Methods for reducing background binding in antibody preparations

In accordance with the present invention, there are provided novel methods and reagents for reducing background binding in antibody preparations having an unwanted affinity for intracellular protein(s). The invention method comprises treating an antibody preparation with permeabilized cells, then separating the antibody preparation from the permeabilized cells. The reagents are useful for reducing the level of background binding in antibody preparations. Also provided are antibody preparations that are substantially free of background binding.

FIELD OF THE INVENTION 
The present invention relates to methods and reagents for reducing the 
level of background binding in antibody preparations having unwanted 
affinity for intracellular protein(s). The invention also relates to 
antibody preparations that are substantially free of background binding. 
BACKGROUND OF THE INVENTION 
Immunological applications that demand specific binding between antibody 
and antigen may be compromised by the presence of high concentrations of 
other macromolecules. For example, a good polyclonal serum will contain 
multiple antibodies directed against different epitopes on an antigen. In 
addition to specific, desirable antibodies, polyclonal sera generally also 
contain relatively high concentrations of irrelevant antibodies of unknown 
specificity. These will include the entire repertoire of antibodies in the 
animal when the serum was collected. These antibodies create troublesome 
problems in applications such as, for example, efforts to detect specific 
antigen/antibody binding on cultured cells and tissue sections. The 
presence of unwanted binding by antibodies of unknown specificity can make 
it difficult or impossible to detect specific binding by the antibody of 
interest. This unwanted binding is referred to as "background." 
For example, background binding is very high in polyclonal antibodies 
raised in rabbits against the reporter gene product, firefly luciferase. 
Reporter genes are routinely used in cell biology for the study of gene 
function and related cellular events. The gene encoding firefly luciferase 
has proven to be highly effective for this purpose. This enzyme catalyzes 
the oxidation of beetle luciferin with the concomitant production of a 
photon. The firefly luciferase gene is a widely used reporter gene because 
the luciferase protein: (1) produces light with the highest quantum 
efficiency known for any chemiluminescent reaction; (2) remains 
intracellular; and (3) requires no post-translational processing for 
enzyme activity and can therefore function immediately upon translation. 
At this time, the only method for reliable detection of luciferase is to 
monitor the light produced by the enzyme. While this type of assay 
provides information about gene function and relative rates of 
transfection, it provides no information about specific transfection 
efficiency, which is critical information in many instances. An antibody 
preparation free from background-creating antibodies, that could reliably 
and specifically detect the gene product luciferase in a wide variety of 
species and tissues, could provide this information. At this time, no such 
antibody preparation exists. 
Methods for removing background-creating contaminants from antibody 
preparations typically are based on differences in physicochemical 
attributes such as electric charge, size and shape, hydrophobicity, and/or 
binding specificity. Most purification methods are based on 
chromatographic separations that are designed to produce specific 
antibodies. Although effective, the development of these methods is often 
very time consuming and hence, costly, because, for example, they require 
the empirical development of the requisite conditions for achieving the 
separation of product and contaminant antibodies. 
For example, ion exchange chromatography is used to separate contaminants 
from antibody preparations based on differences in charge. The electric 
charge of proteins and antibodies is conferred by free side chain groups 
present on arginine, aspartic acid, cysteine, glutamic acid, histidine, 
lysine, and tyrosine. The net charge is low but sufficient to enable 
interaction with the charged side groups of ion exchange resins. 
Resolution of antibodies and contaminants is based on differences in 
charge and chromatography pH. Operation at pHs above or below the 
isoelectric point may enable acceptable separation of product from 
background antibodies. In this method, a protein-containing solution is 
passed over or through appropriate ion exchange material at a solution pH 
which facilitates binding of the desired protein. This is commonly 
followed by a washing step and an elution step at a different ionic 
strength or pH, which facilitates the release of the protein. In general, 
altering the pH of a protein towards its isoelectric point causes it to 
lose net charge and elute from an ion exchanger. One drawback of using ion 
exchange chromatography to purify antibodies is that the pH required for 
distinguishing between product and contaminant charge may lie outside the 
stable range of the antibody. Moreover, a key disadvantage of ion exchange 
chromatography is that the separation process may be very slow because of 
slow intraparticle diffusion through traditionally used porous supports. 
See, for example, Papoutsakis et al., Recombinant DNA Technology and 
Applications, 357 (New York 1991). 
Affinity chromatography is a separation technique based on the highly 
selective binding exhibited by many biological molecules to ligands. As 
the product stream flows past beads containing immobilized ligand, the 
ligand reacts specifically with the product, which is retained on the 
column. The column is then washed to remove nonspecifically adsorbed 
proteins. After washing, conditions in the column are changed (e.g., 
temperature, pH, or ionic strength) to encourage elution of the product 
from the column. Although effective, affinity chromatography is a very 
time consuming method to develop. This is because the appropriate 
conditions for separation must be identified from a number of variables 
(e.g., temperature, pH, ionic strength of mobile phase, solid phase 
composition, and mobile phase composition). In addition, the high cost of 
the immobilized ligand makes affinity chromatography better suited for 
relatively small scale final stage purification processing. 
Proteins and antibodies can also be purified by selective precipitation of 
either product or contaminant antibodies. This is generally accomplished 
by salting out with ammonium sulfate, polyethylene glycol (PEG), or 
specific precipitants, such as calcium ions. However, a major limitation 
of the precipitation method is that purification is only possible when the 
product and contaminant exhibit different precipitation behavior within 
the same system. Determination of an appropriate precipitation system 
depends on variables such as solution temperature, pH, composition, ionic 
strength, dielectric constant, the precipitant selected, etc. Even when 
separation is possible, in many cases, the protein and antibody yields are 
less than desired. 
Accordingly, a need clearly exists for cost effective methods for reducing 
the level of background binding in antibody preparations. A need also 
exists for antibody preparations that are substantially free of background 
binding. 
BRIEF DESCRIPTION OF THE INVENTION 
In accordance with the present invention, we have developed a method for 
reducing the level of background binding in antibody preparations having 
unwanted affinity for intracellular protein(s). The invention method 
comprises treating the antibody preparation with permeabilized cells, then 
separating the treated antibody preparation from the permeabilized cells. 
In accordance with another embodiment of the present invention, we have 
developed an anti-luciferase polyclonal composition that is substantially 
free of background binding. 
In yet another embodiment of the present invention, there is provided a 
reagent for removing background binding from antibody preparations having 
unwanted affinity for intracellular proteins. 
DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, a method for reducing background 
binding in antibody preparations having unwanted affinity for 
intracellular protein(s) is provided. The invention method comprises 
(i) treating an antibody preparation with permeabilized cells to produce a 
treated antibody preparation, 
wherein said permeabilized cells are prepared by contacting whole cells 
with a suitable permeabilizing agent, 
wherein said permeabilizing agent comprises an agent selected from the 
group consisting of alcohols and aqueous alcohols, aldehydes and aqueous 
aldehydes, ketones and aqueous ketones, nonionic surfactants and aqueous 
media containing nonionic surfactants, and combinations of any two or more 
thereof, provided, however, that the permeabilizing agent is not neat 
acetone, then 
(ii) separating the treated antibody preparation from the permeabilized 
cells. 
The term "background binding" refers herein to the occurrence of unwanted 
antibody binding from an antibody preparation containing an antibody of 
interest. The unwanted binding is typically caused by antibody binding to 
intracellular proteins. Unwanted antibody binding interferes with the 
detection and measurement of binding signals attributable to the antibody 
of interest. As used herein, the term "signal" refers to a positive 
indicator of antibody binding. Exemplary signals include the visual 
detection of a change in color, absorbance, reflectance, fluorescence, and 
other like signals. These indicators are used in methodologies for 
detecting the presence of particular antibodies and antigens. Such 
methodologies include cell staining techniques, ELISA assays, and other 
methods and techniques that are well known in the art. For example, in 
antibody binding experiments employing cells, background binding is 
characterized by prevalent and seemingly indiscriminate binding signals. 
Thus, the presence of background binding makes it virtually impossible to 
detect specific binding signals of the antibody of interest. 
As used herein, "antibody preparation" refers to a population of antibodies 
that is heterogeneous with respect to the character of epitopes available 
for binding. Exemplary antibody preparations for use in the practice of 
the present invention include polyclonal antibodies and mixtures of 
polyclonal antibodies. The term "polyclonal antibodies" is used herein to 
refer to antibodies produced in the normal immune response to an antigen 
that includes a number of closely related but non-identical proteins. The 
variation reflects the fact that they are formed by a number of 
lymphocytes. 
As used herein, the term "affinity" refers to a specific attraction between 
molecules, such as for example, the binding of specific antibodies to 
specific epitopes on an antigenic molecule. In antibody preparations, 
unwanted affinity for intracellular proteins is typically caused by the 
presence of background binding antibodies. 
Any animal used to make polyclonal antibodies can yield undesirable 
antibodies that have high background reactivity. Generally, any polyclonal 
raised against a desired antigenic component can potentially contain 
unwanted background binding antibodies. Certain animals are likely to 
generate background, which manifests itself as ubiquitous binding of 
unwanted antibodies to intracellular proteins. For example, polyclonal 
antibodies that are raised in rabbit, rat, mouse, sheep, goat, chicken, 
horse, donkey, and guinea pig appear likely to contain antibodies that are 
specific to cytoskeletal proteins. 
As used herein, the term "permeabilized cells" refers to cells that have 
cell membranes that have been rendered permeable to antibodies. The term 
"permeabilizing agent," as used herein, refers to an agent that renders 
cell membranes permeable to antibodies, provided, however, that 
"permeabilizing agent" does not refer to neat acetone. As contemplated in 
the practice of the present invention, permeabilized cells are prepared by 
contacting whole cells with a suitable permeabilizing agent. 
Exemplary permeabilizing agents include alcohols and aqueous alcohols, 
aldehydes and aqueous aldehydes, ketones and aqueous ketones (excluding 
neat acetone), nonionic surfactants and aqueous media containing nonionic 
surfactants, and combinations of any two or more thereof. Suitable 
alcohols include methanol, ethanol, and the like, and mixtures thereof. 
Suitable aldehydes include formalin, glutaraldehyde, and the like, and 
mixtures thereof. Suitable ketones include acetone, methyl ethyl ketone, 
3-pentanone, and the like, and mixtures thereof. Suitable non-ionic 
surfactants include polyethoxylated alcohols, polyethoxylated 
alkylphenols, polyethoxylated fatty acids, polyalkylene oxide block 
copolymers, carboxylic acid esters, carboxylic amides, and the like, and 
combinations of any two or more thereof. Exemplary non-ionic surfactants 
include Triton.RTM. (Rohm and Haas Co., Philadelphia, Pa.), Tween.RTM. 
(ICI Americas Inc., Wilmington, Del.), and Tergitol.RTM. NP40 (Union 
Carbide Corp., Danbury, Conn.). 
The permeabilizing agents contemplated by the present invention are 
typically used under conditions that are sufficient to render cell 
membranes permeable to antibodies but not sufficient to substantially 
denature intracellular proteins. As used herein, the term "denature" 
refers to a change in the structural configuration of a protein so that a 
denatured protein has fewer epitopes available for binding. 
Suitable permeabilized cells include those prepared from hepatocytes, 
epithelial cells, mesenchymal cells, endothelial cells, muscle cells, 
neural cells, and the like, as well as combinations of any two or more 
thereof. As contemplated in the practice of the present invention, 
exemplary whole cells for permeabilization can be obtained from most any 
vertebrate species, such as, for example, pig, rabbit, mouse, human, 
monkey, and the like. 
The choice of suitable cell type and species for permeabilization will 
depend on the particular antibody preparation being used. If the antibody 
preparation is polyclonal, the antigen against which the polyclonal is 
raised preferably is not endogenous to the cell type and species selected 
for permeabilization. For example, a particularly suitable polyclonal and 
cell type combination is anti-luciferase and pig hepatocytes because the 
antigen, luciferase, is not endogenous to mammalian hepatocytes. 
As used herein, the term "treating" refers to the exposure of an antibody 
preparation containing unwanted background binding antibodies to 
permeabilized cells. The antibody preparation can be treated with 
permeabilized cells either with or without agitation. For example, the 
permeabilized cells can be left to stand in the antibody preparation 
without agitation. Preferably, the antibody preparation and permeabilized 
cells are gently agitated to increase the transport of antibodies into the 
cells, which thus, increases the binding rate of unwanted antibodies to 
intracellular proteins. The permeabilized cells can be gently agitated in 
the antibody preparation by shaking, stirring, rocking, and other 
mechanical mixing methods that are well known in the art. 
The term "contacting," as used herein, refers to the exposure of whole 
cells to a suitable permeabilizing agent under conditions that are 
sufficient to render the cells permeables to antibodies. As contemplated 
in the practice of the present invention, whole cells can be contacted 
with suitable permeabilizing agent either with or without agitation. For 
example, whole cells can be left to stand in permeabilizing agent without 
agitation. Alternatively, whole cells can be gently agitated in 
permeabilizing agent by shaking, stirring, rocking, or other mechanical 
mixing methods that are well known to those skilled in the art. 
Whole cells are typically permeabilized by contacting with a suitable 
quantity of suitable permeabilizing agent for a period ranging from about 
1 minute to about 36 hours at a temperature ranging from about -20.degree. 
C. to about 22.degree. C. However, temperatures may be higher or lower 
depending on a variety of factors, such as the duration of contact with 
the permeabilizing agent, the presence or absence of agitation, etc. For 
most systems, permeabilization occurs by contacting whole cells with 
suitable permeabilizing agent for a period ranging from about 30 minutes 
to about 24 hours at a temperature ranging from about -20.degree.C. to 
about 22.degree. C. Preferably, whole cells are permeabilized by 
contacting with suitable permeabilizing agent for a period ranging from 
about 15 minutes to about 30 minutes at a temperature ranging from about 
-20.degree. C. to about 4.degree. C. Generally, cell permeabilizations 
carried out at lower temperatures are preferred because low temperatures 
are more likely to inhibit the activity of degradative enzymes that might 
alter or damage antigenic epitopes. 
As contemplated in the practice of the present invention, a suitable 
quantity of permeabilizing agent is an amount sufficient to achieve the 
desired level of cell permeability. Generally, cells contacted with 
relatively large quantities of permeabilizing agent over relatively long 
time periods will lead to more highly permeabilized cells as compared to 
cells contacted with smaller quantities of permeabilizing agent over 
relatively short time periods. Those of skill in the art can easily 
determine, without undue experimention, the actual quantity of 
permeabilizing agent, and thus, actual level of cell permeability, 
required to achieve optimal reduction in background binding. 
As contemplated in the practice of the present invention, permeabilized 
cells can be separated from permeabilizing agent by methods that are well 
known in the art. For example, the permeabilized cells can be pelleted by 
centrifugation at forces in the range of about 1,000 to about 50,000 times 
gravitational force (".times.g"). Preferably, the mixture is centrifuged 
at a force of about 10,000.times.g. Centrifugation causes the 
permeabilized cells to fall out of the mixture as a pellet. The 
permeabilizing agent can then be easily separated from the pellet by 
methods that are well known in the art, such as for example, aspiration. 
The permeabilized cells can also be separated from the permeabilizing 
agent by other separation methods that are well known in the art, e.g., 
filtration, and the like. When filtration is employed, filters having a 
nominal pore size rating of about 0.8 microns are preferred. 
Preferably, after permeabilization, the cells are washed in a suitable 
volume of an isotonic saline solution such as phosphate buffered saline 
(PBS). As contemplated in the practice of the present invention, a 
suitable volume of isotonic saline solution is an amount sufficient to 
remove substantially all of the permeabilizing agent from the 
permeabilized cells. The cells may be washed either with or without 
agitation. The saline solution can be separated from the washed and 
permeabilized cells by centrifugation at forces in the range of about 800 
to about 50,000.times.g. Preferably, the permeabilized and washed cells 
are centrifuged at a force of about 10,000.times.g. After centrifugation, 
the saline solution can be easily separated from the resulting pellet of 
cells by, for example, aspiration. The cells can also be separated from 
the saline solution by other separation methods that are well known in the 
art, e.g., filtration, and the like. When filtration is employed, filters 
having a nominal pore size rating of about 0.8 microns are preferred. The 
cells can be used immediately while wet, or optionally, the permeabilized 
and washed cells can be dried (e.g., by vacuum drying, air drying, 
lyophilization, and other like methods) and stored for later use. 
As used herein, the term "lyophilized" refers to the removal of water from 
cells by sublimation, i.e., the direct phase transition of water in the 
form of ice to water vapor. Methods and equipment for lyophilization are 
generally well known in the art. 
As contemplated in the practice of the present invention, the antibody 
preparation is treated with permeabilized and optionally, lyophilized 
cells for a period of time ranging from about 30 minutes to about 24 hours 
at a temperature ranging from about 4.degree. C. to about 37.degree. C. 
Preferably, the antibody preparation and permeabilized cells are contacted 
for a period ranging from about 30 minutes to about 5 hours at a 
temperature ranging from about 22.degree. C. to about 37.degree. C. Most 
preferably, the antibody preparation is treated with permeabilized cells 
for a period ranging from about 30 minutes to about 1 hour at a 
temperature ranging from about 22.degree. C. to about 37.degree. C. Those 
of skill in the art can easily determine, without undue experimentation, 
the most cost effective combination of variables, such as time, 
temperature, and volume ratio of antibody preparation to cells, to achieve 
optimal reduction in background binding. Generally, the antibody 
preparation is treated with permeabilized cells for longer periods at 
lower temperatures and for shorter periods at higher temperatures. For 
example, the antibody preparation can be treated with permeabilized cells 
for about 30 minutes at about 37.degree. C., about 1 hour at about 
22.degree. C. (approximately room temperature), and about 16 hours at 
about 4.degree. C. 
After the antibody preparation has been treated with permeabilized cells 
for a sufficient time, the resulting treated antibody preparation is 
separated from the permeabilized cells. As used herein, the term "treated 
antibody preparation" refers to an antibody preparation that has been 
exposed to permeabilized cells as described herein. The treated antibody 
preparation can be separated from the permeabilized cells by 
centrifugation at forces in the range of about 800 to about 
50,000.times.g. Preferably, the treated antibody preparation is separated 
from the permeabilized cells by centrifugation at a force of about 
10,000.times.g. After the cells have been pelleted by centrifugation, the 
supernatant, which contains the treated antibody preparation, can be 
easily separated from the cells by, for example, aspiration. The treated 
antibody preparation can also be separated from the permeabilized cells by 
other separation methods that are well known in the art, e.g., filtration, 
and the like. When filtration is employed, filters having a nominal pore 
size rating of about 0.8 microns are preferred. 
In accordance with another embodiment of the present invention, there is 
provided a method for reducing background binding in an antibody 
preparation having unwanted affinity for intracellular protein(s) 
comprising: 
(i) permeabilizing optionally immobilized whole cells by contacting the 
whole cells with a suitable permeabilizing agent, 
wherein said permeabilizing agent comprises an agent selected from the 
group consisting of alcohols and aqueous alcohols, aldehydes and aqueous 
aldehydes, ketones and aqueous ketones, nonionic surfactants and aqueous 
media containing nonionic surfactants, and combinations of any two or more 
thereof, provided, however, that the permeabilizing agent is not neat 
acetone; 
(ii) optionally lyophilizing the permeabilized whole cells; 
(iii) treating said antibody preparation with said optionally lyophilized, 
permeabilized whole cells to produce a treated antibody preparation; then 
(iv) separating the treated antibody preparation from the permeabilized 
cells. 
As contemplated by the present invention, the permeabilized cells can 
optionally be immobilized on a solid support. Suitable solid supports are 
made from either polymeric organic or inorganic materials. For example, 
the solid support can be made from inorganic materials such as glass or 
ceramic materials. The solid support can also be made from natural or 
synthetic polymeric materials. Exemplary natural polymeric materials 
include polysaccharides such as dextran, cellulose, agarose, and the like. 
Exemplary synthetic polymeric materials include polyamides, copolymers of 
polystyrene and divinylbenzene, polyacrylates, poly(vinyl alcohol), 
hydroxylated polyethers, and the like. 
Solid supports employed in the practice of the present invention can take a 
variety of shapes, including column packing material, a bead, membrane, 
test tube, centrifugation tube, vial, multi-well plate, tissue culture 
dish/flask, multichambered slide, and the like. 
Cells frequently can be immobilized on a solid support without the use of 
additional reagents. However, cell attachment can be promoted by coating 
the solid support with reagents such as extracellular matrix proteins. 
Exemplary extracellular matrix proteins suitable for immobilizing cells to 
a solid support include collagen, laminin, fibronectin, complex matrix 
proteins (e.g. lung and liver matrix proteins, and the like), and the 
like, as well as combinations of any two or more thereof. Cell attachment 
can also be promoted by chemically modifying the solid support to add a 
charge to the surface of the support. For example, cells are known to have 
an affinity for negative charges. As contemplated in the practice of the 
present invention, the solid support can be modified by the application of 
polyelectrolytes to the surface of the support, chemical derivatization of 
the solid support, and other methods for modifying the surface chemistry 
of solids as are well known in the art. Also contemplated by the present 
invention is the use of both reagent and surface modification of the solid 
support to facilitate immobilization. As contemplated by the present 
invention, cells can be permeabilized either before or after 
immobilization. 
It has been discovered that the treated antibody preparations produced by 
the present invention are substantially free of background binding. An 
antibody preparation that is substantially free of background binding can 
be characterized by both qualitative and quantitative methods. A 
qualitative determination of background binding can be made from antibody 
binding experiments (employing cells) by using standard microscopic 
detection systems. In one such experiment, the antibody preparation of 
interest is used in undiluted form to bind to cells that are known not to 
express the antigen to which the antibody was raised. Background binding 
can be quantified by measuring, for example, a fluorescent signal from 
cell antibody binding experiments using Fluorescence Activated Cell Sorter 
(FACS) analysis. 
Substantially background-free antibody preparations exhibit at least a 
ten-fold reduction in background binding as indicated by a change in 
binding signal of the antibody preparation measured before and after 
treatment with permeabilized cells. Preferably, substantially 
background-free antibody preparations of the present invention exhibit 
about a ten to one thousand-fold reduction in background binding after 
treatment with permeabilized cells. Antibody preparations are considered 
to be "free" of background binding if no binding signal is observed after 
incubating the antibody preparation, in undiluted form, with cells that do 
not produce the antigen against which the antibody preparation was raised. 
As a specific example, substantially background free anti-luciferase 
polyclonal antibody preparation can be prepared by treating commercially 
available anti-luciferase polyclonal antibody preparation with methanol 
permeabilized pig hepatocytes. The treated anti-luciferase polyclonal 
antibody preparations have reduced background levels that are at least 
about ten to one thousand-fold lower than background levels in 
commercially available anti-luciferase polyclonal antibody preparations. 
Typically, anti-luciferase polyclonal antibody preparations treated in 
accordance with the invention are free of background binding and exhibit 
no visible binding signals in cells that have not been transfected with 
the gene encoding luciferase. This large reduction in background binding 
suggests that the configuration of intracellular proteins within the 
permeabilized cells used to remove the unwanted background binding, 
described herein, is not substantially altered by the permeabilization 
process (i.e., there is no substantial level of protein denaturation). 
In yet another embodiment of the present invention, there are provided 
reagents for reducing the level of background binding in an antibody 
preparation. Invention reagents comprise immobilized, lyophilized, 
permeabilized whole cells, wherein said cells contain intracellular 
protein(s) that are not substantially denatured. The reagents contemplated 
by the present invention are effective at reducing the level of background 
binding in antibody preparations.

The invention will now be described in greater detail with reference to the 
following non-limiting examples. 
EXAMPLE I 
Isolation of Pig Hepatocytes 
Hepatocytes were harvested by a two-step ethylenediaminetetraacetic acid 
(`EDTA`)/collagenase liver perfusion. All hepatocyte isolations were 
performed between 11 a.m. and noon. Non-fasted animals were premedicated 
with intramuscular acepromazine (0.6 mg/kg), ketamine (20 mg/kg), and 
atropine (0.05 mg/kg), endotracheally intubated, and anesthetized using 
1-2% isoflurane. The abdomen was entered through a midline incision, the 
hepatoduodenal ligament was dissected, and all its structures were ligated 
and divided, except for the portal vein, which was cannulated with 
silicone tubing (Masterflex, Cole-Palmer, Chicago, Ill.). Heparin (100 
I.U./kg) was injected intravenously. The liver first was perfused with 
prewashing EDTA solution at room temperature at a flow rate of 300 
milliliters (`ml`) per minute by means of a roller pump (Masterflex; 
Cole-Palmer, Chicago, Ill.). Perfusion was initiated in situ and then 
continued for 10 minutes in a sterile stainless steel basin. Next, the 
basin was filled with the collagenase solution, which was recirculated 
through the liver at 300 ml per minute after passage through gas permeable 
silicone tubing (Silastic, Dow Corning, Midland, Mich.) submerged in an 
oxygen saturated water bath at 40.degree. C. Twenty minutes later, the 
liver capsule was disrupted and the digested liver parenchyma was 
suspended in about 300 ml of ice cold 10% Bovine Calf Serum-Dulbecco's 
Modified Eagle Medium (BCS-DMEM). (Morsiani et al., ASAIO J. 42:2 155 
(April-June 1995), incorporated herein by reference). 
For cell filtration, a custom made stainless steel chamber was used. The 
chamber was designed to allow rapid cell filtration and release from the 
digested liver within a closed sterile system. The inner volume of the 
chamber is 5 liters, divided into 4 compartments by 3 stainless steel 
meshes of decreased porosity (i.e. 400, 280, and 100 Lancaster; Tetko 
Inc., Lancaster, N.Y.). The chamber was attached to the platform of a Red 
Rocker orbital shaker (Hoefer Scientific Instrument Inc., San Francisco, 
Calif.). The primary cell suspension was loaded into the chamber, after 
which 500 ml of ice cold 10% DMEM flushed the cells by gravity into the 
upper compartment of the chamber, while the platform rotated at 60 
oscillations per minute. Within 1 to 2 minutes, approximately 1 liter of 
the cell filtrate was collected into a 1 liter disposable plastic transfer 
bag, which was maintained on ice. 
Next, a COBE 2991 blood cell processor (COBE, Blood Component Technology, 
Lakewood, Colo.) was used for automated washing and purification of the 
liver cells. The instrument consists of a centrifuge bowl with a flexible 
membrane at the bottom, into which fits a sealed doughnut-shaped, 
disposable plastic bag. The flexible membrane is connected to a hydraulic 
pump that allows a liquid to be removed from the processing bag. At the 
hub of the bag, a rotating seal allows both filling and emptying of the 
bag as the bag spins. The centrifuge bowl of the cell washer was 
refrigerated by an air conditioning unit attached to the side of the 
instrument. 
The filtered cell suspension was introduced into the centrifuge bag, and 
air was removed from the bag. Centrifugation was carried out at 600 
revolutions per minute (50.times.g) for 2 minutes, and the collagenase 
solution then was removed at a rate of 350 ml per minute. Hepatocytes were 
washed twice with approximately 630 ml of ice cold 10% BCS-DMEM using the 
cell processor. Pelleted hepatocytes then were resuspended in 10% 
BCS-DMEM. 
EXAMPLE II 
Permeabilization of Pig Hepatocvtes with Methanol 
The pig hepatocyte suspension prepared as described in Example I was placed 
in a disposable conical test tube and centrifuged at 1000.times.g for 5 
minutes at room temperature to pellet the cells. The supernatant was 
discarded and the cells gently resuspended in a 2:1 volume ratio of 
ice-cold methanol to cells. The cell suspension was rocked on a test tube 
shaker for 30 minutes. The suspension was then centrifuged at 
10,000.times.g for 5 minutes. The methanol was removed by aspiration and 
the cells were washed three times with a 4:1 volume ratio of phosphate 
buffered saline (PBS) and then pelleted by centrifugation at 1000.times.g 
for 5 minutes at room temperature. 
Example III 
Preparation of Treated Anti-Luciferase Polyclonal 
A stock 1 mg (total protein)/ml solution of an IgG fraction containing the 
rabbit polyclonal antibody anti-luciferase (Cortex Biochem, San Leandro, 
Calif.) was diluted by a factor of 1:50 in 0.25% Bovine Serum Albumin/PBS. 
The anti-luciferase dilution was added to the washed methanol-treated cell 
pellet prepared according to Example II in a volume ratio of 4:1 
(anti-luciferase solution:cells). The mixture was gently rocked on an 
Adams nutator (Clay Adams, Division of Becton Dickinson and Co., 
Parsippany, N.J.) for 20 hours at 4.degree. C. The cells were pelleted by 
centrifugation at 10,000.times.g for 5 minutes at room temperature. The 
antibody suspension was aspirated from the cells, then filtered through a 
0.8 micron Acrodisco.RTM. Supor.RTM. (polyether sulfone) (Gelman Sciences, 
Ann Arbor, Mich.). 
EXAMPLE IV 
Preparation of Lyophilized and Permeabilized Pig Hepatocytes and Treatment 
of Anti-Luciferase Polyclonal Using Lyophilized and Permeabilized Pig 
Hepatocytes 
A pellet of permeabilized pig hepatocytes, prepared as described in Example 
II, is lyophilized in a Virtis lyophilizer (Virtis Company, Inc., 
Gardiner, N.Y.). The cell pellet is lyophilized at a temperature of 
-75.degree. C. for 24 hours. The lyophilized cells (1.times.10.sup.8) are 
added to a 2% solution of the anti-luciferase polyclonal antibody 
preparation to be purified (Cortex Biochem, San Leandro, Calif.) in a 
volume ratio of 4:1 (anti-luciferase solution:cells) and rocked on a test 
tube shaker for 20 hours at 4.degree. C. The cells are pelleted by 
centrifugation in an IEC/Micromax centrifuge (International Equipment Co., 
Needham Heights, Mass.) at 10,000.times.g for 5 minutes at room 
temperature. The antibody suspension is aspirated from the cells, then 
filtered through a 0.8 micron Acrodisc.RTM. Suporo.RTM. (polyether 
sulfone) filter (Gelman Sciences, Ann Arbor, Mich.). 
EXAMPLE V 
Characterization of Background Binding in Untreated Anti-Luciferase 
Polyclonal 
Pig hepatocytes were transfected with the gene encoding luciferase using 
the liposomal preparation, LipofectAmine.RTM. (Life Technologies, Division 
of Gibco BRL, Gaithersburg, Md.), according to the method suggested by the 
manufacturer. The transfected cells were fixed with ice-cold methanol onto 
12 well plates by adding enough methanol to cover the cells. The plates 
were placed in a -20.degree. C. freezer for 5 minutes. After five minutes, 
the plates were removed from the freezer and the fixed cells were washed 
twice with Phosphate Buffered Saline (PBS). The wells were blocked with 
enough of a 1% Bovine Serum Albumin in PBS solution to cover the cells in 
each well. The plates were incubated at 37.degree. C. for 30 minutes. 
After 30 minutes, the fixed cells were washed twice with PBS. 
Dilutions of a 1 mg/ml stock solution of untreated rabbit anti-luciferase 
polyclonal antibody preparation (as purchased from Cortex Biochem, San 
Leandro, Calif.) in 0.25% BSA-PBS were prepared according to the 
manufacturer's recommendation for use (i.e., 1:50, 1:100, and 1:200 
dilutions). A control stock solution of 1 mg/ml of a matching irrelevant 
rabbit IgG antibody preparation (Jackson Immune Research, West Grove, Pa.) 
in 0.25% BSA-PBS was prepared. Corresponding dilutions were prepared from 
the stock control solution (i.e., 1:50, 1:100, and 1:200 dilutions). 
One set of wells containing the fixed cells was filled with the dilutions 
of anti-luciferase polyclonal antibody preparation (2-3 wells/dilution). A 
second set of wells containing fixed cells was filled with the dilutions 
of control IgG antibody preparation (2-3 wells/dilution). The plates were 
incubated for 45 minutes at 37.degree. C., then washed three times with 
rocking on an Adams nutator (Clay Adams, Division of Becton Dickinson and 
Co., Parsippany, N.J.) with a solution of 0.01% Tween 20 in PBS. 
A 1:200 dilution of a 1 mg (total protein)/ml donkey anti-rabbit-FITC 
conjugated F(ab').sub.2 labelled secondary antibody (Jackson 
ImmunoResearch, West Grove, Pa.) in BSA-PBS was prepared. The diluted 
secondary antibody solution was added to the all of the wells. The plates 
were then incubated for 45 minutes at 37.degree. C. on the nutator. 
Following incubation, the cells were washed three times with rocking on 
the nutator at room temperature with the 0.01 Tween 20 in PBS solution (5 
minutes/wash). The cells were then washed once with PBS for 5 minutes, 
then stored at 4.degree. C. in fresh PBS. 
The fluorescent signal in each well was measured visually using an Olympus 
BH2 fluorescent microscope equipped with epifluorescence optics (Olympus, 
Olympus America Inc., San Jose, Calif.). 
Visual observation of cells incubated with anti-luciferase polyclonal 
preparation indicated pervasive and indiscriminate fluorescent signal 
patterns throughout the samples. These results are consistent with the 
presence of a high level of background binding antibodies in commercially 
available anti-luciferase polyclonal preparation. It was not possible to 
differentiate specific anti-luciferase binding from the background. 
By comparison, visual observation of the control treated cells detected no 
fluorescent signal. Results from the control treated cells indicated that: 
(1) the control irrelevant IgG antibody preparation did not contain 
background binding antibodies and (2) the secondary antibodies were not 
causing the signal in the cells incubated with anti-luciferase polyclonal. 
EXAMPLE VI 
Characterization of Treated Anti-Luciferase Polyclonal 
Pig hepatocytes were transfected with the gene encoding luciferase using 
the liposomal preparation, LipofectAmine.RTM. (Life Technologies, Division 
of Gibco BRL, Gaithersburg, Md.), according to the method suggested by the 
manufacturer. The transfected cells were fixed with ice-cold methanol onto 
12 well plates by adding enough methanol to cover the cells. The plates 
were placed in a -20.degree. C. freezer for 5 minutes. After five minutes, 
the plates were removed from the freezer and the fixed cells were washed 
twice with Phosphate Buffered Saline (PBS). The wells were blocked with 
enough 1% Bovine Serum Albumin in PBS solution to cover the cells in each 
well. The plates were incubated at 37.degree. C. for 30 minutes. The fixed 
cells were then washed twice with PBS. 
A 1:50 dilution of a 1 mg/ml stock solution of untreated rabbit 
anti-luciferase polyclonal antibody preparation (as purchased from Cortex 
Biochem, San Leandro, Calif.) in 0.25% BSA-PBS was treated to remove 
background binding as described in Example III. The treated antibody 
preparation was used at dilutions equivalent to those of the untreated 
antibody preparation in Example V (i.e., 1:50, 1:100, and 1:200 
dilutions). A control stock solution of 1 mg/ml of a matching irrelevant 
rabbit IgG antibody preparation (Jackson Immune Research, West Grove, PA) 
in 0.25% BSA-PBS was also prepared. Corresponding dilutions were prepared 
from the stock control solution (i.e., 1:50, 1:100, and 1:200 dilutions). 
One set of wells containing the fixed cells was filled with the dilutions 
of treated anti-luciferase polyclonal antibody preparation (2-3 
wells/dilution). A second set of wells containing the fixed cells was 
filled with the dilutions of control irrelevant IgG antibody preparation 
(2-3 wells/dilution). The plates were incubated for 45 minutes at 
37.degree. C., then washed three times with rocking on an Adams nutator 
(Clay Adams, Division of Becton Dickinson and Co., Parsippany, N.J.) with 
a solution of 0.01% Tween 20 in PBS (5 minutes/wash). 
A 1:200 dilution of a 1 mg (total protein)/ml donkey anti-rabbit-FITC 
conjugated F(ab').sub.2 labelled secondary antibody (Jackson 
ImmunoResearch, West Grove, Pa.) in BSA-PBS was prepared. The diluted 
secondary antibody solution was added to the all of the wells. The plates 
were then incubated for 45 minutes at 37.degree. C. on the nutator. 
Following incubation, the cells were washed three times with rocking on 
the nutator at room temperature with the 0.01% Tween 20 in PBS solution (5 
minutes/wash). The cells were then washed once with PBS for 5 minutes, 
then stored at 4.degree. C. in fresh PBS. 
The fluorescent signal in each well was measured visually using an Olympus 
BH2 fluorescent microscope equipped with epifluorescence optics (Olympus, 
Olympus America Inc., San Jose, Calif.). 
Visual observation of cells incubated with treated anti-luciferase 
polyclonal preparation, prepared as described in Example III, displayed a 
discrete fluorescent signal consistent with the specific binding of 
anti-luciferase. The pervasive and indiscriminate fluorescent signal 
patterns observed with untreated anti-luciferase (as described in Example 
V) was notably absent. The control did not exhibit any fluorescent 
signals. 
These results demonstrate that the treatment of anti-luciferase polyclonal 
in Example III was effective at significantly reducing the level of 
background binding without also removing the desired specific binding of 
anti-luciferase. 
While the invention has been described in detail with reference to certain 
preferred embodiments thereof, it will be understood that modifications 
and variations are within the spirit and scope of that which is described 
and claimed.