Microflotation devices used for immunoassays and cell/molecular fractionation

This invention relates to a flotation immunoassay employing a novel buoyant matrix to which an antigen or antibody is coupled and which separates the bound and free products of the assay by floating to the surface of the reaction liquid. The novel flotation device which makes it possible to detect and to quantitate either antigen or antibody can also be used to fractionate cells and molecules.

FIELD OF THE INVENTION 
This invention relates to novel microflotation devices for use in 
immunoassay and cell/molecular fractionation. More specifically, the 
present invention relates to the use of a novel lipid matrix which 
preferably is a milk fat globule (MFG), that is, a cell derivative 
obtained from whole milk whose plasma membrane surrounds the densely 
packed lipid which is responsible for its characteristic ability to float 
in water or buffer. 
BACKGROUND OF THE INVENTION 
In the past twenty years, a great deal of emphasis in the field of 
immunochemistry has been placed on the development of new and improved 
techniques for immunoassay. Immunoassay relies, in principle, on the 
natural reactions of the body's immune system to the presence of foreign 
substances introduced into the body. The immune system is provoked by 
these foreign materials, for example, infectious organisms such as 
bacteria or viruses, to produce antibodies which react specifically with 
the foreign substance (or antigen) and which, if effective, aid in the 
elimination of the organisms from the body. The production of antibodies, 
of course, is not limited to the presence of infectious microorganisms but 
is also observed in response to many materials which are not normally 
found in circulation in the body. 
The use of immunoassay techniques for diagnostic testing, that is detection 
of antigen or antibody, has recently become very widespread and now is so 
frequently employed in both research and clinical environments that it may 
be considered commonplace. 
The relative specificity of antibody for a particular antigen has provided 
the basis for highly specific and accurate diagnostic testing for various 
physiological conditions such as infectious diseases, pregnancy and 
presence of drugs in the body. In practice, the test operates by exposing 
a test sample suspected of containing a particular antigen such as a 
bacterium or antibody to a particular microorganism such as the AIDS virus 
to a detectably labelled corresponding "immunological partner," i.e., the 
complementary antibody or antigen. The specificity of the antigen-antibody 
reaction is thus exploited in such well-known immunodiagnostic techniques 
as precipitation reactions, immunodiffusion, agglutination or hemolysis of 
antigen coated red cells, bacteria or latex particles, complement 
fixation, fluorescent immunoassays, immunoelectrophoresis, 
radioimmunoassay (RIA), and enzyme immunoassay (EIA), including 
enzyme-linked immunosorbent assay (ELISA). Two techniques of immunoassay, 
RIA and EIA, have become particularly popular because of their generally 
superior sensitivity, and the greater safety involved in EIA. 
Many of these assays are sensitive and specific, but all are limited by one 
or more of the following: requirements of expensive laboratory equipment 
(e.g. gamma counter, fluorescence microscope, high speed centrifuge, 
spectrophotometer, etc.), a technician trained in the operation of these 
instruments, and reagents which present some hazard or require 
refrigeration. In addition, some of the assays are relatively insensitive, 
not quantitative and require several hours or even days to complete. 
It would be highly desirable, therefore, to provide a cost effective, 
simple and sensitive diagnostic immunoassay which can provide an 
unequivocal positive and negative end point within a matter of minutes, 
which assay can be used quantitatively as well as qualitatively, that 
requires minimal laboratory equipment and that the components of the assay 
can be made shelf stable easily. 
BRIEF SUMMARY OF THE INVENTION 
The present invention provides a novel diagnostic immunoassay having the 
foregoing advantages which involves the use of a buoyant matrix which 
readily separates the bound and free products of the assay by floating to 
the surface of the reaction fluid. Specifically, the preferred matrix for 
use in the present invention is composed of milk fat globules (MFG) which 
comprise the cream fraction of milk and consist of fat droplets which are 
stabilized by an external membrane derived mainly from the apical plasma 
membrane of mammary secretory cells. The milk fat globule thus provides a 
natural, abundant and inexpensive microflotation device with the desired 
characteristics that readily permits the coupling of antigens or 
antibodies. 
The novel flotation immunoassay of the present invention, therefore, offers 
several advantages over existing assays. The present flotation assay 
generates an end point within minutes which is simple to read and requires 
no laboratory equipment or at most a simple clinical centrifuge. Secondly, 
the raw materials for the assay are extremely inexpensive, can be 
stabilized by glutaraldehyde and have no biohazardous elements, such as 
radioactivity. Thirdly, the use of the present flotation immunoassay makes 
it possible to design assays that are homogeneous, i.e. they require no 
separation techniques such as centrifugation or washing of 
antigen-antibody pellets. 
In addition, the flotation concept of the present invention provides a 
means for the separation of heterogeneous mixtures of cells or molecules. 
In this approach, the requirements are that MFG be coupled with an antigen 
or antibody, which reacts with the surface of the cell to be separated or 
with the molecule to be separated. 
The novel flotation immunoassay can be applied to a wide range of 
clinically relevant molecules including drugs, pathogenic bacteria, fungi, 
viruses, cellular antigens, such as blood group antigens and leukocyte 
antigens and tumor specific antigens. The only requirements are that the 
antigen or the antigen-specific antibodies are able to be linked to the 
buoyant matrix and the indicator. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is based upon the discovery that it is possible to 
provide a highly useful flotation assay which floats in water or buffer 
and can serve as a matrix for covalent attachment of antigens or 
antibodies. Milk fat globule (MFG), which is our preferred matrix, 
provides a natural, abundant and inexpensive flotation device with the 
desired characteristics. 
As stated earlier, milk fat globules (MFG) comprise the cream fraction of 
milk and consist of fat droplets that are stabilized by an external 
membrane derived mainly from the apical plasma membrane of mammary 
secretory cells. Keenan, et al. J. Dairy Science, 54:295, 1971. As such, 
these milk fat globules consist of a triglyceride core surrounded by a 
lipid bilayer containing integral membrane proteins. Mather and Keenan, J. 
Membrane Biol. 21:65, 1975. The globules range in size from 1 to 10 .mu.m 
diameter surrounded by a thin membrane called the milk fat globule 
membrane (MFGM). This membrane (approximately 10 nm in cross-section) 
consists of a complex mixture of proteins, phospholipids, glycoproteins, 
triglycerides, cholesterol, enzymes and other minor components. McPherson, 
et al. J. Dairy Research (1983) 50:107-133. Studies of the membrane 
proteins have revealed that several of these are glycoproteins and that 
the proteins are asymmetrically arranged in the membrane such that 
portions of them are exposed on the external surface of the MFG. Kobylka, 
et al. Biochim. Biophys. Acta 288:282, 1972, Huang, et al. Biochim. 
Biophys. Acta 332:59, 1973 and Anderson, et al. Biochem. J. 139:653, 1974. 
The milk fat globules consist of 99% triglycerides which are synthesized in 
the mammary gland secretory cell from precursors in the blood (i.e. 
glucose, acetate, low density serum lipoproteins) This high lipid content 
gives the globules a density less than that of water so that they float to 
the top of an aqueous suspension. MFG can thus be readily isolated by 
simply allowing them to rise to the top of a suspension upon standing, or 
by centrifugation at low speed in a clinical centrifuge, or for large 
scale preparations, by separating in a mechanical cream separator. 
We have found that the stability of the MFG is improved by immediate 
washing of fresh milk in PBS, by storage at room temperature and by gentle 
fixation by dialysis against 1% glutaraldehyde. We have also found that 
the MFG is unaffected by the tonicity of the solution to which it is 
exposed, in that exposure to hypotonic or hypertonic solutions has no 
visible effect on the MFG. 
In the flotation immunoassay of the present invention, the antigen or 
antibody is covalently coupled to the glycoproteins of the MFG by a 
variety of chemical techniques as, for example, the techniques described 
by Jou, et al. in Methods in Enzymology, Vol. 92: 257 (1983), which is 
incorporated herein by reference. 
As therein described the coupling of proteins and haptens to erythrocytes, 
for example, involves the use of a heterobifunctional reagent, e.g. 
N-succinimidyl 3-(2-pyridyldithio)propionate to link proteins to sheep red 
blood cells (SRBC) through disulfide bond formation. A second method takes 
advantage of succinylation of hapten-protein conjugates to facilitate the 
coupling to the surface of SRBC by carbodiimide. These two methods are 
designed to couple haptens to the surface of SRBC by using another 
heterobifunctional reagent, methyl-p-hydroxybenzimidate or a 
multifunctional reagent, 1,3,5-trichlorotriazine. A third method employs 
the noncovalent attachment of proteins and aminohaptens to the surface of 
SRBC via a synthetic lipopolysaccharide reagent. 
This latter method attaches the antigen to the surface of a cell without a 
covalent bond to the membrane molecules. A lipopolysaccharide, 
myristol-oxidized dextran, (MOD) has been designed to which haptens and 
proteins can be covalently coupled. The antigen-MOD directly attaches to 
the surface of the cells via a stable hydrophobic interaction with the 
plasma membrane. It is believed that the antigen-MOD is attached to the 
surface of red blood cells via the intercalation of the lipid moiety of 
the lipopoly-saccharide into the hydrophobic portion of the plasma 
membrane. The covalent attachment of haptens and proteins to the MOD 
occurs between free amino groups present on the haptens or proteins and 
reactive aldehyde groups on the MOD. The synthetic lipopolysaccharide can 
be used as a general method for coupling haptens or proteins to red blood 
cells. 
Thus, we have been able to couple antibodies to MFG indirectly by first 
linking the antibody to oxidized dextran after coupling dextran to 
myristoyl chloride. The myristic acid moiety intercalates into the MFG 
membrane and the bound antibody can be detected on the surface by 
agglutination of the MFG by antigen or anti-Ig antisera. 
We have also been able to couple N-hydroxy-succinimide biotin to the MFG 
surface and to detect this coupling by fluorescence microscopy after 
adding fluorescently labelled avidin. 
Similarly, dextran has been coupled to red blood cells, MFG, bacteria (E. 
coli and B. subtilis) and horseradish peroxidase by first oxidizing the 
dextran using sodium periodate followed by the formation of a Schiff base 
with amino groups on the cells or HRP. The reaction products are then 
stabilized with sodium borohydride. 
As stated earlier, the antigen or antibody is coupled to the glycoproteins 
of the MFG by using one of the three methods heretofore described. 
Complementary antibody or antigen is attached to the surface of a colored 
indicator such as erythrocytes, which would provide a red color, stained 
bacteria or some other readily visible indicator. An indicator visible to 
the naked eye is preferred. Interaction between the complementary ligands 
of the MFG and the indicator create a complex which rises to the top of 
the reaction mixture in a test tube, for example, by virtue of the 
flotation property of the MFG. 
When such an interaction has occurred, the presence of the colored 
indicator with the MFG at the top of the reaction tube can be seen (e.g. 
red blood cells, stained bacteria, enzyme). In the absence of reaction 
between the MFG and an indicator no color will be seen in the MFG layer, 
but rather when cells have been used as indicator, these are seen as a 
residue at the bottom of the tube. This standard assay can be modified in 
a variety of ways to perform quantitative analyses and to analyze the 
presence of antigen (Ag) or antibody (Ab) in a test sample by inhibition 
of the flotation of the indicator. 
Thus, when Ag coated MFG are reactively contacted with a suspension of 
antibody-conjugated red blood cells (Ab-RBC) in buffer, the Ab-RBC bind to 
the MFG and rise to the top of the reaction vessel forming a red ring at 
the surface. When assaying for the presence of the antibody the test 
sample is first reactively contacted with the Ag-coated MFG. If the sample 
contains antibody, it will bind to the Ag on the MFG and thereby inhibit 
the binding of the Ab-RBC when the indicator system is added subsequently. 
The MFG again rises to the top but in the absence of bound Ab-RBC the 
color of the ring at the top of the reaction vessel is white and the 
Ab-RBC fall to the bottom of the tube if not bound to the MFG. 
The operation of the hereinafter described flotation immunoassay is 
schematically shown below: 
______________________________________ 
Anticipated Outcome 
______________________________________ 
A. Assay for Presence of Antibody 
(1) Ag-MFG + Test Sample (Ab) 
In the presence of anti- 
body the complex 
formed between Ag- 
MFG and Ab from test 
(2) Ab-RBC (added subsequently) 
sample rises to top of 
reaction vessel forming 
a white ring, and the 
unbound indicators (Ab- 
RBC) form a red spot 
on bottom of reaction 
vessel. 
OR 
(1) Ag-MFG + Test Sample (No Ab) 
In absence of Ab in test 
sample, the complex 
formed between Ag- 
(2) Ab-RBC (added subsequently) 
MFG and Ab-RBC rises 
to top of reaction vessel 
forming a red ring. 
B. Assay for Presence of Antigen 
(1) Ab-MFG + Test Sample (Ag) 
In the presence of anti- 
gen the complex formed 
between Ab-MFG and 
Ag from test sample 
rises to top of reaction 
vessel forming a white 
(2) Ag-RBC (added subsequently) 
ring and the unbound 
indicators (Ag-RBC) 
form a red spot on bot- 
tom of reaction vessel. 
OR 
(1) Ab-MFG + Test Sample (No Ag) 
In absence of Ag in test 
sample, the complex 
formed between Ab- 
(2) Ag-RBC (added subsequently) 
MFG and Ag-RBC rises 
to top of reaction vessel 
forming a red ring. 
C. Assay for Antibody 
(1) Ag-MFG + Ag-RBC + Test 
In the presence of Ab in 
Sample (Ab) the test sample a bridge 
(present simultaneously) 
complex formed bet- 
ween MFG-Ag:Ab:Ag- 
RBC rises to the top of 
the reaction vessel form- 
ing a red ring. 
OR 
(1) Ag-MFG + Ag-RBC + Test 
In the absence of Ab in 
Sample (No Ab) test sample, unbound 
(present simultaneously) 
Ag-MFG forms a white 
ring at the top of the 
reaction vessel and un- 
bound Ag-RBC forms a 
red spot on the bottom. 
D. Assay for Antigen 
(1) Ab-MFG + Ab-RBC + Test 
In the presence of anti- 
Sample (Ag) gen in the test sample a 
(present simultaneously) 
bridge complex formed 
between MFG-Ab:Ag: 
Ab-RBC rises to the top 
of the reaction vessel 
forming a red ring. 
OR 
(1) Ab-MFG + Ab-RBC + Test 
In the absence of Ag in 
Sample (No Ag) test sample, unbound 
(present simultaneously) 
Ab-MFG forms a white 
ring at the top of the 
reaction vessel and un- 
bound Ab-RBC forms a 
red spot on the bottom. 
______________________________________ 
In the examples hereinafter a well defined system of pure antigen and 
monoclonal antibody are shown. The antigen used is a dextran derived from 
the bacterium Leuconostoc mesenteroides. This antigen is a 
well-characterized molecule consisting primarily of .alpha.-1,3 and 
.alpha.-1,6 sugar linkages. Its high molecular weight and physico-chemical 
characteristics combine to make this molecule highly immunogenic and easy 
to couple to the surface glycoproteins of either MFG or to the indicator 
system. This molecule is easily purified Finally this antigen is present 
in the cell wall of several bacteria and is associated with Aspergillus. 
The antibodies are monoclonal antibodies specific for the .alpha.-1,3 
linkage groups of the dextran. 
In addition to the novel flotation immunoassay described above, we have 
also found that MFG can be used to fractionate cells and molecules. This 
is demonstrated by the fact that sheep red blood cells (SRBC) to which 
dextran has been covalently linked bind to MFG which have on their surface 
anti-dextran or dextran In the latter case anti-dextran is added to link 
dex-MFG and dex-SRBC.

The invention will be described in greater detail in conjunction with the 
following specific examples which demonstrate the feasibility of the 
hereinabove described flotation immunoassay. 
EXAMPLE A 
Assay for the Presence of Antibody 
Dextran to be coupled to milk fat globules is first oxidized by treating a 
solution of dextran at a concentration of 10 mg/ml in phosphate buffered 
saline, pH 7.4 (PBS) with sodium periodate at a final concentration of 10 
mM for one hour at room temperature. The oxidized dextran is subjected to 
extensive dialysis against 100 volumes PBS at 40.degree. C. The milk fat 
globules are obtained from raw bovine milk by centrifugation at 
1500.times.g for 10 min at room temperature followed by removal of the 
underlying (sub-natant) aqueous material. The milk fat globules are washed 
three times by the addition of PBS to the original volume followed by 
centrifugation at 1500.times.g for 10 min and removal of the sub-natant 
fluid To 0.9 ml packed MFG is added 0.1 ml oxidized dextran and the 
mixture is incubated overnight at room temperature. The oxidized dextran 
binds to amino groups on the surface of the MFG forming a Schiff base. The 
resulting dextran coupled milk fat globules are transferred to a 10 ml 
syringe fitted with a stopcock, brought to 10 ml with PBS and centrifuged 
at 1500.times.g for 10 min. The sub-natant fluid is removed by opening the 
stopcock and the milk fat globule layer is washed three times by gentle 
suspension in 10 ml PBS followed by centrifugation. The final milk fat 
globule layer is brought to a final concentration of 20 percent (v/v) by 
the addition of approximately 4 ml PBS for storage at room temperature. 
The Schiff base so formed between oxidized dextran and the MFG can be 
stabilized by the addition of 10 mM sodium borohydride prior to washing of 
the dex-MFG. The reduced dex-MFG can be stabilized further by dialysis 
overnight against 100 volumes of a solution of 1 percent glutaraldehyde in 
PBS. Partial sterilization of the dex-MFG can be obtained by treatment 
with 10,000 rads of gamma irradiation without loss of the flotation 
properties or of the antigenicity of the dextran. 
The efficiency of coupling can be assessed by incubating dex-MFG with 
anti-dextran antibodies and microscopically observing agglutination of the 
dex-MFG. 
The antibodies, to be coupled to the indicator red blood cells, are treated 
with the heterobifunctional reagent, 
N-succinimydyl-3-(2-pyridyldithio)propionate (SPDP). Modification of the 
antibody is achieved by incubating with SPDP at room temperature with 
stirring at a molar ratio of 25 to 1:100. This yields approximately 5-20 
pyridyldithiopropionate (PDTP) molecules per antibody molecule, assuming 
an efficiency of coupling of 20 percent. The required ratio is derived 
empirically for each protein. In the case of the MOPC 104E monoclonal 
anti-dextran antibody used here, a ratio of 1:100 is used. Extensive 
dialysis of the resultant modified antibodies is performed against 1,000 
volumes of PBS at 4.degree. C. 
The red blood cells (RBC) to which antibody is to be coupled are obtained 
by venous puncture of sheep, are defibrinated and washed three times and 
stored as a 50 percent (v/v) suspension in PBS. Prior to treatment with 
PDTP modified antibody, 12.5 ml of a 2 percent suspension of RBC in PBS 
are incubated with 0.5 ml of freshly prepared 1M dithiothreitol (DTT) for 
1 hour at room temperature in order to reduce disulfide groups on the cell 
surface to thiol groups. Free DTT is then removed by washing the reduced 
RBC four times by the addition of 15 ml PBS, centrifugation at 
1,500.times.g for 10 min and removal of the supernatant fluid. To the 
resultant 0.25 ml packed reduced RBC is added 0.5 ml PDTP modified 
anti-dextran antibodies, and the mixture incubated on a rotating shaker 
overnight at room temperature. The resultant anti-dex-RBC are washed four 
times by the addition of 15 ml of PBS, centrifugation at 1,500.times.g for 
10 min and removal of the supernatant fluid. The final RBC pellet is 
brought to a final concentration of 10 percent (v/v) by the addition of 
approximately 2.25 ml PBS for storage at 4.degree. C. 
The anti-dex RBC can be stabilized, as above, using 1 percent 
glutaraldehyde and sterilized, as above. 
The efficiency of coupling can be assessed by incubating anti-dex-RBC with 
an antibody prepared against the MOPC 104E protein and microscopically 
observing agglutination of the anti-dex-RBC. 
The addition in a 6.times.60 mM tube of 50 .mu.l of a 2% (v/v) suspension 
of the thus-prepared anti-dex-RBC to 250 .mu.l of a 4% (v/v) dex-MFG 
suspension in a total volume of 500 .mu.l followed by gentle mixing and 
incubation at room temperature for 30-60 minutes results in a strongly 
positive "red ring" at the top of the tube. When control RBCs (i.e. no 
dextran on the surface) are added, the red color (i.e. the hemoglobin 
associated with the RBC) appears at the bottom of the reaction tube. 
The addition of a test sample containing antidextran antibodies results in 
the antibody binding to the dex-MFG and inhibits the binding of the 
indicator-RBC to the milk fat globules, that is to say, in the presence of 
anti-dextran antibodies the MFG ring at the top of the reaction tube is 
white and all of the indicator cells fall to the bottom of the reaction 
tube. This demonstrates the specificity of the immunoassay and it 
demonstrates the ability of the immunoassay to detect the antibody 
(antidextran) in a test sample. 
EXAMPLE B 
Assay for the Presence of Antigen 
Antibody is covalently coupled to the surface of MFG following the 
procedure of Example A. The complementary antigen, dextran, is coupled to 
the indicator RBC by the procedure in Example A. When the reaction is 
performed as in Example A, and antigen is present in the test sample it 
binds to the antibody MFG and inhibits the binding of the indicator, 
dex-RBC. Accordingly, the dex-RBCs fall to the bottom of the reaction 
vessel (test tube) forming a red spot and the Ab-MFG rises to the top 
forming a white ring. See C of the photograph shown in FIG. 1 of the 
annexed drawing. When no antigen is present in the test sample, the 
indicator dex-RBCs bind to the MFG and rise to the top of the reaction 
vessel forming a positive red ring. See B of the photograph shown in FIG. 
1 of the annexed drawing. When control RBCs are used (with no dextran on 
the cell surface) the indicator cells fail to bind to the Ab-MFG and fall 
to the bottom of the tube forming a red pellet at the bottom of the tube 
and a white ring of Ab-MFG at the top. See A of the photograph shown in 
FIG. 1 of the annexed drawing. 
EXAMPLE C 
Assay for the Presence of Antibody 
Milk fat globules (MFG) coupled to dextran (dex-MFG) following the 
procedure outlined in Example A and red blood cells (RBC) coupled to 
dextran (dex-RBC) following the procedure of Example A, are mixed in a 1:1 
ratio in a reaction vessel in the presence of a monoclonal anti-dextran 
antibody. Microscopic observation reveals clumps of mixed dex-RBC and 
dex-MFG. When this experiment is carried out in a 6.times.60 mm tube in a 
total volume of 500 ul containing equal volumes of 2% dextran-MFG and 1% 
dextran-RBC, the indicator red cells bind to the MFG and rise to the top 
of the tube forming a red ring. No RBCs are present in the MFG layer in 
the absence of added antibody or when either the MFG or RBC used in the 
assay have no dextran coupled to them. Thus the indicator cells that are 
bound to the MFG rise to the top of the tube immediately after a low speed 
spin in a clinical centrifuge (500.times.g) and can be readily visualized 
grossly. The same result occurs when the reaction tubes are allowed to 
settle without centrifugation. 
EXAMPLE D 
Assay for Presence of Antigen 
Antibody is covalently coupled to both MFG and indicator SRBC following the 
procedure of Example A. When the antigen is present in the test sample it 
forms a bridge between the Ab-MFG and the Ab-SRBC so that the indicator 
red cells rise to the top of the test tube forming a red ring. In the 
absence of antigen in the test sample, no red cells bind to the MFG and 
the ring at the top of the assay tube is white, that is, only the MFG is 
present. 
While the invention has been described hereinabove particularly with regard 
to milk fat globules as the lipid source and red blood cells as the 
indicator, it is within the scope of the present invention to employ other 
materials in the described immunoassay. Thus, we may substitute stained 
bacteria, e.g. E. coli and B. subtilis, horseradish peroxidase, etc. for 
the indicator red blood cells. 
It is expected that phospholipid formulations can be used to prepare lipid 
vesicles with densities less than water to replace MFGs as the buoyant 
matrix. It is also possible to use synthetic polymers to prepare tiny 
spheres or beads with chemically reactive groups on their surface for the 
attachment of antibodies or antigens. 
It is also within the scope of the present invention to use many different 
antigens and polyclonal antisera or monoclonal antibodies directed against 
them. Among the antigens which can be used are: 1) macromolecular 
antigens, including proteins, carbohydrates and nucleic acids, 2) haptens, 
such as N-.epsilon.-dinitrophenyl-lysine, 3-aminopyridine, 
4-aminopyridine, 4-aminophthalate and 5-aminoisophthalate, 3) antigens 
associated with microbial cells, both pathogenic and non-pathogenic, 
including bacteria (e.g. Pseudomonas, Mycobacterium), fungi (e.g. Candida) 
viruses (e.g. AIDS virus, retroviruses), and protozoa (e.g. Schistosoma), 
4) cell surface antigens, such as blood group antigens, specific T cell 
antigens (e.g. Thy-1, lyt2, L3T4, T4, T8, etc.), 5) tumor specific 
antigens, e.g. the 160,000 molecular weight glycoprotein (gp160), a cell 
surface molecule associated with human lung tumor cells. Many of these 
molecules have been or can readily be coupled to RBC and/or MFG using the 
techniques outlined above and described in Jou et al., Methods in 
Enzymology 92: 257-275 (1983). For example, in addition to coupling 
dextran as described, the above-mentioned haptens have been successfully 
conjugated to membrane using myristoyl-oxidized dextran as the coupling 
agent. Proteins that have been successfully coupled, in addition to the 
monoclonal anti-dextran antibodies, include Bence-Jones protein, human 
.gamma.-globulin, bovine .gamma.-globulin, rabbit anti-human Fab antibody, 
mouse anti-phthalate antibodies and monoclonal anti-phthalate antibody. 
The invention as described herein can also be used in a quantitative 
fashion, although the examples given have for clarity been described for 
the qualitative determination of the presence or absence of antigen or 
antibody. For a quantitative assay, standard samples containing a known 
amount of antigen or antibody can be diluted serially two-fold and 
incubated with the appropriately coupled MFG and indicator RBC. In this 
way, it is possible to determine the end-point, i.e. the smallest amount 
of antigen or antibody capable of inhibiting binding of the indicator RBCs 
to MFGs and thus preventing the appearance of red color at the top of the 
tube. Similar dilution of the test sample containing unknown quantity of 
antigen or antibody to an end point would allow the calculation of the 
amount of antigen or antibody in the test sample. One example which 
demonstrates the feasibility of this is shown in FIG. 2 of the 
accompanying drawings in which known quantities of free dextran are added 
to inhibit the binding of anti-dextran RBC to dex-MFG. Here it is 
established that inhibition occurs with 500 ng of dextran but not at 50 ng 
(FIGS. 2d and 2e). By constructing a standard curve using data derived in 
this fashion one is able to quantitate the amount of antigen (or antibody) 
in a test sample. 
The file of this patent contains at least one drawing executed in color. 
Copies of this patent with color drawing(s) will be provided by the Patent 
and Trademark Office upon request and payment of the necessary fee. 
The invention also encompasses the use of antigen or antibody coupled MFG 
as a device for the separation by flotation of cells or molecules. In this 
instance, an antigen or antibody which specifically interacts with a 
complementary molecule on the surface of a cell or free in solution is 
coupled to MFG. Reaction of these MFG with a suspension of such cells or 
with a solution of such molecules will result in the flotation of the 
bound cells or molecules to the top of the tube. Molecules or cells thus 
separated, may be recovered from the MFG layer, unbound cells may be 
recovered from the cell pellet, and unbound molecules may be recovered 
from the underlying solution