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Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.

A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVB1, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVB1 to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10(3) PFU/ml, with 50% inhibition at approx. 3 x 10(3) PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents.

Hemophilia A is a congenital bleeding disorder which is characterized by a functional deficiency of the coagulation protein factor VIII. We have developed sensitive enzyme-linked immunosorbent assays (ELISAs) for measuring the antigenic reactivity of factor VIII. The assays utilize dot immunobinding techniques, commercial monoclonal antibodies, and a detection system enhanced by the interaction of avidin and biotin. The dot immunobinding ELISAs were optimized for measuring factor VIII in normal and hemophilic plasma, and in partially and highly purified preparations of factor VIII. Linear standard curves were established for all samples, defining the range for accurate measurement. Factor VIII was detected at concentrations as low as 0.0005 U/ml, which represents 0.1 pg of protein.

Low molecular weight Factor VIII is thought to exist either as an integral part of a high molecular weight glycoprotein or as a noncovalently linked component which is dissociated from the large parent molecule in the presence of high calcium concentrations. However, all of this previous work on the molecular structure of Factor VIII has been done using blood which was collected into an anticoagulant that acts by chelating calcium. In order to study the structure of Factor VIII under conditions where the calcium concentrations are maintained at physiological levels during the entire period of collection, blood was taken directly into heparin. This anticoagulant inhibits clot formation by inactivating thrombin but leaves the ionic composition of the plasma unaltered. Under these conditions, more than 50% of the Factor VIII activity is associated with a low molecular weight fraction. This low molecular weight fraction may be effectively separated from the high molecular weight glycoprotein by either gel chromatography or sucrose gradient ultracentrifugation. Further chromatography on a calibrated Sephadex G-100 column shows that the procoagulant molecule has a molecular weight of approximately 20,000. The data support the “two molecule” theory for the structure of Factor VIII and suggest that the many inconsistencies in the literature reflect differences in the ionic environment of the molecule.

Human factor VIII was purified from heparinized blood by cryoprecipitation, poly(ethyleneglycol) precipitation, Affi-Gel blue, aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. A purification of 280,000-fold over plasma with a specific activity over 5300 units/mg was achieved. Analyses of factor VIII using HPLC indicated a molecular mass of 280-340 kDa. Variation in the native mass may reflect heterogeneity of the protein due to associated lipid since structural analysis confirmed that factor VIII contained variable amounts of free fatty acids and diglycerides and triglycerides, but no phospholipids. Additional characterization by denaturing polyacrylamide gel electrophoresis under reducing conditions, followed by silver staining, showed a major single-chain polypeptide of factor VIII with a mass of approximately 260 kDa. To determine whether proteolyzed forms of factor VIII were present during fractionation, we analysed earlier steps in purification. This revealed additional species of factor VIII eluting faster than the single-chain form during chromatography on polyelectrolyte E5. Gel electrophoresis showed that these species of factor VIII consisted of multiple polypeptide chains, and partial peptide mapping using Staphylococcus aureus V8 protease indicated that they were structurally related. Monoclonal and hemophilic antibodies were used in immunoadsorption experiments to demonstrate that the purified factor VIII was composed predominantly of the 260-kDa factor VIII chain.

Loss of Factor VIII procoagulant activity (VIII:C) following blood collection is a major problem in providing sufficient amounts for therapeutic use and biochemical analyses. We have examined the effects of inhibition of plasma proteases and maintenance of physiological calcium ion on plasma VIII:C stability. The addition of protease inhibitors such as benzamidine, phenylmethylsulfonyl fluoride (PMSF), aprotinin, or soybean trypsin inhibitor (SBTI) to CPD plasma provided no significant protection against decay of VIII:C activity. Neither the rate of decay in the first 24 hours nor the final VIII:C activity observed after storage for 48-72 hours were significantly altered. On the other hand, addition of DFP or heparin to CPD plasma resulted in a marked improvement in VIII:C stability over 24 hours. This demonstrated that these two inhibitors are effective in preventing VIII:C degradation during storage. In addition to protease inhibition, the importance of maintaining physiological calcium ion was demonstrated by 100% stabilization of VIII:C in heparin plasma. Plasma obtained from CPD plus heparin blood could also be stabilized provided free calcium ion levels were restored to physiological concentrations. The inactivation of VIII:C in CPD plus heparin plasma was completely reversible up to 4 hours after collection. Studies on the recovery of activity after recalcification of CPD plus heparin plasma provided kinetic data which support a renaturation process of VIII:C rather than one due to enzymatic activation. The use of a thrombin-specific chromogenic substrate revealed that after recalcification and during the recovery of VIII:C activity, there was no significant thrombin activity. Although the data suggest that proteolytic degradation plays some part in VIII:C decay, only the maintenance of physiologic calcium ion levels under cover of an effective non-chelating anticoagulant and protease inhibitor allows preservation of VIII:C activity.

The well-known two phase decay curve of Factor VIII which has been attributed to enzyme degradation is not altered by the addition of specific protease inhibitors. However, complete stability of Factor VIII, for at least 24 hours, is accomplished when physiological levels of calcium are maintained. Addition of calcium plus heparin to whole blood at 0, 2, and even 4 hours, effectively restores Factor VIII activity to 0 time values and maintains this activity throughout the next 24 hours. The restoration of activity noted after 2 and 4 hours.of incubation is very rapid with the Factor VIII activity increasing within 15 minutes of addition of calcium. Data obtained from column chromatography indicates that the molecular distribution of Factor VIII between high and low molecular weight forms is affected both by time and by the level of calcium present in blood. When blood is collected either into heparin or a mixture of CPD plus heparin plus calcium chloride, the elution profile indicates that Factor VIII activity is equally distributed between high and low molecular weight forms, both at 0 and 24 hours with the total activity remaining unchanged during this time interval. However, when Factor VIII activity at 0 time is in the high molecular weight form with less than 10% in the low molecular weight form. By 24 hours there is virtually no low molecular weight activity remaining in this citrated plasma. The data demonstrate an absolute requirement for calcium in the maintenance of Factor VIII: C activity and suggests that the relative lability of Factor VIII during the first phase of the decay curve is due to the loss of VIII: C activity when calcium is not present at physiological concentrations to stabilize the molecule.

The high molecular weight complex of Factor VIII is known to dissociate into two subunits when chromatographed in a high ionic strength solution containing either calcium or sodium salts. Calcium also has an essential role in maintaining Factor VIII activity during storage (AABB 1980). These two findings suggest ah integral involvement of calcium in the basic structure-function relationship of the Factor VIII molecule. Consequently, we have examined the binding of 45Ca to both the low molecular weight (LMW) procoagulant subunit of Factor VIII and the high molecular weight (HMW) complex using PAGE and column chromatography on Sepharose 4B. When the HMW complex of Factor VIII was isolated from cryoprecipitate by standard chromatographic procedures, incubated with 45CaCl2 and subjected to polyacrylamide gel electrophoresis, the resultant autoradiogram demonstrated that all of the 45Ca was associated with the HMW material which did not enter the gel. When this HMW Factor VIII was incubated with 45Ca and then dissociated by column chromatography in a buffer containing 0.25 M CaCl2, all of the 45Ca was associated with the material eluting in the 2.3 Vo region. This corresponds to the elution volume of the LMW, procoagulant subunit of Factor VIII. Dialysis against buffer was not effective in removing the 45Ca. The data indicate that calcium is tightly associated with the low molecular weight subunit of Factor VIII and presents further corroborative evidence that calcium has an essential role in determining both the chemical and physical properties of the Factor VIII molecule.

We have recently shown that in heparinized plasma, approximately 50% of the Factor VIII procoagulant activity has a very low molecular weight (VLMW) of approximately 20,000. The balance of the procoagulant activity is in a high molecular weight form (HMW) which is associated with the carrier protein (VIII:RAG). On the other hand, Factor VIII which has been purified from citrated plasma is known to have a molecular weight of about 10 6 daltons and can be separated after CaCl 2 dissociation into a high molecular weight carrier (VIII:RAG, molecular weight 10 6 ) and a low molecular weight procoagulant component (LMW, molecular weight 150,000). We have now studied the factors which influence the aggregation of the native VLMW form of Factor VIII. The ratio of VLMW:HMW in heparinized plasma is 1:1 and decreases to 1:5 when CPD is used as anticoagulant. VLMW is not detectable in EDTA plasma. The collection of blood into heparin and graduated amounts of CPD results in an increased proportion of VLMW as the CPD concentration is reduced. The ratio of VLMW:HMW remains constant at 1:1 during storage of the heparinized plasma but decreases to almost 0:1 after 24 hours in citrated plasma. When separated from HMW, the VLMW alone aggregates on storage at −80°C for 72 hours producing forms of intermediate molecular weight although the majority of the activity is in a high molecular weight form. This aggregated VLMW material is devoid of VIII:RAG and dissociates into low and high molecular weight peaks following chromatography with buffer containing 0.25 M CaCl 2 . Both cryoprecipitation and PEG precipitation of heparinized plasma shift the VLMW:HMW ratio from 1:1 to 0:1. These results show that the usual conditions of storage and processing of plasma result in the aggregation of the normal VLMW to high molecular weight forms which are artifacts of the isolation process, and are further evidence against the “one molecule” theory of Factor VIII structure.