Enzyme amplification can enhance both the speed and the sensitivity of immunoassays

Stanley, Christopher J.; Johannsson, A.; Self, Colin H.

doi:10.1016/0022-1759(85)90061-4

Cited by 137 publications

(36 citation statements)

References 7 publications

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“…The color was developed using an amplification system (Gibco BRL, Grand Island, NY, USA). 9 Bound TPO was quantified colorimetrically. The absorbance of each sample was subtracted from that of the sample incubated with TN1.…”

Section: Patientsmentioning

confidence: 99%

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia

et al. 1998

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We studied serum thrombopoietin (TPO) levels and circulating numbers of platelet during five courses of myelosuppressive post-remission chemotherapy in three patients with acute leukemia in complete remission. Serum TPO levels were measured by a newly developed and sensitive sandwich enzyme-linked immunosorbent assay. In all courses, serum TPO levels changed reciprocally with the platelet counts. When platelets were transfused into patients near the time of platelet nadir, the TPO levels dropped temporarily, while platelet counts temporarily increased. In addition, platelets obtained after transfusion in a thrombocytopenic patient showed lower binding to biotinylated TPO than donor platelets prior to the transfusion. The finding indicated that the TPO receptors were saturated with endogenous TPO of the patient with a high serum TPO level. These results suggest that the platelet mass directly regulates serum TPO levels by receptor-mediated absorption and is one of the major regulators of serum TPO levels in humans.

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Section: Patientsmentioning

confidence: 99%

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia

et al. 1998

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“…The technique uses one antibody bound to a solid phase with sequential addition of samples, second antibody-enzyme conjugate and substrate. The assay was based on the technique of enzyme amplification (12) to improve sensitivity and reduce assay time (13). The performance of this assay has been compared t o two RIAs for CRF-41 in the measurement of rat hypothalamic CRF-41 content and release.…”

mentioning

confidence: 99%

Corticotrophin‐Releasing Factor‐41 in the Human and Rat—Utility of a Highly Sensitive Enzyme Amplified Immunometric Assay

Milton

Hillhouse

Fuller³

et al. 1990

J Neuroendocrinology

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RbstractW e have developed a highly sensitive and specific immunoassay for humanlrat corticotrophin-releasing factor-41 (CRF-41) to enable determination of immunoreactive CRF-41 levels in biological samples. To achieve high specificity, sensitivity and s p e e dwe have used two antisera in a sandwich enzyme immunoassay combined with enzyme amplification. The assay h a s a sensitivity of 0.08 fmollwell compared with radioimmunoassay sensitivities of 0.5 fmol/tube and is highly specific for the intact CRF-41 molecule. Measurement of samples is complete within 24 h compared with the 5 days required to obtain sensitive radioimmunoassay measurement. The assay has been used to measure both rat hypothalamic CRF-41 tissue content and release in vitro with good correlation when compared to radioimmunoassay measurement using antisera rC70 (0.983) or R1 (0.953). The assay only measures immunoreactive CRF-41 coeluting with humanlrat CRF-41 and its oxidized form Met [02'~38]CRF-41 in human and rat tissue extracts separated by high-performance liquid chromatography. The ability to measure immunoreactive CRF-41 in unextracted plasma allows rapid measurement and eliminates multiple extraction steps.( 'orticotrophin-releasing factor-41 (CRF-41) is a 41 amino-acid pcptide originally isolated from ovine hypothalamic tissue ( I ) . 7hc amino-acid sequences of human and rat CRF-41 are identical and differ from the ovine sequence at 7 residues (2, 3). CRF-41 cilso shares considerable sequence homology with sauvagine, a fi.ogskin peptide which has corticotrophin-releasing activity (4). 1,'arly assays for C R F relied on bioassay techniques ( 5 ) and were, therefore, susceptible to interfercnce from substances such as :idrenaline or arginine vasopressin which also have corticotrophinlcleasing activity (4). The development of RIAs for CRF-41 (6) allowed more specific and sensitive assay. However, the RIA tcchnique is time consuming and can give elevated measurements in the presence of CRF-41 peptide fragments or peptides with dcgrees of sequence homology with CRF-41 (4). Such RIAs also 1-cquire extraction of the material from biological fluids to reduce interference (7-9). The development of a 'two-site' immunoradiometric assay (IRMA) for CRF-41 (10) was a significant advance, reducing interference from fragments and allowing direct measurement of the peptide in biological fluids including plasma (1 1) In this study, we sought to construct an enzyme amplified ilnmunometric assay (EAIA) for human/rat CRF-41 using two polyclonal antisera raised against the whole CRF-41 molecule. The technique uses one antibody bound to a solid phase with sequential addition of samples, second antibody-enzyme conjugate and substrate. The assay was based on the technique of enzyme amplification (12) to improve sensitivity and reduce assay time (13). The performance of this assay has been compared t o two RIAs for CRF-41 in the measurement of rat hypothalamic CRF-41 content and release. The ability of the EAIA to measure human CRF-41 in both unextracted gest...

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“…Bound MAb was detected using a murine anti-idiotypic antibody (IgG2b) (Bosslet et al, 1990), selective for the murine as well as the humanised anti-CEA MAb BW 431, and an alkaline phosphatase labelled goat anti-mouse-IgG2b antibody (Southern, Birmingham, USA) combined with the alcohol dehydrogenase-diaphorase amplification system (Stanley et al, 1985; system III). Optical density was measured at 493 nm using a Titertek multiscan type 3100 (Flow Laboratories).…”

Section: Methodsmentioning

confidence: 99%

Characterisation of a humanised bispecific monoclonal antibody for cancer therapy

Bruynck

Seemann²,

Bosslet³

1993

Br J Cancer

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Enzyme amplification can enhance both the speed and the sensitivity of immunoassays

Cited by 137 publications

References 7 publications

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia

Corticotrophin‐Releasing Factor‐41 in the Human and Rat—Utility of a Highly Sensitive Enzyme Amplified Immunometric Assay

Characterisation of a humanised bispecific monoclonal antibody for cancer therapy

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