Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly removed by the reticuloendothelial system.

A simple method for the separation of cardiac myosin light chains.

A simple and efficient procedure is presented for the separation of pig cardiac myosin light chains. The method employs only three repetitive isoelectric precipitations without column chromatography, and is suitable for use with large quantities of material. The method also permits the recovery of homogeneous light chains with good yields.

A microprecipitation test for rapid detection and identification of Venezuelan, eastern and western equine encephalomyelitis viruses.

The development of a new diagnostic procedure for the identification of Venezvelan, eastern and western equine encephalomyelitis (VEE, EEE, WEE) viruses is described. The procedure utilizes virus precipitation with reference fluorescein-conjugated gamma globulin, followed by cellulose acetate electrophoresis. Clinical specimens containing varying concentrations of virus yielded, in primary duck embryo cell culture, sufficient virus for detection within 22 to 44 hours. Identification of VEE, EEE and WEE virus in specimens was accomplished by microprecipitation within this time. In contrast to conventional identification methods, our procedure eliminates the cost of utilizing laboratory animals and considerably reduces the time required for virus identification.

Interference by pro-apolipoprotein A-I in apolipoprotein E phenotyping using chemical precipitation procedures.

Lipoproteins of density, d less than 1.063, isolated by polyanion-cation precipitation methods, gave isoelectric focussing patterns of apolipoprotein E isoforms by rod-gel electrophoresis which differed from the corresponding patterns obtained from ultracentrifugally-derived very low density lipoproteins. The differences were sporadic and variable but the most common effect was an increased frequency of the E3 isoform. Two-dimensional analyses involving sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis against anti-apolipoprotein A-I indicated that contamination of precipitated lipoproteins with pro-apolipoprotein A-I was responsible for this phenomenon. It is suggested that two-dimensional techniques should be applied for definitive phenotyping if precipitated lipoproteins are used as source material.

Extended experimental and preliminary surgical findings with autologous fibrin tissue adhesive made from patients own blood.

A surgical tissue adhesive can be made from the patient's own blood. We have been refining the procedures for manufacturing autologous fibrin tissue adhesive to facilitate its use in the operating room and to increase its bonding strength. Fibrin tissue adhesive efficacy depends on fibrinogen concentration. We found that fibrinogen precipitation using the ammonium sulfate method produced the highest concentration. Bonding power was compared with that of the commercial glue 10 minutes and 30 minutes after glueing two pieces of 1 X 1 cm2 human dura together. Bonding strength of the autologous product was close to that of the commercial product. Comparisons of fibrinolysis inhibition time of autologous fibrin tissue adhesive and commercial glue in experiments on rats over a period of one hour to six days after subcutaneous injection are described.

High-density lipoprotein subfractions as measured by differential polyanionic precipitation and rate zonal ultracentrifugation.

Recent interest in the putative protective role of high-density lipoprotein (HDL) and its subfractions against atherosclerosis has highlighted the need for a rapid, simple subfractionation procedure. Here we compared HDL subfractionation by two recently developed polyanionic-precipitation methods with the values obtained by rate zonal ultracentrifugation. A similar result for total HDL cholesterol was obtained by all three methods. However, HDL2 cholesterol as measured by the precipitation procedures was significantly higher than the zonal value, and HDL3 was lower. This reflects the different underlying principles involved in the separations and highlights the need for a clearer understanding of the functional roles of the HDL fractions.

[Clinical use of latex tests. II. Evaluation of latex tests in C-reactive protein determination]. / L'impiego clinico dei tests al lattice. II. Valutazione dei metodi al lattice per la determinazione della proteina C reattiva (PCR).

We compared the most reliable and practical laboratory methods for the quantitative determination of serum C-reactive protein that is polistyrene latex particles together with purified anti-C-reactive protein rabbit globulin, the precipitation test using anti-C-reactive protein serum and finally, radial immunodiffusion on agar-gel plates. We examined 163 serum specimens from ambulatory patients and from various hospitalized patients in Istituti Clinici di Perfezionamento of Milan (Italy). All the methods tested showed a good qualitative discrimination for serum C-reactive protein presence or absence at pathological levels. We also compared the two latex methods. In conclusion, radial immunodiffusion confirmed to be the best method for accurate C-reactive protein dosage during inflammatory and degenerative processes.

A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoprotein cholesterol.

The accurate quantitation of high density lipoproteins has recently assumed greater importance in view of studies suggesting their negative correlation with coronary heart disease. High density lipoproteins may be estimated by measuring cholesterol in the plasma fraction of d > 1.063 g/ml. A more practical approach is the specific precipitation of apolipoprotein B (apoB)-containing lipoproteins by sulfated polysaccharides and divalent cations, heparin-Mn(2+) being the most commonly used combination. The present heparin-Mn(2+) procedure was found to be reasonably specific and not often subject to large errors; however, 9% (primarily hypertriglyceridemic samples) of the 966 plasma samples treated with heparin-Mn(2+) had obvious supernatant turbidity, indicating incomplete sedimentation of apoB-associated lipoproteins. Furthermore, 48% of the nonturbid supernates contained more than 1 mg/dl (mean 2.5 mg/dl) of apoB-associated cholesterol when measured by a radial immunodiffusion procedure, indicating slight overestimation of HDL cholesterol. Determination of the extent of the unprecipitated apoB-associated lipoproteins by sensitive radioimmunoassay and of the amount of precipitated high density lipoprotein by radial immunodiffusion assay of apolipoproteins A-I and A-II at various heparin and Mn(2+) concentrations indicated that the usual heparin level (approximately 1.3 mg/ml) was adequate. However, a twofold increase in Mn(2+) concentration to 0.092 M improved precipitation of the apoB-associated lipoproteins without excessive precipitation of high density lipoprotein from plasma. This increased Mn(2+) level also provided improved sedimentation of the apoB-associated lipoproteins from hypertriglyceridemic plasma. Additional observations suggested that, for convenience, the heparin and Mn(2+) can be added simultaneously as a combined reagent, that samples can be incubated for 10 minutes at room temperature before centrifugation, and that turbid supernates from hypertriglyceridemic samples can usually be made free of apoB-associated lipoproteins by centrifugation at 12,000 g for 10 minutes.

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG.

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.

Changes induced in antibodies by isolation methods.

IgG was isolated from plasma at low temperatures using the Cohn fractionation method, and then processed to three different products in order to evaluate how chemical and physical manipulations affect antibodies. Evaluation of the antibodies was done by measuring their ability to bind human immunodeficiency virus, cytomegalovirus, Herpes simplex virus, and rubella virus. In addition, the IgG products were compared by crossed immunoelectrophoresis, and circular dichroism and ultra violet spectroscopy. It was found that exposure of purified IgG to physiological pH altered the molecular conformation of IgG and induced various degrees of irreversible loss in antibody binding to viruses. These observations indicate that more efficacious antibodies may be obtained for clinical use if isolation at their isoelectric point is avoided.

[Serum assay of anti-wasp venom IgG by radioimmunoprecipitation. Application to patients during desensitization]. / Dosage sérique des IgG anti-venin de guêpe par radioimmunoprecipitation (RIP-IgG). Application à des sujets en cours de désensibilisation.

Rapid, specific and reproducible radioimmunoprecipitation assay (RIP) for measuring IgG antibodies against Yellow Jacket Venom (YJV) has been developed. The YJV-I125 is added to serum sample and the immune complexes are precipitated by a second antibody with polyethylene glycol. IgG antibodies were measured by RIP in 24 patients receiving Yellow Jacket Venom immunotherapy. RIP assays were compared for 159 sera to a solid phase procedure (RAST IgG).

Evaluation and comparison of immunoinhibition and immunoprecipitation methods for differentiating MB and BB from macro forms of creatine kinase isoenzymes in patients and healthy individuals.

We compared results for measurements of creatine kinase isoenzyme MB (CK-MB) by immunoinhibition vs immunoprecipitation, using sera from 53 normal healthy individuals, 55 patients with increased CK-MB associated with acute myocardial infarction, and 42 patients whose blood exhibited one or more abnormal forms of CK by electrophoresis. These last 42 patients, selected from a group of 91 cases exhibiting abnormal forms as detected in a screening of 5000 hospitalized and clinic patients, include: (a) CK-BB bound to IgG (macro CK type 1), (b) a polymeric complex of mitochondrial CK (macro CK type 2), (c) abnormally high activity of free CK-BB isoenzyme, and (d) persistent increases of CK-MB from patients without myocardial infarction. These abnormal forms occur in less than 2% of all patients and are exceedingly rare in patients with acute myocardial infarction. Therefore, the vast majority of CK-MB analyses can be performed rapidly and efficiently by immunoinhibition, which has analytical sensitivity, is associated with high clinical sensitivity, and is easily automated for a low cost per test. In contrast, immunoprecipitation is a more specific analytical measurement of CK-MB but is less efficient and more costly.

Diagnosis of human immunodeficiency virus infection by immunoassay using a molecularly cloned and expressed virus envelope polypeptide. Comparison to Western blot on 2707 consecutive serum samples.

To detect human immunodeficiency virus (HIV) antibodies in a simple enzyme-linked immunoassay (CBre3-EIA), we used an Escherichia coli-expressed polypeptide antigen, representing the carboxy-terminal third of the external membrane glycoprotein gene fused with the amino-terminal half of the transmembrane glycoprotein gene. Over a 3-month period, 2707 consecutive serum samples referred for confirmatory testing for human T-lymphotrophic virus type III (HTLV-III) antibodies were evaluated by both Western blot and CBre3-EIA. On a single determination for each sample, the CBre3-EIA was found to have an estimated sensitivity (99.9%) and specificity (99.1%) similar or superior to the more cumbersome Western blot method. This study shows that all HIV-seropositive subjects have antibodies to the virus envelope protein; no other virus antigens are required for construction of highly sensitive immunoassays.

Comparison of four procedures for separating apolipoprotein A- and apolipoprotein B-containing lipoproteins in plasma.

Because lipoproteins containing apolipoprotein A (ApoA-I + ApoA-II) or apolipoprotein B (ApoB) seem to exert opposite effects as risk factors for coronary heart disease, we decided to determine the separability of these two major plasma lipoproteins by procedures originally designed to separate high-density from low- and very-low-density lipoproteins. The presumably ApoB-free lipoproteins isolated from normal plasma by (a) ultracentrifugation at d = 1.063; precipitation with (b) heparin-Mn2+ or (c) phosphotungstate-Mg2+; or (d) immunoprecipitation with antibodies to ApoB were characterized by quantifying cholesterol and apolipoproteins A-I, A-II, B, C-II, C-III, D, E, F, and Lp(a). ApoA- and ApoB-containing lipoproteins were completely separated only by immunoprecipitation with antibodies to ApoB. The ApoB-containing lipoproteins isolated by other procedures always contained 4% to 20% of total plasma ApoA-I and differed substantially from one another with respect to the content of some of the minor apolipoproteins. Measuring apolipoproteins was more reliable than measuring cholesterol for monitoring this separation and for expressing the concentrations of ApoA- and ApoB-containing lipoproteins.

Measurement of glycated protein by a rapid and specific method for absolute quantification of lysine-bound glucose.

We modified the liquid-chromatographic assay of Schleicher and Wieland (J Clin Chem Clin Biochem 1981; 19:81-7) for measuring lysine-bound glucose in serum proteins, increasing its performance and practicality. After precipitating serum proteins from 10- to 50-microL samples with ethanol (700 mL/L) and hydrolyzing these in 6 mol/L HCl, we inject 20 microL of the diluted hydrolysate directly into the chromatograph, which consists of an acid-resistant C18 precolumn combined with a high-resolution C18 main column. The eluent is 3.5 mmol/L H3PO4 solution containing 30 mL of acetonitrile per liter. These modifications increase sensitivity, provide excellent resolution and longevity of stationary phases, shorten assay times to 15 to 20 min, and are suited for automation. The assay is highly sensitive and highly specific, quantifying nanomoles of lysine-bound glucose per milligram of protein. A precision (CV) of 5.1% is achievable at physiological and supra-physiological glucose concentrations, and analytical recovery is 99%. This inexpensive method has been applied to serum albumin, bulk serum proteins, and preparations of low-density lipoproteins and immunoglobulins.

Phase-separation immunoassays.

Solid-phase-based immunoassays have traditionally been plagued by nonspecific binding to the solid phase and by slow reaction kinetics relative to reactants that are free to diffuse in solution. We have developed two novel immunoassays in which the solid phase is generated in situ after the specific binding reaction has occurred, thereby enhancing reaction kinetics and minimizing the opportunities for non-specific binding. In the first system, the capture antibody is conjugated to an organic monomer, polymerization of which to form insoluble polymer particles is initiated by a reaction involving free radicals. The amount of signal-labeled antibody incorporated into the resulting particles is directly proportional to the concentration of antigen. The principle is illustrated for the simultaneous assay of IgG and IgM in a single sample. In the second system, capture antibody is conjugated to a polymer, the solubility of which is a function of temperature. Specific binding is conducted below the critical solution temperature of the polymer, which is then separated from solution by increasing the temperature above the critical temperature. The incorporation of signal-labeled antibody into the precipitated polymer is directly proportional to the concentration of antigen. This principle is illustrated for the assay of hepatitis B surface antigen and Chlamydia trachomatis.