

Cited by 68 publications

(40 citation statements)

References 58 publications

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“…Various surface effects have been considered as possible explanations for differences in affinities in solid-phase assays [12][13][14]. Coating of protein on microtitre plate wells may change conformation or even lead to denaturation [15].…”

Section: Discussionmentioning

confidence: 99%

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Tangemann

Engel

1995

FEBS Letters

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To clarify the question as to why different solid-phase assays yield different results in terms of interaction strength, we used fihrinogen binding to immobilized aIIbfl3 integrin as a test system. A classical 'three step' enzyme-linked (ELISA), a 'two step' biotin enzyme-linked streptavidin and a 'one step' radioligand assay were compared under otherwise identical conditions. Only the last assay yielded binding constants comparable to earlier data by total internal reflection fluorescence microscopy while the other assays yielded apparent binding constants 5-to 1000-fold too high. These effects are explained by non-linearity of detection signals.

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Section: Discussionmentioning

confidence: 99%

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Tangemann

Engel

1995

FEBS Letters

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“…The test described in this paper detects Ͻ1 ng of small proteins using Dynalbeads, whereas 3-mm-diameter modified glass beads will detect up to 4 g. These levels are sensitive enough to detect and quantify the toxin of interest. However, the test becomes inadequate at higher concentrations because BSA will bind nonspecifically to the beads in addition to specific antibody capture (32).…”

Section: Discussionmentioning

confidence: 99%

“…This may also help to explain the high background level seen in the control. The protein present in the slush tank, which contained whey and other cheese production byproducts, can coat the surface of the modified beads and thus prohibit the interactions between the capture molecule and the target (32). The capture of E. coli O157:H7 in flow with 3-mm-diameter glass beads containing a PEG spacer in two sample types was demonstrated (Fig.…”

Section: Discussionmentioning

confidence: 99%

Solid-Phase Capture of Proteins, Spores, and Bacteria

Weimer

Walsh

Beer

et al. 2001

Appl Environ Microbiol

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Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (antiEscherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 g) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25°C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.

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“…The methods for antigen preparation are important, because denaturation of the tertiary structure of PR3 leads to the loss of epitopes. Coating of proteins onto a solid phase also leads to denaturation of proteins [21], and we therefore developed capture assays to present PR3 in a better preserved form. These seem to work in the expected way, as more IIF-positive sera were positive in the capture assays than in the direct PR3-ANCA ELISA.…”

Section: Discussionmentioning

confidence: 99%

“…3). A disadvantage of using a capture ELISA with mouse antibody is that some patients may have developed human anti-mouse antibodies, either through such an event as a rodent bite or treatment with MoAb [21]. Another interfering factor producing false-positive results is rheumatoid factors [22].…”

Section: Discussionmentioning

confidence: 99%

Screening for anti-neutrophil cytoplasmic antibodies (ANCA): is indirect immunofluorescence the method of choice?

Baslund

Segelmark

Wiik

et al. 1995

Clinical and Experimental Immunology

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SUMMARYDetection of ANCA has become an important tool for the diagnosis and monitoring of disease activity in Wegener's granulomatosis (WG). Unfortunately, a group of sera positive by the standard method for ANCA detection, indirect immunofluorescence (IIF), are negative when more specific tests with purified proteins are used. In order to examine this discrepancy we examined groups of sera selected for being (i) C-ANCA-positive by IIF; (ii) positive in proteinase 3 (PR3)-ANCA ELISA; and (iii) from 24 patients with WG. The following assays were used: IIF, PR3-ANCA ELISA and capture PR3-ANCA ELISA using MoAbs against PR3. Furthermore, since granule enzymes are released during coagulation, we also measured ANCA in complex with PR3. To test if granule enzyme release had any influence on ANCA detection, both serum and EDTA-plasma were collected from a patient with active WG. No difference, however, was found. In the IIF-positive group (n = 60) 68% of the sera were positive in PR3-ANCA ELISA, 86% in capture PR3-ANCA ELISA and 80% were positive for the PR3/IgG-ANCA complex. In the PR3-ANCA ELISA group (n = 105) 88% of the sera were positive by IIF, 98% in capture PR3-ANCA ELISA and 53% in the PR3/IgG-ANCA assay. To evaluate the tests clinically sera from 24 patients with WG were examined. In the remission group (n = 10) two patients were positive by IIF, four in the PR3-ANCA ELISA, and five in the capture PR3-ANCA ELISA. Fourteen had active disease, and in this group 11/14 were positive by IIF, 10/14 in PR3-ANCA ELISA and 12/14 by capture-ELISA. The correlation between IIF and capture PR3-ANCA ELISA titre (r = 0-72, P = 0 0095) was better than between PR3-ANCA ELISA and IIF (r = 0-56, P = 0 043). It is concluded that the capture PR3-ANCA ELISA is more sensitive than PR3-ANCA ELISA, and that the capture ELISA can be used for screening of PR3-ANCA.

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Artifacts and limitations of enzyme immunoassay

Cited by 68 publications

References 58 publications

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Solid-Phase Capture of Proteins, Spores, and Bacteria

Screening for anti-neutrophil cytoplasmic antibodies (ANCA): is indirect immunofluorescence the method of choice?

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