Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/545—Synthetic resin
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase

Definitions

• This invention relates to the treatment of a sample to remove immunoreactive substances therefrom. More particularly, this invention relates to the removal of immunoreactive substances from blood and from samples to be assayed for analyte.
• a process for removing at least one immunoreactive component from a sample which comprises contacting the sample with at least one antibody supported on a solid support.
• the solid support is a fluorocarbon polymer.
• the antibody is immunoreactive with the at least one immunoreactive component to effect immunobinding thereof.
• the process of the present invention is applicable to the removal of immunoreactive substances from blood.
• the process of the present invention may be used in connection with a plasmapheresis process.
• Plasmapheresis is employed for treating a human patient's blood.
• blood is removed from a patient and separated into cellular and fluid components with the cellular components being reintroduced into the patient.
• the fluid portion of the blood may then be treated prior to reintroducing the blood to the patient.
• a fluid portion of blood is contacted with at least one antibody which immunoreacts with the substance(s) to be removed from the blood to immunobind the immunoreactive substance(s) to the antibody wherein at least one antibody is supported on a fluorocarbon polymer support.
• the antibody supported on the fluorocarbon polymer support may be a polyclonal antibody or a monoclonal antibody and the monoclonal antibody could also be employed as a hybrid form of monoclonal antibody (one which recognizes two different substances often referred to as a bispecific antibody) or as a chimeric monoclonal antibody.
• the antibody which is employed is dependent upon the immunoreactive substance to be removed from the blood.
• the term antibody or monoclonal antibo'dy as used herein also encompasses an antibody fragment.
• the antibody fragment may be an Fab portion of the antibody or a portion of the Fab portion, or any other portion of the antibody (including a single peptide chain) which is recognized by or recognizes the substance to be removed.
• the supported antibody may be a single antibody or may be two or more antibodies .
• the supported antibody may be employed for removing a single immunoreactive substance from blood or two or more immunoreactive substances from blood.
• the supported antibody may be a murine monoclonal antibody which is recognized by human anti-murine antibody (HAMA) and which recognizes carcinoembrionic antigen (CEA) , whereby both CEA and HAMA may be removed from blood.
• HAMA human anti-murine antibody
• CEA carcinoembrionic antigen
• the at least one antibody used in the treatment is supported on a fluorocarbon polymer support as described in United States Patent No. 3,843,443, which is incorporated herein by reference.
• the support is a fluorocarbon polymer having an atomic ratio of carbon to fluorine of from about 0.5 to about 2.0.
• fluorocarbon polymers there may be mentioned polytetrafluoroethylene, polychlorotrifluoroethylene, polyvinylfluoride, and polyvinylidene fluoride, with polyvinylidene fluoride being preferred.
• polyvinylidenefluoride is sold under the mark Kynar.
• fluorocarbon polymers are unsintered (the fluorocarbon polymer has not been heat treated at or above its crystaline melting point).
• the support is preferably in particulate form; however, other forms may be employed, e.g., as a tube, sheet, etc.
• the particles having antibody supported thereon are preferably added to the sample; however, other forms of treatment are possible.
• the antibody may be supported on the solid support by a variety of procedures , with a representative procedure being described in Example 1.
• the antibody is supported on the solid support by adsorption; however, other techniques may be employed.
• This embodiment is applicable to removing a wide variety of immunoreactive substances from blood.
• the process may be employed for removal of tumor-associated antigens, such as CEA, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) , colon-specific antigen-protein (CSAp), microbial organisms and toxins (viruses, bacteria, endotoxins, fungi, parasites); other toxic substances (poisons, venoms, etc.); immune complexes, etc.; antibodies, e.g., HAMA, etc.
• tumor-associated antigens such as CEA, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) , colon-specific antigen-protein (CSAp), microbial organisms and toxins (viruses, bacteria, endotoxins, fungi, parasites); other toxic substances (poisons, venoms, etc.); immune complexes, etc.; antibodies, e.g., HAMA,
• This embodiment has particular applicability to removing HAMA and/or tumor marker antigens and also blood-phases of microorganisms and microbial toxins , as well as other toxic substances in the blood against which antibodies can be developed or are available.
• CEA circulating in the blood may prematurely bind the monoclonal antibody employed for such treatment.
• the quantity of CEA present in the blood may be reduced.
• the monoclonal antibody employed for diagnostic or therapeutic purposes is a murine monoclonal antibody
• any HAMA present in the blood may prematurely bind the murine monoclonal antibody.
• the quantity of HAMA in the blood may be reduced.
• a murine monoclonal antibody which recognizes CEA may be employed as the supported antibody to reduce the quantities of both HAMA and CEA.
• NP-1 or NP-3 murine anti-CEA monoclonal antibody may be employed for removing CEA and HAMA (NP-1 and NP-3 are described in Cancer Research Vol. 43 pages 686-92 Feb. 1983).
• the supported antibody would be one which recognizes AFP, preferably a monoclonal antibody.
• the supported antibody could be antibody which recognizes AFP and antibody which recognizes CEA; for example, a hybrid antibody which recognizes both CEA and AFP or two antibodies one., of which recognizes CEA and the other of which recognizes AFP. If such antibody or antibody mixture is a murine antibody, HAMA would also be removed.
• antigens such as of microorganisms and microbial products, toxic substances, and/or antibodies may be removed by an appropriate selection of the supported antibody which is deemed to be within the scope of those skilled in the art from the teachings herein.
• an antibody preferably a monoclonal antibody, which is recognized by the human anti-foreign species antibody, would be employed as the supported antibody.
• the immunoreactive substance to be removed may be an immunoreactive complex; e.g., a complex of antibody and complement.
• the supported antibody may be an anti-activated complement antibody.
• immune complexes may be removed in accordance with the present invention by use of supported anti-activated complement antibody to prevent accumulation of the complex in the kidney.
• the at least one antibody supported on a fluorocarbon polymer support preferably polyvinylidene fluoride such as sold under the mark KYNAR
• a fluorocarbon polymer support preferably polyvinylidene fluoride such as sold under the mark KYNAR
• the supported antibody is generally employed in an amount of from 5.0% to 30%, by weight, of the plasma which is treated. In most cases, the amount is from 10% to 20%, by weight.
• the contact time is generally from 5 to 20 minutes.
• the supported antibody and plasma are then separated from each other under sterile conditions and the separated treated plasma is reintroduced into a patient.
• whole blood is removed from a patient and separated into cells and plasma; e.g, by centrifugation.
• the red blood cells are then transfused back to the patient.
• the separated plasma may then be introduced into a plasmapheresis bag, including the appropriate antibody or antibodies supported on finely divided fluorocarbon polymer. After such treatment, the plasma is separated from the supported antibody (e.g., by centrifugation) , and the treated plasma reinfused into the patient.
• a treatment kit or package which includes an antibody, as hereinabove described, supported on a fluorocarbon polymer support, as hereinabove described.
• the reagent kit or package may include a plasmapheresis bag having the supported antibody therein, whereby treatment may be accomplished by adding plasma to the bag.
• the process of the present invention may be used in connection with an assay for an analyte, wherein at least one immunoreactive component is removed from the sample to be assayed.
• this embodiment is applicable to the removal of human anti-foreign species antibody from a sample (e.g., human anti-murine antibodies (HAMA), human anti-goat antibodies (HAGA) , human anti-rabbit antibodies (HARA) , etc.)
• Murine monoclonal antibodies are being evaluated with increasing frequency as therapeutic agents either unmodified or as conjugates (drugs, radionuclides, toxins, etc.). Repeated injection of MAbs induce human anti-murine antibodies (HAMA) with high frequency. It has been found that HAMA can cause false-positive and false-negative results in sandwich assays for carcinoembryonic antigen (CEA) (JNM, 28 . 615, 1987 and Clin. Chem 34/2 261-64, 1988). As a result, it has been proposed to destroy HAMA with a simple heat treatment prior to an assay.
• CEA carcinoembryonic antigen
• an improvement in an assay for an analyte wherein prior to the assay the sample to be assayed is contacted with at least one antibody which is recognized by one or more human anti-foreign species antibody which interferes with the assay results (i.e., human anti-foreign species antibody which binds to the assay binder for the analyte) wherein the at least one antibody is supported on a fluorocarbon polymer support.
• the antibody which is supported on the fluorocarbon polymer support for pretreating the sample to be assayed is one which does not recognize the analyte to be assayed.
• the human anti-foreign species antibody which is removed may be one or more of human antibodies raised in response to antibodies such as goat antibody, rabbit antibody, horse antibody, simian antibody (for example, baboon antibody), sheep antibody, avian antibody (for example, chicken antibody), bovine antibody, murine antibody, etc.
• the human anti-foreign species antibody could be a heterophile antibody. This embodiment has particular applicability to the removal of human anti-murine antibody (HAMA).
• the antibody which is supported on the solid support is dependent upon the antibodies to be removed from the sample, prior to the assay.
• the antibody may be a human antibody, in which case, human antibodies including human anti-foreign species antibodies may be removed from the sample.
• the supported antibody may be a murine antibody.
• the supported antibody may be a goat antibody.
• the antibody on the support is dependent upon the human anti-foreign species antibody to be removed.
• more than one human anti-foreign species antibody may be removed, in which case, different antibodies may be supported on the solid support which are recognized by the different anti-foreign species antibodies to be removed.
• two or more different antibodies which are recognized by human antibodies elicited from antibodies of the same foreign species may be supported on the solid support.
• two different murine monoclonal antibodies may be supported on the solid support for removing HAMA from a sample prior to an assay.
• the supported antibody may be a monoclonal antibody or a polyclonal antibody, or a chimeric antibody or a bispecific antibody, sometimes called a hybrid antibody, with a monoclonal antibody being preferred. Chimeric antibodies are described in Clin. Chem 34/9, 668-75 (1988).
• human anti-foreign species antibody may be elicited in response to a chimeric antibody whereby a sample to be assayed, derived from a human, which includes such elicited antibodies, may be treated with a supported chimeric antibody which is recognized by the different human anti-foreign species antibodies elicited in response to the chimeric antibody.
• two antibodies may be employed as the supported pretreatment reagent, one of which is an antibody derived from a first foreign species portion of the chimeric antibody which elicited the human antibody to be removed and the other of which is derived from a second foreign species portion of the chimeric antibody which elicited the human antibody to be removed.
• human antibodies elicited in response to anti-goat, anti-rabbit chimeric antibody may be removed by the use of supported rabbit and goat antibody.
• the antibody employed in the pretreatment is supported on a fluorocarbon polymer support as hereinabove described in connection with the embodiment wherein a fluid portion of blood is treated.
• the murine antibody or a mixture thereof which is supported on the solid support may be a polyclonal or a monoclonal antibody, preferably a monoclonal antibody. As hereinabove described, such supported antibody may be a chimeric or hybrid monoclonal antibody.
• the antibody which is employed is one which is recognized by the human antibody to be removed and which does not recognize the analyte to be determined in the subsequent assay.
• the antibody may be supported on the solid support by a variety of procedures as hereinabove described.
• a murine antibody which is recognized by HAMA and which does not recognize and/or is not recognized by the analyte to be assayed which is supported on a particulate fluorocarbon polymer support (preferably a murine monoclonal antibody supported on polyvinylidene fluoride) is contacted with a sample, such as a blood sample, and incubated at room temperature.
• a sample such as a blood sample
• the contact time and quantity of the supported murine antibody is in an amount effective for removing HAMA from the sample.
• the particles, which now have any HAMA present in the sample bound thereto are separated from the sample whereby the sample may be subjected to an immunoassay procedure, without potential interference by HAMA which may have been present in the sample.
• the supported antibody is generally employed in an amount of from 10.0% to 30.0%, by weight, of the sample to be treated.
• the treatment may be generally accomplished in a period of from 5-20 minutes.
• the human sample which is to be assayed is preferably blood or derived from blood (for example, serum or plasma) ; however other samples may be employed; e.g., samples derived from sputum, feces , spinal fluid, etc.
• the pretreatment may be effected prior to an assay for a wide variety of analytes , such as peptides, hormones, steroids, proteins, haptens , viral antigens, etc.
• analytes are hCG, CEA, TSH, LH, FSH, T, , hepatitis antigen, etc.
• the present invention has particular applicability to assays for tumor markers; e.g., alpha-fetoprotein (AFP), CEA, colon-specific antigen-protein (CSAp); hCG, prostatic acid phosphatase (PAP) etc.
• the murine antibodies employed in the assay procedure, as well as the procedure for removing immunoreactive substances from blood may be obtained by techniques known in the art.
• a polyclonal antibody is employed, such polyclonal antibody is preferably an affinity-purified polyclonal antibody.
• affinity-purified polyclonal antibodies are available from Jackson Research Laboratory in Avondale, Pennsylvania.
• the murine antibody as hereinabove described, is preferably a monoclonal antibody, and such monoclonal antibodies are either available in the art, or may be prepared by procedures known in the art.
• the antibody which is employed is preferably a monoclonal antibody and such monoclonal antibody should not cross-react with the analyte to be assayed to prevent a change in assay values.
• the supported antibody may be a murine anti-alpha-fetoprotein monoclonal antibody or a murine anti-colon-specific antigen- protein monoclonal antibody. If the assay is for an analyte such as alpha-fetoprotein, then the supported antibody may be a murine anti-CEA monoclonal antibody. The selection of a particular supported antibody is deemed to be within the scope of those skilled in the art from the teachings herein.
• the sample may be subjected to an assay for determining the analyte by procedures known in the art.
• an assay procedure which employs a murine monoclonal antibody which recognizes the analyte
• the murine monoclonal antibody will also be recognized by HAMA, which can adversely affect assay results.
• HAMA is removed prior to the assay and such removal is effected in a manner which does not interfere with analyte to be assayed.
• the assay for determining the analyte may be a sandwich assay in which the binder for the analyte is a murine monoclonal antibody.
• the binder for the analyte is a murine monoclonal antibody.
• murine monoclonal antibody is supported on a solid support.
• the tracer employed in such a sandwich assay may or may not be a monoclonal antibody and may or may not be a murine antibody.
• the assay procedure may be a competitive assay procedure in which the analyte and a tracer compete for binding sites on a murine monoclonal antibody which recognizes the analyte and tracer.
• the presence of HAMA may adversely affect the assay results in that HAMA recognizes the binder used in the assay.
• removal of HAMA prior to the competitive assay, as hereinabove described prevents HAMA interference.
• an improvement in an assay procedure wherein a sample to be assayed for an analyte is initially contacted with an antibody supported on a fluorocarbon polymer support to remove from the sample any human anti-foreign species antibody which may be present in the sample, and which may affect assay results.
• HAMA may intefere with the assay, and such HAMA may be removed from the sample, prior to the assay, by use of a supported murine antibody.
• HAMA human anti-rabbit antibody
• HARA may be removed from the sample, prior to the assay, by use of a supported rabbit antibody, etc.
• an assay or reagent kit which includes in a kit or reagent package, an antibody, preferably a monoclonal antibody, supported on a fluorocarbon polymer support of the type hereinbove described.
• the assay or reagent kit may also include a binder for the analyte and a tracer for the analyte.
• the kit may further include additional reagents to be used in the assay, such as buffers, standards, etc.
• additional reagents to be used in the assay such as buffers, standards, etc.
• the supported antibody or antibodies are murine antibody to remove HAMA.
• the use of antibodies supported on a fluorocarbon polymer support results in effective removal of various immunoreactive substances from a sample.
• the use of antibodies supported on a fluorocarbon support is of particular value in removing human anti-foreign species antibody, and in particular HAMA.
• a procedure is an improvement over heat treatment in that some analytes are adversely affected by heat.
• such a procedure is an improvement over one using antibody supported on a cellulose support.
• fluorocarbon polymer support in combination with the antibody offers the following advantages: oriented rather than random attachment to the solid phase; rapid binding at room temperature; high capacity and does not leach antibody; good pelleting properties at low g force; inert, does not bind or trap analytes; no sample dilution due to minimal pellet volume; adheres well to glass for decanting and draining with no loss; no special equipment is needed; total sample pretreatment takes less than 30 minutes; supported antibody does not interfere in an assay for analyte; and is easily scaled down when the sample volume is limited.
• cellulose supported antibody has been used to remove heterophilic antibody from serum to prevent interference in immunoassays, this reagent is inferior to fluorocarbon (in particular polyvinylidene fluoride) supported antibody for several reasons.
• Polyvinylidene fluoride is hydrophobic while cellulose is hydrophilic.
• the pelleted polyvinylidene fluoride contains minimal aqueous buffer to dilute the sera while the cellulose pellet contains a large volume of aqueous buffer that dilutes the specimen.
• the amount of immunoglobulin that is bound per unit weight of polyvinylidene fluoride vs. cellulose is many fold the weight of immunoglobulin that can be coupled to cellulose.
• the use of cellulose supported antibody may not be sufficient to remove an antibody which one desires to remove; e.g., the level of HAMA induced by injection of foreign antibody is normally at levels higher than that which could be removed by use of antibody supported on cellulose.
• Antibody must be covalently coupled to cellulose but is adsorbed to polyvinylidene fluoride in its native state. Covalent coupling results in loss of many immunoglobulin sites due to steric blocking of these sites by the large cellulose polymer chains.
• Cellulose supported antibody settles rapidly, which requires shaking or rotation, whereas polyvinylidene fluoride supported antibody settles slowly and removes human-foreign species antibody without the need of shaking or rotation of the tubes.
• the use of supported antibody is also an improvement over an attempt to use unsupported antibody.
• the addition of IgG to specimens that contain 100 ⁇ g/ml of HAMA results in the formation of immune complexes that crosslink the enzyme labeled tracer to the solid phase.
• addition of excessive levels of a murine monoclonal antibody to the reagents still demonstrates false-positive results when HAMA is present (a plasma containing 2.0 ng/ml of CEA gave a CEA result of 11.6 ng/ml).
• a plasma- containing 2.0 ng/ml of CEA gave a false-positive value of 87 ng/ml of CEA.
• EXAMPLE 1 GENERAL PROCEDURE FOR SUPPORTED ANTIBODY PREPARATION A suspension was prepared by dispersing 2.0g of Kynar (unsintered vinylidene fluoride resin powder, grade 301F Penwalt Corp.) in 100 ml of 2-propanol. The suspension was homogenized by a Brinkman POLYTRON for 5 min at a pulse-frequency of 4000 c.p.s., transferred to a cylinder containing a liter of normal saline or phosphate of normal saline or buffered saline at pH 7.0 and stirred until dispersed.
• Kynar unsintered vinylidene fluoride resin powder, grade 301F Penwalt Corp.
• the suspension was homogenized by a Brinkman POLYTRON for 5 min at a pulse-frequency of 4000 c.p.s., transferred to a cylinder containing a liter of normal saline or phosphate of normal saline
• the floccules were allowed to settle out, washed twice with PBS, and resuspended in PBS containing 0.1% merthiolate to yield a 2% (w/v) suspension.
• Two and one half mg of murine antibody per gram of activated Kynar was added with stirring.
• the mixture was homogenized by the POLYTRON as before stirred at room temperature for 3-4 h and at 4°C for a minimum of 12 h.
• the suspension was ashed twice by PBS (pH 7.0) containing 0.1% merthiolate, and by centrifugation (1,500 x g for 10 min), resuspended to 2% (w/v) in PBS; 0.25 ml of 25% HSA per g of Kynar was added and the polytron step repeated.
• Murine antibody-KYNAR suspension was ready to use or stored at 4°C.
• Plasma does not need to be separated from the Kynar pellet and can be kept at 4°C. If Kynar pellet is dispersed, by accident, recentrifuge.
• a plasma sample was treated as described in Example 3 wherein the Kynar murine IgG suspension was prepared as in Example 1 using a murine anti-fetal protein monoclonal antibody as the murine IgG.
• the murine anti-fetal protein monoclonal antibody was prepared by the procedure disclosed by Kohler and Milstein (Nature 256 p. 495 1975). Such monoclonal antibody is available at Immunomedics, Warren, N.J. , and is called AFP-7-31.
• the plasma sample is then subjected to a sandwich CEA assay as follows using NPl monoclonal antibody as a supported capture antibody and horseradish peroxidase labeled NP-3 monoclonal antibody as the tracer (NP-1 and NP-3 monoclonal antibody are described in Cancer Research. Vol. 43 Pages 686-92 Feb. 1983).
• PBS-Polysorbate Completely dissolve the contents of one packet of PBS in one liter of glass-distilled or deionized water. Add 0.5 ml of Polysorbate 20 and mix. This solution is used to wash plates and to dilute the conjugate. PBS-Polysorbate is stable for one month when stored at room temperature. For longer storage (up to two months), keep at 2°C to 8°C. Discard PBS-Polysorbate if visibly contaminated.
• Disposable glass or plastic ware must be used.
• the tracer is diluted with PBS-Polysorbate just prior to use.
• Assay Procedure 1 Appropriately label the plates. All samples should be run in duplicate.
• microtitier plate reader uses an appropriate microtitier plate reader to read and record the optical density at 488 to 492 nm within 10 minutes of test completion. If the plate is not read immediately, it should be stored in the dark for not longer than 30 minutes.
• the CEA value of the assay after pretreatment in accordance with the invention is 2.64 ng/ml.
• the CEA value of the same plasma sample without pretreatment is 40 ng/ml.
• the CEA value of the same plasma sample, which is subjected to heat treatment prior to the assay, is 3.0 ng/ml.
• Example 4 was repeated except that the Kynar murine IgG suspension was prepared using murine anti-colon-specific antigen-protein (CSAp) monoclonal antibody.
• CSAp murine anti-colon-specific antigen-protein
• the monoclonal antibody was prepared by conventional hybridoma procedures and is available at Immunomedics and is called MU-9).
• the CEA assay values were as follows:

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