Biodegradable particle coatings having a protein covalently immobilized by means of a crosslinking agent and processes for making same

The invention relates generally to colloidal particles having a crosslinked coating with pendent functional groups attached thereto. Magnetic and non-magnetic particles have a biodegradable, crosslinked gelatin coating to which is covalently attached pendent biological substances or molecules, especially monoclonal antibodies. The monoclonal antibodies so attached are useful in a variety of positive and negative biological assays.

RELATED INVENTION 
This invention is related to U.S. Pat. No. 5,062,991, filed Jun. 4, 1990 
and issued Nov. 5, 1991, and entitled "IN SITU USE OF GELATIN IN THE 
PREATION OF UNIFORM FERRITE TICLES". These applications are owned by 
a common assignee. 
FIELD OF THE INVENTION 
This invention relates generally to colloidal sized particles having a 
crosslinked gelatin coating with pendent functional groups attached 
thereto. Specifically, this invention relates to colloidal particles 
having a crosslinked gelatin coating that is functionalized to bind a 
pendent protein such as an antibody, to the method of making such 
particles and to the use of such particles in biological assays. 
BACKGROUND OF THE INVENTION 
The use of polymeric particles and magnetic particles to bind a compound 
has long been known and used in industrial and laboratory procedures. For 
example, the Merrifield resins, crosslinked styrene-divinylbenzene 
spheroidal beads, were among the earliest and most widely used modern 
substrate particles. They were used in organic synthesis, for 
heterogenizing homogeneous catalysts and in biochemical reactions. Since 
the Merrifield resins were fairly large, they could easily be separated by 
filtration. In some fields, however, it is desirable to use colloidal 
sized particles because the material to be bound is scarce, expensive or 
is to be used in a procedure where larger particles are not desirable. 
This is particularly true in the biochemical field. When particles are of 
colloidal size, however, their separation from liquid medium by filtration 
can become lengthy and difficult. In particular, colloidal particles tend 
to coat the surface of the filter and slow the process. The use of 
magnetic particles, specifically magnetic particles having a polymeric 
coating, has found great utility because such particles can be 
magnetically gathered to one side of a reaction vessel and the bulk of the 
reaction medium simply decanted. (The word "particles" as used herein 
encompasses spheres, spheroids, beads and other shapes as well. These 
words are used interchangeably unless otherwise specified.) The use of 
coated magnetic particles has found a particular utility in biological 
applications, especially where antibodies are bound to the surface coating 
of the particles. The bound antibodies may then be used to capture a 
specific biological substance from a test sample containing numerous 
biological samples or to capture undesired species from the test sample, 
leaving the desired species in the sample. 
The categories of coated magnetic particles, also known as magnetic spheres 
or beads, can be divided into four general classes. 
1. Core-and-shell beads with a magnetic core and a hard shell coating of 
polymerized monomer or a silanizing agent. See Rembaum U.S. Pat. No. 
4,267,234 (polyglutaraldehyde shell around ferrofluid core particles); 
Czerlinski U.S. Pat. No. 4,454,234 (suspension or emulsion polymerized 
coating around submicron magnetic particles); Whitehead et al. U.S. Pat. 
No. 4,554,088 (silanized magnetic oxide particles of polydisperse size and 
shape); and Margel et al. U.S. Pat. No. 4,783,336 (suspension polymerized 
polyacrolein around ferrofluid particles). 
2. Core-and-shell beads with a magnetic core and a loose shell of random 
coil or globular polymer which may or may not be crosslinked. See Molday 
U.S. Pat. No. 4,452,773 (dextran coating around ferrofluid particles) and 
Owen et al. U.S. Pat. No. 4,795,698 (protein such as bovine serum albumin 
around ferrofluid particles. 
3. Magnetic latex materials formed by uniformly embedding ferrofluid 
particles in polystyrene latex particles. See Daniel et al. U.S. Pat. No. 
4,358,388. 
4. Porous polymer particles filled with magnetic materials such as 
polymer-ferrite or polymer maghemite composite systems. See K. Nustad et 
al. "Monodisperse Polymer Particles In Immunoassays And Cell Separation", 
Microspheres: Medical and Biological Applications, A. Rembaum and Z. 
Tokes, eds. (Boca Raton, Fla.: CRC Press, 1988) pages 53-75; C. D. 
Platsoucas et al., "The Use Of Magnetic Monosized Polymer Particles For 
The Removal Of T Cells From Human Bone Marrow Cell Suspensions", ibid. at 
pages 89-99; and International Patent Publication No. WO 83/03920, 
Ughelstad et al. (polymer coated magnetic particles prepared by treating 
compact or porous particles with a solution of iron salts and the use of 
such particles for medical, diagnostic or other purposes). 
The usefulness of most polymer coated magnetic beads in medical and 
biological applications has been limited by practical considerations such 
as the uniformity of particle size and shape, the need for the biological 
reagent to be strongly bound to the particle, a preference for hydrophilic 
polymer coatings as opposed to hydrophobic coatings, and whether or not 
the coating is biodegradable. While biodegradability is of particular 
importance where a biological reagent is to administered in vivo, it is 
also important in various cell sorting, separation and assay procedures. 
The most desirable coated magnetic particles would have the following 
features. 
1. The particles should be as small as possible in order to maximize the 
surface area on which the biological reagent is coated, but the particles 
should still be easily separable with a small magnet. Small size and large 
surface area are desirable in order to use the least possible quantity of 
particles to remove the targeted substance; e.g., to interact with on the 
order of 10.sup.6 cells per sample in one step, thereby avoiding 
sequential additions and work-ups. 
2. There should be a low non-specific binding of the antibody-coated 
particles to cell surfaces. The particle surface should be hydrophilic or 
covered with a coating of a hydrophilic substance to which the antibody is 
attached. 
3. The polymer and antibody layers on the particles should be covalently 
bound to each other in order to reduce dissociation and conformational 
changes. 
4. The coating on the magnetic particles and any molecular chains which 
link an antibody to the polymer surface should be metabolizable. 
5. In positive selection of cells, a mechanism for quickly and easily 
recovering viable cells from the magnetic particles should be available in 
order that recovered cells can be cultured. 
6. In the negative selection of cells, the antibody-coated particles should 
be sterile so that the remaining cells can be cultured. 
In addition to magnetic particles, there is also a need for polystyrene 
latex (PSL) particles which have been coated with hydrophilic polymer 
coatings to which antibodies can be subsequently bound. These polymer 
coated PSL particles can be used in bead-based cell population analyses 
and immunoassays. However, non-magnetic PSL particles, as made, usually 
have a relatively low density of various functional groups such as 
carboxyl or amino groups. Consequently, covalent coupling of coating 
materials such as dextran or gelatin to the surface of PSL particles is 
not satisfactory. 
The various particles described above have been used in the biological arts 
to immobilize a variety of biological substances, particularly antibodies. 
In using such particles, immobilization of antibodies by covalent coupling 
is preferred to immobilization by antibody adsorption which requires 
careful and separate adjustment of pH and antibody concentration for each 
monoclonal antibody used. P. Bagchi et al., J. Colloid Interface Sci., 
83:460-478 (1981); J. Lyklema, Colloids and Surfaces, 10:33-42 (1984); M. 
D. Bale et al., J. Colloid Interface Sci., 125:516-525 (1988); C. C. Ho et 
al., ibid., 121:564-570 (1988); "Proteins at Interfaces: Physicochemical 
and Biochemical Studies", ACS Symposium Series, No. 343, J. L. Brash and 
T. A. Horbett, Eds. (Washington: Amer. Chem. Soc., 1987); W. Norde, Adv. 
Coll. Interface Sci., 25:267-340 (1986); A. V. Elgersma et al., Abstracts 
of the 198th Amer. Chem. Soc. Meeting, Miami Beach, Fla., Sept. 10-15, 
1989, COLL 0131; and D. E. Brooks, Annenberg Center for Health Sciences 
and H. B. Wallis Research Facility at Eisenhower Latex Conference, 
Orlando, Fla., Dec. 4-5, 1989. However, even when the pH and antibody are 
carefully controlled, there is little assurance that the orientation of 
adsorbed antibody will be such that an active adsorbed antibody will 
result. Adsorbed antibodies also have long term storage problems arising 
from antibody desorption from the particles' surfaces. Furthermore, 
proteins, such as antibodies, tend to achieve maximum adsorption on 
hydrophobic surfaces at or near the pI of the protein. However, if 
electrostatic interactions between charge groups are important, then the 
adsorbing surface and the adsorbate should have net opposite charges. 
Covalent coupling methods, on the other hand, are not as sensitive to 
these conditions. 
Covalent coupling methods have been used with particles of magnetite 
embedded in carboxy-modified latex subsequently coated with aminodextran 
and derivitized with a number of antibodies. R. S. Molday et al. FEBS. 
Lett., 170:232-238 (1984). If the antibody is of IgG isotype, the covalent 
coupling method assures that the linkage between the antibody and the 
particles occurs at the antibody's Fc or hinge region, and not at the 
antibody's Fab region. If the antibody is of pentameric IgM isotype which 
has only Fab regions exposed, the coupling of one Fab region to the 
particle will still leave four Fab regions exposed and available for 
reaction. 
This invention provides for the preparation of magnetic and non-magnetic 
particles having a biodegradable coating to which can be attached pendent 
biological substances, such as monoclonal antibodies. The particles of the 
invention can be used in various cell separation and assay methodologies. 
Biodegradability in the coating used on the magnetic or latex core 
material is important in cell separation technology. For example, 
antibodies may be conjugated to gelatin coated magnetic particles such as 
manganese ferrite particles. These particles would thus contain a 
proteinaceous coating and a manganese-iron oxide core, all of which are 
biodegradable. In a positive cell selection procedure using such 
particles, once the desired cell has been isolated from other cells, the 
particles and coating can be allowed to degrade in a manner such that the 
cells are kept viable and can be cultured for further use. Alternatively, 
the enzyme collagenase can be used first to release the core material 
(magnetic or latex) by digestion of the gelatin coating. The core material 
can then be removed from the cell suspension before culturing the cells. 
In the negative selection of cells with such biodegradable beads, the 
beads can be left in the cell suspension from which targeted cells were 
removed without compromising the viability of the remaining cells. For 
example, in bone marrow purging operations using biodegradable magnetic 
beads, there is less concern about leaving behind some beads in the purged 
marrow that is to be transplanted in a patient. Currently, synthetic 
polymer-magnetite particles prepared by Ughelstad et al, International 
Patent Publication No. WO 83/03920, and conjugated with antibody are being 
used in bone marrow purging. The polymer is not biodegradable and imparts 
a hydrophobic surface to these beads. This hydrophobicity, which is not 
present in the gelatin coated particles of the claimed invention, is 
responsible for non-specific interactions between the beads and cells. As 
a result of this non-specific interaction, the selectivity is poor and 
more beads must be used to attain the desired level of treatment. The 
claimed invention avoids these problems. 
SUMMARY OF THE INVENTION 
The invention provides a method for the preparation of discrete colloidal 
particles having a solid core and coated with a water soluble gelatin or 
derivative thereof, said coating being crosslinked or fixed by the action 
of a chemical crosslinking agent and having a plurality of pendent 
functional groups. The pendent functional groups may be or have terminal 
aldehyde or carboxylate groups, amine groups, sulfhydryl groups or 
maleimidyl groups, and polyclonal or monoclonal antibodies. 
The invention provides discrete colloidal particles having pendent 
biological functional groups such as polyclonal and monoclonal antibodies 
covalently attached to the crosslinked gelatin coating by means of a 
derivatized short diamine or polyamine chain so as to enable advantageous 
use of said antibody functionalized particles in biological separations 
and assays. The derivatized diamine or polyamine chain acts as a bridging 
group between the biological substance or functional group and the 
crosslinked gelatin. 
The invention provides a process for the preparation of discrete colloidal 
particles having a solid core coated with a biodegradable, crosslinked 
gelatin or gelatin derivative having pendent functional groups. The 
process comprises coating a solid core material which has a hydrophobic 
surface with gelatin or a gelatin derivative, crosslinking the adsorbed 
gelatin and derivatizing the crosslinked gelatin to obtain a product 
having a desired reactive species covalently bound to said crosslinked 
gelatin surface. The invention further provides a process for the 
preparation of particle bound polyclonal and monoclonal antibodies. 
The invention provides a process for the separation, either positive or 
negative, and analysis of biological substances comprising contacting a 
solution containing a biological substance with an antibody covalently 
bound to the surface of a crosslinked gelatin coated solid core particle, 
incubating the resultant mixture at a temperature and for a time 
sufficient to form a complex between said antibody and said substance, 
separating the particles from the solution and analyzing the particles or 
the solution for the presence and/or absence of the desired substance.

DETAILED DESCRIPTION OF THE INVENTION 
In the Detailed Description Of The Invention and Preferred Embodiments 
which follow, applicants place reactive maleimidyl groups on the 
crosslinked gelatin coated particles and reactive sulfhydryl groups on the 
antibodies. These may be reversed such that the maleimidyl groups are 
attached to the antibodies and the sulfhydryl groups are attached to the 
crosslinked gelatin. Applicants have also elected to use 2-iminothiolane 
hydrochloride as the model for the sulfhydryl reagent and sulfo-SMCC 
(described below) as the model for the maleimidyl reagent. Other reagents 
enumerated or of like nature and result may also be used. 
Glossary of Biological Reagents 
All of the monoclonal antibodies (Ab) referred to herein are identifying 
designations used by Coulter Corporation, Hialeah, Florida for monoclonal 
antibodies made by Coulter Corporation. The following information further 
identifies the antibodies used herein. The use of these monoclonal 
antibodies is by way of example only and is not to be understood as 
limiting the invention. The term "CD" refers to "Cluster Designation" 
adopted by the International Workshops on Human Leukocyte Differentiation 
Antigens. A.T.C.C. is the American Type Culture Collection, Rockville, 
Maryland. 
______________________________________ 
Antibody 
CD Description or Reference 
______________________________________ 
T11 CD2 Derived from hybridization of mouse 
NS/1-AG4 cells with spleen cells of 
BALB/cJ mice immunized with T cell 
chronic lymphocytic leukemia cells. 
T4 CD4 As T11, but immunized with peripheral 
human T lymphocytes. 
T8 CD8 As T11, but immunized with human 
thymocytes. 
KC16 -- U.S. Pat. No. 4,752,563; 
A.T.C.C. Deposit No. CRL 8994. 
1D3 -- U.S. Pat. No. 4,931,395; 
A.T.C.C. Deposit No. HB 9445 
KC48 -- U.S. Pat. No. 4,865,971; 
A.T.C.C. Deposit No. HB 9584 
MO2 CD14 R. F. Todd et al, 
J. Immunol., 126:1435 (1981). 
PLT-1 -- R. F. Todd et al., Blood, 59:775 (1982); 
Griffith et al., Blood, 61:85 (1983). 
______________________________________ 
Other reagents used herein and commercially obtainable from Coulter 
Corporation are: 
______________________________________ 
MsIgG1-RD1/MsIgG1-FITC: 
Mouse IgG1-phycoerythrin 
[RD1]/Mouse IgG1-Fluorescein 
Isothiocyanate [FITC]. 
T11-RD1/B4-FITC: Ab T11-phycoerythrin/Ab 
B4-FITC. 
T4-RD1/T8-FITC: Ab T4-phycoerythrin/Ab 
T8-FITC. 
______________________________________ 
Detailed Description 
In using the method of the invention, uniform particles (the core material) 
in the size range of 0.1 to 5.0 microns are coated with gelatin or a 
gelatin derivative, and the coating is fixed by means of a chemical fixing 
agent. The uncoated particles have a hydrophobic or partially hydrophobic 
surface. The preferred size of the particles is in the range of 0.1 to 1.0 
microns. 
The magnetic particles used in the claimed invention may be preformed 
magnetic particles that are dispersible in a gelatin solution or they may 
be magnetic particles prepared by the in situ use of gelatin in the 
preparation of said magnetic particles. The in situ method for the 
preparation of monodispersed colloidal particles of ferrites of manganese, 
zinc, mixed manganese-zinc, iron, barium, cobalt and nickel involves the 
use of an aqueous metal hydroxide gel first formed by mixing ferrous and 
other metal salts in an aqueous gelatin solution with potassium or sodium 
hydroxide and potassium or sodium nitrate solution, all solutions being 
purged with nitrogen gas. The conversion of the gel to the metal oxide sol 
is achieved by mild thermal treatment at 90.degree. C. (low temperature) 
for 4-72 hours, during which nitrate oxidation of ferrous iron occurs. The 
magnetic particles in the hydrosol are then washed and resuspended in a 1% 
aqueous solution of gelatin of the type described below prior to further 
treatment as described herein. In preparing magnetic particles using in 
situ gelatin as described herein, only one type of gelatin has been found 
optimal for such use. This is type B or alkali-cured gelatin with a pI 
range of 4.75 to 5.0. The procedures for the preparation of magnetic 
particles using in situ gelatin are fully described in U.S. Pat. No. 
5,062,991, the teachings of which is incorporated here by reference, and 
also described herein. The gelatins which are crosslinked according to the 
present invention are given below. 
Gelatin is obtained from highly crosslinked collagen in fibrous tissue, 
such as skin or bone, which has been acid or base cured and then thermally 
degraded at or above 39.degree. C. The collagen molecule combines the 
helical structure of the .alpha.-type proteins with the inter-chain 
hydrogen bonding of the .beta.-type proteins. The three collagen peptide 
chains, each in the form of a left handed helix, are twisted about each 
other to form a superhelix. Upon treatment, the three peptide strands of 
the superhelix are separated by the breaking of inter-chain hydrogen bonds 
and replacing them with hydrogen bonds to water molecules. The separated 
peptides have random coil configurations. "The Theory of the Photographic 
Process", T. H. James, Ed., (New York: MacMillan Press, 1977). The 
.alpha.-1 peptide chain has been sequenced and found to have over 1000 
residues. D. J. S. Hulmes et al., J. Mol. Biol., 79:137 (1973). They 
contain extensive segments of mainly non-polar residues; and the polar 
residues which are present are not localized into acidic or basic regions. 
Furthermore, in contrast to globular proteins which tend to expose their 
hydrophilic residues on their surfaces and bury their hydrophobic residues 
within their structure {see R. E. Dickerson et al., "The Structure and 
Action of Proteins", (Menlo Park: Benjamin, 1969)}, random coil gelatin 
has exposed hydrophobic residues readily available for adsorption onto the 
surface of hydrophobic particles such as polystyrene latex particles or 
magnetite and ferrite particles. When aqueous gelatin is adsorbed onto the 
surface of a particle, its hydrophilic side chains (aspartyl, glutamyl and 
lysyl residues) tend to be directed externally to the aqueous medium. The 
lysyl groups, which function as the intramolecular crosslinkage points in 
collagen, will be accessible for cross linking in the adsorbed gelatin. 
Glutaraldehyde is frequently used as the crosslinking agent. Van Der Merwe 
et al. U.S. Pat. No. 4,478,946 and S. B. Sato et al., J. Biochem., 
100:1481-1492 (1986). 
A number of different, usually bifunctional, crosslinking agents such as 
bis [2-(succinimidooxycarbonyloxy)-ethyl]sulfone, disuccinimidyl 
tartarate, ethylene glycol bis (succinimidylsuccinate), disuccinimidyl 
suberate and glutaraldehyde may be used in the claimed invention. 
Glutaraldehyde, the preferred gelatin crosslinking agent, as commercially 
available, contains mainly monomer absorbing at 280 nm (nanometers). 
However, there is present in the commercial product a significant amount 
of polymeric material which gives rise to an absorbance at 235 nm. The 
polymeric species, probably trimers or linear oligomers, are of sufficient 
length to form intra- and inter-molecular bridges between amino groups 
present on the adsorbed gelatin. By judiciously selecting the reaction 
time between the adsorbed gelatin and glutaraldehyde, the gelatin can be 
suitably fixed on the core particles so that it will not be removed during 
subsequent separation, reaction and washing steps. Large flocs created by 
excessive crosslinking of free gelatin can thereby be avoided and 
interparticle crosslinking is negated. 
Several types of gelatin are available for use in the present invention, 
such as type A, acid cured, isoelectric point pH 8.3-8.5 and type B, 
alkali cured, isoelectric point, pH 4.75-5.0. Each type is available in a 
variety of Bloom Numbers which indicate gel strength. Type A gelatin Bloom 
Numbers useful in the claimed invention range from 60 to 300. Type B Bloom 
Numbers useful in the claimed invention range from 60 to 225. The type A, 
175 Bloom gelatin used in the preferred embodiment of the claimed 
invention is preferred and was selected for its relatively large number of 
lysyl residues and its lower Bloom number in order to minimize 
intermolecular interactions between gelatin molecules. For optimum 
adsorption on magnetite and ferrite particles, it was buffered to pH 8.4, 
the middle of its isoelectric point range, at which pH it is most soluble 
in water and gives the least viscous solution. The instability of gelatin 
adsorbed on ferrite particles, which instability arises when 
glutaraldehyde is added, was overcome by the present invention by the use 
of more dilute particle and gelatin concentrations [0.1% weight/volume 
(w/v) instead of the 2.5% w/v solids suspension that was used in other 
reactions herein] in conjunction with an inert polymeric stabilizer, 
polyvinylpyrrolidone (PVP), that does not react with glutaraldehyde. The 
use of the stabilizer and the 25-fold lower gelatin concentrations avoids 
interparticle crosslinking during the glutaraldehyde fixation reaction. 
Since polymer desorption is a very slow process relative to the time of 
the glutaraldehyde fixation reaction, approximately 6 minutes, a stable 
gelatin coating around the core particle was produced. 
In order to be useful in the biological and medical arts, the fixed 
(crosslinked) gelatin coating should contain functional groups which can 
be conjugated with biologically active substances such as antibodies to 
produce immobilized biologically active substances attached to the 
particle surface. Covalent coupling of biological substances to the 
particle surface is preferred over simple adsorption. The coupling of an 
antibody, either polyclonal or monoclonal, to the crosslinked gelatin 
surface is accomplished by the use of "short chain" diamines or polyamines 
and a heterobifunctional reagent. (Hereafter, the word polyamine includes 
diamines). The polyamine is reacted with residual aldehyde or carboxyate 
groups, either naturally occurring or present by the steps of this 
invention, present on the crosslinked gelatin surface. The use of 
polyamine serves not only to block aldehyde/carboxylate groups, but also 
serves to replenish gelatin amino groups such as lysyl amino groups which 
were depleted during the crosslinking process. This procedure is generally 
accomplished in two steps. In the first step, unreacted terminal aldehyde 
groups are reacted with a polyamine followed by sodium borohydride 
(NaBH.sub.4) reduction of the resulting Schiff's base to create stable, 
saturated C-N linkages. In the second step, exposed carboxylic acid 
residues (glutamic, aspartic) of gelatin are coupled to polyamine in the 
presence of a water soluble carbodiimide such as 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC). 
Short chain diamines or polyamines are preferred in order to avoid 
crosslinking neighboring aldehyde or carboxylic acid groups on the same 
particle or to avoid linking such groups on different particles. One 
polyamine amine group reacts with the gelatin surface and the other(s) 
remains unreacted and available for coupling, directly or indirectly, to a 
biological substance. Examples of `short chain` diamines or polyamines 
include ethylenediamine, phenylenediamine, propylenediamine, 
1,4-cyclohexanediamine, cyclohexenediamine, tetramethylenediamine, 
diethylenetriamine, 1,5-diamino-3-(2-aminoethyl)pentane [(H.sub.2 
NCH.sub.2 CH.sub.2).sub.3 C] and the like. Ethylenediamine is preferred. 
The coupling of the biological substance to the particle involves 
activation of the free amino groups of the gelatin-coated particles with a 
water soluble heterobifunctional reagent such as 2-iminothiolane 
hydrochloride (IT), 
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate 
(sulfo-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester, 
N-succinimidyl-3-(2-pyridyldithio)propionate, 
succinimidyl-4-(p-maleimidophenyl)butyrate, 
N-succinimidyl-(4-iodoacetyl)aminobenzoate, the reagents listed above as 
substitutes for glutaraldehyde and the like. The 2-iminothiolane 
hydrochloride and the maleimidyl/succinimidyl reagents are preferred. E. 
Ishikawa, Immunoassay Supp., 1:1-16 (1980) and J. Immunoassay, 4:209-227 
(1983); M. Imagawa et al., J. Appl. Biochem., 4:41-57 (1982); and M. D. 
Partis, J. Protein Chem., 2:263-277 (1983). When using sulfo-SMCC, the 
active sulfosuccinimidyl ester end of sulfo-SMCC will react at pH 7.0-7.5 
with amines to give peptide bonds. The sulfo-SMCC/diamine bridging unit 
which results is approximately 16 Angstroms in length. 
When performing the polyamine and sulfo-SMCC reactions, particle 
aggregation was monitored by microscopic examination (1000.times. 
magnification) and by light scattering analysis using a Coulter N.sub.4 MD 
submicron particle size analyzer (COULTER CORPORATION, Hialeah, Florida), 
or similar instrument. 
The maleimidyl group of sulfo-SMCC will react at pH 6.5-7.5 with free 
sulfhydryl groups to form a stable, covalent thioether bond. However, it 
is essential that the coated particles with which sulfo-SMCC is reacted 
contain no free sulfhydryl groups which could react with the maleimidyl 
end of sulfo-SMCC. Sulfhydryl groups are found on or generated from 
cystine and cysteine amino acid residues of which gelatin has very few. 
Consequently, the crosslinked gelatin particles of the claimed invention 
do not require a protein modifier to block free sulfhydryl groups prior to 
reaction with sulfo-SMCC. 
Biological substances, particularly either monoclonal or polyclonal 
antibodies, can be covalently linked to the maleimidyl end of sulfo-SMCC 
functionalized particles by means of sulfhydryl groups present, either 
naturally or by derivatization, on said biological substances. Biological 
substances which have cysteinyl residues inherently contain sulfhydryl 
groups. To introduce additional sulfhydryl groups, the biological 
substances' amine groups are activated with Traut's reagent, 
2-iminothiolane hydrochloride (IT), at a pH in the range of 7-10. M. 
Erecinska, Biochem. Biophys. Res. Commun., 76:495-500 (1977); J. M. 
Lambert et al., Biochemistry, 17:5406-5416 (1978); and M. E. Birnbaumer et 
al., Biochem J., 181:201-213 (1979). When the bio-substances are 
antibodies, antibody lysyl and terminal amine groups are activated by IT. 
In the present invention, reaction conditions and the concentration of 
reactants were varied to determine the optimal coupling so that the 
bio-substance, especially antibody, when conjugated with the substrate 
particles, retains its maximum functional activity. Although maleimides 
react quite rapidly with sulfhydryl groups in solution, the same groups 
immobilized on particles were given longer reaction periods to react with 
protein. Particle and antibody concentrations during antibody conjugation 
were optimized to avoid aggregation, particularly when IgM antibodies were 
used. The procedures optimized for IgM antibodies can be used for all 
monoclonal antibodies with an isoelectric point range of about 5.0 to 
about 9.0. Generally, about 30-fold less antibody was required to achieve 
covalent coupling than is required for simple adsorption; a consequence of 
importance where expensive or hard to obtain antibodies are involved. 
The optimum concentration of iminothiolane-activated antibody to use in 
conjugation reactions with maleimidyl-activated particles was determined 
by the use of activated antibody binding curves (Total Antibody vs Surface 
Antibody Concentration). After a typical conjugation period, a sample is 
taken and filtered through a 0.2 .mu.m low-protein binding filter. The 
filtrate is analyzed spectrophotometrically. The surface antibody is 
determined by the difference between the total antibody in the starting 
solution and the antibody in the filtrate (Total Antibody--Filtrate 
Antibody). The binding data in antibody (Ab) concentration dependent runs 
show Langmuir isotherm-type characteristics; i.e., a linear low 
concentration region for total antibody versus surface antibody 
concentration, a smooth inflection point and a plateau indicating 
saturation at the particle surface at high concentrations. The antibody 
concentrations actually used were those at the inflection point or at 
concentrations slightly above the infection point. Binding constants were 
obtained graphically by recasting the equation of a hyperbola into one for 
a straight line. A double reciprocal plot of 1/n.sub.2.sup.2 versus 
1/C.sub.2 was constructed, where n.sub.2.sup.s is the number of moles of 
IT-Ab bound per gram of particles and C.sub.2 is the molar concentration 
of free IT-Ab at equilibrium. Linear plots are indicative of Langmuir-type 
binding behavior. The binding constants K.sub.1 =n.sup.s K of IT-Ab for 
sulfo-SMCC-activated ferrite particles were calculated using the equation 
1/n.sub.2 =1/(n.sup.s KC.sub.2)+1/n.sup.s, where K is the intrinsic 
binding constant and ns is the number of moles of binding sites per gram 
of ferrite particles. Linear regression analysis of plots for various 
monoclonal antibodies gave the following results: 
______________________________________ 
Ab T11: K = 1.3 .times. 10.sup.6 M.sup.-1 
n.sup.s = 5.9 .times. 10.sup.-8 mol/g 
Ab KC16: K = 6.4 .times. 10.sup.6 M.sup.-1 
n.sup.s = 5.1 .times. 10.sup.-7 mol/g 
Ab 1D3: K = 2.7 .times. 10.sup.6 M.sup.-1 
n.sup.s = 2.0 .times. 10.sup.-7 mol/g 
Ab MO2: K = 1.8 .times. 10.sup.7 M.sup.-1 
n.sup.s = 7.1 .times. 10.sup.-7 
______________________________________ 
mol/g 
The results for the ferrite particles compare favorably with similar data 
for commercially available carboxy-modified latex beads (23% magnetite, 
0.980 .mu.m dia., obtained from Rhone-Poulenc) covalently coated with 
aminodextran and conjugated to monoclonal antibodies and protein. These 
results are: 
______________________________________ 
Ab T11: K = 6.5 .times. 10.sup.5 M.sup.-1 
n.sup.s = 1.1 .times. 10.sup.-7 mol/g 
Ab KC16: K = 3.2 .times. 10.sup.6 M.sup.-1 
n.sup.s = 6.9 .times. 10.sup.-8 mol/g 
Ab 1D3: K = 3.2 .times. 10.sup.5 M.sup.-1 
n.sup.s = 1.7 .times. 10.sup.-7 mol/g 
Ab MO2: K = 2.0 .times. 10.sup.6 M.sup.-1 
n.sup.s = 1.6 .times. 10.sup.-7 mol/g 
Ab KC48: K = 2.5 .times. 10.sup.5 M.sup.-1 
n.sup.s = 7.6 .times. 10.sup.-8 mol/g 
Ab PLT-1: 
K = 2.8 .times. 10.sup.5 M.sup.-1 
n.sup.s = 2.2 .times. 10.sup.-7 mol/g 
Streptavidin: 
K = 1.3 .times. 10.sup.6 M.sup.-1 
n.sup.s = 9.5 .times. 10.sup.-8 
______________________________________ 
mol/g 
In addition to ferrite core beads, the present invention was also evaluated 
using monoclonal antibodies conjugated to crosslinked gelatin-coated 
polystyrene beads. The binding constants for these antibodies, which 
compare favorably to both evaluations given above, are: 
______________________________________ 
Ab T8; K = 1.7 .times. 10.sup.6 M.sup.-1 
n.sup.s = 9.5 .times. 10.sup.-8 mol/g 
Ab T4: K = 2.5 .times. 10.sup.7 M.sup.-1 
n.sup.s = 3.5 .times. 10.sup.-8 mol/g 
______________________________________ 
The results with the polystyrene beads indicate that the method of the 
present invention is not limited to magnetic spheres, but may be used with 
any colloidal particles that have a hydrophobic surface. 
DESCRIPTION OF THE PREFERRED EMBODIMENT USING MAGNETIC BEADS 
Preparation of Magnetite and Other Magnetic Particles in Gelatin Solution 
10 mmol (5 mL) of 2M KNO.sub.3 solution, 12.5 mmol (2.5 mL) of 5M KOH 
solution and 11.25 mL of double distilled water (DDW) were mixed and 
purged with N.sub.2 gas for 10 minutes (Solution A). 6.25 mmol (6.25 mL) 
of 1M FeSO.sub.4 solution and 25 mL of freshly prepared, N.sub.2 purged, 
2% type B, 225 Bloom, bovine skin gelatin solution [useful gelatin 
solution range is from about 0.8% to about 2.0%] were then added to 
Solution A in a Pyrex.RTM. bottle, mixed, swept with N.sub.2 gas, capped 
tightly, and placed undisturbed in an oven at 90.degree. C. for 4 hours. 
After the suspension of black magnetite particles had reached room 
temperature, they were sonicated for 1/2 hour, washed with 1% type B, 225 
Bloom gelatin solution, and then contacted with a large excess of 1% w/v 
gelatin as is the next step. 
Metal ferrites may also be prepared using gelatin in situ in their 
preparation. In trials with other metals, namely Mn.sup.2+, Zn.sup.2+, 
Co.sup.2+, Ni.sup.2+, and, the molar ratio of M.sup.2+ :Fe.sup.2+ was kept 
at 1:2, but nitrate was used instead of sulfate for Co.sup.2+ and 
Ni.sup.2+. The total metal-to-hydroxide molar ratio was maintained at 1:2; 
but the relative KNO.sub.3 to total metal and KNO.sub.3 to KOH molar ratios 
were altered. In preparing the mixed Mn/Zn ferrite, a 1:1 molar ratio of 
manganese sulfate to zinc sulfate and the same total molar amount of 
non-ferrous metal ions were used. The following is an example. 
10 mmol (5 mL) of 2M KNO.sub.3 solution, 18.75 mmol (3.75 mL) of 5M KOH 
solution and 6.875 mL DDW were mixed and purged with N.sub.2 gas for 10 
minutes (Solution C). 6.25 mmol (6.25 mL) 1M DeSO.sub.4 solution, 3.125 
mmol (3.125 mL) of 1M Co(NO.sub.3) solution and 25 mL of type B, 225 
Bloom, bovine skin gelatin solution were mixed and purged with N.sub.2 gas 
for 10 minutes. (Solution D). Solution D was added to Solution C in a 
Pyrex.RTM. bottle, mixed, swept with N.sub.2 gas, capped tightly, and 
placed undisturbed in an oven at 90.degree. C. for 5 hours. After the 
suspension of brown particles had reached room temperature, they were 
sonicated for 1/2 hour, washed with 1% type B, 225 Bloom gelatin solution 
and then contacted with a large excess of 1% w/v gelatin as in th next 
step. 
Using the methods described above, cobalt and nickel ferrite particles of 
about 0.1 and 0.2 .mu.m in diameter and of spherical shape were formed in 
large, loosely-held brown aggregates. Zinc gave low yields of light brown 
magnetic material of less than 0.2 .mu.m diameter even after 72 hours of 
heat treatment. Dark brown manganese ferrite particles of uniform, 
spherical shape and 0.3 .mu.m diameter were obtained as single particles 
in 83-88% yields. Similar light brown manganese-zinc ferrite particles 
were produced in 49-55% yield after 72 hours of heat treatment at 
90.degree. C. For barium, the procedure was modified since BaSO.sub.4 is 
insoluble in water. (Except for the case where barium is present, the 
divalent metals may be used as their chlorides or sulfates as well as 
their nitrates). Thus 6.25 mmol (6.25 mL) of 1M FeCl.sub.2 solution, 0.5 
mmol (5.0 mL) of 0.1 Ba(NO.sub.3).sub.2 solution and 25 mL of 2% gelatin 
were mixed and purged with N.sub.2 gas for 10 minutes (Solution D). 
Solution C and the remainder of the ferrite preparation procedure was 
unchanged except 10 mmol KOH solution (2 mL) was used and the heat 
treatment was continued for 20 hours. Black barium ferrite particles of 
uniform non-spherical shape with a 0.2 .mu.m diameter were produced. 
Preparation of Gelatin Coated Magnetic Particles 
A quantity of magnetic particles, for example, manganese ferrite particles, 
of uniform size (0.3 .mu.m) and spherical shape and prepared using in situ 
gelatin according to the procedures described above were contacted with a 
large excess of 1% w/v, type B, 225 Bloom aqueous gelatin solution. 
Alternately, preformed (i.e., formed by methods other than the in situ use 
of gelatin), dispersible magnetic particles, for example, manganese ferrite 
particles, of uniform size (0.3 .mu.m) and spherical shape were contacted 
with a large excess of 1% w/v, type B, 225 Bloom gelatin solution at 
ambient temperature for approximately 60 minutes. The particles (either of 
the above) were then magnetically separated and washed five times with a 2% 
w/v, type A, 175 Bloom gelatin solution in 0.2M aqueous sodium chloride, pH 
8.4. After washing, the particles were stored at ambient temperatures for 
up to several months as 2.5% w/v (weight/volume) solids suspension in a 2% 
w/v aqueous solution of the type A gelatin containing 0.2M sodium chloride, 
0.1% w/v sodium azide at pH 8.4. Provided the azide content of the storage 
solution is maintained, the suspension can be stored for up to about 3 
months. The magnetic particles so formed have two gelatin layers, a first 
layer of type B gelatin and a second layer of type A gelatin. 
Crosslinking the Adsorbed Gelatin 
62.5 L of 25% aqueous glutaraldehyde (0.156 mmol) solution was added to 120 
ml of 1% aqueous polyvinylpyrrolidone (MW=40,000) in 0.2M aqueous sodium 
chloride, pH 7.2. To this, 5 ml of the 2.5% solid suspension prepared 
above was added to the glutaraldehyde solution and the resulting 
suspension was mixed at ambient temperature for a time in the range of 
3-15 minutes, preferably about 6 minutes. 
Blocking of Unreacted Aldehyde Groups 
0.105 ml of 99% ethylenediamine (1.56 mmol) was added to a 125 ml 
suspension of the fixed, gelatin coated magnetic particles (0.1% w/v 
solids) in 1% PVP solution, 0.2M in sodium chloride, pH 7.2. The resulting 
suspension was mixed for a time in the range of about 1 to 4 hours, 
preferably about 2 hours, in a 250 ml tissue culture flask. At the end of 
the mixing time, 1.25 ml of a 10 mg/ml solution of sodium borohydride 
(NaBH.sub.4) in 0.1 mM KOH was added to the magnetic particles and the 
resulting suspension was mixed for an additional 15 minutes. The particles 
were then magnetically separated and washed a plurality, preferably three, 
times with 0.2M aqueous sodium chloride. 
Reaction with Fixed Gelatin's Carboxylate Residues 
2.11 ml of 99% ethylenediamine were added to an 118 ml suspension of the 
aldehyde-blocked beads, 0.1% w/v solids, in 0.2M aqueous NaCl. The 
resulting suspension was physically and sonically mixed for approximately 
15 minutes. After this mixing, 4.5 ml of 10 mg/ml EDAC in 0.2M NaCl was 
added and the suspension was first physically and sonically mixed for 
approximately 15 minutes, and finally physically mixed for a time in the 
range of about 8-16 hours. The contents of the flask were then 
magnetically separated, washed a plurality of times with 1.times.PBS, 
sonically mixed in 1.times.PBS or approximately 30 minutes, and finally 
concentrated to 5 ml of 2.5% w/v solids in 1.times.PBS. For large scale 
(100.times.) preparations, the previous aldehyde blocking step and the 
EDAC coupling step have been combined to avoid multiple separations and 
washings. The combination of steps did not result in any loss of activity 
in the final antibody-conjugated beads. 
Activation of Diamine Treated Particles with Sulfo-SMCC 
In general, 27 .mu.L of freshly prepared 10 mg/ml sulfo-SMCC in 1.times.PBS 
was used per milliliter of 2.5% w/v magnetic particle suspension. In a 
typical preparation, 135 .mu.L of the sulfo-SMCC solution was added to 5 
ml of 2.5% w/v particles. The mixture was then roller mixed in a 15 ml 
plastic centrifuge tube for approximately one hour, sonically mixed for 
approximately 5 minutes, magnetically separated, and washed a plurality of 
times with 1.times.PBS. 
The functionalized, crosslinked, gelatin coated particles resulting from 
the above series of steps have pendent maleimidyl groups and are suitable 
for a variety of medical and/or biological uses. If the substance which is 
desired to be conjugated to the particles has a sufficiency of active 
sulfhydryl groups, activation of that substance is not necessary, and the 
following step may be skipped. 
Antibody Activation with 2-iminothiolane Hydrochloride 
A 51.24 mg/ml concentrate of T11 monoclonal antibody in 1.times.PBS 
containing 0.1% NaN.sub.3 was prepared. For 10 mg of T11 antibody and 15 
mg/ml antibody concentration during coupling, the total reaction volume 
should be 0.667 ml. Using a 15:1::IT:T11 activation ratio, 0.9375 .mu.mol 
(0.129 mg) IT (65 .mu.L of 2 mg/ml IT) in 1.times.PBS is required. 
Therefore, 0.407ml of 1.times.PBS solution was added to 0.195 ml of T11 
concentrate, to which resulting solution an additional 65 .mu.L of 2 mg/ml 
IT solution was added. The net resulting solution was roller mixed in a 
tube reactor for 1 hour. The content of the reaction tube was then applied 
to the top of a 20 ml G-50 Sephadex column, equilibrated and washed with 
100 ml 1.times. x PBS. The derivatized antibody was eluted using 
1.times.PBS and a plurality of 2.5 ml fractions were collected with the 
aid of a UV monitor. Fractions in the middle of the band absorbing at 280 
nm were pooled and the A.sub.280 value was used to determine T11/IT 
antibody concentration. Typically, the T11/IT concentration was about 3.0 
mg/ml. The T11/IT solution may be concentrated by solvent removal. 
Conjugation of T11/IT with Sulfo-SMCC Derivatized Particles 
In a laboratory scale conjugation, total volume 5 ml, the concentration of 
particles was 2.5% w/v solids and the T11/IT concentration was 0.9 mg/ml. 
In one sample, when the purified T11/IT solution concentration was 1.850 
mg/ml, then 2.392 ml of T11/IT antibody solution in 1.times.PBS was added 
to 5 ml of 2.5% w/v solids sulfo-SMCC activated particles which had been 
preconcentrated by the removal of 2.432 ml of supernatant. The T11/IT 
solution was added to the particles in 0.5 ml increments with sonic and 
rapid physical mixing between additions. The resultant solution was then 
roller mixed in a 15 ml tube for approximately two hours. A 1 ml test 
sample was then taken, filtered through a low-protein binding 0.2 .mu.m 
filter, and the filtrate analyzed spectrophotometrically for T11 antibody 
by measuring the absorbance at 280 nm; A.sub.280 =c (supernatant) =0.3986 
mg/ml. [measurement by difference, c (surface)=c (total)-c (supernatant)]. 
Thus c (surface)=0.9 mg/ml-0.3986 mg/ml=0.501 mg/ml. This translates to a 
T11 surface loading of 20 mg T11 per gram particles or, for a specific 
surface area of 4.89 m.sup.2 /g for manganese ferrite particles, a 4.1 mg 
T11/m.sup.2 particle surface area. Similar procedures with 2- and 3-fold 
dilutions of particle concentration, but the same total antibody 
concentration during conjugation, gave higher surface antibody loading. 
However, a limitation was reached when a 4-fold dilution of the particles 
concentration did not yield higher surface coverage of antibody. 
Blocking Unreacted Maleimidyl and Sulfhydryl Groups 
Unreacted maleimidyl groups on the sulfo-SMCC activated particles were 
blocked with L-cysteine after antibody conjugation. Typically, 0.480 ml of 
5 mg/ml L-cysteine in 1.times.PBS was added to remaining 4 ml of the 
conjugation mixture of the previous step and the resulting solution was 
roller mixed for 15 minutes. Unreacted sulfhydryl groups were blocked by 
the addition of 0.534 ml of 20 mg/ml iodoacetamide in 1.times.PBS followed 
by the addition of 0.100 ml of 1M, pH 9.8 sodium borate buffer solution. 
The resulting solution was roller mixed for 30 minutes, the blocked 
conjugation mixture was magnetically separated and the particles washed 
three times with 1.times.PBS containing 1% bovine serum albumin (fraction 
V, heat shock) and 0.1% NaN.sub.3 (BSA buffer solution). After washing, 4 
ml of the foregoing BSA solution was added to the particles, the particles 
roller mixed for approximately 1 hour, stored at 4.degree. C. for a time in 
the range of about 8-16 hours, magnetically separated and washed three 
additional times with BSA buffer. 
Antibody containing particles prepared according to the method described 
herein have been found useful in various cell separation assays. The 
biological substances used in assays utilizing the invention may be 
selected from the groups consisting of normal or non-normal T-cells, 
B-cells, leukocytes, viruses, erythrocytes, cells of the breast, uterus, 
colon, kidney, liver, lung, testes, stomach, thyroid and parathyroid, and 
the like; provided that the biological substance contains an antigenic 
determinant capable of binding to an antibody. 
In an embodiment of the invention equivalent to the magnetic particle 
embodiment described above, the maleimidyl groups and the sulfhydryl 
groups are transposed. That is, the crosslinked gelatin coated particles 
are derivatized to have pendent groups ending in reactive sulfhydryl 
groups in place of the maleimidyl groups described above and the 
antibodies are derivatized to have reactive maleimidyl groups in place of 
the sulfhydryl groups described above. The methods used to prepare this 
equivalent embodiment are the same as described above. In both cases, the 
antibody is connected to the gelatin surface by a molecular bridge 
prepared as described. 
The following examples are given to illustrate the utility of the claimed 
invention and are not to be taken as limiting said invention. 
EXAMPLE 1 
Protocol for Magnetic Bead Depletion of T-cell and B- cell Populations. 
Mononuclear cells (MNC) were obtained from whole blood samples by density 
isolation on Ficoll-hypaque gradients and washed in PBS. 1.times.10.sup.6 
MNC in 1 ml PBS were added to a series of tubes containing 5, 10, 25, 50 
and 100 .mu.L of the monoclonal antibody (MoAb) conjugated magnetic 
particle suspension (2.5% w/v) being tested. Two tubes were set up for 
each depletion and for the undepleted control. The resulting suspensions 
were then nutated for 3 minutes in a multi-tube vortexer or a single tube 
nutator. At the end of incubation, the cell suspension was placed for a 
total of 2 minutes in the magnetic field provided by a single tube 
magnetic rack. At the end of the magnetic separation, unbound cells were 
extracted by withdrawing all the clear liquid from the center of the tube 
with a Pasteur pipet. 
For T- or B-cells (T11, T3, T4, T8, B1, B4), the cell suspension collected 
after depletion was compared directly to the original cell suspension 
prior to particle depletion. The samples, original and depleted, were 
centrifuged for 5 minutes at 1200 rpm and the supernatant decanted to 
leave approximately 100 .mu.L of PBS remaining in each tube. One tube of 
each pair of depletion tubes was then stained with 10 .mu.L CYTO-STAT.RTM. 
MsIgGl-RDl/MsIgGl-FlTC control reagent (MS) and the other tube was stained 
with 10 .mu.L CYTO-STAT.RTM.T11-RDl/B4-FlTC reagent (for T11, T3, B1 or B4 
depletions) or with 10 .mu.L of T4-RDl/T8-FlTC reagent (for T4 or T8 
depletions) at room temperature for 10 minutes. At the end of incubation, 
500 .mu.L of PBS were added to each sample and the samples were analyzed 
by flow cytometry. The samples were analyzed on the EPICS.RTM. Profile 
using the MBead 2-Color program. (EPICS and CYTO-STAT.RTM. are registered 
trademarks of Coulter Corporation). As the original sample stained with MS 
control reagent was being run, it was checked to determine whether the 
lymphocyte population was fully incorporated in Bitmap 1, and adjustments 
were made if necessary. The left side of discriminator 2 was set for each 
fluorescence histogram on the channel which would give &lt;1% positive 
staining. This was done for each sample stained with MS control reagent 
and then the corresponding tube stained with specific antibody was 
analyzed. The data were collected and recorded as the absolute number of 
positive staining cells in the red and green histograms (T and B or T4 and 
T8) not percent positive. Test results are summarized below. 
EXAMPLE 2 
Protocol for Magnetic Bead Depletion of Red Blood Cells (RBC). 
100 .mu.L of Na.sub.4 EDTA-anticoagulated whole blood was placed in a 
series of reaction tubes. To each tube, 25 to 150 .mu.L of KC-16 
conjugated magnetic particles suspension (2.5% w/v) was added and the 
total volume was adjusted to 250 .mu.L using PBS. The suspensions were 
nutated for 3-5 minutes in a multitube vortexer or a single tube nutator 
at low mixing speed. When nutation was completed, 1 ml of PBS was added to 
each sample tube which was then placed on a magnetic rack for 2-5 minutes. 
All the supernatant was removed from each tube using a Pasteur pipet and 
saved in labelled tubes. Samples were analyzed on a Coulter S-plus IV or 
similar rbc counter as total rbc number/ml whole blood. The positive 
control was 100 .mu.L whole blood plus 1.150 ml PBS to give 100% rbc count 
and the negative control was 100 .mu.L whole blood plus 1.150 ml of Batch 
lyse or similar lysing agent to give 0% rbc count. Percentage of rbc 
depleted=100%- [(rbo count in sample tube)/(100% rbc count)]. 
EXAMPLE 3 
Protocol for Magnetic Bead Depletion of Leukocytes 
100 ml of Na.sub.4 EDTA-anticoagulated whole blood were collected, divided 
among a number of centrifuge tubes and centrifuged at 500 g for 10 
minutes. The majority of plasma was removed and the buff colored layer of 
cells from each tube was removed, pooled together and centrifuged at 500 g 
for an additional 10 minutes. The buff colored cells and the plasma 
constitute the leuko-rich whole blood which should have an rbc count no 
greater than 8.0.times.10.sup.9 /ml and a white blood cell (wbc) count of 
2-4.times.10.sup.7 /ml. 
100 .mu.L of leuko-rich whole blood was pipetted into a number of reaction 
tubes. An amount of 10 to 160 .mu.L of magnetic bead suspension (2.5% w/v) 
was then pipetted into each tube followed by the addition of 200 .mu.L of 
1.times.PBS. (N.B. Lower titer points with 10 to 40 .mu.L of beads should 
be run first. Additional beads were added only if endpoint depletion was 
not obtained at 40 .mu.L). Each tube was nutated for 3-5 minutes at low 
speed. 2 ml of 1.times.PBS was then added, the contents of a tube mixed 
and then magnetically separated for 2 minutes. All supernatant liquid was 
removed and placed in a duplicate tube which was then centrifuged at 400 g 
for 5 minutes. The resulting supernatant was then carefully removed by 
pipette and analyzed. 
The leuko-rich or the leuko-depleted whole blood samples were analyzed by 
the addition of 10 .mu.L of single or dual color antibody preparation 
designed to discriminate for the depletion of specific cells from a 
mixture of cells. For example, when T11-conjugated magnetic beads were 
used in depletion, T11-B4 dual color was used to discriminate between 
actual T11+ cell depletion and the non-specific depletion of T11-cells 
(i.e. B-cells). The mixture was vortexed and incubated for 10 minutes at 
room temperature in the dark. Controls were isotype control and antibody 
control with undepleted cells. The tubes were then placed on a Coulter 
EPICS.RTM. Q-prep, or similar instrument, and run on the 35 second lyse 
mode. After the rbc were lysed and the samples fixed (Q-prep), all samples 
were analysed on a Coulter EPICS.RTM. Profile flow cytometer or similar 
instrument. This procedure is required to obtain data as actual number of 
cells per volume of sample. Programs available on Profile were used to 
analyze lymphocytes and monocyte-myeloid populations. 
Summary of Test Results using the Protocols of Examples 1-3 
1. In a T11/B4 lymphoid cell assay, the undepleted control gave 97,209 
T11+, 18,240 B4+, 19,717 monocyte and 25,381 granulocyte counts. After 
depletion with 10 .mu.L of 2.5% w/v solids magnetic beads conjugated with 
T11 antibody, the counts were 15,826, 20,181, 19,954 and 30,972 
respectively. Depletion with 20 .mu.L T11 antibody conjugated beads gave 
2,256, 20,989, 20,874 and 31,965 counts; 30 .mu.L gave 1,150, 21,428, 
20,697 and 35,362 counts; and 40 .mu.L gave 644, 21,232, 19,817 and 33,935 
counts, all respectively. 
2. In a T4/T8 lymphoid cell assay, the undepleted control, which contained 
4.1.times.10.sup.5 T8 and 7.9.times.10.sup.5 T4 cells, gave 54,415 T4 and 
27,906 T8 counts. After depletion with 10, 20 and 30 .mu.L of 2.5% w/v 
solids magnetic beads conjugated with T8 antibody the counts were 57,030 
and 12, 59,538 and 6, and 60,905 and 5, respectively. 
3. In an erythrocyte/thrombocyte assay, the undepleted control contained 
4.5.times.10.sup.6 wbc, 4.4.times.10.sup.8 rbc and 4.7.times.10.sup.7 
platelets. Depletion experiments were conducted using 20, 40, 60 and 80 
.mu.L of 2.5% w/v solids magnetic beads conjugated with KC-16 antibody. 
The wbc, rbc and platelets remaining after depletion were 20 
.mu.L:4.4.times.10.sup.6 wbc, 1.6.times.10.sup.8 rbc and 
4.3.times.10.sup.7 platelets; 40 .mu.L:4.6.times.10.sup.6 wbc, 
1.times.10.sup.7 rbc and 4.5.times.10.sup.7 platelets; 60 
.mu.L:4.5.times.10.sup.6 wbc, 1.times.10.sup.7 rbc and 4.3.times.10.sup.7 
platelets; and 80 .mu.L:4.5.times.10.sup.6 wbc, 1.times.10.sup.7 rbc and 
4.3.times.10.sup.7 platelets. The results indicate that 40 .mu.L of 2.5% 
solids beads which contained 1.85.times.10.sup.10 particles removed 
4.3.times.10.sup.8 rbc, thus giving a particle-to-rbc ratio of 43. 
4. In a myeloid cell assay, the undepleted control gave 73,821 lymphocyte, 
13,426 monocyte and 55,661 granulocyte counts. Depletion studies were 
conducted using 10, 20, 30 and 40 .mu.L of 2.5% w/v solids magnetic beads 
conjugated with KC-48 antibody. The results were 10 .mu.L:70,330, 9,309 
and 340 counts; 20 .mu.L:68,414, 2,006 and 1,332 counts; 30 .mu.L:62,966, 
1,597, and 922 counts; and 40 .mu.L:59,340, 1,546 and 899 counts, all 
respectively. 
A similar depletion study was conducted using 10, 20, 30 and 40 .mu.l of 
2.5% w/v solids magnetic beads conjugated with 1D3 antibody. The results 
were 10 .mu.L:13,839 and 1,597 counts; 20 .mu.L:73,198, 8,653 and 1,216 
counts; 30 .mu.L:65,667, 2,590 and 2,130; and 40 .mu.L:66,276, 1,906 and 
1,686 counts, all respectively. 
A further depletion study was conducted using 10, 20, 30 and 40 .mu.L of 
2.5% w/v solids magnetic beads conjugated with MO2 antibody. The results 
were 10 .mu.L:72,563, 3,107 and 56,520 counts; 20 .mu.L:72,905, 3,616 and 
34,533 counts; 30 .mu.L:69,644, 1,618 and 32,313 counts; and 
40.mu.L:69,477, 1,210 and 30,899 counts, all respectively. 
5. In an erythrocyte/thrombocyte assay, the undepleted control contained 
7.times.10.sup.6 wbc, 4.9.times.10.sup.10 rbc and 3.0.times.10.sup.7 
platelets. Depletion studies were conducted using 20, 40, 60 and 80 .mu.L 
of 2.5% w/v solids magnetic beads conjugated with PLT-1 antibody. The 
results, after depletion, were 20 .mu.L: 10.times.10.sup.6 wbc 
5.4.times.10.sup.10 rbc and 1.times.10.sup.6 platelets; 40 
.mu.L:10.times.10.sup.6 wbc, 5.8.times.10.sup.10 rbc and 1.times.10.sup.6 
platelets; 60 .mu.L:7.times.10 6 wbc, 5.1.times.10.sup.10 rbc and 
1.times.10.sup.6 platelets; and 80 .mu.L:10.times.10.sup.6 wbc, 
5.6.times.10.sup.10 rbc and 0 platelets. 
DESCRIPTION OF THE PREFERRED EMBODIMENT USING POLYSTYRENE LATEX TICLES 
Preparation of Gelatin Coated Polystyrene Latex Particles 
Sulfate polystyrene latex particles (IDC Corporation, Portand, Oregon) of 
uniform size (2.17 .mu.L.+-.3.0%) and spherical shape were dispersed in 
distilled water and centrifuged for 10 minutes at 3000 rpm. The 
supernatant liquid was discarded and the particles were resuspended in 1% 
aqueous, type A, 175 Bloom gelatin at 2.5% w/v solids, sonically mixed for 
1 minute to aid redispersion and roller mixed for 8-16 hours. 
Crosslinking the Adsorbed Gelatin and Blocking Unreacted Aldehyde Groups 
A 0.300 .mu.L aliquot of 25% aqueous glutaraldehyde (0.749 mmol) was added 
to 575 ml phosphate buffered saline (1.times.PBS) containing 1% 
polyvinylpyrrolidone (40,000 MW). 25 ml of 2.5% w/v solids sulfate 
polystyrene latex particles in 1% gelatin solution were then added to the 
glutaraldehyde solution. The resulting suspension was placed in a 1L 
polypropylene centrifuge bottle and roller mixed for 6 minutes. After 
mixing, 0.505 ml of 99% ethylenediamine (7.49 mmol) was added to the 600 
ml of particles in 1.times.PBS and the resulting suspension was roller 
mixed for about 2-3 hours. 6.0 ml of 10 mg/ml NaBH.sub.4 in 0.1 mM aqueous 
KOH were added and the suspension again roller mixed for 15 minutes. The 
particles were washed three times with 0.2M aqueous NaCl by centrifugation 
and decantation. After washing, the particles were resuspended in 0.2M NaCl 
to yield 24 ml of 2.5% w/v solids suspension. 
Coupling of Ethylenediamine to the Carboxylate Residues of Gelatin Coated 
on Polystyrene Latex Particles 
A 0.404 ml aliquot of 99% ethylenediamine (6.00 mmol) was mixed with 24 ml 
of fixed, aldehyde blocked polystyrene particles, 2.5% w/v solids. A 0.960 
ml sample of 10 mg/ml EDAC (0.050 mmol) in 0.2M NaCl solution was added to 
the particles and roller mixed for 8-16 hours in a 50 ml centrifuge tube. 
The contents of the tube were washed five times with 1.times.PBS by 
centrifugation for 10 minutes at 3000 rpm and decantation. The particles 
were then resuspended in sufficient 1.times.PBS to give a total volume of 
24 ml. 
Activation by Sulfo-SMCC and Antibody Coupling to the Gelatin Coated 
Polystrene Latex Particles 
The coupling of monoclonal antibodies to gelatin coated polystyrene 
particles was carried out using the same procedures as followed for 
magnetic beads, except that separation of the particles was accomplished 
by centrifugation for 10 minutes at 3000 rpm followed by decantation of 
the supernatant liquid. 
EXAMPLE 4 
T4 and T8 Cell Population Assays Using Monoclonal Antibody Covalently Bound 
to Gelatin Coated Polystyrene Latex Particles 
15 .mu.L of KC-48-conjugated magnetic bead suspension (2.5% w/v) were added 
to 50 .mu.L of whole blood. The mixture was gently vortexed for 15 seconds 
and magnetically separated. 28 .mu.L of the supernatant were transferred 
to a new test tube and 15 .mu.L of T4- or T8-conjugated, gelatin coated 
polystyrene latex beads (2.5% w/v) were added to the tube. The contents of 
the tube were then vortexed for 15 seconds. 300 .mu.L of Batch lyse for red 
blood cells were added, the mixture vortexed for 4 seconds, 120 .mu.L of a 
lyse quencher added and the mixture again vortexed for 4 seconds. The 
resulting sample was analyzed on a Coulter VCS or similar instrument for 
the population of shifted T-cells. Controls were whole blood and whole 
blood with granulocytes removed by KC-48-conjugated magnetic beads. The 
percent of T4 or T8 cells in a sample equals the cell population shifted 
into the KC-48 depleted region of DC versus opacity, (RF-85)/DC, 
plot/[(cell population shifted into KC-48 depleted region)+(cell 
population in lymphocyte region)].times.100. 
In an embodiment of the invention equivalent to the latex particle 
embodiment described above, the maleimidyl groups and the sulfhydryl 
groups are transposed. That is, the crosslinked gelatin coated particles 
are derivatized to have pendent groups ending in reactive sulfhydryl 
groups in place of the maleimidyl groups described above and the 
antibodies are derivatized to have reactive maleimidyl groups in place of 
the sulfhydryl groups described above. The methods used to prepare this 
equivalent embodiment are the same as described above.