[4] Immunoradiometric assays for factor VIII antigens: c coagulant protein (antihemophilic factor) and factor VIII-related protein (von willebrand factor)

[4]

IMMUNORADIOMETRIC

ASSAY O F F A C T O R

VIII

ANTIGENS

51

[4] I m m u n o r a d i o m e t r i c Assays for Factor VIII Antigens: Coagulant Protein (Antihemophilic Factor) and Factor VIII-Related Protein (yon Willebrand Factor)

By

LEON

W.

HOYER

and

NORMA

C.

TRABOLD

General Considerations It is now recognized that plasma factor VIII is a complex of two proteins that have distinct immunologic and biochemical properties as well as separate genetic control. Factor VIII procoagulant activity (VIII:C) (antihemophilic factor) is the property of a small glycoprotein that is activated - - a n d subsequently inactivated--by thrombin. Its presence in plasma is under the control of a X chromosome gene and it comprises only a small part of the protein mass of the factor VIII complex. Factor VIII-related protein (VIIIR) (von Willebrand factor) is a polymeric glycoprotein that circulates in large forms (0.85-12 x 10e daltons) and is required for normal platelet interaction with damaged blood vessels. It is synthesized in endothelial cells under the control of an autosomal gene and can be reduced to homogeneous ca. 200,000 dalton subunits when incubated with mercaptoethanol or dithiothreitol. Severe VIIIR deficiency prolongs the bleeding time and a functional in vitro estimate of VIIIR content can be obtained by assays of ristocetin-induced platelet agglutination. Both VIII:C and VIIIR can be measured by immunoassays, and immunoradiometric methods provide an excellent combination of specificity, sensitivity, and simplicity. We have used the same general approach to measure the two different proteins. The VIII:C antigens (VIII:CAg) are measured with high titer human anti-VIII:C derived from patients with autoantibodies or transfused hemophilic patients who have developed inhibitors. VIIIR antigens (VIIIR:Ag) are measured with rabbit antibodies obtained by immunization with purified factor VIII. Both assays use labeled antibody that has been purified from immune complexes. Antigen measurement in these assays is based on the differential solubility of the antigen-antibody complexes and free antibody in ammonium sulfate solutions. M e a s u r e m e n t of Factor VIII-Related Antigen (VIIIR:Ag) Antibody Source

Satisfactory results have been obtained with several different antibodies obtained from rabbits immunized with purified human factor VIII. METHODS IN ENZYMOLOGY, VOL. 84

Copyright © 1962by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181984-1

52

PROTEINS OF THE BLOOD CLOTTING SYSTEM

[4]

While the factor VIII purification method is not critical, if there is trace contamination with other proteins, the antiserum must be absorbed before use. We have used sera prepared from animals immunized with factor VIII obtained by the method of Zimmerman and co-workers 1 and by the method of Bouma and co-workers. 2 A commercial antiserum (Behring Diagnostics, Woodbury, NY) has also been successfully used with this method. The rabbit sera are heated to 56° for 30 min, centrifuged at 2000 g for 15 min at 4 °, absorbed with Ca(PO4)3 (10 mg/ml serum)for 10 min at room temperature, and centrifuged at 2000 g for 15 min at 4 °. When necessary, rabbit sera are absorbed with proteins that are soluble in 3% ethanol at 3° but which are precipitated when the ethanol concentration is brought to 8%. The most efficient use of these proteins is to couple them to agarose (Sepharose 2B-CL) 3 so that they can be reused. In a typical preparation, 490 nag protein derived from 300 ml normal human plasma was incubated with 80 ml of activated agarose and 92% of the protein was coupled. Antisera are absorbed by running them over a column of agarose-protein (2 ml antiserum/ml beads) at 20 ml/hr at room temperature. An equal volume of saturated ammonium sulfate is added to the absorbed serum and the insoluble proteins removed by centrifugation at 16,000 g for 15 min at 4 °. The precipitate is dissolved in a small volume of saline and dialyzed against 0.04 M phosphate, pH 8.0. The absorbed antisera form a single line on Ouchterlony immunodiffusion when tested with a factor VIII-rich concentrate of normal human plasma (cryoprecipitate dissolved in ~ of the volume of plasma from which it was prepared) and have no lines when tested with a similar concentrate of plasma from a patient with severe von Willebrand's disease. -

Antibody Purification IgG can be isolated from the antiserum by a variety of techniques, all of which are satisfactory. We have used DEAE-ion exchange chromatography: the antiserum (or a globulin fraction obtained by precipitation of plasma proteins with an equal volume of saturated ammonium sulfate) and DE-52 (Reeve-Angel, Clifton, N.J.) are equilibrated with 0.04 M phosphate at pH 8.0. A ratio of 100 nag globulin/30 ml DEAE-cellulose has provided satisfactory purification. The IgG fractions (protein not absorbed by the column) have been characterized by immunoelectrophoresis using a potent anti-whole rabbit serum. Although small amounts T. S. Zimmerman, O. D. Ratnoff, and A. E. Powell, J. Clin. Invest. 50, 244 (1971). 2 B. N. Bouma, Y. Wiegednck, J. J. Sixma, J. A. van Mourik, and I. A. Mochtar, Nature (London) New Biol. 236, 104 (1972). 3 j. Porath, R. Axen, and S. Ernback, Nature (London) 215, 1491 (1967).

[4]

IMMUNORADIOMETRIC ASSAY OF FACTOR

VIII

ANTIGENS

53

of transferrin are present in some preparations in addition to the IgG, this is easily removed in subsequent purification steps. The IgG is then dialyzed against borate-buffered saline, 7.854 and stored at - 2 0 °. The purified rabbit IgG has been satisfactorily labeled by both the iodine monochloride and lactoperoxidase methods. The latter technique 5 is now routinely employed in our laboratory. The following reagents are added in sequence to 50/zl IgG anti-VIIIR:Ag (0.2-0.4 rag) in a 12 x 75mm polystyrene tube: 5/zl of 125I (0.5 mCi of carrier-free Na125I for protein labeling), 5/~1 lactoperoxidase (2 mg/ml) in 0.05 M sodium phosphate pH 7.5, and 5/~1 of H~O~ (0.44 mM, freshly prepared by dilution in the phosphate buffer). The mixture is gently vortexed for 1 min at room temperature and the iodination terminated by adding 0.5 ml of the phosphate buffer and 50/.d of normal rabbit serum. The solution is then dialyzed against one liter of borate-buffered saline, pH 7.85. Over 80% of the added 125Iis bound to protein and > 95% of the counts are precipitated by 10% trichloroacetic acid at 4°. Immune complexes are prepared by incubating the labeled antiVIIIR:Ag with 5 ml of normal human plasma (30 min at 37°) and the mixture is gel filtered using a 6% agarose column equilibrated with boratebuffered saline. The flow rate is maintained at 20 ml/hr with a peristaltic pump and 5 ml fractions are collected (Fig. 1A). The void volume fractions contain immune complexes and 3-17% of the labeled IgG. The amount of labeled IgG incorporated into immune complexes depends on the potency of the antibody preparation. Normal rabbit serum (0.25 ml) is added to the pooled void volume fractions and the mixture dialyzed at room temperature for 3 hr against 1 liter of 0.05 M glycine, 0. I M NaC1, pH 2.4. The mixture is then gel filtered on Sephadex G-200 equilibrated with the glycine-saline buffer. The flow rate is maintained at 10 ml/hr with a peristaltic pump with 5 ml fractions are collected in tubes that contain 3 ml of borate buffer, pH 8.44 (Fig. 1B). The fractions containing IgG (the second peak) are pooled for use in the immunoassay and 2 ml normal rabbit serum is added. The dissociated purified anti-VIIIR:Ag is then concentrated by adding an equal volume of saturated ammonium sulfate, adjusting the pH to 8, mixing for 0.5 hr at room temperature, and centrifuging at 16,000 g for 20 min at room temperature. Over 99% of the radioactivity is recovered in the precipitate which is dissolved in 5 ml borate-saline. The antibody is then "precycled" to remove any material insoluble in 25% saturated ammonium sulfate by adding 2.5 ml of normal rabbit plasma and 2.2 ml satu-

4 W. E. Vannicr, W. P. Bryan, and D. H. Campbell, Immunochemistry 2, 1 (1964). 5 j. E. Thorell and B. G. Johansson, Biochim. Biophys. Acta 251, 363 (1971).

54

PROTEINS OF THE BLOOD CLOTTING SYSTEM

4 0

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FIG. 1. Purification of rabbit anti-VIIIR:Ag for use in immunoradiometric assay. (A) Separation of nonantibody IgG from immune complexes composed of t"I-labeled rabbit IgG and human VIIIR:Ag. Five milliliters of normal human plasma and 0.3 mg labeled IgG (0.4 mCi) were incubated together for 30 min prior to gel filtration on a 1.6 x 80-cm column of 6% agarose equilibrated with borate-saline. The bar indicates those fractions that were pooled and dissociated at pH 2.4. (B) Separation of dissociated ~"I-labeled anti-VIIIR:Ag from undissociable immune complexes and denatured IgG. The immune complexes from (A) were acidified prior to gel filtration on a 1.6 x 85-cm column of Sephadex G-200. The fractions containing free antibody (indicated by the bar) were pooled for use in the immunoassay.

rated ammonium sulfate to the dissolved precipitate, mixing for 0.5 hr at room temperature, and centrifuging at 16,000 g for 20 rain at room temperature. The precipitate containing approximately 10% of the radioactivity is then discarded and the supernatant dialyzed against borate-buffered saline. After dialysis, the material is stored in 1 ml aliquots at - 2 0 °. The protein concentration of the purified antibody solution is calculated from the cpm/ml and the specific activity of the initial IgG preparation. The amount of labeled antibody used in the immunoradiometric assay is chosen to permit satisfactory counting statistics within a reasonable period of time. 6 6 L. W. Hoyer, d. Lab. Clio. Med. • , 822 (1972).

[4]

IMMUNORADIOMETRIC ASSAY OF FACTOR VIII ANTIGENS

55

The Immunoradiometric Assay The assay is carried out in 12 x 75-mm polystyrene tubes. To undiluted normal rabbit plasma (0.1 ml) and dilutions of test material in borate-buffered saline (0.1 ml) are added 5-15 ng labeled antibody (0.2 ml containing 2-7000 cpm). The mixture is incubated for 30 min at 37° in most assays since this provides satisfactory sensitivity. Increased sensitivity may be obtained by continuing the incubation at room temperature or 37° overnight. At the end of the incubation, 0.4 ml of 50% saturated ammonium sulfate is added, the contents mixed and the tubes left at room temperature for an additional 30 min. The precipitate is then separated by centrifugation at room temperature (2600 g for 15-30 rain) and washed once with 1 ml of 25% saturated ammonium sulfate. The precipitated radioactivity is determined by counting for 10 min using a crystal scintillation detector. Duplicate determinations are done for each dilution of test material and for dilutions of pooled normal plasma standards that are included in each assay. When a 30-min incubation is used, the standard curve is prepared with dilutions of normal plasma between 1/20 and 1/640 (Fig. 2). Extended incubation increases the sensitivity so that normal plasma dilutions as low as 1/10,000 give values above the blank. ~Y

1500-

is .~ TO00-

2 0 .500-

10-

~. ~0-

O" r /,'

0

d2

o'.s

~

~

;

VIIIR:Ag (units x 103)

FIG. 2. Immunoradiometric assay of VIIIR:Ag. Data are given for a typical experiment. The amount of VIIIR:Ag in the 0.1 ml assay volume is indicated for dilutions of the pooled normal plasma standard between 1/20 and 1/640. The amount of l=SI-labeled antibody precipitated in 25% saturated ammonium sulfate given on the vertical axis: both cpm and percentage of total radioactivity are noted. This assay was carried out by the standard procedure using a 30-rain incubation. Assay sensitivity can be increased 20-fold ff the labeled antibody is incubated with test materials for 16 hr prior to the separation of immune complexes with ammonium sulfate.

56

PROTEINS OF THE BLOOD CLOTTING SYSTEM

[4]

Assay characteristics include sensitivity of 0.002 U/ml when the 30min incubation period is used and 0.001 U/ml with prolonged incubation. The assay reproducibility is such that repeated determinations of a single plasma on different days give a coefficient of variation of 9%. The specificity of the assay has been determined with plasmas from patients with severe von Willebrand's disease; they have less than 0.001 U/ml. There is good correlation of VIII:Ag with VIII:C activity in normal plasma (r = 0.82). 6 Normal values of VIIIR:Ag are found in hemophilic plasmas and reduced levels are found in plasmas from patients with yon Willebrand' s disease. ~ M e a s u r e m e n t of Factor VIII Proeoagulant Antigen (VIII:CAg)

Antibody Source Immunoradiometric assay of VIII:CAg requires a high titer human inhibitor. Both spontaneous autoantibodies and antibodies developing in multitransfused hemophiliacs are satisfactory if their titer is sufficiently high. We have used a very potent (3800 Bethesda U/ml) spontaneous inhibitor in most of our studies, but three other antibodies with titers > 1000 Bethesda units/ml have been satisfactory. 7 There is a theoretical advantage in using material obtained from a patient with an autoantibody who has not received multiple transfusions since it is less likely to be contaminated by antibodies to other human plasma proteins. For this reason, most inhibitor antibodies can be used without absorption.

Antibody Purification IgG has been separated from other serum proteins by the caprylic acid method of Steinbuch and Audran, s and Fab' fragments have been prepared by sequential pepsin digestion, reduction with/3-mercaptoethanol, and Sephadex G-100 gel filtration. 9 In a typical preparation, 10 ml of 0.06 M sodium acetate (pH 4) is added with vigorous stirring to 5 ml of antibody plasma. After thorough mixing, 68 mg of caprylic acid is added per ml of plasma while the solution is vigorously stirred at room temperature. After 30 min incubation, the non-IgG plasma proteins are separated by centrifugation at 12,000 g for 15 min at room temperature. The supernatant is then concentrated 3-fold by negative-pressure ultrafiltration using standard dialysis tubing and dialyzed against 0.1 M sodium acetate, pH 4.5. Pepsin dissolved in the acetate buffer is added to the IgG (0.02 mg pepsin/mg IgG) and the digestion is allowed to proceed at 37° overnight. r j. Lazarchick and L. W. Hoyer, J. Clin. Invest. 62, 1048 (1978). s M. Steinbuch and R. Audran, Arch. Biochem. Biophys. 134, 279 (1969). 9 G. M. Edelman and J. J. Marchalonis, in "Methods in Immunology and Immunochemistry," (C. A. Williams and M. W. Chase, eds.), Vol. 1, p. 405. Academic Press, New York, 1967.

[4]

IMMUNORADIOMETRIC ASSAY OF FACTOR

VIII ANTIGENS

57

After the pH is adjusted to 7.4, a small amount of precipitate is removed by centrifugation (12,000 g for 10 min at room temperature). This protein is then gel filtered over a 1.6 x 80-cm column of Sephadex G-100 in borate-buffered saline. Proteins of the initial peak are pooled and concentrated to 5 ml by negative-pressure ultrafiltration at room temperature. The F(ab')~ is then reduced by adding 2-mercaptoethanolamine-HCl to a final concentration of 0.02 M and incubated at 37° for 75 min. The reaction is stopped by adding iodoacetamide, pH 8, to a final concentration of 0.022 M. The solution remains at pH 7.4 while gently mixed for 15 min at room temperature. The gel filtration is then repeated using the same Sephadex G-100 column. The fractions of the second protein peak (ca. 50,000 daltons) are pooled, concentrated to 2 - 3 mg/ml by negative-pressure ultrafiltration, and stored at - 2 0 ° in 0.5-ml aliquots. There is good separation of Fab' from any residual F(ab'), in this gel filtration since the unreduced material remains at the void volume. Fab' is iodinated for use in the immunoassay by the method described for IgG anti-VIIIR:Ag. The protein concentration and incubation conditions are identical to those already described. Complex formation between labeled Fab' and VIII:C is achieved by incubating 0. I-0.3 mg antibody (70 Bethesda units in 50 ttl) and a Factor VIII concentrate containing 50-70 units of VIII:C. The immune complexes obtained during 4 hr incubation at 37° are separated by gel filtration using 6% agarose in borate-buffered saline (Fig. 3A). Fractions eluting before free Fab' are pooled and dissociated after bovine serum albumin is added to a final concentration of 1 mg/ml. The mixture is brought to pH 3.5 with special precautions taken to avoid any lower pH. This is accomplished by the addition of 0.1 M HCI until the pH is approximately 4 followed by a final adjustment with 0.01 M HCI. The slow addition is done with thorough mixing on a magnetic stirrer. After the mixture has been held at 37° for 30 min, it is returned to pH 7.4 with 0.1 M NaOH, and the mixture is centrifuged to remove precipitated proteins (12,000 g for 10 rain at room temperature). The solution is then concentrated to 5 10 ml by dialysis against Aquacide (Calbiochem, Costa Mesa, CA), and dissociated antibody is separated from undissociated immune complexes and from denatured protein by Sephadex G-200 gel filtration. The purified antibody (late eluting fractions corresponding to free Fab') contains 3060% of the radioactivity (Fig. 3B). It should be noted that the optimum conditions for dissocation of immune complexes may be different for different antibodies. If the antibody is stable at lower pH values, it may be possible to obtain better yields by using a lower pH. We have also used 4 M guanidine, pH 7, as a dissociating agent. 1° Although the yields have 10 B. Furie, K. L. Provost, R. A. Blanchard, and B. C. Furie, J. Biol. Chem. 253, 8980 (1978).

58

PROTEINS OF THE BLOOD CLOTTING SYSTEM

[4]

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F16. 3. Purification of human anti-VIII:CAg for use in immunoradiometric assay. (A) Separation of nonantibody Fab' from immune complexes composed of mI-labeled human Fab' and human VIII:CAg. Fifty units of a factor VIII concentrate and 0.3 nag labeled Fab' (0.4 mCi) were incubated together for 4 hr prior to 6% agarose gel filtration. The bar indicates those fractions that contained immune complexes. They were pooled and dissociated at pH 3.5. (B) Separation of dissociated 125I-labeledanti-VIII:CAg from undissociable immune complexes and denatured Fab'. The immune complexes from (A) were incubated at pH 3.5 for 30 rain and neutralized prior to gel filtration on Sephadex G-200. The fractions containing free Feb' (indicated by the bar) were pooled for use in the immunoassay. been c o m p a r a b l e , the method requires that the second gel filtration be carried out in guanidine and is, therefore, s o m e w h a t m o r e c u m b e r s o m e . As in the V I I I R : A g assay, the a m o u n t of purified a n t i - V I I I C A g used in each assay tube is arbitrary. We have chosen to dilute the antibody so that it has no less than 10,000 c p m / m l , r and 1 m g / m l B S A (final concentration) is added.

The Immunoradiometric Assay for VIII:CAg The assay is carried out in 12 x 75-mm p o l y s t y r e n e tubes to which are added sequentially 0.1 ml o f h u m a n I g G (20 m g / m l in borate-buffered saline) included as a carrier protein so that the precipitate is large enough for e a s y separation, 0.1 ml of test material or dilution o f test material in borate-buffered saline, and 0.1 ml o f the purified radiolabeled F a b ' antiV I I I : C A g . The mixture is incubated at 37 ° for 4 hr to obtain maximal corn-

IMMUNORADIOMETRIC ASSAY OF FACTOR VIII ANTIGENS

[4]

59

plex formation, or at 37° for 0.5 hr and at 4° for 18 hr. The results obtained by the two alternative incubation periods are similar. Subsequently, the bound antibody is separated from free Fab' by adding 0.2 ml of a solution containing 95% saturated ammonium sulfate. The precipitated proteins are separated by centrifugation (2800 g for 30 rain) after a 30-rain incubation at room temperature and are washed twice with 1 ml of 38% saturated ammonium sulfate. The radioactivity of the washed precipitates is then determined. Duplicate determinations are done for at least two dilutions of test material, and the assay values are determined by reference to a standard curve obtained with dilutions of pooled normal plasma (Fig. 4). VIII:C can be detected at levels as low as 0.01 U/ml in most assays, with the limiting factor being nonspecific precipitation of radiolabel in the blank tubes (8-21% depending upon the antibody preparation). The reproducibility of the assay is such that repeated assays for the same plasma on different days have a coefficient of variation between 7 and 12%. The specificity in the assay has been verified by demonstrating no detectable reactivity in plasmas from patients with severe classic hemophilia. As described above, the method is satisfactory for quantitative assay of column fractions, other materials with low protein concentrations, and plasma samples diluted at least 1 to 4 with borate-buffered saline. The incubation mixture has been modified slightly for more concentrated samples to avoid the influence of protein concentration. Thus, when plasmas

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Fx6. 4. Immunoradiometric assay of VIII:CAg. Data are given for a typical experiment. The amount of VIII:CAg in the 0.1 ml assay volme is indicated for dilutions of the pooled normal plasma standard between 1/2 and 1/128. The vertical axis indicates the amount of radioactivity precipitated in 38% saturated ammonium sulfate.

60

PROTEINS OF THE BLOOD CLOTTING SYSTEM

[5]

are assayed at concentrations less that 1 : 4, both standards and unknowns are diluted in hemophilic plasma that has no detectable VIII:CAg. Concentrations of VIII:CAg as low as 0.01 U/ml can be measured in undiluted plasma samples if this precaution is observed. When VIII:CAg values are compared with VIII:C levels in normal human plasmas, a very good correlation is detected (r = 0.90). 7 Plasmas from patients with von WiUebrand's disease also have similar VIII:C and VIII:CAg values. In hemophilia, most patients with severe disease have undetectable (less than 0.01 U/ml) VIII:CAg and VIII:C. Low concentrations of VIII:CAg have been found in 25% of patients with severe classic hemophilia and comparable values for VIII:C and VIII:CAg are found in patients with mild and moderate disease. Some (ca. 10%) of mild and moderate hemophiliacs have normal levels of VIII:CAg even though the VIII:C activity is less than 0.1 U/ml. 7 The assay is sensitive and reproducible and it can detect proteins that have only one VIII:CAg antigenic determinant. This is an important advantage when compared to two-site immunoradiometric assays that require divalent antigens. The assay has considerable potential in studies of VIII:C structure and the characterization of plasma from patients with hemophilia and yon Willebrand's disease. It is of demonstrated value in the prenatal diagnosis of hemophilia using small plasma samples obtained by fetoscopy, n 11 S. I. Firshein, L. W. Hoyer, J. Lazarchick, B. G. Forget, J. C. Hobbins, L. Clyne, F. A. Pitlick, W. A. Muir, I. R. Merkatz, and M. J. Mahoney, N. Eng. J. Med. 300, 937 (1979).

[5] C o n f o r m a t i o n - S p e c i f i c Antibodies: Approach to the Study of the Vitamin K-Dependent Blood Coagulation Proteins

By

BRUCE F U R I E , R I T A A. B L A N C H A R D , D A V I D J. ROBISON, M I N D Y M . T A I , a n d BARBARA C . F U R I E

Introduction The antibody combining site is a sensitive probe of the structure and conformation of a macromolecule against which it is directed. Antibody populations raised against an intact native protein may be purified and antibody subpopulations directed against a particular set of structural determinants on the protein isolated. These subpopulations may serve as useful reagents for the qualitative and quantitative evaluation of the structure and the conformational states of these protein antigens. METHODS IN ENZYMOLOGY, VOL. 84

Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181984-1