An ELISA avoiding interference by heterophilic antibodies in the measurement of components of the plasminogen activation system in blood

Grebenchtchikov, Nicolaï; Sweep, Fred C.G.J.; Geurts-Moespot, Anneke; Piffanelli, A; Foekens, John A.; Benraad, Th. J.

doi:10.1016/s0022-1759(02)00213-2

Cited by 33 publications

(24 citation statements)

References 29 publications

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“…Injections were repeated 12 times for both chickens and rabbits at 2-week intervals. Polyclonal antibodies against BCAR1, isolated from chicken egg yolks and citrate plasma from rabbits, were purified by affinity chromatography on GST-BCAR1/F1R5-coated columns (AffiGel ® 15; Bio-Rad Laboratories) according to previously described procedures (8,10 ). The purified antibodies, marked 328# for chicken anti-BCAR1 and 329# for rabbit anti-BCAR1, were diluted with glycerol (1:1) and stored at Ϫ20°C.…”

Section: Materials and Methods Assay Developmentmentioning

confidence: 99%

“…In these ELISA formats, two antibodies were used for capture (preanalyte stage), and two other antibodies were used for detection (postanalyte stage). The use of exclusively avian antibodies at the preanalyte stage of the assay and mammalian antibodies at the postanalyte stage made the ELISAs insensitive to the presence of heterophilic antibodies, one of the major sources of interference in two-site immunologic measurements (10 ).…”

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confidence: 99%

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Development of an ELISA for Measurement of BCAR1 Protein in Human Breast Cancer Tissue

Grebenchtchikov¹,

Brinkman²,

Broekhoven³

et al. 2004

Clinical Chemistry

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Background: High concentrations of breast cancer antiestrogen resistance 1 (BCAR1) protein measured by Western blotting in primary breast tumor cytosols are associated with early disease progression and failure of tamoxifen therapy. The aim of the present study was to develop an ELISA to measure BCAR1 quantitatively in extracts of human breast cancer tissue. Methods: A recombinant fragment of BCAR1 (the human homolog of murine p130Cas) was produced in bacterial M15 cells, purified, and injected into chickens and rabbits. The generated antibodies were affinitypurified and used for the construction of an ELISA. After validation, the results obtained with the ELISA were compared with Western blot findings on primary breast tumors. Results: The detection limit the BCAR1 ELISA was 0.0031 g/L, and the within-run imprecision (CV) was <20% at concentrations down to 0.004 g/L. The withinrun imprecision (CV) was 1.0 -7.2%, and the betweenrun CV was 3.6 -5.4%. There was no cross-reactivity with family member HEF1. The assay exhibited parallelism of results between serial dilutions and a mean recovery (range) of 96 (79 -118)%. Conclusions: The ELISA measures BCAR1 in human breast cancer cytosols with high sensitivity and specificity. The assay can be used to confirm and to quantitatively extend previous semiquantitative Western blot data on the prognostic and predictive value of BCAR1 in

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Section: Materials and Methods Assay Developmentmentioning

confidence: 99%

mentioning

confidence: 99%

Development of an ELISA for Measurement of BCAR1 Protein in Human Breast Cancer Tissue

Grebenchtchikov¹,

Brinkman²,

Broekhoven³

et al. 2004

Clinical Chemistry

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“…We agree with the authors that screening all clinical samples for all types of possible interferences is not feasible in practice, and we alternatively directed our efforts to devising assays that would be less susceptible to interfering substances (3,4 ). Assessment of the presence of interfering substances is done by use of nonfunctional antibody combinations in a sandwich ELISA format that should not give a true signal because it was designed against a nonexistent analyte (so-called nonsense format; see Fig.…”

Section: To the Editormentioning

confidence: 57%

“…This has been found to essentially preclude all interference in these assays (3,4 ). This effect is obtained because interfering factors that would typically bridge between pre-and postanalyte antibodies in the absence of analyte, leading to false positives, do not bind to avian IgY antibodies.…”

Section: To the Editormentioning

confidence: 99%

Screening for Interference in Immunoassays

Span¹,

Grebenchtchikov²,

Geurts-Moespot³

et al. 2003

Clinical Chemistry

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“…One of the primary requirements for a CRM is the use of a matrix similar to the patient specimen (2 ). We have observed that the calibrator developed for mass spectrometric methods may be a working solution of the analytes prepared in dilute acid or methanol (3 ). This type of calibrator cannot be tested in the Bio-Rad HPLC method because the analytical recovery, in the absence of appropriate concentrations of salts, proteins, and buffers, does not resemble the analytical recovery observed in human urine.…”

mentioning

confidence: 99%