CA2000861A1

CA2000861A1 - Removal of immunoreactive components from a sample

Info

Publication number: CA2000861A1
Application number: CA002000861A
Authority: CA
Inventors: Hans J. Hansen; Edward S. Newman
Original assignee: Immunomedics Inc
Current assignee: Immunomedics Inc
Priority date: 1988-10-26
Filing date: 1989-10-17
Publication date: 1990-04-26
Also published as: EP0440722A4; IL91948A; IL91948A0; EP0440722A1; WO1990004976A1; KR900701313A

Abstract

ABSTRACT OF THE DISCLOSURE
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 at least one antibody is immunoreactive with the at least one immunoreactive component to effect immunobinding thereof. The support is a fluorocarbon polymer, such as polyvinylidene fluoride. Such a process is especially useful in plasmapheresis, and in assays for analyte, wherein antibodies which may, cause false-positive or false-negative results are removed from the sample to be assayed.

Description

MOV~L O:P IMMUNORI~ACTI~E
COMPO~TS FROM A SAMPL13 This invention relates to the treatment of a sample to remo~e immunoreactive sub~tances therefrom.
More particularly, this invention relates to the removal of immunoreactive substances from blood and from samples to be assayed for analyte.
In accordance with an aQpect of the present invention, there i~ provided a process ~or removing at least one immunoreactive component from a sample which comprises contacting the sample with a~ least one antibody supported on a ~olid support. The solid support is a fluorocarbon polymer. The antibod~ is immunoreactive with the at least one immunoreactive component to effect immunobinding thereof.
In one embodiment, the proce~8 of the present invention is applicable to the removal of im~unoreactive substances from blood. In particular, the proces the present invention may be used in connection with a pla~mapheresis process.
Pla~mapheresis is employed for treating ~ human patient's blood. Thus, for example, in such a procedure ~lood is removed from a~patient and separated into cellular and fluid components with the cellular component~ being reintroduced into the ,;

, i,., .~ ~

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patient. The fluid portion of the blood may then be treatad prior to reintroducing the blood to the patient.
Thu~, in accordance with one embodiment, a fluid portion of blood i9 contacted with at :least one antibody which immunoreacts with the substance(s) to be removed from the blood to immunobind the immunoreactive s~bstance(s) to the antibody wherein at least one antibody i~ supported on a fluorocarbon polymer upport.
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 a~ a bispecific antibody) or a~ 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 monoc~onal antibody as used herein al30 encompasses an antibody fragment.
The an~ibody 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 recogni~es the sub3tance to be removed.
The supported antibody may be a single antibody or may be two or more antibodie~. The supported a~tibody may be employed for remo~ing a sin~le immunoreactiYe sub~tance from blood or ~wo or more immunoreactive gubstance3 from blood. Thu~, for example, the supported antibody may be a murine monoclonal antibod~ which is recognized by human anti-murine antibody tHAMA) and which recognize~

6~l.

carcinoembrionic antigen ~CEA), whereby both CEA and HAMA may be removed from blood.
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.
More particularly, the support is a fluorocarbon polymer having an atomic ratio of carbon to fluorine of from about Q.5 to about 2Ø As representative examples of such fluorocarbon polymers, there may be mentioned polytetrafluoroethylene, polychlorotrifluoroethylene, polyvinyl~luoride, and polyvinylidene fluoride, with polyvinylidene fluoride being preferred. Such polyvinylidenefluoride is sold under the mark Kynar.
Such fluorocarbon polymers are unsintered (the fluorocarbon polymer ha~ 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 form~ o~ treatment are pos~ible.
The antibody may be supported on the solid support by a variety of procedures, with a repre~ent~tive procedure being described in Example 1. Thus, in a preferred embodiment, the antibody is supported on the solid support by adsorption;
however, other technique~ may be employed.
This embodiment is applicable to removing a wide variety of immunoreacti~e sub~tanoes from blood.
Thus, for example, the process may be employed for remoYal of tumor-associated antigen~, such as CEA, alpha-fetoprotein ~AFP), human chorionic gonadotropin ~ I .

(hCG), colon-specific antigen-protein (CSAp), microbial organism~ and toxins (viruses, bacteria, endotoxins, ungi, parasites); other toxic substances ~poison~, venoms, etc. ); immune comple~es, etc.;
antibodie~, e.g., HAMA, etc.
This embodiment haq particular applicab.ility to removing HAMA and/or tumor marker antigens and also blood-pha~e~ of microorganisms and microbial toxins, as well a~ other to~ic sub~tance~ in the blood against which antibodie~ can be developed or are available. Thus, for exampl~, in treating a patient with a monoclonal antibody which recognize~ CEA for diagno~tic or therapeutic purposes, CEA circulating in the bloot may prematurely bind the monoclonal antibody employed for such treatment. By ~se o~ the present invention, the quantity of CEA present in the blood may be reduced.
Similarly, if the monoclonal antibody employed for diagnostic or therapeutic purpoqes is a murine monoclonal antibody, then any HAMA present in the blood may prematurely bind the murine monoclonal antibody. ~y employing the proce~s for removing immunoreactive substance~ from blood a~ herein above de~cribe~, the quantity of HAMA in the blood may be reduced.
A9 hereinabove indicated, if levels of both HAMA
and CEA in blood are such as to pre~ent a potential problem, a murine monoclonal antibody which reco~nize~ CEA may be employed as the supported antibody to reduce the quantities o~ both HAMA and CEA.
Thus, for example, NP-l or NP-3 murine anti-CEA
monoclonal antibody ma~ be employed for removing CEA
and HAMA (NP-l and NP-3 are described in Cancer Research Vol. 43 page~ 686-g2 Feb. 1983).

Similarly, if it is desired to remove AFP, ~he supported antibody would be one which recognizes AFP, prPferably a monoclonal antibody.
If it wer~ de~ired to remove both AFP and CEA, the supported antibody could be antibody which recognize~ AFP and antibody which recognizes CEA; for example, a hybrid antibody which recognizes both CEA
and AFP or two antibodie3 one o$ which recognize~ CEA
and the other of which recogniæes AFP. If such antibody or antibody mixture is a murine antibody, HAMA would also ba removed.
O~her antigen~ ~uch a~ of microor~anism~ and microbial products, toxic substance~, ~nd/or antibodies may be removed by an appropriate selection o~ the supported antibody which is deemed to be within the scope of tho~e skilled in the art from the teaching~ herein.
Similarly, if it wer~ desired to remove human anti-foreign species antibody other than HAMA, then 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 3ub~tance to be removed may be an immunoreactive complex; e.g., a complex o antibody and complement. In such a case, the supported antibody may be an anti-activated complement antibody. Thus, for example, in an autoimmune di~ease such as Lupu~ erythemsto~u~, i~mune complexes may be removed in acsordance with t~e present invention by use of supported anti-activa~ed complement an~ibody to prevent accumulation of the complex in the kidney.
In a preferred embodiment, the at lea~t one antibody ~upported on a fluorocarbon polymer support (preferably polyvinylidene fluoride such as sold under the mark KYNAR3, in parti~ulate form, is mixed with the fluid components of the blood. The supported antibody i~ generally employ,ed in an amount of from 5.0% to 30%, by weight, of the plasma which i9 treated. In most ca~e~, the amount i3 from 10% to 20%, by weight. The contact time i9 generally from 5 to 20 minute~. The supported antibody and plasma are then ~eparated rom each other under ~terile condition~ and the separated treated p:Lasma i~
reintroduced into a patient.
It i3 po~sible to combine this treatment with other treatment~ to be performed with respect to the patient's plasma.
In a representative treatment, whole '~lood is removed from a patient and separated into cellY and plasma; e.g, by centrifugation. The red blood cells are then tran~fused back to the patient.
The separated pla~ma may then be introduced into a plas~aphere~is bag~ including the appropria~e antibody or an~ibodies s~pported on finely divided fluorocarbon polymer. After ~uch treatment, the pla ma i~ separated from the supported antibody ~e.g., by centrifugation), and the treated pla3ma reinfused into the patient.
In accordance with another embodiment, there i~
provided a treatment kit or package which includes an antibody, as hercinabove de~cribed, ~upported on a fluorocarbon polymer support, a~ hereinabove described. In one embodiment 7 the reagent kit or package may include a pla~maphere~i~ bag having the supported antibody therein, whereby treatment may be acco~pli3hed by addin~ pla~ma to the bag.
In accordance with another embodiment of the pre~ent invention, the proce~ of the pre~ent invention may be used in connection with an assay for an analyte, wherein at lea3t one immunoreactive component is removed frvm the sampl~ to be as~ayed.
In particular9 this embodiment i~ applicable to the remo~al of human anti-foreign species antibody from a sample (e.g., human anti-murine antibodies (HAMA), h~lman anti-goat antibodie (~AGA), huma:n anti-rabbit antibodie~ ~HARA), etc.) Murine monoclonal antibodies (MAbs) ar~ being evaluated with increasing frequency as therapeutic agent~ either unmodified or as conjugates (drug~, radionuclides, toxin~ 9 etc.). Repeated in~ection of MAb3 induce human anti-murine antibodies ~HAMA) with high frequency. It has been found that HAMA can cause false-po~itive and false-negative result~ in sandwich assay~ 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 as~ay.
It has also been propo~ed t~ remove HAMA by the use of mouse ~onoclonal antibody ~uppor~ed on finely divided cellulose prior to an assay for creatine-kinase MB Isoenzyme, Clin Chem. Vol. 32/3 476-81 (198~
There ha~e also been reports of human anti-foreign species antibodies interfering with as~ays for other analytes.
For example, Vladita et al. JAMA 248/19 p.
2489-90 (1982) di~clo~e~ that human anti-goa~ and anti-bovine antibodies interfered with an hCG assay.
Thus~ in accordance with another embodiment of tbe present invention, there is provided an improvement in an as~ay for an analyte wherein prior tQ the a~say the ~ample to be as~ayed is contacted with at lea~t one antibody which is recognized by one 2 ~ 6 ~

or more human anti-foreign species antibody which interferes with the assay result~ (i.e., human anti-foreign species antibody which binds to the a~say binder or the analyte~ wherein the at least one antibody i~ ~upported on a fluorocarbon polymer support. The antibody which i~ support:ed on the fluorocarbon polymer support for pretreating the sample to be a~ayed i9 one which does not re~ognize the analyte to be assayed.
The human anti-foreign ~pecies antibody which is removed may be one or more of human antibodies raised in re~ponse to an~ibodies ~uch as goat antibody, rabbit antibody, horse antibody, ~imian antibody tfor example, baboon antibody), ~heep antibody, avian antibody (for example, chicken antibody), bovine antibody, murine antibody, etc. The iluman anti-foreign specie~ antibody could be a heterophile antibody. This embodiment has particular applicabili~y to the removal of human anti-murine antibody (HAMA).
The antibody which i~ ~upported on the ~olid ~upport i9 dependent upon the antibodies to be removed from the sampIe, prior to th~ assay. Thu~, for example, the an~ibody may be a human antibody, in which ca~e, human antibodies including human anti-foreign ~pecies antibodie~ may be removed from the ~ample.
I~ HAMA is to be removed from the sample, then the 3upported antibody may be a murine antibody.
If human anti-goat antibody (HAGA3 is to be removed, then the supported antibody may be a goat antibody.
Thu~, a~ should be apparent, the antibody on the support is dependent upon the human anti-foreign species antibody to be removed.

~-9 -In some ca~es, 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 di:Ef~rent anti-f~relgn qpecies antibodie~ to be removed.
Alternatively, two or ~ore different antibodie3 which are recognized by human antibodies elicited from antibodies of the same foreign species may be supported on the solid support.
For example, 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 mo~oclonal an~ibody or a polyclonal ~ntibody, or a chimeric antibody or a bispecific antibody, ~ometimes called a hybrid antibody, with a monoclonal antibody being preferred. Chimeric antibodies are described in Clin. Chem 34/9, 668-75 (1988).
Thus, for example, human anti-foreign species antibody may be elicited in respon~e to a chimeric antibody whereby a sample to be assayed, derived from a human9 which includes such elicited antibodies, may be ~reated with a supported chimeric antibody which i recognized by the different human anti-foreign species antibodies elicited in response to the chimeric antibody. Alternatively, two antibodie~ may be employed as the supported pretreatment reagent, one of which is an antibody derived from a firs~
foreign ~pecies portion of the chimeric antibody which elicited ~he human antibody to be removed and the other of which is derived from a second foreign species portion of the chimeric antibody which e~icited the human antibody to be re~oved. For example, human antibodies ellcited in response to anti-goat, anti-rabbit chimeric antibody may be i removed by the use of supported rabbit and goat antibody.
The antibody employed in the pretreatment is supported on a fluorocarbon polymr support as hereinabove described in connection with the embodiment wherein a fluid portion of blood i8 treated.
The murine antibody or a mixture thereof which is supported on the solid ~upport may be a polyclonal or a monoclonal antibody, preferably a monoclonal antibody. As hereinabove de~cribed, such supported antibody may be a chimeric or hybrid monoclonal antibody. The antibody which i9 employed i9 one which i9 recognized by the human antibody to be removed and whi.ch does not recognize the analyte to be determined in the ~ubsequent assay.
The antibody may be supported on the solid support by a variety of procedures a~ hereinabove described.
This embodiment will be further described with re~pect to a preferred embodiment for removing HAMA;
however, the ~eachings are equally applicable to removing other human anti-foreign species antibodies.
In accordance with a preferred embodiment, a murine antibody which i~ recognized by HAMA and which does not recognize and/or is not recognized by the analy~e to be assayedJ which i~ supported on a particulate fluorocarbon polymer support (preferably a murina monoclonal antibody supported on polyvinylidene fluoride) is contacted with a sample, such as a blood sample, and incubated at room temperature. The contact ti~e and quantity of the supported murine antibody is in an amount effective for removing HAMA from the sample. After the incubation, the particle~, which now have any HAMA

pr~sent in the ~ample bound thereto a~e separated from the ~ample whereby the sample may be ~ubject~d to an immunoass~y procedure, without potential interference by HAMA which may have b~!en pre~ent in the ~ample.
In the treatment, the supported antibody i~
generally employed in an amou~t of from lO.OZ to 30.0%, by weigh~, of the sample to be treated. The treatment may be generally accomplished in a period of from 5-20 minutes.
The human ~ample which i3 to be assayed is preferably blood or derived from blood (for example, serum or plasma); however other samples may be employed; e.g., ~amples derived from sputum, feces, 9pinal fluidl etc.
The pretreatment may be effected prior to an assay for a wide variety of analytes, ~uch as peptides, hormone~, ~teroids, proteins, haptens, viral antigens, etc. Representative analyte~ are hCG, CEA, TSH, LH, FSH, T4, hepatitis antigen, etc.
The present invention has particular applicability to assay~ for tumor marker~; e.g., alpha-fetoprotein (AFP), C~A, colon-specific anti~en-protein (CSAp);
hCG, prostatic acid pho~phatase (PAP) etc.
The murine antibodie~ employed in the assay procedure, as well as the procedure for removing immunoreactive sub~tances from blood, may be obtained by techniques known in the art. Thu9, for example, if a polyclonal antibody is employed, such polyclonal antibody i~ preferably an affinity-purified polyclonal antibody. Such affinity-purified polyclonal antibodies are available from Jackqon Research Laboratory in Avondale, Penn~yl~nia.
The murine antibody, as hereinabove described, is preferably a monoclonal antibody, and ~uch 6~.

monoclonal antibodies are either available in the art, or may be prepared by procedure~ known in the art.
As hereinabove indicated, 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 a~say values. Thus, for example, in removing HAMA from a sample to be assayed for CEA, the supported antibody may be a murine anti-alpha-fetoprotein monoclonal antibody or a murine anti-colon-~pecific 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 teachin~s herein.
After the pretreatment, the sample may be subJec~ed ~o an assay for determining the analyte by procedures known in the art. As he~einabove described, in an assay procedure which employs a murine monoclonal antibcdy which recognizes the analyte, the murine monoclonal antibody will also be recognized by HAMA, which can adversely affect assay r~sultq. By proceeding in accordance with the pre~ent invention, HAMA is removed prior to the assay and such removal i~ effected in a manner which does not interfere with analyte to be assayed.
The as~ay for determining the analyte may be a sandwich assay in which the binder for the analyte is a murine monoclonal antibody. In accordance with a preferred as~ay procedure, such murine monoclonal an~ibody 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.
In some cases, the assay procedure may be a eompetitiv~ assay procedure in which the analyte and a tracer compete for binding si.tes on a murine monoclonal antibody which recognizes the analyte and tracer. In such an assay, the presence of HAMA may adversely affect the assay results in that HAMA
recognize3 the binder u~ed i~ the assay. Thus, removal of HAMA prior to the competitiv~ a~say, a~
hereinabove described, prevents HAMA interference.
Thus, in accordance with an embodiment of the pre~ent invention, there is provided an improvement in an a~ay procedure wherein a ~ample to be as~ayed for an analyte is initially contacted with an antibody ~upported 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 result~.
rhus, for example, if the binding or trapping antibody to be used in the aqsay is a murine antibody, then HAMA may inte.fere with the as~ay, and such HAMA may b~ remov~d from the sample, prior to the as~ay, by use of a supported murine antibody. If the binding or ~rapping antibody ~o be u~ed in the assay i~ a rabbit antibody, then human anti-rabbit antibody (HARA~ may inte~fere with the assay, and such HARA may be removed from the sample, prior to the a~say, by u~e of a supported rabbit antibody, etc.
In accordance with another embodiment, there is provided an as~ay or reagent ~it which includes in a kit or reagent pac~age, an antibody, preferably a monoclonal antibody, ~upported on a fluorocarbon polymer suppor~ of ~he type hereinbove de~cribed.

The assay or resgent kit may also include a binder for the analy~e and a tracer for the analyte. The kit may further include additional reagent~ to be used in the as~ay~ su~h a~ buffer~ J standard~ etc.
Thu3, for example, in a kit employing a murine antibody a~ the binder for the a~say, the supported antibvdy or antibodies are murine antibody to remove HA~ . .
The use of antibodie~ supported on a fluorocarbon polymer supp~rt results in effective removal of variou~ immunoreactive ~ub~tance from a sample. The use of antibodie~ ~upported on a fluorocarbon support is of particular value in removillg human anti-foreign specie~ antibody, and in particular HAMA. A~ applied to an assay as hereinabove de~cribed, 8ch a procedure i~ an improvement over heat treat~nent in that some analy~es are adversely affected by heat. In addition such a procedure i9 an improvement over one u~ing antibody supported on a celluloYe support..
Further, the use of fluorocarbon polymer support in combination with the antibody offers the following ad~antage~: oriented ra~her than random attachment to the solid phase; rapid binding at room temperature; high capacity and does not leach antibody; good pelleting propertie~ at low g force;
inert, does not bind or trap analytes; no sample dilution due to minimal pellet volume; adhere3 well to gla~s for decanting ~nd draining with no loss; no special equip~ent i~ needed; total sa~ple pretreatment takes le~ than 30 minute~; supported antibody does not interfere in an assay for analy~e;
and is easily scaled down when the ~ample ~olume i~
limited.

.

Although cellulose supported antibody has been used to remove heterophilic antibody from serum to prevent interference in immunoa~say~, thi~ reagent is inferior to fluorocarbon (in particular polyvinylidene fluoride) ~upported anti.body for several reasons. Polyvinylidene fluori.de is hydrophobic while cellulose i9 hydrophi.lic. Thu~, the pelleted polyvinylidene fluoride contains minimal aqueous buffer to dilute the ~era while the cellulose pellet contain~ a large volume o aqueous buffer that dilutes the specimen. In addition, the amount of immunoglobulin that i8 bound per unit weight of polyvinylidene fluoride vs. cellulose is many fold the weight of immunoglobulin that can be coupled to cellulose. Thus, 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 in~ection of foreign antibody is normally at levels higher than that which could be removed by use of antibody support~d on cellulose.
.Antibody mu~t be covalently coupled to cellulose but is ad~orbed to polyvinylidene fluoride in it~
native state. Covalent coupling results in loss of many immunoglobulin sites due to ~teric blocking of these sites by the large cellulose polymer chains.
Cellulose supported antibody settle3 rapidly, which require~ shaking or rotation, wherea~
polyvi~ylidene fluoride ~upported antibody se~tles slowly and removes human-foreign species antibody without the need of shaking or rotation of the tubes.
The u~e of supported antibody is also an improvement over an attempt to use un~upported antibody. For example, the addi~ion of IgG to ~pecimens that contain lO0 ~gjml o HAMA results in the formation of immune complexes that crosslink the 8~.

-lB-enzyme labeled tracer to the solid pha~e. In a cGmmercial CEA assay, addition of ~ces~ive levels of a murine monoclonal antibody to the reagents still demon~trates false-positive re3ults whlen HAMA i~
pre~ent (a plasma containing 2.0 ng/ml of CEA gave a CEA result of 11.6 ng/ml). When it ~a~ attempted to prevent false-positiv~ re~ult~ by addition of exce~sive level of murine IgG 7 a plasma containing 2.0 ng/ml of CEA gave a fal~e-positive value of 87 ng/ml of CEA.
The present invention will be furth~r described with respect to the following examples; however, the scope of the invention i~ not to be limited thereby.

E~AMPLE 1 GENERAL PROCEDURE FOR SUPPORTED
ANTI~ODY PREPARATION
A ~uspension wa~ prepared by dispersing 2.0g of Kynar (un~intered vinylidene fluoride re~in powder, grade 301F Penwalt Corp~) in 100 ml of 2-propanol.
The su~pension wa~ 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 nor~al saline or phosphate of normal ~aline or buffered ~aline at pH 7.Q and stirred until dispersed. The floccules were allowed to settle out, wa~hed twice with P~S, and resuspended in PBS
containing 0.1% merthiolate to yield a 2% (w/v) suspension. Two and one half mg of murine antibody per ~ram of activated Kynar was added with stirring.
The mi~ture was homogenized by the POLYTRON as before ~tirred at room tempera~ure for 3-4 h a~d at 4C for a minimum of 12 h. The su~pension was wa~hed twice by PBS (pH 7.Q~ containing 0.1% merthiolate, and by centrifu~ation (1,500 x ~ for 10 min), re~uspended to , :-2% (w/v~ in PBS; 0.25 ml of 25% HSA p~r g of Kynar was added and the polytron ~tep repeated. Murine antibody-KYNAR ~uspension was ready to use or stored at 4C

E~AMPLE 2 Procedure or Plasmapheresis U3ing Supported Antibody.
1. Remo~e 400-1200 ml of blood into a pla mapheresis bag containing anticoagulant.
2. C~ntrifuge plasmapheresis bag to ~ep~rate cellular and fluid elements. Save cellular material for reinfusion.

3. Transfer the plasma into a second plasmaphoresi~
bag containing 100-500 ml of pelleted and decanted treatment material prepared as in Example 1 at lOX
concentration.

4. Di~perse treatment material throughout the plasma. Maintain the susp~nsion on a rotary shaker for 10 minute~ at room temperature.

5. Centrifuge the treated plasma, to pellet the treatment material, at 1500 rpm for 5-10 minutes at room temperature.

6. Take a S-10 ml ~ample of the treated plasma.
Mark the sample as post 1st. The treated plasma and cellular raction are infused back into patient.

7. After infu~ion has started, remove 400-1200 ml of blood and perform all the steps described above.
Steps 2-6.

8. A~er the econd infu~ion has started remove 400-1200 ml of blood and perform all of the ~tep~
de~cribed above. Step~ 2-6 are repeated until substaDce to be renoved is reduced to desired level.

2~ 6~

E~A~PLE 3 - GENERAL PROCEDURE FOR
PRETREATMENT OF SAMPLES FOR USE IN AN
IN VITRO ASSAY
1. 5.0 ml of a 2% Xynar murine IgG susp~n~ion prepared as in ~xample 1 wa~ pipetted into 13xlOO mm glass te~t tubes.
2. Centrifu~e at 2000 rpm, 10 min. at room temp~r~ture.
3. Decant and discard the supernatant - drain tubes well.
4. Add 1.0 ml of patient pla~ma to the Kynar murine I~G pellet.
5. Vortex to resu~pend the Kynar murine-IgG pallet~
6. Incubate at room temperature for 10-15 min.
7. Repeat ~tep 2.
8. Tske an aliyuot of the plasma for assay purposes or decant plasma. Plasma does not need to be separated from the Kynar pellet and can be kept at 4C. If Kynar pellet i~ disp~rsed, by accident, recentrifuge.

; A pla ma sample was treated as d~scribed in Example 3 wherein the Kynar murine IgG su~pen~ion was prepared as in Example 1 u~ing a murine anti-fetal protein monoclonal antibody as the murine IgG. The murine anti-fetal protein monoclonal antibody wa~
prepared by the procedure disclo~ed by Kohler and Mil~tein ~ature 256 p. 495 1975). Such loonoclonal antibody is a~ailabl~ at Immunomedics, Warren, N.J., and is called AFP-7-31.
The plasma sample is then sub~ected to a sandwick CEA assay as follows u~ing NP1 monoclonal antibody as a supported capture antibody and hor~eradi3h peroxidase labeled NP-3 monoclonal , , ~

.

-antibody as the tracer (NP-l and NP-3 monoclonal antibody are de~cribed in Cancer Research, Vol. 43 Page~ 686-92 Feb. 1983).

PREPARATION OF REAGENTS

1. Preparation of PBS-Polysorbate Completely dis301ve the content~ of one packet of PBS in one liter of glass-di~tilled or deionized water. Add 0.5 ml of Poly~orbate 20 and mi~. This solution is u~ed to wash plate~ and to dilute the con~ugate. PBS-Polysorbate is stable for one month when ~tored at room temperature. For lon~er storage ~up to two months), keep at 2C to 8C. Discard PBS-Polysorbate if viYibly contaminated.

2. Preparation of Specimen Diluent Prepare a 1:10 dilu~ion of specimen concentrate with PBS-Poly~orbate. For example, add 1 ml of specimen concentrate to 9 ml of PBS-Poly~orbate. Specimen diluent i~ stable for ~ight hours at room temperature.

3. Preparation of Tracer Di~pos~ble gla~s or pla~tic ware mu~t be u~ed. The tracer is diluted with PBS-Polysorbate ~ust prior to u~e.
A3qay Procedure ~g~

1. Appropriately label the plates. All sample~
~hould be run in duplicate.

2. Add 0.100 ml of CEA diluent to each well of a microtiter plate to be used in the as~ay. The wells of the microtiter plate are coated with the capture antibody.

3. Add 0.100 ml of each ~ample and each standard to the appropriate wells on the plate.

4. Incubate the plate~ at 34 to 37C in a moi~qt enviro~ment for 90 minutes.

5. Approximately 10 minutes before use, prepare the appropriate dilution(s) of the tracer in PBS
Poly~orbate 20.

6. Aspirate or decant the sample from the microtiter pIate at the end of incubation and ;~ wash the plate.

7. Add 0.200 ml diluted tracer9 to appropiate well~.
;

8. Incubate the plate at 34 to 37C in a moist environment as for 30 minutes.

. Prepare an~OPD substrate approximately 10 minutes before use. Store the substrate in a dark environment until u~e.

10. Di~card the tracer from the plate after incube~ion, and wa~h the plate.

36~.

11. Add 0 . 200 ml of substrate to a:Ll the wells including the well~ needed for blanking the plate reader.

12. Incubate the plates, uncovered, a~ 15 to 30C
in the dark for 30 minute~.

13. Stop rea~tion by adding 0 . 050 ml of 4N sulfuric acid to all the well~ co2ltaining substrate.

14. Using an appropriate microtitier plate r~sder, read and record the optical denqity at 488 to 492 nm within 10 minute~ of test completion. If the plate is not read immediately, it should be stored in the darlc for not longer than 30 min~tes.

The CEA value of the assay after pretreatment in accordance with the invention i~ 2.64 ng/ml.
The CE~ value of the same plasma sample withou~
pretreatment is 40 ng/ml.
The CEA value of the same plasma sample, which is sub~ected to heat treatmen~ prior to the as~ay, is 3.0 ng/ml.

EXAMPLE S
Examplc 4 was repeated e~cept that the Kynar ~-murine IgG suspQnsion wa~ prepared using murine anti-colon-specific antigen-protein (CSAp) monoclonal antibody. (The monoclonal antibody wa prepared by conventional hybridoma procedures and is available at Immunomedics and i~ called MU-9).:

.~ , ,.

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The CEA assay value~ were as follows:
1. No Pretreatment - 40 ng/ml.
2. Heat Pretreatment 3.0 ng/ml.
3. Pretreatment in accordance with the invention 3.88 ng/ml.
Numerous modifications and variations of the pre3ent invention are po~ible ~n light of the above teachings; therefore, within the scope of the appended claim~, the invention may be practiced otherwise than as particularly de~cribed.

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Claims

1. A process for removing at least one immunoreactive component from a sample, comprising:
contacting said sample with at least one antibody supported on a solid support, said at least one antibody being immunoreactive with aid at least one immunoreactive component to effect immunobinding thereof, said solid support being a fluorocarbon polymer.

2. The process of Claim 1 wherein the support is polyvinylidene fluoride.

3. The process of Claim 2 wherein the support is in particulate form.

4. The process of Claim 1 whreein said at least one antibody is a monoclonal antibody.

5. The process of Claim 4 wherein the at least one antibody is a murine monoclonal antibody.

6. The process of Claim 5 wherein the at least one antibody is a monoclonal antibody which recognizes a tumor marker.

7. The process of Claim 5 wherein the at least one antibody is a murine monoclonal antibody selected from the group consisting of murine anti-CEA
monoclonal antibody, murine anti-AFP monoclonal antibody, murine anti-colon-specific antigen protein monoclonal antibody, and murine anti-hCG monoclonal antibody.

8. The process of Claim 1 wherein said at least one antibody is an antibody mixture.

9. The process of Claim 1 wherein said at least one antibody is a bispecific antibody.

10. The process of Claim 1 wherein said at least one antibody is a chimeric antibody.

11. The process of Claim 1 wherein said at least one antibody is a hybrid antibody.

12. The process of Claim 1 wherein said at least one antibody is an antibody fragment.

13. A process for removing at least one immunoreactive component from blood, comprising:
contacting a fluid portion of blood with at least one antibody supported on a solid support, said at least one antibody being immunoreactive with said at least one immunoreactive component to effect immunobinding thereof, said solid support being a fluorocarbon polymer; and separating the fluid portion of blood from the at least one supported antibody.

14 The process of Claim 13 wherein the at least one antibody is an anti-activated complement antibody.

15. In plasmapheresis wherein blood is removed from a patient and separated into cellular and fluid components, the fluid components are treated and the cellular component and treated fluid component are reinfused into a patient, the improvement comprising:
treating a fluid portion of blood with at least one antibody supported on a solid support, said at least one antibody being immunoreactive with said at least one immunoreactive component to effect immunobinding thereof, said solid support being a fluorocarbon polymer; and separating the fluid portion of blood from the at least one supported antibody.

16. The process of Claim 15 wherein the treating is effected with two or more supported antibodies.

17. The process of Claim 15 wherein said treatment is effected with supported antibody in an amount of from 5.0% to 30.0% by weight of the fluid component.

18. A product comprising:
a plasmapheresis bag, and at least one antibody supported on a particulate support in said plasmapheresis bag, said particulate support being a fluorocarbon polymer.

19. The process of Claim 13 wherein said at least one antibody is an antibody against a toxic substance.

20. The process of Claim 13 wherein said at least one antibody is an antibody against a microbial organism or a microbial toxin.

21. In an assay for an analyte wherein in the assay the analyte is bound to an assay binder for the analyte, the improvement comprising:
pretreating a sample to be assayed for analyte with at least one antibody which is recognized by human antibody which binds to an assay binder to be used for assaying the analyte and which antibody does not recognize analyte to be assayed, said at least one antibody being supported on a fluorocarbon support.

22. The improvement of Claim 21 wherein the at least one antibody is a murine antibody which is recognized by HAMA.

23. The improvement of Claim 22 wherein the murine antibody is a monoclonal antibody.

24. The improvement of Claim 21 wherein the assay binder is a murine monoclonal antibody and the supported antibody is a murine monoclonal antibody.

25. The improvement of Claim 21 wherein said assay binder recognizes an antigen selected from the group consisting of CEA, AFP, and colon-specific antigen-protein.

26. The improvement of Claim 21 wherein in the supported antibody is selected from the group consisting of goat antibody, rabbit antibody, bovine antibody, simian antibody, horse antibody, murine antibody, sheep antibody, avian antibody and mixtures of one or more of said antibodies.

27. The improvement of Claim 21 wherein the assay binder is a monoclonal antibody.

28. The improvement of Claim 21 wherein the assay is a sandwich assay.

29. The improvement of Claim 21 wherein said assay binder recognizes at least one member selected from the groups consisting of tumor marker antigens, peptides, proteins, viral antigens, steroids, and haptens.

30. The improvement of Claim 29 wherein the assay binder recognizes a tumor marker antigen.

31. The improvement of Claim 21 wherein said at least one antibody is an antibody which recognizes a human heterophilic antibody.

32. The improvement of Claim 21 wherein the assay binder recognizes a member selected from the group consisting of hCG, PAP, TSH, LH, FSH, T4, and hepatitis antigen.

33. In a reagent kit for determining analyte, the improvement comprising:
an assay binder for the analyte; and at least one antibody supported on a solid support for pretreating sample to be assayed, said at least one antibody recognizing human antibody in a sample to be assayed which human antibody binds to the assay binder, said at least one antibody being an antibody which does not recognize analyte, said solid support being a fluorocarbon polymer support.

34. A composition, comprising:
a non-human monoclonal antibody supported on particulate polyvinylidene fluoride.

35. The composition of Claim 34 wherein the antibody is a murine monoclonal antibody.