Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
  • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
  • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
  • G01N2333/96433—Serine endopeptidases (3.4.21)
  • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
  • G01N2333/96444—Factor X (3.4.21.6)

Definitions

• This invention relates to a process to prepare factor X depleted plasma.
• this invention relates to a process to prepare factor X depleted plasma using a monoclonal antibody coupled to a solid phase.
• This invention also relates to a monoclonal antibody to factor X light chain.
• Factor X or Stuart factor is a vitamin K dependent clotting protein.
• Factor X has an approximate molecular weight of about 59,000 and is composed of a heavy chain (Mr 42,100) and a light chain (Mr 16,900) held together by a disulfide bond.
• Factor X participates in the middle phase of the intrinsic and extrinsic pathways of the blood coagulation cascade. In the intrinsic pathway, factor X is converted to factor Xa by proteolysis by factor LXa in the presence of factor Villa, calcium, and phospholipid. In the extrinsic pathways, factor X is converted to factor Xa by factor Vila in the presence of tissue factor. Factor Xa converts prothrombin to thrombin. Thrombin then converts fibrinogen to fibrin.
• factor Xa The cleavage of factor X to factor Xa is thought to occur at a specific Arg-He bond near the amino terminus of the factor X heavy chain. The light chain remains uncleaved. A tentative structure of factor X has been proposed. Leytres, Stephen
• the light chain contains the Gla domain and the potential growth factor domain. In addition, the light chain contains a number of g-carboxyglutamic acid residues and b- hydroxy aspartic acid residues.
• Factor X deficiency in humans causes various clotting abnormalities.
• Factor X deficiency may be either congenital or may be due to treatment with such drugs as coumadin or warfarin.
• Patients with an inherited deficiency of factor X express a variable bleeding pattern.
• the factor X deficiency is transmitted as an autosomal recessive trait. Severely decreased values (i.e. less than one percent factor X activity) are seen only in homozygotes and levels of less than about 20% are seen in heterozygotes.
• Factor X depleted or deficient plasma is a plasma which is suitable for coagulation investigations and is essentially free of factor X, but contains, in essentially normal concentrations, all the other clotting factors normally present in the plasma.
• a factor X deficient plasma is suitable for determining the activity of factor X in factor X containing fluids such as plasma.
• Factor X deficiency may be determined by such means as the prothrombin time (PT) test or the partial thromboplastin time (PTT) test because the PT and PTT are both prolonged when using such tests.
• PT prothrombin time
• PTT partial thromboplastin time
• a patient plasma is mixed with factor X-deficient plasma and the PT value is determined; the degree of correction of the PT is proportional to the level of factor X in the patient plasma.
• Functional factor X is quantitated using a dose-response curve obtained by plotting the log of the clotting time as a function of the log of a dilution of a calibrated reference plasma or fresh normal plasma pool.
• the assay must be sensitive to less than about one percent (1%) of factor X activity. In addition, the assay must be sensitive to a borderline normal percent factor X activity which is about fifty percent (50%) factor X activity. Much of the sensitivity of the assay is determined by the amount of factor X activity in the factor X deficient plasma. For example, if the factor X deficient plasma contains more than one percent (1%) of active factor X, the standard curve generated during an assay is not sensitive at less than about five to fifteen percent (5 - 15%) factor X activity. See, Figure 1. Thus, the residual factor X activity in the factor X deficient plasma is critical to the determination of factor X activity in plasma.
• Factor X deficient plasma is available in compositions having less than 1% factor X activity from Baxter Diagnostics Inc., Sigma Diagnostics and other sources.
• the composition available from Baxter Diagnostics Inc. is an immunoadsorbed factor X deficient plasma prepared by removing factor X from pooled normal human plasma with polyclonal antibodies
• the composition from Sigma is a lyophilized human plasma congenitally deficient in factor X.
• the polyclonal antibodies do not have a high of an affinity for factor X, thus more of the antibody must be utilized to strip the plasma.
• immunoadsorption columns prepared with cannot be regenerated more than four or five times without an unacceptable increase in the amount of factor X activity in the final preparation. Thus, the usefulness is limited.
• the antibody source is not constant and new sources of antibodies must continually be provided. Thus, the use of polyclonal antibodies is expensive and time consuming.
• Factor X deficient plasma obtained from donors whose plasma has less than one percent factor X activity is difficult , for obvious reasons, to obtain.
• Factor X monoclonal antibodies are known in the art. For example, Church, William R and Mann, Kenneth G., A Simple Purification of Human Factor X Using a High Affinity Monoclonal Antibody
• Immunoadsorban Thrombosis Research, 38:417-424 (1985) describe a monoclonal antibody (FX-2b) having an epitope expressed on the heavy chain of human factor X and factor Xa.
• the binding constant to factor Xa is 9xl0 M- 1 .
• the antibody is useful for purifying factor X when bound to an immunoadsorbant.
• the BFX2b antibody was prepared using the method described in Foster W.B. et al., Monoclonal antibodies selective for the functional states of bovine factor V and factor Va: Thrombosis Research 28: 649-662 (1982). See Church, W. et. al.
• FX52 and FX64 monoclonal antibodies that bind to factor X but not factor Xa were developed (FX52 and FX64). See, Hoad, Richard B. and Gez, Carolyn L., Characterization of monoclonal antibodies to factor X/Xa: Initial observations with a quantitative ELISA procedure: Journal of Immunological Methods 136: 269-278 (1991). In addition Hoad and Gez also developed monoclonal antibodies specific for factor Xa (FXa24). They found that the antibody FX52 may be useful to quantitate factor X in plasma and to capture factor X from plasma. Hoad and Gez reported that they were unable to produce monoclonal antibodies to factor X using conventional methods. Therefore, they used footpad immunizations and fusion of LNC.
• the antibody To be successful as an antibody to prepare a factor X deficient plasma, the antibody must have a low cross reactivity with other factors and must bind factor X. To be practicable, the binding of the factor X to the antibody should be reversible under conditions that are mild enough so that the antibody is not denatured. Thus, the factor X antibody coupled to a solid support should bind the factor X reversibly so after the plasma has been stripped, .the immunoadsorbant can be regenerated. Then, the factor X can be removed without denaturing the antibody and the immunoadsorbant can be reused.
• the factor X deficient plasma obtained from the immunoadsorption process should have less than one percent (1%) factor X activity. Moreover, the sensitivity of an assay using the factor X deficient plasma should be at less than one percent (1%) on a standard curve.
• a monoclonal antibody to factor X is used to prepare factor X depleted plasma.
• the present invention is a process to prepare a factor X deficient plasma from a normal starting plasma using immobilized monoclonal antibody against Factor X.
• the monoclonal antibody is immobilized onto a solid support.
• starting plasma is contacted with the solid support so that substantially all the factor X binds to the antibody to Factor X.
• the factor X depleted plasma is collected.
• the invention further comprises a factor X depleted plasma having less than about one percent (1% )factor X activity and can give an assay sensitivity of less than one percent (1%) of factor X activity on a standard curve.
• the invention also comprises monoclonal antibodies to the light chain of factor X.
• Figure 1 is a graph of the clotting times of serial dilutions from a normal plasma pool diluted in a buffer to give various % Factor X Activities as determined in a one stage-coagualtion assay for factor X using the factor X deficient plasma of this invention as the substrate.
• Figure 2 is a graph of the clotting times of the same samples tested in figure 1 except that the factor X deficient plasma substrate was spiked to give 1% residual factor X activity.
• Figure 3 is a graph of patient samples in a one-stage factor X assay using as a substrate the factor X deficient plasma of the present invention compared with a one-stage clotting assay of the same samples using as a substrate, a congential factor X deficient plasma .
• Figure 4 is a graph of patient samples in a one stage factor X assay using as a substrate, the factor X deficient plasma of the present invention compared with a one-stage clotting assay of the same samples using as a substrate, factor X deficient plasma commercially available from Baxter Diagnostics Inc.
• Monoclonal antibodies are prepared using standard techniques. See,
• the antibodies against human factor X are prepared by immunizing BALB/c mice with an effective amount of factor X in Freund's adjuvant or other suitable adjuvant, such as Hunter's Titermax using an intraperitoneal (IP) injection.
• the preferred amount of factor X is from about 5 to 50 micrograms and most preferably the amount of factor X is about 10 micrograms. If the amount of antigen is too high, the mice could develop an intolerance.
• Mice may be boosted by IP injection with a sufficient amount of human factor X in incomplete Freund's adjuvant or other suitable adjuvant.
• the amount of factor X is about 10 micrograms.
• the boost occurs about three weeks after the first immunization. Mice are boosted by IP injection once again after about two more weeks and then again after about a month with an effective amount of factor X in phosphate buffered saline or other suitable buffer.
• the amount of factor X is about 10 micrograms of factor X.
• the mice are boosted by IP injection with an effective amount of factor X.
• the mice are boosted daily with about 50 micrograms of factor X per day. Fusions are carried out using standard techniques. See, Harlow and
• Lane. Lymphocytes from the spleens of the nnmunized mice are fused with mouse myeloma cells.
• the type of mouse myeloma cell is not critical.
• a preferred mouse myeloma cell line is X-63-Ag8.653 also known as C5 mouse myeloma cells
• the fused cells are plated at about 2 x IO 6 cells using a media high in growth factors.
• a suitable media is Dulbecco's Modified Essential Media (DMEM) with about 20% of serums such as fetal bovine serum. It is preferable to add growth factors such as mitogens, macrophages or other suitable growth factors to stimulate cell growth.
• the plates are incubated under C0 2 at 37C.
• the cells may be tested when at least about 50% of the plate (e.g. microtitire well) is covered. Since numerous colonies are formed, a strict selection criteria is needed.
• the hybridomas are screened using an ELISA assay for factor X antibodies. Microtitre plates are coated with factor X at about 0.5 micrograms/mL to 8 micrograrns/mL, 1 microgram/mL is preferred.
• the buffer used to coat the plates contains calcium ion at about 4 to 40 mM, preferably about 15 mM.
• the preferred buffers are any buffer system that will buffer at pHs greater than about 7 pH units. See, Friefelder, D. Physical Biochemistry, Second Edition, W.H. Freeman and Company Chapter 4 (1982).
• the most preferred buffer is .01 M Tris pH 8.5.
• the ELISA may be carried out by adding a sample of the hybridoma supernatant, IgG/IgM conjugated enzyme and substrate to the enzyme, with any necessary washes occurring between steps. It may be convenient to add stop solution. Samples containing no supernatant (i.e. blanks or background) are also evaluated. Hybridomas giving absorbance values of greater than about twice the blank value in the ELISA assay are noted. Antibodies are further selected by evaluating the cross reactivity of the hybridoma with other factors.
• the antibodies from the hybridomas may be evaluated by coating microtitre plates with about 1 microgram/mL of other factors such as prothrombin, thrombin, fibrinogen, factor V, factor, XI, factor XII, factor IX , factor VII and factor VIII.
• Antibodies to factor X are tested in ELISA format for each antigen. Antibodies that have cross reactivity with these other blood coagulation factors are noted.
• the antibodies are subtype. They may be IgG or IgM. Preferred antibodies are of the IgG class and most preferably the IgGl subclass. Types and subtypes are noted.
• One or more limiting dilution study should be performed to ensure that there is only clone and that all of the clones are secretors of antibody.
• a limiting dilution study is performed by counting the cells and diluting the cells out to statistically one or fewer cells per microtitre well.
• Antibodies that are particularly suitable are designated Al.A; A 20.E; A 41.V; C 28.L; D 43.G; B 21.A; and H 28.C. These antibodies are all IgG subtype Gl, give absorbance values of greater than about seven times the blank in an ELISA assay and exhibit negligible to no cross reactivity with other factors.
• the P.I. of these antibodies under isoelectric focusing is about 5 to 6.5.
• affinity constants of about 10-° - lO- ⁇ M- 1 Thus, from a selection of more than 1000 hybridomas, the choice of an appropriate antibody can be narrowed to less than 10 antibodies.
• the antibodies are all evaluated for there ability to immunodeplete factor X from normal human plasma.
• the most preferred antibody is FX7- D43-G.
• the clones that are selected are injected into pristane primed female
• Ascites material may be collected using standard techniques. .
• the ascites may be reintroduced into tissue culture in 10% RPMI medium or other simple media.
• the antibodies may be purified from ascites using Protein A Sepharose (available from Bio-Rad). Binding Buffers and Elution Buffers, both commercially available from Bio-Rad, may be used in the purification.
• the ascites is applied using a binding buffer.
• the antibody elutes using an eluting buffer.
• the eluting buffer has a pH of about 3 to 5, preferably 5. Thus, the eluants are neutralized immediately.
• the purified material is buffer exchanged into a bicarbonate buffer and concentrated to about 4 to 5 mg/mL. Antibody at about 5 mg/mL protein is immobilized onto agarose.
• a preferred method of coupling the antibody is to use cyanogen bromide activated Sepharose. CL-4B.
• the Sepharose CL-4B may be activated with cyanogen bromide in a fume hood and a controlled temperature. See, Kohn and Wilchek, Biochemical and Biophysical Research Communications, 107 (3): 878-8
• the activated gel is washed with deionized water and resuspended in sodium bicarbonate buffer, pH 8.5, and immediately added to the antibody.
• the antibody is diluted in the same buffer. About 2 milligrams of antibody per gram of gel are combined. After the antibody has reacted with the gel at about 2-8°C for about 8 hours, the suspension is filtered and resuspended in about 1M ethanolamine at about pH 8.6. The suspension is rotated for about 2-4 hours at room temperature.
• the gel is recovered in a sintered glass funnel, and washed with a) 0.8 M sodium chloride, b) 0.1 M glycine, 0.5 M sodium chloride, pH 3.5, c) 0.1 M glycine, 0.5 M sodium chloride, pH 8.5 and 4) equilibration buffers such as 3 M glycine with about 1 M NaCl at about pH 9..
• the Anti-Factor X Coupled Sepharose CL-4B is suspended in buffers such as HEPES or the like (see, Freifelder).
• the preferred buffer is 10 mM HEPES, pH 7.3.
• the buffer may contain antipain and polybrene at 1.8 micrograms /mL.
• the gel is packed in a column.
• Immunoadsorbed factor X deficient plasma is prepared from citrated normal human plasma to which buffers and stabilizers have been added within two hours of collection.
• the factor X from about two hundred milliliters of plasma can be depleted using a gel column volume of about 20 mL.
• the plasma is passed through the column at slow flow rates. Preferably the flow rate is less than about .4 mL/minute but it may be as high as 0.7 mL/minute.
• the plasma is preferably pumped through the bottom of the column. Fractions are collected and the fractions that have an activity less than 1% , preferably about less than 0.5%, and most preferably less than about 0.3% are pooled.
• volume equal to about the first column volume are discarded and the next five to ten column volumes are collected and pooled.
• Glycine and sorbitol or other stabilizers may be added and the product is lyophilized.
• concentration of the other factors should be within normal range. Factors may be added if required. Preferably, it is not necessary to add any factors.
• the antibody that is most preferred is FX7-D43-G.
• the antibody reacts with the light chain of factor X. Normal human plasma immunodepleted with this antibody gives a factor X deficient plasma having less than 0.3% factor X activity and an assay performed using this deficient plasma is sensitivite at less than 1% factor X activity on a standard curve.
• the column can be regenerated by removing the bound factor X.
• the most preferred method to regenerate the column is to wash the column with three separate solutions.
• First the column is washed with a salt buffer with concentrations of NaCl or the like that are sufficient to remove any non specifically bound material.. Concentrations of less than about 1 M NaCl are adequate.
• Next the column is washed alternatively with high and low pH buffers to remove any proteins that are specifically bound to the column.
• the high pH buffer is preferably about 0.1 M glycine with about 0.5 M NaCl, pH 8 to 9, preferably pH 8.5.
• the low pH buffer is preferably about 0.1 M glycine with about 0.5 M NaCl, pH 3 to 4, preferably 3.5. Washing with the high and low buffers may be repeated.
• the gel may be stored in 10 mM HEPES, pH 7.0 or other suitable storage buffer. If the column is to be regenerated, the process should begin as soon as possible to avoid clot formation.
• the invention may be illustrated by the following examples, which are purely exemplary, and should not be taken as limiting the true scope of the present invention as described in the claims.
• mice Ten BALB/c mice were injected with about 12 micrograms of human factor X from Enzyme Research Laboratories in either Freund's complete adjuvant by intraperitoneal injection. Three weeks after the first boost, the mice that were injected with Freund's adjuvant were boosted by intraperitoneal injection with 12 micrograms of human factor X in Freund's incomplete adjuvant. All mice received two final boosts by intraperitoneal injection of about 12 micrograms of human factor X in phosphate buffered saline. Two of the mice were selected for fusion based on the titers. Both of the mice had the initial injection using Freund's adjuvant. Three days prior to fusion, both mice were injected by intraperitoneal injection with about 50 micrograms of human factor X per day.
• the spleens from the two mice were harvested, teased into a single cell suspension and counted.
• the spleen cells are washed in a serum free media.
• Mouse myeloma cells (C5) were collected and counted and the viability was determined. The viability of the myeloma cells was greater than at least 85%.
• the myeloma cells were washed in a serum free media
• About 4 x IO 8 spleen cells were added to about 4 x IO 8 myeloma cells.
• the total volume was about 30 milliliters.
• the cells were spun at 1200 rpms for about 10 minutes and the supernatant was removed.
• PEG 1500 in Whittaker RPMI 50% PEG 1500 in Whittaker RPMI was added with gentle mixing. Mixing was continued for a few more minutes. The cells were spun at 1200 rpms for about two minutes and then spun at 1500 rpms for about a minute. The PEG was removed and about 3 milliliters of serum was added. The cells were resuspended in the serum and incubated at about 37 C for about an hour.
• the complete media contained Dulbecco's Modified Essential Media with about 25 mM HEPES buffer and 2.5 g/L glucose with 2 mM L-glutamine, about .1% sodium bicarbonate, about 20% heat inactivated fetal bovine serum, about 1% glycine , about 1%HT Supplement, 1% GMS-S (Gibco Laboratories) and preservatives.
• the STM is a B-cell mitogen from S.lyphimurium for in vitro fusion and is available from RIBI Immunochem Research, Inc.
• the HT Supplement is commercially available from Whittaker and contains hypoxanthine (136 mg/dL) and thymidine (76 mg/dL). About 100 microliters of the fused material containing about 2-3 x IO 6 spleen cells were plated in a microtitre plates. The cells are incubated under C0 2 at 37° C. The cells are ready to be tested when at least 50% of the plated area is covered by cell growth. A sufficient amount of cell growth was seen after about two weeks. Screening of Hybridoma Colonies About 1000 colonies were generated. Each colony was evaluated in an ELISA assay. The assay was carried out by coating about 1 microgram/mL of human factor X onto ELISA plates using a coating buffer of about 0.01 M Tris at about pH 8.5 and containing about 15 mM calcium ion.
• Serial dilutions of the supernatant secreted from the colonies were prepared in about a 0.02 M Tris Buffer containing about 0.25 M NaCl at about pH 7.4 and also containing about 2% gelatin and 0.1% Tween-20 (TBS). A serial dilution of about 1:5 was found to be adequate. About 100 microliters of sample or blank control (i.e. buffer only) was added to each well and incubated for about 30 minutes. The plates were washed five to six times with a rinse buffer. The rinse buffer contains about 0.002 imidazole buffered saline with .02% Tween 20. Rinse buffers are commercially available from Kiekegaard & Perry Laboratories Inc. as Catalog No. 50-63-00.
• ABTS 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] Peroxidase Substrate System from Kirkegaard & Perry Laboratories Inc. Cat. NO. 50- 62-00.
• 100 microliters of a Stop Solution was added. Stop Solution for ABTS Peroxidase is commercially available from KirkEgaard & Perry Laboratories Inc. Catalog No. 50-85-01.
• the color development is usually adequate after about 10 minutes. The amount of color developed was read at 405 nanometers on a spectrophotometer.
• Results were compared with that of the blank control. Values that were at least two times the value of the blank control were considered positive. In addition, since the number of hybridomas was so high only those clones giving a value of more than seven times the blank were selected for further testing. Other cell lines were frozen down for storage.
• hybridomas Over 30 hybridomas were selected on the basis of the results from the ELISA. Each of these hybridomas were tested for cross-reactivity with the following antigens: Prothrombin (F1.2 fragment), fibrinogen, factor V, factor XI, factor XII, factor IX, factor VIII, and factor VII. Each of the antigens were coated at about 1 microgram/mL onto microtitre plates using the coating buffer. The assay procedure was carried out using the method and reagents described above. Of the hybridomas that were evaluated ten cross-reacted with the F 1.2 fragment of prothrombin and 2 of those ten cross-reacted with factor VII. These hybridomas were excluded from further evaluation.
• Hybridomas were subtyped using standard subtyping procedures, about twenty-five clones were evaluated. Nineteen clones were subtyped as IgGl K, two clones were IgG2a, two clones were IgG2b, and one clone was an IgM. Only the IgGl clones were selected for further evaluation. Of these clones two were excluded from further evaluation. One clone was excluded because of a slight cross-reactivity with other subtypes. The other clone was excluded because it appeared to be secreting an insufficient amount of antibody. Of these seventeen antibodies seven were selected because they had good overall growth and appeared to give good antibody production.
• the ascites for each clone was purified using a protein A, 10 mL column commercially available as Bio-Rad Affi-Prep Maps II Protein A. Binding Buffers and Elution Buffers are also available commercially from Bio-Rad. See, U.S. Patent No. 4,704,366.
• the column was equilibrated with six columns volumes of Binding Buffer. About 40 mgs (about 10 mLs of ascites fluid diluted 1:2 in Binding Buffer) of unpurified antibody were applied to the column. Elute was continually reapplied to the column using Binding Buffer for a total of two reapplications.
• the antibody was eluted using Elution Buffer and the fractions containing the antibody were collected and neutralized immediately using 50 microliters of 1 M Tris per 1 milliliter of collected fluid. Fractions containing the antibody were pooled. The pooled fractions were buffer exchanged into phosphate buffered saline at neutral pH for storage. The final concentration of protein was about 4 to 5 milligrams/milliliter. The final volume was about 6 to 6.5 milliliters. Isoelectric focusing was performed on each antibody using a Bio-
• the Phast gel type was IEF 3-9.
• the PI of each of the antibodies was between 5.1 and 6.5.
• Sepharose CL-4B gel was activated with cyanogen bromide in a fume hood using the procedure described by Kohn and Wilchek, Biochemical & Biophysical Research Communications, 107 (3) 878-884 (1982). An ultra low temperature (about -40 C) circulating bath was used to control temperature.
• the activated gel was washed with deionized water and resuspended in about 0.1 M sodium bicarbonate buffer at about pH 8.5.
• the antibody is buffer exchanged into about 0.1 M sodium bicarbonate buffer, pH about 8.5.
• the gel was immediately added to purified anti- factor X antibody at 2 milligrams of antibody per gram of gel.
• the gel antibody mixture was mixed overnight at about 2 to 8 C.
• the gel was separated by filtration and resuspended in about 1 M ethanolamine, pH about 8.6. The suspension was rotated for between about 2 to 4 hours at room temperature. The gel was recovered in a sintered glass funnel, and was washed in order with about five column volumes of about 0.8 M NaCl, then with about two column volumes of 0.1 M glycine having about 0.5 M NaCl at pH about 3.5, then with about two column volumes of 0.1 M glycine having about 0.5 M NaCl at pH about 8.5, and then with about five column volumes of 10 mM HEPES at about pH 7.0. The gel can be stored until required. The coupling efficiency was determined to be greater than seventy percent (70%).
• the anti-factor X antibody coupled to the gel was prepared into a column using about 100 mL of gel per liter of plasma.
• the column is equilibrated with about 10 mM HEPES pH about 7.3. Citrated plasma was added to the column .
• the lmear flow rate of the column was calculated by dividing the bed height by a residency time of at least thirty minutes.
• the plasma was eluted from the bottom of the column .
• the first column volume was discarded.
• the plasma from the next ten column volumes was collected. This plasma had a factor X activity less than 0.3%, although plasma less than 0.9% is acceptable.
• Stabilizers such as glycine (about 9 grams/liter) and sorbitol (about 9 grams/liter) were added.
• the factor X deficient plasma should be stored between 2-8 C.
• the plasma may be aliquoted into separate vials containing about 1 mL of the plasma . Then, the vials were lyophilized.
• the anti-factor X column was regenerated immediately after use.
• the column was washed with about six column volumes of about 0.8 M NaCl, then with three column volumes of a glycine buffer at pH 3.5 containing 0.1 M glycine, then with three column volumes of the glycine buffer at pH 8.5. The wash with high and low buffers may be repeated. The column was stored in 10 mM HEPES at pH 7.0 containing 0.1% sodium azide.
• Example 4
• % Factor Activity was determined using a Factor X clottin assay for the factors listed in Table 1 for each of the subclones of D43.G and compared with IP 10-31, an immunodepleted factor X deficient plasma prepared using polyclonal antibodies and with congenitally deficient plasmas. The PT and APTT were also measured.
• a Standard curve of a One-Stage Coagulation Assay for Factor X was determined.
• a normal plasma pool was diluted with Owner's buffer to give various % factor X activities, the one stage factor X clotting time of each diluted plasma was obtained by using factor X deficient plasma.
• the results are presented in Table 3 and Figure 1.
• Factor X was added to the factor X deficient plasma to give residual 1% factor X activity.
• the results are presented in Table 3 and Figure 2.
• a comparison of Figures 1 and Figure 2 shows the loss of sensitivity at the low end of the curve in Figure 2. Thus, it is important that the activity of factor X in the plasma is less than one percent.
• the figures showed high correlation between the monoclonal immunoadsorbed plasma and other commercial substrates for factor X.

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