Normal Human Pooled Plasma Research Articles

Radioimmunoassay for quantitative measurement of factor VIII-heavy chain.

An immunoassay has been developed for the measurement of factor VIII heavy chain (FVIII-HC). IgG from a human inhibitor plasma with specificity for FVIII-HC and FVIII-light chain (FVIII-LC) was coated to microplates with loose wells. After washing, the plates were incubated with sample and after another wash 125I-FVIII-HC was added so that the amount of bound tracer was dependent on the amount of FVIII-HC in sample. When EDTA was included in the assay buffer the assay response was increased 3-fold for normal human plasma. This indicated that the antibody was reactive with a determinant hidden in the FVIII-HC/FVIII-LC complex as EDTA splits the complex. The sensitivity of the assay was 0.004 units/ml (1 unit/ml in normal human plasma pool). Together with a previously published assay for FVIII-LC it has now become possible to measure the relative amounts of FVIII-HC and FVIII-LC in haemophilia A plasma and to study the separate expression of FVIII-HC and FVIII-LC by recombinant DNA technology. Patients with severe haemophilia A had FVIII-HC levels below 0.01 units/ml. However, inhibitor patients in high dose FVIII-treatment showed up to 50 times higher levels of FVIII-HC than FVIII-LC and FVIII:C, indicating the presence of FVIII/anti-FVIII-LC immune complexes. Thus, dependent on assay specificity plasma samples can show very variable content of FVIII:antigen.

A subfraction of human fibrinogen with high sialic acid content and elongated gamma chains.

Fibrinogen isolated from normal human single donor or pool plasma was fractionated by DEAE-cellulose chromatography. Three different fibrinogen subfractions were obtained. The most acidic fraction comprising 22% of the whole fibrinogen pool was prominent by two special features: 1) its sialic acid content was significantly higher than that of bulk fibrinogen, namely 8 mol of sialic acid/mol of fibrinogen versus 6 mol in bulk fibrinogen. 2) Two-dimensional electrophoresis of the polypeptide chains obtained after reduction revealed a preferential accumulation in this subfraction of the elongated gamma chains previously described as gamma chain heterogeneity of whole human fibrinogen.

Laser nephelometric quantitation of antithrombin-III (AT-III) development of a new assay

Antithrombin-III (AT-III) concentration in normal healthy volunteers and in an out-patient population was determined by a radial immunodiffusion (RID) and by a laser nephelometric technique, developed in our laboratory. In 43 normal individuals tested, the mean AT-III concentration by laser nephelometry was 24.1 ± 3.3 mg/dL or 108 ± 13% normal human plasma pool (NHP). The coefficient of variation (CV) of the assay was in the order of 7–10% and there was a good correlation (r=0.81) between RID and laser nephelometric method. Some developmental aspects of the laser nephelometric procedure and its advantages over the RID method are discussed.

Determination of Hageman factor (HG, factor XII) and plasma prekallikrein (Fletcher factor) by radioimmunoassays.

The titers of components of the plasma kallikrein-kinin system have been measured conventionally by their biological functions. The functional assays are, however, antagonized by the presence of inhibitors and/or the absence of potentiators in test samples. Immunologic assays obviate these difficulties. We have developed specific, sensitive and reproducible radioimmunoassays (RIA) for HF and prekallikrein, and have applied these assays to some clinical conditions. Normal pooled human plasma contained approximately 40 microgram of HF and 50 microgram of prekallikrein per ml. RIA were able to measure concentrations of HF and prekallikrein as low as 0.1% and 0.3% that of normal pooled plasma respectively. A good correlation existed between titers measured by clotting and radioimmunoassays among 40 normal subjects (correlation co-efficient = 0.82 for HF and 0.71 for prekallifrein). There was no significantly reduced in plasmas of patients with advanced liver cirrhosis or disseminated intravascular coagulation (DIC) and in cord serums, but they were normal in plasmas obtained after strenuous physical exercise and in plasmas of patients under treatment with warfarin.

Purification and properties of crystalline human ceruloplasmin.

Crystalline human ceruloplasmin was prepared from a Cohn's fraction F‐IV‐1 of normal human pooled plasma, by procedures based on acetone fractionation and chromatography on DEAE‐sephadex A‐50.Needle‐shaped crystals of the human ceruloplasmin showed a single component on ultracentrifugation, electrophoresis and immunoelectrophoresis and had a sedimentation constant of 7.04 S and an electrophoretic mobility of 5.2 × 10–5 cm2 volt‐1 sec‐1. Light scattering of the ceruloplasmin gave a molecular weight of 160,000. Absorption coefficient at 610 nm was E1%1 cm 0.66, at 280 nm, 14.7 and the ratio E610 nm/E280 nm, 0.045. This protein provided 0.31% copper and contained serine, aspartic acid and valine residues as an amino terminal amino acid and showed an oxidase activity of 62 arbitrary units.