Enzyme immunoassay for antibodies to membrane associated antigen of varicella zoster virus.

An in situ enzyme immunoassay to viral membrane antigen was developed to enable the specific estimation of antibodies to varicella zoster (VZ) virus. The technique was compared with a modified fluorescent antibody to membrane antigen (FAMA) procedure and with the complement fixation (CF) test by parallel assay of 352 plasma samples. The enzyme immunoassay (EIA) procedure showed very good correlation with the modified FAMA procedure, and both were far more specific than the CF test. This specificity was achieved by the use, in the EIA, of VZ virus-infected cells grown and fixed in situ with glutaraldehyde. Thus the only virus antigens accessible to antibody were the VZ-specific antigens expressed at the cell membrane, cross-reactions with herpes simplex virus antibodies thereby being avoided.

Immune response and reactions to various dose regimens for raising hyperimmune antisera in sheep.

To determine the optimum procedure for raising hyperimmune sera to tetanus toxin, three adjuvants, four antigen preparations and two routes of administration in various combinations were investigated in sheep. Oil-in-water adjuvants alone or in combination with aluminum gels were superior to aluminium gels on their own. This disadvantage of aluminium gels was partially but not completely abrogated when the frequency of doses was increased to three per week. Intensity of local reaction was strongly correlated with immune response; the more immunogenic a dose, the more reactive. Reactivity of oily adjuvants could be lessened by use of a more suitable route of administration, thus oily adjuvants appeared suitable for use when administered by the intraperitoneal route even though moderate to severe reactions resulted from subcutaneous injections. Of other variables investigated, toxin did not confer any advantage over toxoid as an immunogen, purified toxoid was a significantly better immunogen than unpurified toxoid and two large bleeds (30% of total blood volume each) every six weeks rather than 20 ml test bleeds did not affect the titre of the hyperimmune serum produced.

Albumin solutions--their production and quality control.

Analysis of all clinical reactions (complaints) reported to CSL in the past decade has revealed that "hypotension syndrome" continues to be the predominant side effect of infusion of albumin solutions. Although the incidence of clinical reactions to albumin solutions reported to CSL is approximately 1 in every 20,000 bottles issued, the virtual elimination of PKA from SPPS has not reduced the number of adverse clinical reports received by CSL. It is concluded that factors other than PKA contamination are responsible for hypotensive effects during or after infusion of albumin solutions. Other possible causes of "hypotension syndrome" are discussed.

Effect of virucidal heat treatment on proteins in human factor VIII concentrates.

Intermediate-purity and fibrinogen-poor factor VIII concentrates were heated in the lyophilized state at 60 degrees C for up to 72 hours to inactivate blood-borne viruses. The effect of heat treatment on factor VIII, von Willebrand factor (vWf), and other proteins present in the concentrates (albumin, fibrinogen, fibronectin, IgG, and IgM) was evaluated. Heat-induced protein aggregation, particularly of fibrinogen and fibronectin, occurred within 48 hours in the intermediate-purity concentrates and correlated well with decreased solubility of these products. Heated fibrinogen-poor concentrates were readily soluble and did not show protein aggregation even after 72 hours at 60 degrees C. Neither concentrate developed detectable neoantigens when tested against antisera to whole human plasma and to heated and unheated concentrates. Aggregation of the vWf molecule, detected by altered mobility in crossed immunoelectrophoresis and multimeric analysis in SDS agarose gels, occurred in heated intermediate-purity concentrates but not in fibrinogen-poor concentrates. Thus, higher-purity factor VIII concentrates withstand heat treatment better than concentrates that contain greater levels of contaminating proteins, particularly fibrinogen.

Effect of delayed blood processing on the yield of factor VIII in cryoprecipitate and factor VIII concentrate.

Current standards for the preparation of factor VIII (FVIII) concentrates from human plasma recommend separation of plasma from red cells (RBCs) within 6 hours of blood donation, thereby reducing the volume of plasma from donated whole blood available for processing to FVIII concentrate. The decay of FVIII clotting activity (FVIII:C) in whole blood and plasma stored at 22 and 4 degrees C and the recovery of FVIII:C in cryoprecipitate and FVIII concentrate prepared from plasma separated from whole blood stored overnight at 4 degrees C were investigated. In whole blood stored at 22 degrees C and plasma stored at either 4 or 22 degrees C, 90 percent of the original FVIII:C was present at 6 hours, 80 percent at 12 hours, and 65 to 70 percent at 18 hours. At these times lower levels of FVIII:C were recovered from whole blood stored at 4 degrees C, that is, 84, 68, and 56 percent, respectively. In cryoprecipitates prepared from plasma separated from RBCs after 18 hours' storage at 4 degrees C (18-hour plasma), 43 percent of FVIII:C activity was recovered, as compared with 61 percent recovered from standard plasma separated within 6 hours of donation (6-hour plasma), p less than 0.05. With large-scale preparation of FVIII concentrates, however, the yield of FVIII:C was similar whether 18- or 6-hour plasma was used. Thus FVIII concentrates--but not cryoprecipitates--can be prepared from plasma separated from whole blood stored at 4 degrees C for up to 18 hours without undue loss of potency.

Use of heat-treated clotting-factor concentrates in patients with haemophilia and a high exposure to HTLV-III.

In a group of 126 Australian patients with haemophilia, who were receiving lyophilized clotting-factor concentrates prepared from locally collected plasma, a high prevalence of antibody to human T-cell lymphotropic virus III (HTLV-III) was demonstrated in those with severe disease. Patients with moderate or mild disease had a much lower prevalence of HTLV-III antibody. After heat treatment of lyophilized factor VIII and factor IX concentrates (60 degrees C for 72 hours) to inactivate the virus, the losses of activity of an intermediate-purity and of a fibrinogen-poor factor VIII concentrate, and of the coagulant activity of a factor IX concentrate, were within acceptable limits. The solubility of the intermediate-purity factor VIII concentrate was markedly decreased; the fibrinogen-poor factor VIII concentrate and the factor IX concentrate were readily soluble. In-vivo recovery and survival of heated concentrates were equivalent to those of the unheated products, and they were effective in the treatment of spontaneous and traumatic haemorrhages.