New factor VIII coagulant polypeptides

Active factor VIII:C coagulant polypeptides identified by partial amino acid sequences are disclosed.

BACKGROUND OF THE INVENTION 
This invention relates to new factor VIII polypeptides, that is, proteins, 
exhibiting coagulant activity. The invention therefore has utility in the 
therapy for classic hemophilia, and in the further study and 
characterization of the polypeptide or polypeptide complexes which provide 
desired clotting behavior to the blood of humans and other mammals. 
It has long been known that plasma factor VIII plays a crucial role in 
blood coagulation, and that thrombin activates the coagulant effect of 
factor VIII. Recent attempts to characterize factor VIII have postulated 
that factor VIII is a complex of at least two polypeptides, which are 
known as VIII:C and VIII:R, and have found coagulant activity to reside in 
the VIII:C portion. Studies of the effect of thrombin on factor VIII:C 
have led to the conclusion that thrombin activates this factor by breaking 
it down into several smaller polypeptides. However, no prior studies have 
been able to associate thrombin-induced factor VIII activation in humans 
with any defined polypeptides formed from human factor VIII:C. 
For instance, Hoyer and Trabold, in "The effect of thrombin on human factor 
VIII", J. Lab. Clin. Med. 97:50-64 (1981), sought to purify human factor 
VIII:C by immunoadsorbent chromatography using a polyclonal antibody to 
factor VIII:R raised in the rabbit. They then incubated the factor VIII:C 
with purified human .alpha.-thrombin, and determined that small amounts of 
the thrombin activated the factor VIII:C whereas larger amounts activated 
it less or not at all. They also concluded that thrombin activation is 
accompanied by a decrease in the size of the protein, and they proposed a 
molecular weight of about 116,000 for the activated factor VIII:C. Most 
significantly, they could not identify specific polypeptides that retained 
factor VIII:C activity and VIII:C antigen determinants. 
Fulcher, C. A. and Zimmerman, T. S. in "Characterization of the human 
factor VIII procoagulant protein with a heterologous precipitating 
antibody", Proc. Natl. Acad. of Sci. USA, 79:1648-1652 (1982) obtained 
highly purified human factor VIII:C from plasma concentrate by passing the 
concentrate through a column containing a monoclonal antibody to factor 
VIII:R, eluting the VIII:C from the adsorbed VIII:C/VIII:R complex, and 
concentrating the factor VIII:C on a second column. The purified factor 
VIII:C was then analyzed by sodium dodecyl sulfate/polyacrylamide gel 
electrophoresis (hereafter, "SDS-PAGE"), both before and after addition of 
thrombin to the purified material. The purified factor VIII:C prior to 
thrombin addition showed a wide array of bands on SDS-PAGE corresponding 
to polypeptides of various molecular weights (M.sub.r), including a 
relatively strong doublet at M.sub.r of 80,000 and 79,000, and at least 
six additional polypeptides with larger M.sub.r including one at M.sub.r 
about 92,000. Addition of thrombin to the purified factor VIII:C caused 
the diminution or disappearance of all of the polypeptides shown prior to 
thrombin addition. 
The coagulant activity of the plasma concentrate rose following thrombin 
addition to a maximum of three times that of the material prior to 
thrombin addition, and then diminished. The coagulant activity of the 
purified factor VIII:C also rose to a maximum of three times that of the 
pre-activated material. That is, the thrombin had essentially the same 
activating effect on the factor VIII:C in each case. Thus, whereas the 
purified factor VIII:C possessed a reported specific activity some 3280 
times that of the starting material, one skilled in this art would 
conclude that the increase in specific activity was due to the high degree 
of purification achieved. There is no basis in this article for ascribing 
activated coagulant activity to any specific one or more of the large 
number of polypeptides associated with the bands observed in the purified 
factor VIII:C prior to activation with thrombin. 
SUMMARY OF THE INVENTION 
One aspect of the present invention is a factor VIII:C coagulant 
polypeptide complex characterized in that: 
(i) the complex has one or more polypeptides which exhibit a band at a 
point corresponding to an M.sub.r of about 92,000; or bands at points 
corresponding to M.sub.r values of about 92,000, about 80,000, and about 
79,000; or of about 92,000, about 72,000 and about 71,000; or of about 
92,000, about 80,000, about 79,000, about 72,000, and about 71,000; when 
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 
accordance with Procedure A described in the Example hereinbelow; 
(ii) the complex exhibits specific coagulant activity higher than 1800 
Units/mg, and preferably higher than 5400 Units/mg; 
(iii) the complex exhibits the activity in step (ii) over a continuous 
period of at least about 10 minutes; and 
(iv) the complex binds to an antibody for human factor VIII:C. 
Other aspects of the invention include biological preparations containing 
the complex, and the treatment of the clotting disorders of hemophilia by 
administering the complex or preparations thereof. Yet another aspect of 
the present invention comprises making the complex, or a concentrated 
preparation thereof, by digesting human factor VIII:C with 
.alpha.-thrombin, discontinuing the digestion while the complex described 
above is present, and concentrating the complex. A further aspect of the 
invention is a process for recovering VIII:C polypeptides, without losing 
coagulant activity, from an immunoadsorbent containing monoclonal 
antibodies to factor VIII:C. 
DETAILED DESCRIPTION OF THE INVENTION 
As indicated, the present invention encompasses polypeptide complexes which 
exhibit factor VIII:C coagulant activity and factor VIII:C immunological 
behavior, and which show characteristic M.sub.r value(s) when analyzed by 
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereafter 
"SDS-PAGE"). By "polypeptide complex" is meant not only combinations of 
two or more polypeptides known to be physically distinct, but also 
preparations containing only one discernible polypeptide, i.e., that which 
exhibits a band at an M.sub.r of 92,000. 
The description below showing the preparation of the claimed complex 
utilizes human factor VIII:C which has been highly purified, in order to 
free the identification and characterization of the novel product from the 
effects of extraneous polypeptides. It should be recognized, though, that 
the invention itself is not dependent on how the starting material has 
previously been treated, except where specifically indicated. The active 
factor VIII:C polypeptides of the present invention can be prepared not 
only by digestion with thrombin as described in greater detail below, or 
with other similarly acting proteases such as Factor Xa, Factor IXa, or 
Russell's viper venom-V, but also by recombinant DNA techniques in which 
the polypeptides of interest are produced by bacteria, yeast, or other 
cells into which one or more genes for producing the polypeptides of 
interest are inserted by techniques known to those of ordinary skill in 
the art. Either process for producing the complex of interest can be 
expected to produce the complex in a mixture with one or more other 
polypeptides. 
Any plasma or plasma concentrate containing human factor VIII:C can be 
employed to advantage. The novel coagulant polypeptide complex can be 
prepared from human factor VIII:C which has been ultrapurified in 
accordance with the process described in U.S. Pat. No. 4,361,509, issued 
Nov. 30, 1982, the disclosure of which is hereby incorporated herein by 
reference. In that process, a source of factor VIII:C such as plasma or a 
plasma concentrate is passed through an immunoadsorbent column to which 
monoclonal antibodies to factor VIII:R have been attached. The factor 
VIII:R/VIII:C complex is adsorbed on the column, and then the factor 
VIII:C is eluted and passed through a second column such as aminohexyl 
agarose. It should be noted that the second column can also be an 
immunoadsorbent containing antibodies to factor VIII:C. The factor VIII:C 
should be kept free of .alpha.-thrombin and other proteases. The factor 
VIII:C is conveniently stored in a saline, buffered solution containing, 
e.g., 0.3M calcium chloride, at a pH of about 6.8 to about 7.4. 
The human factor VIII:C is then digested with .alpha.-thrombin under 
conditions effective to form the polypeptide complex described below. The 
purified .alpha.-thrombin can be prepared by the procedure described by 
Fenton, J. W. II; Fasco, M. J.; Stackrow, A. B.; Aronson, D. L.; Young, A. 
M.; and Finlayson, J. S. "Human Thrombin. Production, Evaluation, and 
Properties of .alpha.-Thrombin". J. Biol. Chem. 252: 3587-3598 (1977). 
The .alpha.-thrombin and the factor VIII:C are combined in an aqueous 
system, preferably buffered at a pH of about 6.8 to about 7.4. The 
thrombin should be present in an amount relative to the factor VIII:C that 
is sufficient to permit reaction with the factor VIII:C, but not so high 
that the factor VIII:C degrades to inactive polypeptides before the 
desired active polypeptide complex can be recovered. As an illustration, a 
preparation containing 200-400 units (0.2 mg/ml) of factor VIII:C per ml 
should be digested with about 0.1 to about 0.5 units/ml of 
.alpha.-thrombin. The digestion can proceed at room temperature; too high 
a temperature can denature the polypeptides, and too low a temperature can 
retard the progress of the digestion. 
The digestion is allowed to proceed for a time long enough to permit the 
formation of the desired polypeptide complex. The optimum time will be 
from 0.1 to about 60 minutes, with times from 0.1 to 30 minutes preferred. 
Times from 1 to 10 minutes have been found highly satisfactory, although 
it will be recognized that optimum times can be identified with minimal 
experimentation using aliquots of the factor VIII:C starting material 
being treated. An optimum time is that which forms the maximal amount of 
the polypeptide complex exhibiting an M.sub.r of about 92,000, accompanied 
by the formation of a protein complex exhibiting an M.sub.r doublet of 
about 79,000 and about 80,000, without degrading the M.sub.r 92,000 
polypeptide significantly. The M.sub.r 79,000-80,000 doublet may possibly 
function after it has undergone degradation to a complex exhibitng a 
doublet at M.sub.r values of 71,000-72,000. The 71,000-72,000 doublet 
alone does not exhibit factor VIII:C activity. 
The digestion is then discontinued by adding to the reaction mixture an 
effective amount of (p-amidinophenyl) methanesulfonyl fluoride (hereafter, 
"p-APMSF") or another thrombin inhibitor. The p-APMSF prevents the 
.alpha.-thrombin from reacting further with the factor VIII:C proteins, 
without itself degrading those proteins. The amount of p-APMSF to add 
should comprise about 1.5 to about 2.5 millimoles per unit of 
.alpha.-thrombin activity initially present in the reaction mixture. 
p-APMSF can be obtained through California Medicinal Chemistry Company, San 
Francisco, Calif., and its preparation is described in Laura, R.; 
Robinson, D. J.; and Bing, D. H. "(p-Amidinophenyl)methanesulfonyl 
Fluoride, an Irreversible Inhibitor of Serine Proteases", Biochemistry 
(1980) 19, 4859-4864, at 4861. 
The reaction mixture can then be treated to concentrate the polypeptide 
complex comprising the present invention. Preferably, the polypeptide 
complex is concentrated with respect to other factor VIII and non-factor 
VIII proteinaceous material, to provide the complex in a form which 
affords the very high activity possessed by the complex in the purified 
form. Purification techniques include, for example, ultrafiltration, 
ultracentrifugation, ion exchange, gel permeation chromatography, 
preparative electrophoresis, isoelectric focusing, and gel affinity 
chromatography. 
The desired complex can also be concentrated and/or recovered by passing 
the reaction mixture, which can already have been concentrated by another 
technique, through an immunoadsorbent column containing anti-human VIII:C 
antibodies, or equivalent antibodies against VIII:C other than human, that 
react with the polypeptide(s) of the complex. The antibodies are attached 
to agarose (see Example I below). Several antibodies which can be used to 
concentrate the complex and/or isolate certain of the polypeptides thereof 
are described below. The active VIII:C complex adsorbs preferentially to 
the column, and is then eluted from the column with a solution of calcium 
ions (e.g., CaCl.sub.2) which can optionally also contain a non-ionic 
surfactant. Suitable non-ionic surfactants include alkyl phenyl 
polyoxyethylenes such as Triton-X-100, -N-101, or -X-405 (Eastman Chemical 
Co.); Tween-20, -60 or -80 (Sigma Chemical Co.); and Nonidet P-40 (Sigma 
Chemical Co.); all of these are well-known articles of commerce having 
known chemical formulas. 
The amounts of calcium ion and surfactant to use should be high enough to 
desorb the polypeptide complex, but not so high that the eluant 
inactivates the polypeptide. A calcium ion concentration of up to about 
0.5M or even up to 1.0M is satisfactory, and 0.25M is preferred. A 
surfactant concentration of up to about 1 wt. % is satisfactory, and about 
0.1 wt. % is preferred. The eluant is applied to the immunoadsorbent 
column at about 1 to about 8 bed volumes per hour, and preferably about 3 
to about 4 per hour. Too high a flow rate risks disruption of the column 
and non-absorption of the polypeptide complex. The skilled practitioner 
will readily adapt these guidelines to immunoadsorbent processes other 
than fixed-bed columns. 
The VIII:C polypeptide complex is recovered in a suitable buffer, at a pH 
of about 6.8 to 7.4, which also contains calcium ion and surfactant from 
the eluant solution. The calcium and surfactant concentrations can be 
lowered, and the surfactant preferably removed, such as by dialyzing the 
solution against a buffer such as the VIII:C buffer used in Example 1 
which contains a lower amount of calcium ion. The complex of this 
invention can be stored in this solution, or lyophilized. The complex can 
be administered to patients with hemophilic clotting disorders by 
adjusting the calcium content to be physiologically compatible and 
injecting a sterile saline solution thereof. 
When analyzed by SDS-PAGE as shown below, the complex of this invention 
exhibits an M.sub.r of about 92,000, and can normally contain material 
exhibiting an M.sub.r doublet of about 79,000 and about 80,000, some of 
which can have undergone degradation to exhibit an M.sub.r doublet of 
about 71,000 and about 72,000. In its purified form, this band or the 
group of these bands are essentially the only bands that appear. However, 
it will be appreciated that the present invention also encompasses 
biological preparations in which less than 100%, i.e., 95%, 90%, or even 
80%, 70% or 60%, or even as little as 30%, 20%, 10%, or 1% of the 
proteinaceous matter present comprises the complex of the present 
invention. This invention thus encompasses preparations in which the 
factor VIII:C activity is due to the presence of the complex. 
The protein complex of the present invention possesses specific VIII:C 
coagulant activity (that is, activity per milligram of all protein 
present) higher than that exhibited by purified human factor VIII:C, for 
instance human factor VIII:C purified by the process disclosed and claimed 
in the aforementioned U.S. Pat. No. 4,361,509. Indeed, the activity of the 
purified complex should be several times, e.g., 3 to 5 times, that of 
purified human factor VIII:C, and is advantageously at least 10 times or 
even 50 times as active. Likewise, biological preparations comprising the 
complex of this invention in association with one or more other proteins 
can be prepared which exhiblit higher specific activity than that afforded 
in previously known coagulant preparations. The specific activity of the 
complex, and of biological preparations containing it, is higher than 1800 
Units/mg, advantageously higher than 5400 Units/mg, and in more 
advantageous embodiments exceeds 7,500 and even 10,000 Units/mg. 
Preferably, the specific activity of the inventive preparations exceeds 
that of the purified human factor VIII:C used herein by a factor of 3 to 
5, and more advantageously by at least 10 times, by 50, or even by 100 
times. 
The protein complex of the present invention, and biological preparations 
thereof, are characterized in that the enhanced activity described above 
remains present over a continuous period of at least about 10 minutes and 
preferably at least about 30 minutes. Of course, activity will generally 
be stable for much longer. The complex also possesses the immunological 
characteristics of a factor VIII:C protein, i.e., it binds to an antibody 
for human factor VIII:C. This can be ascertained, for instance, by growing 
a monoclonal antibody to factor VIII:C as described below, attaching the 
antibody to an agarose column, passing an aqueous solution of the complex 
through the column, and assaying the resultant solution for factor VIII:C 
activity. 
A further desirable attribute of this invention is that the polypeptides of 
M.sub.r =92,000 and M.sub.r =79,000-80,000 are stable, relative to native 
human factor VIII:C which is notoriously susceptible to proteolysis, 
degradation, and resultant loss of coagulant activity. This stability is 
demonstrated by the polypeptides' ability to survive the treatement steps 
described herein. 
The polypeptide described above, i.e., that polypeptide which exhibits a 
band at an M.sub.r of 92,000 and which is associated with the increase in 
VIII:C activity is further identified by its active, partial protein 
sequence as follows: 
##STR1## 
wherein X and Y are amino acids. 
Preferably, X is ALA (alanine) and Y is SER (serine) in the above amino 
acid sequence. 
For purposes of this disclosure, accepted short-hand designations of the 
amino acids have been used. A complete listing is provided hereinbelow: 
______________________________________ 
The 20 Amino Acids in Proteins 
______________________________________ 
Glycine GLY Lysine LYS 
Alanine ALA Arginine ARG 
Valine VAL Asparagine ASN 
Isoleucine ILE Glutamine GLN 
Leucine LEU Cysteine CYS 
Serine SER Methionine MET 
Threonine THR Tryptophan TRP 
Proline PRO Phenylalanine 
PHE 
Aspartic acid 
ASP Tyrosine TYR 
Glutamic acid 
GLU Histidine HIS 
______________________________________ 
While only the partial amino acid sequence is identified for the 92,000 
molecular weight segment, that information is sufficient to disclose the 
active portion of the molecule insofar as coagulant activity is concerned. 
In fact, the fragmented polypeptide protein having that amino acid 
sequence is active per se. By "active", is meant it has procoagulant 
activitiy. Such activity is typically found when the 92,000 segment is 
associated with the 79,000 or 71,000 segments or combinations therewith. 
The earlier referenced polypeptide having a molecular weight in the range 
of about 79,000-80,000 also exhibits coagulant activity and is further 
defined as having a partial protein amino acid sequence as follows: 
##STR2## 
wherein Z, A, B, C, D and E are amino acids. 
Preferably, the amino acid sequence is as shown wherein Z is serine (SER), 
C is serin (SER), D is serine (SER) and E is phenylalanine (PHE). 
The fragmented polypeptide having such amino acid sequence is similarly 
active per se. 
The amino acid sequences found in the coagulant-active 92,000 and 
79,000-80,000 molecular weight polypeptides were determined by 
art-accepted methods. In general, the identification was carried out in 
the following manner: 
The preparation was subjected to thrombin proteolysis as described in 
Example I so that the M.sub.r =92,000 was cleaved into the M.sub.r =54,000 
and M.sub.r =44,000 fragments and the 79,000-80,000 fragment was cleaved 
into the M.sub.r = 71,000-72,000 fragment. The mixtures containing these 
fragments were subjected to SDS polyacrylamide gel electrophoresis as 
described in Example I so that each fragment was separated from the other. 
The M.sub.r =54,000 fragment and the M.sub.r =71,000-72,000 fragment were 
then eluted according to the procedure described in "Isolation of 
Microgram Quantities of Proteins from Polyacrylamide Gels for Amino Acid 
Sequence Analysis" by M. W. Hunkapiller, E. Lujan, F. Ostrande and L. E. 
Hood, Methods in Enzymology, Vol. 91, Chapter 17, 1983. The peptides so 
obtained were then subjected to amino acid sequencing as described in 
"High-Sensitivity Sequencing with a Gas-Phase Sequenator" by M. W. 
Hunkapiller, R. M. Hewick, W. J. Dreyer, and L. E. Hood, Methods in 
Enzymology, Vol. 91, Chapter 36, 1983.

EXAMPLE I 
This Example shows how factor VIII:C was purified from commercial 
concentrate and digested with purified .alpha.-thrombin. A monoclonal 
antibody to factor VIII:C was produced and used to identify VIII:C 
polypeptides. At several selected points during the digestion, portions of 
the digestion mixture were assayed for VIII:C (coagulant) activity, and 
for protein bands using SDS-PAGE. 
Purification of VIII:C 
All steps were at room temperature. Chemicals were reagent grade. 40 
bottles of commercial factor VIII concentrate (provided by Armour 
Pharmaceutical) were reconstituted in 1000 ml of VIII:C buffer (0.02M 
imidazole/0.15M sodium chloride/0.1M L-lysine HCl/0.02% sodium azide, pH 
6.8). This sample, which contained a total of 17,000 units of VIII:C 
activity, was applied to a 2.5-3.0 liter bed volume immunoadsorbent 
column. The column was cyanogen bromide-activated agarose (Sepharose 4B, 
Pharmacia, Piscataway, N.J.), to which monoclonal antibodies to VIII:R had 
been covalently bonded. The antibodies were raised and attached to the 
column as described in the aforementioned U.S. Pat. No. 4,361,509. The 
antibodies were precipitated from ascites fluid using 50% ammonium 
sulfate, reprecipitated two more times, and then attached to the column at 
a density of 2-4 mg/ml of Sepharose. The immunoadsorbent was pre-eluted 
with 3M sodium thiocyanate, washed with VIII:C buffer (0.02M imidazole HCl 
pH 7.0, 0.15M NaCl, 0.1M L-lysine-HCl, 0.02% sodium azide), treated twice 
with 2 mM di-isopropyl fluorophosphate, and then the concentrate was 
added. 
The column was washed with 20 liters of VIII:C buffer containing 0.15M 
sodium chloride, and VIII:C was then eluted from the VIII:R with VIII:C 
buffer containing 0.35M calcium chloride. Active fractions were pooled and 
concentrated under nitrogen pressure 100-fold in an Amicon stirred cell 
with a YM10 membrane. The concentrate was then diluted 1:10 in VIII:C 
buffer and applied to a 4 ml column of aminohexyl-Sepharose (Pharmacia) 
equilibrated in VIII:C buffer containing 0.025M calcium chloride, VIII:C 
was eluted in high concentration with VIII:C buffer containing 0.3M 
calcium chloride at a flow rate of 10 ml/hr. The concentrated 
immunoadsorbent pool was adjusted to 0.25M calcium chloride and adsorbed 
twice for 1 h each time with 1/10 vol/vol of a mixture of monoclonal 
anti-fibrinogen, anti-fibronectin and anti-vWF antibodies which had been 
coupled to cyanogen bromide-activated Sepharose. 
Production of monoclonal antibody against VIII:C 
Monoclonal antibodies were produced as described in U.S. Pat. No. 4,361,509 
using purified VIII:C as immunogen. The antibodies were selected with a 
solid-phase assay in Linbro-Titertek (Flow Laboratories, Inglewood, CA) 
plates and an enzyme-linked immunoadsorbent (ELISA) detection system 
described in Engvall, E. and Perlmann, P. "Enzyme-linked immunoadsorbent 
assay (ELISA), Quantitative assay of immunoglobulin G" Immunochemistry 
8:871-874 (1971) using a peroxidase-antibody conjugate (Zymed 
Laboratories, Burlingame, CA). The plates were coated with 100 ng of 
purified VIII:C per well. The ELISA-positive culture supernatant of the 
clone selected for use in this study also inhibited plasma VIII:C 
activity. 
Thrombin activation time course analysis of purified VIII:C 
Purified human .alpha.-thrombin(sp. act. 2534 U/mg, final concentration 0.5 
U/ml), was added to the purified VIII:C (final concentration 167 .mu.g/ml) 
in imidazole saline buffer containing 0.04M CaCl.sub.2. Buffer alone was 
added to a control aliquot. The solutions were incubated at room 
temperature and at various time intervals samples of the VIII:C-thrombin 
mixture were added to tubes containing p-APMSF (California Medicinal 
Chemistry Co.) to inactivate the thrombin rapidly and irreversibly. In 
order to minimize hydrolysis of p-APMSF, it was diluted 1:10 from a stock 
solution (100 mM in methanol) into imidazole saline buffer 60 seconds 
before reaction with the VIII:C-thrombin samples. The final p-APMSF 
concentration was 1 mM. The control aliquot was treated similarly with 
p-APMSF at the start of the experiment. At the end of the 60 minute time 
course, all VII:C samples were assayed for VIII:C activity using an 
activated partial thromboplastin time assay described in the literature 
and then prepared for SDS-PAGE. 
SDS-PAGE "Procedure A" 
Discontinuous SDS polyacrylamide slab gel electrophoresis was performed 
based on the procedure of Laemmli, U.K., Nature 227, 680-685, 1970. The 
"Procedure A" followed is: 
I. Sample Preparation 
1. Dialyze the protein sample (ideally 50-100 microliters containing 5-60 
micrograms of protein) against sample buffer overnight at room 
temperature. If the sample contains calcium ion, include 10 millimolar 
ethylenediamine tetracetic acid (EDTA) in the sample buffer. 
2. Place the dialyzed sample in a tube and add 1/10 volume of 10% SDS. 
Cover the tube with aluminum foil. Heat the sample in a boiling waterbath 
for 10 minutes. 
3. Remove the sample from the waterbath and add to it 1/10 volume of 500 
millimolar dithiothreitol. Incubate it at 56.degree. C. for 4 hours. 
4. Allow the sample to cool to room temperature and prepare it for layering 
onto the gel by adding stock glycerol solution to 10% final concentration 
and stock bromophenol blue dye solution to 0.05% final concentration. 
II. Preparaion of gel solutions (use deionized, distilled water) 
1. Stock glycerol solution: 50% glycerol 
2. Stock bromophenol blue dye solution: 0.5% bromophenol blue 
3. Lower gel stock solution: 
18.2 grams of Tris base 
4 ml of 10% SDS 
final volume 100 ml. 
Adjust pH to 8.8 with concentrated hydrochloric acid. Filter. 
4. Upper gel stock solution: 
6.1 grams of Tris base 
4 ml of 10% SDS 
Final volume 100 ml 
Adjust pH to 6.8 with concentrated hydrochloric acid. Filter. 
5. Sample buffer 
0.01M sodium phosphate 
1.0% SDS 
10 millimolar disodium EDTA 
Final volume 1 liter 
pH adjusted to 7.0 with sodium hydroxide or phosphoric acid. 
6. Acrylamide stock solution: 
Dissolve 30 g of acrylamide in 50 ml of water and add 0.8 g of 
bisacrylamide and dissolve. Bring to 100 ml final volume. Filter the 
solution and store in the dark at 4.degree. C. 
7. Stock electrode buffer solution; 
30.3 g Tris base 
144.1 g glycine 
final volume 1 liter 
8. Electrode buffer 
100 ml of stock electrode buffer solution 
890 ml water 
10 ml of 10% SDS 
9. Stock Coomassie blue dye solution 
1% Coomassie blue R 250 in water 
Dissolve with stirring for at least 30 minutes at room temperature and 
filter. 
10. Ammonium persulfate solution 
10% ammonium persulfate. Stored in dark at 4.degree. C. and made fresh 
every week. 
III. Gel Preparation and Running: Final acrylamide concentration=7.6% 
1. Lower gel solution 
20 ml of lower gel 
______________________________________ 
Stock lower gel solution 
5 ml 
Stock acrylamide solution 
5 ml 
Water 10 ml 
N,N,N',N'--tetramethylethylenediamine 
.005 ml 
(TEMED) 
10% ammonium persulfate 
0.1 ml 
______________________________________ 
2. Upper gel solution 
10 ml of upper gel 
______________________________________ 
Stock upper gel solution 
2.5 ml 
Stock acrylamide solution 
1.0 ml 
Water 6.5 ml 
N,N,N',N'--tetramethylethylenediamine 
0.01 ml 
(TEMED) 
10% ammonium persulfate 
0.03 ml 
______________________________________ 
3. Procedure: 
a. Prepare the slab gel apparatus for a 14.5 cm.times.9.0 cm.times.0.8 mm 
slab gel. The apparatus is a standard gel electrophoresis apparatus, 
available, for instance, from Hoeffer Scientific Instruments, San 
Francisco, Calif. 
b. Mix all lower gel ingredients except the TEMED and ammonium persulfate 
in a 50 ml vacuum flask and de-aerate. Then add the TEMED and ammonium 
persulfate, mix gently and pour the lower gel immediately. Layer the lower 
gel with water-saturated butanol and allow it to polymerize undisturbed 
for at least 1 hour, preferably 2-6 hours. 
c. Pour off the butanol layer and rinse the top of the lower gel with the 
complete upper gel mixture. (The upper gel mixture is prepared, de-aerated 
and TEMED and ammonium persulfate are added as for the lower gel above). 
d. Pour the upper gel and insert the comb into the upper gel allowing at 
least 1.0 cm between the bottom of the comb teeth and the upper gel-lower 
gel interface. Fill with upper gel solution as full as possible. Allow 
upper gel to polymerize at least 1 hour before running the gel. 
e. To remove the comb, pipet electrode buffer over the top of the upper gel 
and gently remove the comb. Rinse the upper gel wells with electrode 
buffer several times. 
f. Assemble the apparatus for running and add electrode buffer. Apply the 
sample(s) by layering it into the upper gel wells underneath the buffer 
layer. 
g. Run the gel using constant current: 8 milliamperes while samples are in 
the upper gel and 15 milliamperes while samples are in the lower gel. Stop 
the electrophoresis when the bromophenol blue dye front is 1.0 cm from the 
bottom of the lower gel. 
IV. Fixing and Staining the gel 
Reference: Fairbanks, G., Steck, T. L., and Wallach, D. F. N., Biochemistry 
10, 2606-2617, 1971. 
1. Fix the gel at least overnight in a sealed chamber in a solution 
containing 25% isopropanol, 10% acetic acid, 10 ml of 1% Coomassie blue 
stock solution, final volume 400 ml. 
2. Next, soak the gel at least 1 hour in 10% isopropanol, 10% acetic acid 
and 1.0 ml of 1% Coomassie blue stock solution, final volume 400 ml. 
3. Soak the gel about 4 hours in 10% acetic acid with changes or until 
completely destained. 
4. The destained gel can be dried onto filter paper using a gel dryer to 
increase contrast. 
Approximately 5-20 g of protein was applied to the gels. VIII:C M.sub.r s 
were calculated for reduced samples by semilogarithmic plots of M.sub.r 
versus migration distance, using reduced fibronectin (M.sub.r 200,000), 
phosphorylase b (M.sub.r 95,000), bovine serum albumin (M.sub.r 68,000), 
and IgG heavy chain (M.sub.r 50,000), and ovalbumin (M.sub.r 43,000) as 
standards. 
Scanning and integration of a photographic print of the finished gel was 
done using a Zeineh soft laser scanning densitometer. 
Results 
The specific activity of the purified factor VIII:C was 2000 units/mg. 
Thrombin activation of purified VIII:C activity was analyzed over a 60 
minute time course. Before thrombin exposure the untreated VIII:C sample 
showed the characteristic array of VIII:C forms ranging from a doublet at 
M.sub.r =79,000-80,000 to a band at M.sub.r =188,000. A band above M.sub.r 
=188,000 and two bands below M.sub.r =79,000 did not bind to the 
monoclonal anti-VIII:C antibody immunoadsorbent. The bands between M.sub.r 
=79,000 and M.sub.r =188,000 did bind to the anti-VIII:C antibody. 
During the first 5 minutes of the thrombin activation time course, all but 
one of the monoclonal anti-VIII:C antibody reactive bands with an M.sub.r 
greater than 92,000 gradually disappeared and were undetectable when 
VIII:C activity reached its peak at 5 minutes. 
A band at M.sub.r =122,000 appeared thrombin-resistant in only some 
experiments, but after extensive thrombin treatment neither this band nor 
any other band was reactive with the immobilized monoclonal anti-VIII:C 
antibody. 
A band at M.sub.r =92,000 increased in intensity as VIII:C activity 
increased. A doublet at M.sub.r =79,000 and 80,000 appeared to be 
converted to a doublet at M.sub.r =71,000-72,000, with the latter form 
predominant from 5 to 60 min. as VIII:C activity decreased. Two bands, at 
M.sub.r =54,000 and M.sub.r =44,000, became clearly visible from 5 to 60 
min. The M.sub.r =44,000 band also appeared as a doublet in some 
experiments. The M.sub.r =71,000-72,000 doublet, the M.sub.r =54,000 band 
and the M.sub.r =44,000 band were not removed to a significant degree by 
the immobilized monoclonal anti-VIII:C antibody. 
Scanning and integration of the gel discussed above allowed correlation of 
changes in polypeptide concentration with changes in VIII:C activity. The 
results are shown in the Table. As shown, the M.sub.r =92,000 band 
increased and then decreased in concentration in parallel with VIII:C 
activity. This suggests that the M.sub.r =92,000 band is an active form of 
VIII:C whose concentration increased with thrombin activation. The M.sub.r 
=54,000 and M.sub.r =44,000 bands increased steadily in concentration 
between 1 and 40 min., even after activity of the mixture declined. 
Most of the M.sub.r =79,000-80,000 doublet was lost during the first 0.1 to 
10 min. as VIII:C activity peaked, while most of the M.sub.r 
=71,000-72,000 doublet appeared during this time and predominated even as 
VIII:C activity decreased. These data suggest that the M.sub.r 
=71,000-72,000 doublet was derived from the M.sub.r =79,000-80,000 doublet 
and that the M.sub.r =71,000-72,000 doublet is inactive by itself. These 
data are consistant with the retention by the M.sub.r =71,000-72,000 
doublet of the ability to complex with the M.sub.r =92,000 polypeptide; 
this complex would also have activity. 
Direct evidence that the M.sub.r =92,000 polypeptide is complexed with the 
M.sub.r =79,000-80,000 doublet derives from experiments utilizing the 
anti-VIII:C monoclonal immunoadsorbent. It was first shown that the 
monoclonal antibody reacts predominantly with the M.sub.r =79,000-80,000 
doublet and not with the M.sub.r =92,000 polypeptide. This was shown by 
electrophoretic transfer experiments. Then it was shown that the 
monoclonal anti-immunoadsorbent removed both the M.sub.r =79,000-80,000 
doublet and the M.sub.r =92,000 polypeptide from the solution. The M.sub.r 
=92,000 polypeptide could be eluted from the anti-VIII:C immunoadsorbent 
column with 10 mM EDTA, whereas the M.sub.r =79,000-80,000 doublet 
remained bound. The doublet could be subsequently eluted with 3M sodium 
thiocyanate. These experiments demonstrated that the M.sub.r =92,000 
polypeptide bound to the immunoadsorbent because it was complexed with the 
M.sub.r =79,000-80,000 doublet and that it was this doublet which bound 
directly to the immunoadsorbent. 
__________________________________________________________________________ 
Time (min) from 
addition of thrombin 
to addition of p-APMSF: 
0* 0.1 
1 2 5 10 20 30 40 60 
__________________________________________________________________________ 
Activity of 300 
900 
1300 
1350 
1400 
1250 
800 
375 
250 
100 
digestion mixture 
Amount of each poly- 
peptide (M.sub.r) present, 
as % of total 
protein present 
M.sub.r = 92,000 
7.3 
9.5 
11.1 
11.5 
11.8 
9.6 
7.3 
6.7 
4.9 
4.4 
M.sub.r = 79-80,000 
21.9 
18.8 
16.1 
12.9 
11.0 
8.4 
9.0 
8.6 
7.4 
7.4 
M.sub.r = 71-72,000 
0.0 
9.0 
11.2 
12.9 
16.5 
15.9 
15.7 
18.8 
18.2 
20.4 
M.sub.r = 54,000 
4.9 
5.9 
5.1 
5.7 
7.1 
9.4 
10.2 
11.8 
13.7 
13.5 
M.sub.r = 44,000 
0.0 
0.0 
0.0 
3.1 
3.7 
5.9 
6.1 
7.2 
7.8 
7.6 
__________________________________________________________________________ 
*Measurements just prior to addition of thrombin 
Activity is in Factor VIII: C units/ml 
EXAMPLE II 
This Example describes the results of treating purified human Factor VIII:C 
with purified human activated protein C (hereafter, "APC"), a known 
anticoagulant enzyme. Human Factor VIII:C was purified as described above. 
APC was purified by the procedure described in Marlar, R. A. et al., 
"Mechanism of action of human activated protein C, a thrombin-dependent 
anticoagulant enzyme." Blood, Vol. 59, 1067 (1982), except that a mono S 
column of a Pharmacia FPLC system was used to separate APC from thrombin. 
Assays: Samples were assayed for VIII:C activity as described above using 
an activated partial thromboplastin time assay with hemophilia A plasma 
substrate. 
Preparation of samples for electrophoresis 
Since calcium ions are required for APC activity, APC was stopped at 
various times by addition of 1/10 volume of 100 mM EDTA containing 10 uM 
DAPA to the VIII:C+APC aliquots as well as to the control VIII:C and APC 
aliquots. A 1/10 volume of 10% sodium dodecyl sulfate was then added to 
these aliquots. They were then heated in a boiling waterbath for 5 minutes 
and subsequently dialyzed for SDS-PAGE as described above. 
Discontinuous sodium dodecyl sulfate 7.5% polyacrylamide gel 
electrophoresis (PAGE) of reduced VIII:C, staining with Coomassie blue 
R250 and scanning and integration of the gel were as described above. 
Sample Preparation 
A 339 .mu.g sample of VIII:C in 0.3 ml of VIII:C buffer containing 0.3M 
calcium chloride was dialyzed overnight against buffer (50 mM 
Tris-chloride, 0.15M sodium chloride, 5 mM calcium chloride, 0.02% sodium 
azide, pH 7.4). To the dialyzed VIII:C sample was added 1.095 ml of 
buffer, 90 .mu.l of rabbit brain cephalin (Sigma Chemical Co., St. Louis, 
MD, reconstituted, stored and thawed according to manufacturer's 
instructions) and 15 .mu.l of 1 mM dansylarginine 
N-(3-Ethyl-1,5-pentanediyl)amide (DAPA), to give a final DAPA 
concentration of 10 .mu.M. The DAPA was included to inhibit any trace 
amounts of thrombin present in the APC since 10 .mu.M DAPA does not 
significantly inhibit APC. The final volume of the sample was 1.5 ml and 
the final VIII:C concentration was 226 .mu.g/ml. Four hundred microliters 
of the 1.5 ml VIII:C sample were withdrawn and set aside as a control 
(designated VIII:C). To the remaining 1.1 ml was added 20 .mu.l (10 ug) of 
APC giving a final APC concentration of 9 .mu.g/ml (designated 
VIII:C+APC). A second comtrol sample was prepared containing all 
components at similar concentrations except that VIII:C was omitted 
(designated APC). 
Timepoints 
VIII:C alone, the mixture of VIII:C and APC, and APC alone were placed in a 
37.degree. C. waterbath and at given timepoints aliquots were withdrawn 
for SDS-PAGE and/or VIII:C activity assay. It was also determined in 
control that APC remained active in the hydrolysis of the synthetic 
substrate S-2238 after prolonged incubation at 37.degree. C. 
Results 
Digestion of purified VIII:C with APC was associated with loss of 
approximately 85% of the control VIII:C activity. APC inactivation of 
VIII:C activity resulted in the diminution of all VIII:C polypeptides of 
M.sub.r between 92,000 and 188,000 and generation of a polypeptide of 
M.sub.r =45,000, while leaving the doublet of M.sub.r =79-80,000 intact. 
A time course inactivation of purified VIII:C by APC showed the progressive 
disappearance of specific VIII:C polypeptides as VIII:C activity decreased 
over a 360 minute time course. Scanning and integration of the gel showed 
that the polypeptide M.sub.r =188,000 and the polypeptide of M.sub.r 
=92,000 decreased in parallel with VIII:C activity. 
One other polypeptide of intermediate M.sub.r was cleaved by APC but it was 
not easily quantitated by gel scanning. In this experiment unlike the 
digestion with APC, some VIII:C polypeptides of M.sub.r =92,000-188,000 
were resistant to the APC digestion. Howver, as in the digestion with APC, 
the doublet polypeptide at M.sub.r =79-80,000 was not proteolyzed by APC. 
A polypeptide of M.sub.r =45,000 appeared to increase in concentration as 
the polypeptides of M.sub.r =188,000 and 92,000 decreased suggesting that 
it is a proteolytic fragment derived from them. No other digestion 
products were visualized with Coomassie blue. 
As shown in Example I, during thrombin activation of VIII:C the M.sub.r 
=92,000 polypeptide increased and decreased in parallel with VIII:C 
activity. In order to determine whether a linear relationship existed 
between proteolysis of the polypeptide of M.sub.r =92,000 and the loss of 
VIII:C activity, the data of this time course inactivation run were 
re-plotted to examine percent VIII:C activity versus percent of 
polypeptide of M.sub.r =92,000. The amount of VIII:C activity appeared to 
be proportional to the amount of the polypeptide of M.sub.r =92,000. 
A further aspect of the present invention comprises monoclonal antibodies 
which are specific in an unforeseeable way to the various polypeptides 
which are formed by the reaction of factor VIII:C with a protease such as 
alpha-thrombin. Each antibody reacts with human factor VIII:C which has 
not been activated nor digested with thrombin or equivalent proteases. The 
antibodies are further characterized by their individual properties, as 
follows: 
(A) One reacts with the M.sub.r =92,000 polypeptide described herein, with 
polypeptides of M.sub.r =108,000 and larger, and with the polypeptide of 
M.sub.r =44,000 which is present in terminal-thrombin digests. It does not 
react with the polypeptide described herein which exhibits a doublet of 
M.sub.r =79,000-80,000, nor with the polypeptide described herein which 
exhibits a doublet of M.sub.r =71,000-72,000. 
(B) One reacts with the M.sub.r =92,000 polypeptide described herein, with 
polypeptides of M.sub.r =108,000 and larger, and with the polypeptide of 
M.sub.r =54,000 which is present in terminal thrombin digests. It does not 
react with the polypeptide described herein which exhibits a doublet of 
M.sub.r =79,000-80,000, nor with the polypeptide described herein which 
exhibits a doublet of M.sub.r =71,000-72,000. 
(C) One reacts with the doublet of M.sub.r =79,000-80,000 and polypeptides 
of M.sub.r =108,000 and greater, but not with the polypeptide of M.sub.r 
=92,000, nor with the polypeptides of M.sub.r =71,000-72,000, M.sub.r 
=54,000, nor M.sub.r =44,000. 
(D) One reacts only with polypeptides of M.sub.r =108,000 and greater. 
Each of these antibodies can be used to concentrate the complex described 
above, from mixtures which also contain other polypeptides. One such 
mixture is produced by partial digestion of human factor VIII:C with 
.alpha.-thrombin or an equivalent protease. Another such mixture is that 
produced by recombinant DNA techniques in which a desired polypeptide or 
complex is expressed by a microorganism and must be recovered from a 
mixture with other proteinaceous compounds. Antibody (A), (B), (C) or (D), 
or a combination of two, three, or all of them, can be attached to an 
immunoadsorbent column in the manner described in Example 1, and a feed 
solution of the mixture containing the polypeptide(s) comprising the 
complex is poured through the column. Those polypeptides having M.sub.r of 
92,000, 79,000-80,000, which are present in the feed solution adsorb onto 
the column, from which they can be eluted as taught hereinabove after the 
source solution has been washed through the column. The resulting eluted 
solution is thereby concentrated in the desired activated VIII:C complex 
compared to the feed solution. 
The new antibodies are also useful for analytical purposes, to detect the 
occurrence of the reaction of human VIII:C with thrombin or other 
proteases, because of their ability to react with products of that 
reaction. An antibody having profile (B) and an antibody having profile 
(C) have been found which neutralize VIII:C coagulant activity when either 
is bound to factor VIII:C. This additional property can be of value in the 
diagnosis of hemophilia-related disorders. 
The discovery of these antibodies also permits further characterization of 
the components of the polypeptide complex described herein. Thus, the 
polypeptide which exhibits a band of M.sub.r = 92,000 contains (at least) 
two epitopes (i.e. antibody binding sites) that are not destroyed by 
thrombin digestion and are not present on the polypeptides which exhibit 
the doublet of M.sub.r = 79,000-80,000 nor the doublet of M.sub.r 
=71,000-72,000. One of these epitopes is also present on the polypeptide 
of M.sub.r =44,000, and the other on the polypeptide of M.sub.r =54,000. 
Thus, the M.sub.r =54,000 and M.sub.r =44,000 polypeptides derive from the 
M.sub.r =92,000 polypeptide. Also, the M.sub.r =92,000 and M.sub.r 
=79,000-80,000 polypeptides are derived from a common precursor or 
precursors. The discovery of antibodies having profile (B) and profile (C) 
each of which neutralizes factor VIII:C coagulant activity is further 
support that the M.sub.r =92,000 and M.sub.r =79,000-80,000 polypeptides 
are important to the coagulant function. The polypeptides exhibiting the 
doublet of M.sub.r = 79,000-80,000 contain an epitope which is destroyed 
by thrombin digestion and which is not present on the polypeptide of 
M.sub.r =92,000. 
These monoclonal antibodies can be prepared by the general steps of 
purification of human factor VIII:C; raising monoclonal antibodies to the 
purified VIII:C; partial digestion and activation of purified VIII:C to 
produce the polypeptide complex described above, including identification 
of the specific polypeptides; reaction of the anti-VIII:C antibodies with 
the products of the activation; and characterization of an antibody by 
identification of the polypeptide(s) with which it reacted. This sequence 
is set forth in more detail in Example III below. Alternatively, one can 
prepare an antibody by isolating the particular polypeptide of interest 
from the partial thrombin digestion products, for instance by 
immunoadsorbtion onto and then elution off of a column comprising agarose 
to which is coupled a monoclonal antibody known to react with the 
polypeptide of interest, and then raising a monoclonal antibody against 
the polypeptide using the procedure described in Example III. 
EXAMPLE III 
Monoclonal antibodies to human Factor VIII:C were raised using the 
following procedure, starting from highly purified VIII:C which had been 
prepared by the process of U.S. Pat. No. 4,361,509. 
Mice were injected with highly purified factor VIII:C according to the 
following procedure. On day zero, the mice were injected intraperitoneally 
with a composition prepared by dissolving (or suspending) 10 .mu.g of the 
protein in 0.1 ml of buffer containing 0.05M Tris, 0.15M sodium chloride, 
0.02% sodium azide, 1 mM phenyl methyl sulfonyl fluoride, trasylol 10 
units/ml at pH 7.3, and shaking with an equal volume of complete Freund's 
adjuvant. On day 14, the mice were again injected with the same material 
except that incomplete Freund's adjuvant was substituted for complete 
Freund's adjuvant. On day 21, the injection of day 14 was repeated. On day 
38, the mice were injected with purified VIII:C only. On day 42, the 
spleens of the mice were removed and fused according to a standard 
procedure, of the type described by J. P. Brown et al "Protein Antigens of 
Normal and Malignant Human Cells Identified by Immunoprocipitation with 
Monoclonal Antibodies", JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 225, pp. 
4980-4983 (1980). The standard technique was varied only to the extent 
that 35% polyethylene glycol 1000 was substituted for 50% polyethylene 
glycol. 
The antibodies were selected using the assay procedure described above in 
Example I, in the paragraph with the heading "Production of monoclonal 
antibody against VIII:C", except that antibodies which did not neutralize 
VIII:C activity were also subcloned and treated as described below. 
The clones which were positive were subcloned twice and stable clones 
producing antibody to VIII:C were then injected into the peritoneal 
cavities of Balb/C mice which had been pretreated intraperitoneally with 
0.5 ml of pristane at least four days prior to injection of cells. 
Hybridoma cells were injected at concentrations of approximately 
5.times.10.sup.6 cells per mouse in 0.5 ml of Delbecco's modified Eagle's 
medium without fetal bovine serum. The mice were tapped when bloated and 
ascites fluid was collected in heparin at approximately 10 units/ml. 
Ascites fluid from multiple mice was pooled to provide a convenient volume 
for subsequent isolation of the monoclonal IgG. The antibodies were 
precipitated from ascites fluid using 50% ammonium sulfate, and then 
reprecipitated two more times. The anti-VIII:C antibodies so raised 
corresponding to (B), (C) and (D) above were bound to agarose beads and 
shown to bind purified VIII:C from solution. 
Separate batches of VIII:C, one untreated and one subjected to thrombin 
proteolysis, were then analyzed by the SDS-PAGE steps described in Example 
1. Then, each band was transferred electrophoretically (Western transfers) 
from the gel onto a nitrocellulose sheet. The apparatus used was Bio-Rad 
"Trans-Blot"cell, and Bio-Rad Model 160.1.6 power supply (Bio-Rad 
Laboratories, Richmond, Calif.). The transfer buffer was 25 mM Tris with 
glycine added to pH 8.3, 20% methanol. Transfers were carried out over 
16.gtoreq.24 hours at 90 volts and 100 milliamperes. 
The specific polypeptides with which each of the antibodies reacted were 
determined using a procedure adapted from W. M. Burnette, "Western 
Blotting: Electrophoretic Transfer of Proteins from Sodium Dodecyl Sulfate 
Polyacrylamide Gels to Unmodified Nitrocellulose and Radiographic 
Detection with Antibodies and Radioiodinated Protein A", Analytical 
Biochemistry, Vol. 112, pp. 195-203 (1981). "Buffer D" was 10 mM Tris 
chloride, 0.15M NaCl, 0.02% sodium azide, pH 7.4. All reactions were 
carried out at room temperature. 
1. Place the nitrocellulose sheet with the transferred protein in a tray 
containing 100 ml of Buffer D and 0.25% gelatin. Place the tray on a 
rotary shaker and shake slowly for 30 minutes. 
2. Then add the monoclonal antibody to the tray (either 0.1%-1% of ascites 
fluid or 1 mg of purified IgG). Shake for 120 minutes. 
3. Wash the nitrocellulose sheet as follows: 
(a) 10 minutes with 100 ml of Buffer D. 
(b) 30 minutes with 100 ml of Buffer D and 0.05% of Nonidet-P-40, with 
changes at 10 and 20 minutes. 
(c) 10 minutes with 100 ml of Buffer D. 
4. Soak the nitrocellulose sheet in Buffer D plus 0.25% gelatin and 
I.sup.125 -labelled purified rabbit anti-mouse IgG for 30 minutes. 
5. Wash the nitrocellulose sheet as follows: 
(a) 10 minutes with 100 ml of Buffer D. 
(b) 16-24 hours with 100 ml of Buffer D plus 0.1% Nonidet P-40 and 0.5M 
NaCl. 
(c) 10 minutes with 100 ml of Buffer D. 
6. Blot the nitrocellulose sheet between two sheets of filter paper and 
then store the nitrocellulose sheet in a sealed plastic bag. 
7. To determine which antibodies and polypeptides had reacted, 
(a) Prepare an autoradiograph of the nitrocellulose sheet, using standard 
procedures known in the literature. 
(b) Compare the autoradiograph with a nitrocellulose sheet onto which 
VIII:C was transferred but which had been stained with Coomassie blue R250 
rather than reacted with monclonal antibody. 
These steps demonstrated that the following distinct antibodies had been 
raised. 
Four antibodies reacted with the polypeptide of M.sub.r =92,000, the 
M.sub.r =108,000 and larger polypeptides, one or the other of the 
polypeptides of M.sub.r =54,000 or 44,000 present in terminal thrombin 
digests, and no other polypeptides. This demonstrates the origin of these 
two latter polypeptides from the polypeptide of M.sub.r =92,000 as the 
result of thrombin cleavage. It also shows that two epitopes on the 
M.sub.r =92,000 polypeptide survive that cleavage, and that these epitopes 
are not present on the M.sub.r =79,000-80,000 doublet. 
A fifth antibody reacted with the doublet of M.sub.r =79,000-80,000, and 
with polypeptides of M.sub.r =108,000 and greater, but not with the 
polypeptide of M.sub.r =92,000 nor with any of the polypeptides present in 
terminal thrombin digests. This demonstrates that the M.sub.r 
=79,000-80,000 doublet possesses an epitope which is not present on the 
M.sub.r =92,000 polypeptide; and that the epitope is destroyed by thrombin 
digestion of the M.sub.r =79,000-80,000 doublet. 
The reaction profiles of these five antibodies indicate that the M.sub.r 
=92,000 and M.sub.r =79,000-80,000 polypeptides derive from a common 
precursor or precursors. 
A sixth antibody reacted only with polypeptides of M.sub.r =108,000 and 
greater. 
To prepare and store a biological preparation of one or more of these 
antibodies, the corresponding monoclonal IgG may be isolated from 
heparinized pooled ascites fluid immediately after collection or a frozen 
portion of the stored solution may be thawed. Regardless of whether fresh 
or frozen material is used, the solution is brought to 4.degree. C. and 
treated with an equal volume of phosphate buffered saline solution (PBS) 
(PBS: 1.6 g sodium phosphate, monobasic monohydrate; 8.4 g sodium 
phosphate, dibasic anhydrous; 61.4 g sodium chloride; water to 7 liters; 
pH=7.2). The diluted ascites is precipitated by dropwise addition with 
stirring at 4.degree. C. Centrifugations are preferably carried out at 
14,000 rpm for 60 minutes (30,000.times.g). The supernatant solution of 
ascites is precipitated twice more with SAS and the mixture of precipitate 
and supernatant liquid stirred and centrifuged in the same manner as in 
the first cycle. The pellets resulting from the third precipitation are 
resuspended in a volume of PBS equal to that of the diluted ascites fluid 
and then dialyzed exhaustively against PBS. Clots appearing in the 
dialysis bags are removed by centrifugation at 20.degree. C. The dialyzed 
IgG is adsorbed by stirring it with a 5% aqueous solution of aluminum 
hydroxide at room temperature and centrifuging at 20.degree. C. after 
adsorption. The adsorption treatment is repeated at least three more times 
using 2.5% aluminum hydroxide solution for each treatment after the first. 
The adsorbed IgG is brought to 4.degree. C. and reprecipitated once with 
SAS as described above. The precipitated pellets may be stored at 
-20.degree. C. until used. 
Two preferred procedures for purifying the monoclonal antibodies and 
maintaining biological preparations containing them are described in P. L. 
Ey et al., "Isolation of Pure IgG.sub.1, IgG.sub.2a, and IgG.sub.2b 
Immunoglobulins from Mouse Serum Using Protein A-Sepharose." 
Immunochemistry, Vol. 15, pp. 429-436; and in C. Bruck et al., "One-Step 
Purification of Mouse Monoclonal Antibodies from Ascitic Fluid by DEAE 
Affi-Gel Blue Chromatography." J. Immunological Methods, Vol. 53, pp. 
313-319 (1982). 
It should be understood by those skilled in the art that various 
modifications may be made in the present invention without departing from 
the spirit and scope thereof as described in the specification and defined 
in the appended claims.