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Results of assays of recombinant FVIII concentrates have been reviewed over a 10-year period. Initially there was wide variability between laboratories but this was minimised by the development of standardised assay methodology, in particular the use of haemophilic plasma for pre-dilution and 1% albumin in assay buffers. Using this standardised methodology and concentrate standards, there were no major differences in potency between one-stage, two-stage and chromogenic assays on the two full-length recombinant FVIII concentrates. However, using a plasma standard, the chromogenic method gave much higher potencies than the one-stage method on the same concentrates, and this explains a similar discrepancy found in patients' post-infusion samples after injection of recombinant concentrates. It is suggested that concentrate standards be used for such post-infusion samples in order to minimise this discrepancy.

Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E. Surface Plasmon Resonance (SPR) technology, employed to further evaluate the epitopes in fibrin, showed that MAb 1H10 had a higher affinity for fragment-E (KD = 8.04 x 10(-9) M) than MAb 5F3 (KD = 1.13 x 10(-8) M). Individual association and dissociation rate constants of 7.97 x 10(5) M-1s-1 and 3.97 x 10(-3)s-1, respectively, for MAb 1HAb 1H10, and 5.16 x 10(5) M-1s-1 and 3.62 x 10(-3)s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However individual isolated polypeptide chains of fibrinogen fragment E (E-A alpha, E-B beta, E-gamma) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures. Furthermore, SPR pair-wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E. These distinct epitopes appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.

Heparin is widely used as an antithrombotic drug, having excellent anticoagulant properties. However in certain clinical situations heparin's efficacy seems to be somewhat limited. Despite the administration of heparin there is a high incidence of reocclusion of coronary arteries following thrombolytic therapy, and it has been observed that a significant number of patients receiving heparin treatment still exhibit thrombus extension. Although it is well established that the in vitro and in vivo anticoagulant activities of heparin is mediated via the potentiation of the major coagulation inhibitor, antithrombin III (ATIII), some in vivo antithrombotic mechanisms are not fully understood. There is poor correlation between the anticoagulant activity of heparin as measured by in vitro assays and their in vivo antithrombotic efficacy. This may be due to heparin being targeted to many blood constituents whose resultant activities on the coagulation system have not been measured as yet. The antithrombotic activity of heparin as well as the pathogenesis of bleeding complications during heparin treatment cannot be completely explained by the inhibition of blood coagulation factors. Platelet dysfunction and acceleration of fibrinolytic process have been implicated as additional factors involved. Recently a number of reports have suggested that the inhibition of the antithrombotic activity of heparin in these clinical situations may be due to the interaction of heparin with other plasma proteins specifically with fibrin(ogen) present in the thrombus. Despite the possible pathophysiological significance of heparin-fibrin(ogen) interaction, little is known about the physicochemical aspects of this reaction. In this study an attempt was made to locate where heparin binds to fibrin(ogen), using various isolated structural domains from the plasmin-mediated digests of fibrinogen and the individual chains of fibrinogen. The BIALITE system (Pharmacia Biosensor AB, Uppsala, Sweden) was employed for such a study. This utilises the Surface Plasmon Resonance (SPR) phenomenon and allows a direct quantitative analysis of the label-free molecular interaction, in real-time, from which association and dissociation rate constants can readily be obtained.

The purpose of the study was to determine the effects of early plasmin-mediated digests of rat fibrinogen on the vascular tone of rat pulmonary artery in order to compare with reported vasoactive effects of high levels of isolated human peptides in various rat vascular beds. Isometric tension was monitored in isolated rings of rat pulmonary artery precontracted with phenylephrine (4 x 10(-8) mol). Fibrinogen degradation products (FgDPs) were produced by digestion of rat fibrinogen with plasmin to 1, 2, 3 and 60 minutes whereupon the digestion was stopped by addition of Trasylol. These FgDPs were added to the tissue bath to achieve final concentrations of 6.7, 13.3, 26.7 and 53.3 micrograms/mL i.e. values similar to those found in vivo during thrombolytic therapy for myocardial infarction. Results were expressed as a percentage of a maximal contraction elicited in each ring in response to phenylephrine (10(-5) M). The early FgDP fractions, at these pathophysiological concentrations, did not induce significant change in isometric tension in the isolated pulmonary arterial rings (P > 0.05, ANOVA).

In order to study the epitopes on fibrin to which monoclonal antibodies are directed, we required pure individual polypeptide chains of human fibrinogen in milligram quantities. High purity chains of human fibrinogen were rapidly obtained, in under 3 minutes, by the novel procedure of reversed-phase perfusion chromatography and these chains were subjected to immunological characterisation using monoclonal antibodies specific to the individual chains. Cross-reactivity against these antibodies in both immunoblotting and enzyme linked immunospecific assay (ELISA) procedures showed that these isolated fibrinogen chains were of high purity and retained high immunoreactivity. These chains were employed to initiate studies to define the epitopes in fibrin to which four fibrin specific monoclonal antibodies, B10, A11, 5F3 and 1H10 are targeted. Two of these antibodies, B10 and A11, were shown to be directed to a linear sequence on the A alpha chain, although the binding profiles for the two antibodies suggested that different epitopes may be involved for each of these two antibodies. MAbs, 1H10 and 5F3, however, did not bind to any of the three fibrinogen chains, suggesting that conformational epitopes in fibrin are likely to be involved in the binding of these two antibodies to fibrin.

In order to study the epitopes in fibrin towards which monoclonal antibodies are directed we needed the pure individual polypeptide chains of human fibrinogen in reasonable quantity. We report here a simplified, rapid method of separation of high-purity human fibrinogen chains. Following reduction and S-carboxymethylation of human fibrinogen, the sample was injected directly onto a column of the polymeric reversed-phase perfusion packing POROS 20-R2, and the chains were completely resolved in less than 3 min at a flow-rate of 10 ml/min. The capacity was equivalent to that of a similar sized conventional silica-based column. However the throughput was approximately five to ten times as high. The column was durable and robust in day-to-day use.

Paired maternal and cord blood samples were collected at delivery from 150 women who received varying doses of tetanus toxoid during pregnancy. Tetanus specific IgM and IgG antibodies were measured in them by standard ELISA with a sensitivity for IgM of 0.001 mg/ml, and for IgG of 0.0003 IU/ml. In 22 infants an additional estimation of tetanus antibody was made 1 month after birth. The presence of specific IgM in 78% of cord samples established an active foetal immune response. The titre did not alter significantly with the number of TT doses given to the mother. Foetal IgM rose in 60% of cases at one month of age compared to cord blood levels. At this time IgG levels were uniformly diminished in accord with a maternally derived passively transferred antibody. No switch of foetal IgM to IgG production was evident. The foetal immune response thus did not confer active protection against tetanus.

A virulent variant of the normally avirulent Kunz strain of influenza virus was obtained by serial passage in mice, and the pathogenesis of the infections caused by the two strains was studied. The virulence of the passaged variant did not appear to result from increased growth in lungs, from acquisition of resistance to non-specific inhibitors or from inadequate immunogenicity, but from its greater ability to replicate in alveolar cells. The apparent adequacy of defence mechanisms to protect against mucosal infection but their failure to protect against infection of alveolar cells in discussed.