Cyclosporine derivatives and uses thereof

The present invention provides novel cyclosporine C (CsC) derivatives having improved protein conjugatibility and hydrolytic stability. The present invention further provides a CsC derivative conjugated to a carrier, e.g., a solid support. Preferably, the solid support is a latex or magnetic particle.

BACKGROUND OF THE INVENTION 
1. Field of the Invention. 
The present invention relates to novel cyclosporine derivatives that have 
improved protein and solid surface conjugatibility and hydrolytic 
stability. The cyclosporine derivatives of the present invention are 
useful in assays measurement of cyclosporin A levels, as well as in the 
production of cyclosporine immunogens and capture conjugates. 
2. Background 
Cyclosporine A (cyclosporine) is a potent immuno-suppressant that has been 
widely used in the United States and other countries to prevent the 
rejection of transplanted organs such as kidney, heart, bone marrow and 
liver, in humans. 
To prevent allograft rejections, minimum level cyclosporine A in the blood 
is required throughout the lifetime of the patient. Chronic high doses can 
result in kidney and liver damage. Distribution and metabolism of the drug 
varies greatly between individuals, as well as in a single individual 
during the course of therapy. Accordingly, monitoring cyclosporine A 
levels in the blood or serum of allograph recipients is considered 
essential. 
Laboratory methods for detection of cyclosporine have been developed. These 
techniques typically involve high performance liquid chromatography 
(HPLC), radioimmunoassay (RIA) and non-radioimmunoassay. 
It has been reported that CsA, itself, is non-immunogenic (Donatsch, p. et 
al., J. Immuno Assay 1981; 2:19). To obtain antibodies, therefore, it is 
necessary to link CsA to a protein carrier. The side chain of CsA, 
however, consists most of alliphatic groups. Few of the functional groups 
customarily used to link a hapten to a carrier. Previous workers have made 
immunogenic cyclosporine C (CsC) protein conjugates because the CsC has a 
threonine residue in position 2. Linkage to a protein was via a 
hemisuccinate linker through an ester group (U.S. Pat. No. 5,169,773). In 
addition, hemisuccinate coupling chemistry has been used to immobilize CsC 
to a solid support such as stabilized chromium dioxide particles (U.S. 
Pat. No. 5,151,348). Due to a number of factors including, for example, 
short chain length of the hemisuccinate linker, hydrophobicity of the 
cyclosporine-C hemisuccinate molecule and hydrolytic instability of the 
hemisuccinate ester linkage, the CsC hemisuccinate derivatives conjugate 
poorly to protein and solid surface. Furthermore, CsC protein conjugates 
and CsC immobilized on a solid support by hemisuccinate coupling, are 
hydrolyticly unstable. Thus, immunoassays developed by using such 
hemisuccinate CsC derivatives suffer from low sensitivity and poor reagent 
stability. There is a strong desire to replace the widely used 
radioimmunoassays and HPLC methods with a more robust and sensitive 
immunoassay for CsA. Accordingly, there is a need in the art for 
cyclosporine derivatives that are capable of being conjugated to solid 
supports and carriers more efficiently and stably. 
SUMMARY OF THE INVENTION 
The present invention provides novel cyclosporine C (CsC) derivatives 
having improved protein conjugatibility and hydrolytic stability. The 
present invention further provides a CsC derivative conjugated to a 
carrier, e.g., a solid support. Preferably, the solid support is a latex 
or magnetic particle. 
The invention also provides improvements in assays for the determination of 
cyclosporin levels in a sample, e.g., whole blood, suspected of containing 
cyclosporin. 
Furthermore, the invention includes kits for conducting an assay for the 
determination of cyclosporin. The present invention also provides for the 
production of cyclosporine immunogens and capture conjugates comprising 
the CsC derivatives of the present invention. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to cyclosporine C (CsC) derivatives having 
the structure: 
##STR1## 
x is selected from the group consisting of: 
##STR2## 
Y is selected from the group consisting of: 
##STR3## 
wherein Z is selected from the group consisting of; 
##STR4## 
wherein R.sub.1 and R.sub.2 are each a C1-C8 alkyl group; wherein R.sub.3 
is a C.sub.0 -C.sub.8 alkyl group; and 
wherein m is 1-200 
A particular embodiment of the present invention includes CsC derivatives 
having the following structures: 
##STR5## 
The CsC derivatives of the present invention were prepared by activation of 
the CsC position 2 hydroxy group using disuccinimidyl carbonate followed 
by coupling with linkers such as diamine linkers, e.g., ethylene glycol 
bis(2-aminoethyl)ether (DA- 10). The following scheme illustrates the 
application of this procedure to the synthesis of the CsC derivatives of 
this invention: 
##STR6## 
Starting materials used in the above-described scheme are either known or 
commercially available. 
The CsC derivatives of the present invention may be used in an immunoassay 
for the measurement of cyclosporin A levels in whole blood samples. An 
example of such an assay comprises the steps of: 
(a) lysing red blood cells in a sample of whole blood containing 
cyclosporin A; 
(b) contacting the lysed whole blood sample with excess labeled 
anti-cyclosporin antibody, e.g., beta -D-galactosidase labeled, to form a 
labeled antibody-cyclosporin A complex; 
(c) separating unbound antibody from the complex by contacting the mixture 
formed in step (b) with a solid phase comprising a cyclosporin derivative 
of the present invention immobilized on a solid support; and 
(d) determining the amount of the label in the complex as a measure of 
cyclosporin A, using, for example, a beta -D-galactosidase substrate 
selected from the group consisting of chlorophenol red- beta 
-D-galactopyranoside (CPRG) and resorufin- beta -D-galactopyranoside (ReG) 
if a beta -D-galactosidase label is used. 
The enzyme-linked immunoassay of this invention is useful for measuring 
cyclosporin A levels in whole blood samples of patients receiving 
cyclosporin A. Monitoring of cyclosporin A blood levels and subsequent 
cyclosporin A dosage adjustment are necessary to prevent toxic effects 
caused by high cyclosporin A blood levels and to prevent organ rejection 
caused by low cyclosporin A blood levels. 
The immunoassay of the present invention is performed by contacting a lysed 
whole blood sample containing cyclosporin A with excess labeled 
anti-cyclosporin antibody, e.g., beta -D-galactosidase-labeled, to form a 
reaction mixture containing a complex of cyclosporin A with labeled 
antibody and free labeled antibody, separating free antibody from the 
reaction mixture by contacting the reaction mixture with a solid phase 
comprising an immobilized CsC derivative of the present invention on a 
solid support, e.g. magnetic particles, separating the solid phase from 
the liquid phase, and measuring the amount of the bound label in the 
liquid phase by adding, for example, to the liquid phase CPRG or ReG as a 
beta -D-galactosidase substrate if a beta -D-galactosidase label is used. 
Specifically, the red blood cells of a whole blood sample containing 
cyclosporin A must be lysed to release cyclosporin A. Red blood cell lysis 
can be accomplished by many methods, such as sonication, detergent lysis 
and distilled water lysis. The lytic agent chosen should be compatible 
with the labeled anti-cyclosporin antibody. Although some detergents can 
denature beta -D-galactosidase, it has been found that by using CPRG and 
ReG as beta -D-galactosidase substrates, the sample volume can be made to 
be sufficiently small to minimize the denaturing effect of the detergent. 
The preferred lysis method uses detergent. 
After lysis, a reaction mixture is formed by contacting the lysed whole 
blood sample with excess labeled anti-cyclosporin antibody and incubating 
the reaction mixture for a time and at a temperature sufficient to permit 
the labeled antibody to form a complex with all of the cyclosporin A in 
the sample. This usually takes 1-5 minutes at room temperature. 
Anti-cyclosporin antibody can be obtained commercially, prepared by known 
methods, or prepared using the derivatives of the present invention. The 
anti-cyclosporin antibody can be polyclonal or monoclonal. A monoclonal 
anti-cyclosporin antibody specific for cyclosporin A is preferred. The 
anti-cyclosporin antibody can be labeled using standard techniques with 
any molecule that can be detected, including, for example, radioactive 
isotopes, a catalyst such as an enzyme (e.g., beta -D-galactosidase), a 
co-enzyme, a chromogen such as a fluorescer, dye or chemiluminescer, a 
dispersible particle that can be non-magnetic or magnetic, a solid 
support, a liposome, a ligand, a hapten, and so forth. 
The unbound anti-cyclosporin antibody is separated from the reaction 
mixture by contacting the reaction mixture with a solid phase comprising a 
CsC derivative of the present invention immobilized on a solid support for 
a time sufficient to permit the unbound labeled antibody to form a complex 
with the immobilized CsC derivative. This usually occurs in approximately 
one minute. 
The immobilization of the CsC derivative of the present invention can be 
accomplished by a number of known immobilization techniques. The preferred 
immobilization technique for derivatives of the present invention is to 
activate the terminal carboxy group, using for example, 
2-Fluoro-lmethylpyridinium p-toluenesulfonate (FMPT), and then coupling to 
a protein, such as albumin or globulin, which can be covalently coupled to 
a solid support. 
The CsC derivative can be immobilized on a variety of solid supports such 
as beaded dextran, beaded agarose, polyacrylamide, or glass. A preferred 
solid support useful in the immunoassay of this invention is described in 
U.S. Pat. No. 5,151,348 and 5,302,532. 
The preferred solid support comprises a stabilized chromium dioxide 
particles having cyclosporin bound to their surfaces. The stabilized 
chromium dioxide particle useful in the preferred solid support are those 
described in U.S. Pat. No. 4,661,408. These particles consist of a core of 
rutile chromium dioxide which has been extensively surface reduced, coated 
with alumina, further coated with silica containing borate and still 
further coated with a silane to which is attached cyclosporin protein 
conjugate, such as bovine gamma globulin. These particles have large 
surface areas, 40-100 m.sup.2 /g, are stable in aqueous solution and can 
be readily coupled to cyclosporin conjugate. 
The support can also be a porous or non-porous water insoluble material. 
The support can be hydrophilic or capable of being rendered hydrophilic 
and includes inorganic powders such as silica, magnesium sulfate, and 
alumina; natural polymeric materials, particularly cellulosic materials 
and materials derived from cellulose, such as fiber containing papers, 
e.g., filter paper, chromatographic paper, etc.; synthetic or modified 
naturally occurring polymers, such as nitrocellulose, cellulose acetate, 
poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose, 
polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), 
polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, 
poly(vinyl butyrate), etc.; either used by themselves or in conjunction 
with other materials; glass, ceramics, metals, and the like. 
The solid phase is separated from the liquid phase by standard separation 
techniques. The preferred separation technique is to use magnet to settle 
the magnetic particle out of liquid phase. 
The amount of cyclosporin A is determined by measuring the amount of the 
bound label in the liquid phase. For example, the amount of bound beta 
-D-galactosidase is determined by adding to the liquid phase either CPRG 
or ReG as a beta -D-galactosidase substrate and measuring 
spectrophotometrically the amount of chromophore produced at 577 nm. 
The immunoassay of this invention can be performed manually or it can be 
adapted to a variety of automated or semi-automated instrumentation, such 
as the Dimension .RTM. RxL (discrete clinical analyzer, a registered 
trademark of Dade International Inc. Deerfield, Ill.). In performing the 
assay on a Dimension .RTM. RxL, a whole blood sample is first lysed and 
preincubated with excess beta -D-galactosidase-labeled anti-cyclosporin 
antibody in a cuvette on the instrument. A known amount of stabilized 
chromium dioxide particle immobilized with CsC derivative of present 
invention is transferred into the cuvette and incubated for certain amount 
time, use magnet to separate the magnetic particle from liquid phase. The 
liquid phase of supernatant contains beta -D-galactosidase-labeled 
anti-cyclosporin antibody complexed with cyclosporin A from the whole 
blood sample. A fraction of the supernatant is pipetted and transferred to 
another cuvette with the addition of CPRG or ReG immediately preceding the 
absorbance measurements at 577 nm. 
The present invention further provides a CsC derivative conjugated to a 
carrier, which is generally a compound of molecular weight greater than 
5,000, or a label. Carriers include polyamino acids, lipopolysaccharides, 
and particles. The CsC conjugate can be used in many applications 
including as a capture conjugate in an assay or as an immunogen. The 
carrier may be immunogenic, i.e,. an immunogenic carrier. 
The poly(amino acids) will generally range from about 5,000 molecular 
weight, having no upper molecular weight limit, normally being less than 
10,000,000, usually not more than about 600,000 daltons. 
Various protein types may be employed as the poly(amino acid) immunogenic 
material. These types include albumins, serum proteins, e.g., globulins, 
ocular lens proteins, lipoproteins, etc. Illustrative proteins include 
bovine serum albumin, keyhole limpet hemocyanin, egg ovalbumin, bovine 
gamma -globulin, etc. Alternatively, synthetic poly(amnino acids) may be 
utilized. 
The immunogenic carrier can also be a polysaccharide, which is a high 
molecular weight polymer built up by repeated condensations of 
monosaccharides. Examples of polysaccharides are starches, glycogen, 
cellulose, carbohydrate gums, such as gum arabic, agar, and so forth. The 
polysaccharide can also contain polyamino acid residues and/or lipid 
residues. 
The immunogenic carrier can also be a nucleic acid either alone or 
conjugated to one of the above mentioned poly(amino acids) or 
polysaccharides. 
The carrier can also be a particle. The particles are generally at least 
about 0.02 microns and not more than about 100 microns, usually at least 
about 0.05 microns and less than about 20 microns, preferably from about 
0.3 to 10 microns diameter. The particle may be organic or inorganic, 
swellable or non-swellable, porous or non-porous, preferably of a density 
approximating water, generally from about 0.7 to about 1.5 g/ml, and 
composed of material that can be transparent, partially transparent, or 
opaque. The particles can be biologic materials such as cells and 
microorganisms, e.g., erythrocytes, leukocytes, lymphocytes, hybridomas, 
streptococcus, staphylococcus aureus, E. coli, viruses, and the like. The 
particles can also be particles comprised of organic and inorganic 
polymers, liposomes, latex particles, phospholipid vesicles, chylomicrons, 
lipoproteins, chrome and the like. 
The particles can be derived from naturally occurring materials, naturally 
occurring materials which are synthetically modified and synthetic 
materials. Among organic polymers of particular interest are 
polysaccharides, particularly cross-linked polysaccharides, such a 
agarose, which is available as Sepharose, dextran, available as Sephadex 
and Sephacryl, cellulose, starch, and the like; addition polymers, such as 
polystyrene, polyvinyl alcohol, homopolymers and copolymers of derivatives 
of acrylate and methacrylate, particularly esters and amides having free 
hydroxyl functionalities, and the like. 
The particles will usually be polyfunctional and will be bound to or be 
capable of binding to the CsC derivative. A wide variety of functional 
groups are available or can be incorporated. Functional groups include 
carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, 
hydroxyl groups, mercapto groups and the like. The manner of linking a 
wide variety of compounds to particles is well known and is amply 
illustrated in the literature. See for example Cautrecasas, J. Biol. Chem. 
(1970) 245:3059. 
The carrier can be an enzyme that is part of a signal producing system. The 
function of the signal producing system is to produce a product which 
provides a detectable signal related to the amount of bound and unbound 
label. Where enzymes are employed, the involved reactions will be, for the 
most part, hydrolysis or redox reactions. Such enzymes that may find use 
are hydrolases, transferases, lyases, isomerases, ligases or synthetases 
and oxidoreductases, preferably hydrolases. Alternatively, luciferases may 
be used such as firefly luciferase and bacterial luciferase. 
A label may be any molecule conjugated to an analyte or an antibody, or to 
another molecule. In the subject invention, the label can be a member of 
the signal producing system that includes a signal producing means. The 
label may be isotopic or nonisotopic, preferably nonisotopic. By way of 
example and not limitation, the label can be a catalyst such as an enzyme, 
a co-enzyme, a chromogen such as a fluorescer, dye or chemiluminescer, a 
dispersible particle that can be non-magnetic or magnetic, a solid 
support, a liposome, a ligand, a hapten, and so forth. 
The signal producing system may have one or more components, at least one 
component being a label. The signal producing system includes all of the 
reagents required to produce a measurable signal including signal 
producing means capable of interacting with the label to produce a signal. 
The signal producing system provides a signal detectable by external means, 
normally by measurement of electromagnetic radiation, desirably by visual 
examination. For the most part, the signal producing system includes a 
chromophoric substrate and enzyme, where chromophoric substrates are 
enzymatically converted to dyes which absorb light in the ultraviolet or 
visible region, phosphors or fluorescers. 
The signal producing means is capable of interacting with the label to 
produce a detectable signal. Such means include, for example, 
electromagnetic radiation, heat, chemical reagents, and the like. Where 
chemical reagents are employed, some of the chemical reagents can be 
included as part of a developer solution. The chemical reagents can 
include substrates, coenzymes, enhancers, second enzymes, activators, 
cofactors, inhibitors, scavengers, metal ions, specific binding substances 
required for binding of signal generating substances, and the like. Some 
of the chemical reagents such as coenzymes, substances that react with 
enzymic products, other enzymes and catalysts, and the like can be bound 
to other molecules or to a support. 
The signal producing system including the label can include one or more 
particles, which are insoluble particles of at least about 50 nm and not 
more than about 50 microns, usually at least about 100 nm and less than 
about 25 microns, preferably from about 0.2 to 5 microns, diameter. The 
particle may be organic or inorganic, porous or non-porous, preferably of 
a density approximating water, generally from about 0.7 to about 1.5 g/ml, 
and composed of material that can be transparent, partially transparent, 
or opaque. 
The label can also be fluorescent either directly or by virtue of 
fluorescent compounds or fluorescers bound to a particle in conventional 
ways. The fluorescers will usually be capable of, or functionalized to 
render them capable of, being bound to the CsC derivative or to the 
particle. 
The CsC derivatives of the present invention can be utilized to prepare 
conjugates using the reactions discussed above and set forth in the 
examples. 
Another aspect of the present invention includes antibodies prepared in 
response to a CsC derivative conjugated to an immunogenic carrier. 
Furthermore, the present invention includes conjugates of such antibodies 
and a label. 
An antibody is an immunoglobulin which specifically binds to and is thereby 
defied as complementary with a particular spatial and polar organization 
of another molecule. The antibody can be monoclonal or polyclonal and can 
be prepared by techniques that are well known in the art such as 
immunization of a host and collection of sera from which the 
immunoglobulin can be separated by known techniques (polyclonal) or by 
preparing continuous hybrid cell lines and collecting the secreted protein 
(monoclonal). Antibodies may include a complete immunoglobulin or fragment 
thereof, which immunoglobulins include the various classes and isotypes, 
such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments 
thereof may include Fab, Fv and F(ab')2, Fab', and the like. 
Monoclonal antibodies can be obtained by the process discussed by Milstein 
and Kohler and reported in Nature, 256:495-497, 1975. 
The antibodies of the present invention recognize the cyclosporins 
including cyclosporin A and derivatives and metabolites of cyclosporins. 
The antibodies of the present invention can be utilized in the 
determination of cyclosporin in a sample suspected of containing 
cyclosporin. The assay can comprise the steps of contacting the sample 
with antibodies for cyclosporin and detecting either directly or 
indirectly immune complexes of the antibodies and cyclosporin. The 
improvement provided in the present invention is the utilization of the 
present antibodies as the antibodies for cyclosporin. The immune complexes 
are detected directly, for example, where the antibodies employed are 
conjugated to a label. The immune complex is detected indirectly by 
examining for the effect of immune complex formation in an assay medium, 
on a signal producing system or by employing a labeled antibody that 
specifically binds to an antibody of the invention. 
In another configuration of an assay for the determination of cyclosporin 
in a sample suspected of containing cyclosporin, the sample is contacted 
with antibodies for cyclosporin and a conjugate of this invention 
recognized by the antibodies. The method further includes detecting either 
directly or indirectly immune complexes of the conjugate and the 
antibodies. The improvement provided in the present invention is employing 
as the CsC derivative conjugated to a label. 
The present assay invention has application to all immunoassays for 
cyclosporin. The assay can be performed either without separation 
(homogeneous) or with separation (heterogeneous) of any of the assay 
components or products. Exemplary of heterogeneous assays are enzyme 
linked immunoassays such as the enzyme linked immunosorbant assay (ELISA), 
see "Enzyme-Immunoassay" by Edward T. Maggio, CRC Press Incorporated, Boca 
Raton, Fla., 1980. Homogeneous immunoassays are exemplified by enzyme 
multiplied immunoassay techniques (e.g. see U.S. Pat. No. 3,817,837), 
immunofluorescence methods such as those disclosed in U.S. Pat. No. 
3,993,345, enzyme channeling techniques such as those disclosed in U.S. 
Pat. No. 4,233,402, and other enzyme immunoassays as discussed in Maggio, 
supra. 
The references cited throughout the specification are herein incorporated 
by reference.

The present invention is further illustrated by the following Examples. 
These Examples are provided to aid in the understanding of the invention 
and are not construed as a limitation thereof. 
EXAMPLE 1 
Synthesis of CsA-DA-10 
In a 25 mL round bottom flask equipped with magnetic stirrer, 540 mg of CsC 
and 454 mg of DSC (disuccinimidyl carbonate) was placed, then 10 mL of dry 
acetonitrile and 1000 .mu.L of triethyl amine were added. The reaction was 
stirred at room temperature for about 16-20 hours until no CsC could be 
found on TLC (thin layer chromatography). If the reaction was not complete 
after 16-20 hours, 50 mg more of DSC were added into the solution and the 
reaction checked again after 2 hours by TLC. (95% EtOAc and 5% MeOH were 
used as TLC solvent, iodine was used to visualize the spots.). To the 
solution was then added 2627 mg of DA-10 (quickly) and 1000 .mu.L of 
triethyl amine. The reaction was stirred at room temperature for another 
24 hours, and then 50 mL of CH.sub.2 CI.sub.2 added. The reaction solution 
was washed with water 3 times. The bottom organic layer was separated and 
dried with sodium sulfate. The white solid of CsA-DA-10 product was 
obtained after removal of all solvent by rotary evaperation and vacuum. 
EXAMPLES 2 
Synthesis of CsA-DA- 10-HemiGlutarnate (HG) 
In a 20 mL vial, about 550 mg of CsA-DA-10, 93.75 mg of glutaric anhydride 
and 10 mL CH.sub.2 CI.sub.2 were placed; 344 .mu.L of triethyl amine was 
added and the reaction solution stirred at room temperature for about 2 
hours. To the reaction solution was then added 40 mL of CH.sub.2 CI.sub.2, 
and washed with 1N HC1 twice and water twice. The organic layer was dried 
with sodium sulfate, and solid CsA-DA- 10-HG was obtained after removal of 
all solvent. 
EXAMPLE 3 
Conjugation of CsA-DA-10-HG with Bovine Gamma Globulin (IgG) 
A: 4 mg/mL of Bovine gamma globulin Solution: 500 mg of IgG dissolved into 
125 mL of 0.1M Na.sub.2 CO.sub.3 (pH 9.5). 
B: Activation of CsA-DA-10-HG with 2-Fluoro- Imethylpyridinium 
p-toluenesulfonate (FM PT). 
134.6 mg of CsA-DA-10-HG (0.0894 mmol) and 38.1 mg of FMPT (0.1345 mmole) 
were weighed. 4800 .mu.L of dried CH.sub.3 CN to dissolve the solids was 
added. 28.1 .mu.L of TEA was then added. The reaction was stirred at room 
temperature for 2 hr. 
C: Coupling of CsA-DA-10-HG to Protein 
The above activated CsA-DA-10-HG solution was added to the 4 mg/mL of 
Bovine gamma globulin solution with stirring, allowing each drop to 
disperse before the next one was added. After the addition was complete, 
the solution was allowed to stir gently for about 18 hours at room 
temperature. The solution was dialyzed (12,000 MW cut-off dialysis tube) 
in a cold room against 6 changes of PBS buffer solution. The dialyzed 
solution was diluted to 2 mg/mL of the protein concentration by adding 
fresh dialysis PBS buffer. 324 mg of brontopol was added to the container 
and agitated until it completely dissolved and was uniformly dispersed. 
EXAMPLE 4 
Coupling CsA-Bovine Gamma Globulin Conjugate to A Magnetic Particle 
40 ml of above CsA-IgG conjugate was added into 40 ml of glutaraldhyde 
activated chromium dioxide solution. (The detailed procedure of 
preparation of activated chromium dioxide particle is disclosed in U.S. 
Pat. No. 4,661,408, the disclosure of which is incorporated herein by 
reference). The reaction was rotated at 4.degree. C. for 24 hours. 32 ml 
of 30% BSA was added into the solution, the reaction rotated at room 
temperature for another 16-20 hours. 112 ml of 2 M glycine buffer was 
added into the above solution to quench the reaction for about 1 hour. The 
solution was washed with water three times and chrome diluent three times. 
The chromium dioxide particle was diluated to 40 ml with chrome diluent. 
______________________________________ 
Chrome diluent: 
______________________________________ 
Polymerized BSA (30%) 
15.1 g/L 
Treholose 28.7 g/L 
Carbowax 4.8 g/L 
Proclin 5 mL/L 
Nemycin Sulfate 0.06 g/L 
______________________________________ 
This invention has been described in detail including the preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art, upon consideration of this disclosure, may make modifications and 
improvements thereon without departing from the spirit and scope of the 
invention as set forth in the claims.