Secretory IgA enzyme immunoassay

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

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Autoantibodies to Thrombomodulin: Development of an Enzyme Immunoassay and a Survey of their Frequency in Patients with the Lupus Anticoagulant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648482 ◽

1992 ◽

Vol 67 (05) ◽

pp. 507-509 ◽

Cited By ~ 16

Author(s):

John Gibson ◽

Margaret Nelson ◽

Ross Brown ◽

Hatem Salem ◽

Harry Kronenberg

Keyword(s):

Enzyme Immunoassay ◽

Lupus Anticoagulant ◽

Specific Binding ◽

Technical Problem ◽

Serum Samples ◽

Citrate Buffer ◽

The Mean ◽

Sample Dilution ◽

Negative Population ◽

Serum Components

SummaryIn order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (± SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 ± 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.

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Antithrombin III Antigen in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660078 ◽

1985 ◽

Vol 54 (03) ◽

pp. 599-602 ◽

Cited By ~ 2

Author(s):

M Léon Alhenc-Gelas ◽

M Aiach ◽

A Gorenflot ◽

J P Andreux

Keyword(s):

Arachidonic Acid ◽

Enzyme Immunoassay ◽

Antithrombin Iii ◽

Human Platelets ◽

Mean Value ◽

Normal Blood ◽

Freezing And Thawing ◽

Healthy Donors ◽

At Iii ◽

Storage Granules

SummaryImmunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 109 platelets with a mean value of 70.3 ± 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

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A Simple Enzyme-Immunoassay (EIA) Test for Factor VIII-Related Antigen (VIIIAGN)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666933 ◽

1979 ◽

Vol 42 (03) ◽

pp. 848-854 ◽

Cited By ~ 22

Author(s):

Paul M Ness ◽

Herbert A Perkins

Keyword(s):

Factor Viii ◽

Enzyme Immunoassay ◽

Hemophilia A ◽

Single Sample ◽

Healthy Controls ◽

Von Willebrand's Disease ◽

Factor Viii Related Antigen ◽

Von Willebrand’S Disease ◽

Increased Sensitivity

SummaryAn enzyme immunoassay (EIA) system has been developed to measure factor VIII- related antigen (VIIIAGN). This assay gives similar results to the commonly used Laurell electroimmunodiffusion (EID) assay for VIIIAGN as shown by comparison of both techniques with samples from healthy controls, patients with hemophilia A, and patients with von Willebrand’s disease. The assay also has a greater precision than the EID technique as demonstrated by multiple assays of aliquots of a single sample. The use of this EIA test for VIIIAGN is simple and employs inexpensive reagents and equipment. The use of expensive antisera is minimized. EIA for VIIIAGN has the advantage of increased sensitivity compared to Laurell EIA.

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