We used monoclonal antibody ELISAs, antigen molecular size distribution, competition ELISA and neonatal mouse immune tolerance methods to detect potential neoantigen formation and increased immunogenicity following severe dry‐heat treatment of high‐purity factor VIII (Liberate) and factor IX concentrates. To provide positive controls, concentrates were heated in solution (70°C for 2 h) to produce denaturation on purpose. The competition ELISA applied to factor IX proved particularly useful for quantifying differences between the positive control and the dry‐heated/unheated concentrates. None of the test systems employed by us indicated any detectable neoantigen formation or any alteration in immunogenicity following terminal severe dry‐heat treatment of the high‐purity concentrates, and this finding is supported by clinical experience so far.

Summary. Aqueous solutions of stroma‐free human haemoglobin are being evaluated as potential oxygen‐carrying resuscitation fluids. There are indications, however, that such solutions may produce toxic side‐effects in vivo. Stroma‐free haemoglobin solution produced a 50% fall in mean arterial pressure when infused into a small animal model despite containing very low levels of non‐haem protein and phospholipid contaminants. This effect was not produced by haemoglobin solutions after extensive dialysis. Red cell‐derived adenine nucleotides were found to be present in concentrations high enough to cause such a response (80–85 μg/ml). We have developed a chromatographic assay capable of predicting hypotension in our animal model and consider that the complete absence of adenine nucleotides must be confirmed in all studies concerning the possible toxic side‐effects of stroma‐free haemoglobin solutions.

A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

Annals of the New York Academy of SciencesVolume 556, Issue 1 p. 292-303 Pharmacokinetic and Biological Activities of Dermatan Sulfate (Mediolanum MF701) in Healthy Human Volunteersa J. DAWES, Corresponding Author J. DAWES MRC/SNBTS Blood Components Assay GroupPresent address: Heart Research Institure, 145–147 Missended Rd., Camperdown, Sydney, NSW 2050, Australia.Search for more papers by this authorB. A. HODSON, B. A. HODSON MRC/SNBTS Blood Components Assay GroupSearch for more papers by this authorI. R. MacGREGOR, I. R. MacGREGOR Scottish National Blood Transfusion Service Headquarters Laborntory, Edinburgh, United KingdomSearch for more papers by this authorD. S. PEPPER, D. S. PEPPER Scottish National Blood Transfusion Service Headquarters Laborntory, Edinburgh, United KingdomSearch for more papers by this authorC. V. PROWSE, C. V. PROWSE South-East Scotland Blood Transfirsion Service, Edinburgh, United KingdomSearch for more papers by this author J. DAWES, Corresponding Author J. DAWES MRC/SNBTS Blood Components Assay GroupPresent address: Heart Research Institure, 145–147 Missended Rd., Camperdown, Sydney, NSW 2050, Australia.Search for more papers by this authorB. A. HODSON, B. A. HODSON MRC/SNBTS Blood Components Assay GroupSearch for more papers by this authorI. R. MacGREGOR, I. R. MacGREGOR Scottish National Blood Transfusion Service Headquarters Laborntory, Edinburgh, United KingdomSearch for more papers by this authorD. S. PEPPER, D. S. PEPPER Scottish National Blood Transfusion Service Headquarters Laborntory, Edinburgh, United KingdomSearch for more papers by this authorC. V. PROWSE, C. V. PROWSE South-East Scotland Blood Transfirsion Service, Edinburgh, United KingdomSearch for more papers by this author First published: June 1989 https://doi.org/10.1111/j.1749-6632.1989.tb22511.xCitations: 4 a This work was supported by Medical Research Council grant nos. PG8218470 and G8607394SA. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume556, Issue1Heparin and Related Polysaccharides: Structure and ActivitiesJune 1989Pages 292-303 RelatedInformation

SummaryAn iodinated derivative of dermatan sulphate was administered by the intravenous, subcutaneous and oral routes to healthy human volunteers in conjunction with unlabelled dermatan sulphate. Following intravenous injection clearance of radiolabel and concentration as measured by competitive binding assay were highly correlated and displayed complex kinetics which were not dose-dependent. Intact 125I-dermatan sulphate was absorbed following both subcutaneous and oral administration, though there appeared to be selective uptake by the gut of a subfraction comprising the smaller or less sulphated molecules. The intact material was subsequently excreted unchanged in the urine. Degradation products of dermatan sulphate were not detected by either gel filtration or affinity chromatography on Polybrene-Sepharose at any time in either plasma or urine, indicating that administered dermatan sulphate is not catabolised by man.

SummaryMurine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125 I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII: C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R.cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII: C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibroneetin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII :C and R. cof. in a single vial whilst using only one specific monoclonal antibody.

SummaryA panel of 10 murine monoclonal antibodies to procoagulant FVIII has been developed from the fusion of a single spleen. Balb/c mice were injected with a purified preparation of FVIII: Ag, and antibody production in sera and hybrid culture supernatants was monitored using a specific radiometric screening assay. The antibodies all inhibit FVIII clotting activity in normal plasma, and when immobilised on agarose retain their ability to recognise and bind the FVIII procoagulant protein. Studies on protein A-purified immunoglobulins demonstrate a range of properties within the panel of antibodies with regard to species crossreactivity, clotting inhibition and immunoadsorption. The panel of antibodies has been used to screen heat-treated FVIII concentrates for the occurrence of heat-induced neoantigens.

Seven mouse monoclonal antibodies have been produced against human melanoma tissue plasminogen activator (t-PA). They were specifically bound to 125I t-PA but not 125I urokinase (u-PA) and inhibited t-PA, but not u-PA, activity in plasminogen-rich 125I fibrin wells. Three of the antibodies directly inhibited the amidolytic activity of t-PA and the two most effective also bound near the active site histidine residue as determined by competition experiments using active site blocking agents. Several antibodies interfered with the fibrin binding properties of t-PA. One antibody neither interacted with the active site nor inhibited fibrin binding but still effectively quenched t-PA activity in fibrin wells suggesting that it masks another region of the molecule necessary for effective biological activity.

SummaryThe competitive binding assay described will specifically and accurately measure concentrations of administered heparin in biological fluids with a sensitivity of 60 ng ml-1. Neither endogenous glycosaminoglycans, nor plasma proteins such as ATIII and PF4 interfere in the assay. Semi-synthetic highly sulphated heparinoids and LMW heparin can also be measured. Using this assay heparin clearance followed simple first-order kinetics over the dose range 100-5,000 units, but the half-life was strongly dose-dependent. There was good correlation with heparin activity measurements by APTT and anti-Xa clotting assays. Plasma concentrations were measurable for at least 5 h following subcutaneous injection of 10,000 units of heparin. Excretion in the urine could be followed after all but the lowest intravenous dose. This assay, used in conjunction with measurements of heparin anticoagulant activity, will be valuable in the elucidation of mechanisms of action of heparin and the heparinoids, and in the assessment and management of problems related to heparin therapy.

Heparan sulphate (HS), a near-relative of heparin, but with much less anticoagulant activity in vitro, is bound to cell surfaces. We examined HS isolated from porcine intestinal mucosa, and found that although the material had low anticoagulant activity by APTT, it had a marked effect in anti-Factor Xa clotting assays, giving anti-Xa/APTT ratios of approximately 4:1 in terms of specific activity. In crossed immunoelectrophoresis experiments, HS bound to antithrombin III, but at higher concentrations than heparin. A heparin analogue (a polysulphated chondroitin), while virtually inactive in vitro, nevertheless when administered s.c. to man potentiated the effect of anti-Factor Xa to an extent comparable to that produced by low-dose heparin, but with an anti-Xa/APTT ratio of 4:1. The analogue also produced a marked release of lipoprotein lipase and a four-fold increase in the level of circulating PF4, as measured by radioimmunoassay. The anti-Xa and APTT effects of HS in vitro are very similar to those produced by the analogue in vivo, and it is suggested that the analogue releases not only lipoprotein lipase and PF4, but also HS. The drug-induced release of an endogenous glycosamino-glycan (probably from the endothelial lining of vessels) with anti-Xa activity may represent a fruitful approach to the prophylaxis of venous thrombosis.