Polymer particles and latices thereof for the immobilization of biologically active substances

Polymer particles dispersible to form a latex and latices of such polymer particles, said particles being adaptable to the fixing or bonding thereto of a biologically active substance and having a core-shell construction wherein the polymer material of the core determines the stability of the form of the latex particles and their redispersibility, and the material of the shell PA1 (1) is so hydrophilic that it would be completely or largely water soluble if it were not anchored to the core material and/or crosslinked, PA1 (2) contains functional groups which are suitable for the covalent fixation or bonding of biological active substances, and PA1 (3) in an anhydrous condition has a T.sub..lambda.max from 20.degree.-250.degree. C., depending on its composition, and methods for bonding a biologically active substance to such particles, for example to prepare a diagnostic reagent.

The present invention relates to certain polymer particles dispersible to 
form a latex, to latices of such polymer particles, and to methods for 
immobilizing (i.e. bonding or fixing) a biologically active substance on 
such particles, which have a core-shell construction and comprise groups 
in the shell region suitable for the covalent fixation to the particle of 
a biologically active substance. 
The problem of fixing biologically active substances onto a carrier is 
posed in manifold aspects in the area of technology, for example in 
biochemistry and biotechnology, and in medicine, particularly in medical 
diagnostics, inter alia. As a rule, the "biologically active substances" 
to be fixed are compounds or functional units which are capable of a 
mutual exchange with biological systems, or are these biological systems 
themselves. 
The fixation of catalysts, especially of enzymes but also of substrates, 
such as are of significance for affinity chromatography for example, is of 
particular interest in technology. In order to explain the existing 
technical problems, the immobilization of such "biologically active 
substances" as can be diagnostically evaluated will be described more in 
detail below. 
In such reactions capable of diagnostic evaluation, the matter is one of 
detecting the mutual interaction of substances which are present in an 
organism, or are produced by an organism, and which are symptomatic of the 
condition which is to be detected diagnostically, with such substances as 
interact as specifically as possible with these "symptomatic" substances. 
An extraordinary high degree of specificity is exhibited by immune 
reactions. As is known, immune reactions occur between antigens and 
antibodies: one of the two reaction partners must be known so that the 
other can be determined qualitatively or quantitatively in a body fluid or 
can be localized in cells and tissues. 
There are various analytic methods for the determination of 
antigen-antibody reactions, for example radioimmunoassay, 
enzymeimmunoassay, immunofluorescence, immunodiffusion, and, particularly, 
immunoagglutination. 
Immunoagglutination makes possible the detection of even low concentrations 
of immunologically active materials, making use of a particulate carrier 
as an indicator, the clumping of which makes evident, visually or 
photometrically, the occurrence of of an immune reaction. 
According to the kind of carrier employed, distinctions are made between 
erythrocyte agglutination (or its inhibition) and latex agglutination (or 
its inhibition). 
Relatively considerable attention has been given to latex agglutination. 
The proposed latices can belong to different polymer types. Frequent use is 
made of latices comprising styrene or styrene-containing copolymers 
(carboxylated polystyrene, carboxylated polystyrene-butadiene copolymers, 
styrene-divinylbenzene, styrene-acrylamide, 
acrylonitrile-butadiene-styrene, styrene-methacrylate), or comprising 
anionic phenolic resins, diazotized aminocellulose in the form of fine 
particles, etc. 
Latices comprising (meth)acrylates have also been proposed. According to 
U.S. Pat. No. 4,138,383, polymers of acrylate monomers containing --OH, 
--NH.sub.2, or --COOH groups have been prepared in the presence of 
crosslinking agents in the form of suspensions of round microspheres 
having a uniform diameter equal to or less than 2000 Angstrom units 
Immunoglobulins G (IgG) have been bound to these latex microspheres using 
carbodiimide or glutaraldehyde as a condensation agent. Experiments for 
modifying the construction of the latex particles have also been 
undertaken. 
Thus, German Offenlegungsschrift No. 28 40 768 proposes carriers to which a 
water soluble polyhydroxy compound is covalently bound. The last-mentioned 
application proposes a particle size in the region from 0.01 to about 0.9 
micron and a density near that of water. The latex materials should be 
inert with respect to immunological diagnostic tests and should have 
active groups which make possible a covalent bonding with a polyhydroxy 
compound To the extent that the described conditions are fulfilled, any 
latex polymer is said to be suitable 
In Belgian Pat. No. 874,588, latex particles having a shell structure and a 
diameter from 0.15-1.5 microns are recommended. In this case, the core is 
to be formed by the polymerization or copolymerization of "hard" monomers 
and the exterior covering is to be prepared by the copolymerization of one 
or more "hard" monomers with an ethylenically unsaturated compound having 
free epoxy groups. 
For example, the free radical polymerization of styrene and glycidyl 
methacrylate in the presence of a polystyrene latex is described. The 
latex so formed can be loaded with, for example, human chorionic 
gonadotropin. 
However, to date, a technical realization of the latex concept in immune 
diagnosis has not gone further than a certain few steps. 
Among the limiting factors are, for example, a bonding of the biologically 
active substances (e.g. of an antibody). Until now, the biologically 
active substances have been predominantly bound onto the latex 
adsorptively. As a result of this, almost inescapable problems arise 
because of the diffusion of the only loosely bound biomacromolecule. 
In certain cases--as already mentioned--use is made of a covalent bonding 
of the biologically active substance. In general, this involves bonding 
functions or groups which must be introduced in several method steps, 
mostly the introduction of --COOH-- or --NH.sub.2 groups by reactions 
analogous to polymerization as well as subsequent coupling with a protein 
with the aid of (soluble) carbodiimides or of glutardialdehyde. As an 
example, the multi-stage covalent immobilization described in German 
Offenlegungsschrift 28 12 845 should be mentioned. 
Latices of particles having a core-shell construction are known from German 
Offenlegungsschrift No. 28 33 510, in which latices the polymer particle 
core is a vinyl-and/or diene-polymer having carboxylic acid and/or 
sulfonic acid groups, and the shell is a vinyl polymer having terminal 
amine-substituted thiophenol ether groups. Activation of the latex 
particles can take place, for example, by means of diazotization. 
Instead of a covalent fixation involving a multi-stage process, attempts 
have also been made to prepare latices of particles having permanent 
groups capable of reaction, for example oxirane groups. However, these 
exhibit only a limited storage stability. The subsequent purification 
steps, which are as complicated as they are indispensable, must be 
considered as perhaps the most serious disadvantage of the latices used 
for the fixation of biologically active systems. For the covalent fixation 
of proteins onto the particles of a latex, for example all auxiliary 
substances (e.g. the urea which is formed in the case of a carbodiimide 
coupling) and above all, unbound protein, must be removed in protracted 
purification steps, for example by ultrafiltration. These time consuming 
and expensive operations practically exclude any meaningful use of first 
rate materials, even those not particularly biologically stable. 
Thus, the problem existed of providing latices the use of which would not 
involve the disadvantages described earlier, or would involve them only to 
a slight degree. 
In any event, certain limits arising from physical phenomena are set on the 
technology of latices. Thus, latex particles are known to represent a 
metastable system which can only be maintained stable in the presence of 
surface active agents and only for a limited period of time. Latex 
particles show instability particularly toward elevated electrolyte 
concentrations. However, since physiologically relevant processes occur in 
solutions containing electrolytes (e.g. in 0.9 percent sodium chloride 
solution), the use of conventional latex particles is very difficult, 
particularly if it is a matter of detecting the small amounts of 
substances which are characteristically involved in diagnostic work. 
Agglutinations can be feigned as soon as there is any drying, for example, 
and even only concentration leads to a precipitation of the latex 
particles. Stabilization by the use of a high concentration of emulsifier 
is not to be recommended because of the denaturing effect of emulsifiers 
on biological systems. To be sure, a stabilizing effect can be evoked in 
the latex particles by the inclusion of strongly ionic groups, but at the 
same time the properties of the latices which are determining for the 
biospecific mutual effect are disadvantageously influenced. 
In this way, a series of demands are made on latices which are proposed to 
be used for the immobilization of biologically active substances: 
the particles of the latices should have reactive groups which make 
possible a covalent bonding of the biologically relevant molecule under 
physiological conditions; 
the particles of the latices should be capable of being stored as anhydrous 
solids in order to assure a constancy in the content of reactive groups 
therein over a long period of time; 
the particles of the latices should be fully redispersible so that their 
drying is not critical to their availability; 
and the latices should be capable of centrifugation, a requirement which is 
fulfilled if the density of the particles therein is sufficiently 
different from the density of the carrier medium or of the continuous 
phase. If the density of the particles is greater than that of the 
surrounding medium, separation of the particles can be effected by 
sedimentation; if their density is less than that of the surrounding 
medium, separation can be by flotation. 
It has now been found that polymer particles having a core-shell 
construction are particularly suitable for the covalent immobilization of 
biologically active substances or structures, particularly from the point 
of view of a diagnostic use. 
According to the invention, the shell of the core-shell polymer particle 
comprises a material capable of being swollen by water. The shell material 
shall be hydrophilic to such a high degree, as a result of its 
composition, that it would be at least partially soluble in water if it 
were not anchored to the core material and/or if it were not crosslinked. 
Thus, the shell can also be crosslinked within itself. The solution of the 
shell of the latex particle in surrounding water is thus hindered by 
bonding to the particle core, e.g. as a result of grafting and/or 
crosslinking. 
Further, the shell has the functional groups which are necessary for the 
covalent fixation of biologically active substances or structures. For 
example, such functional groups, known per se, are used as will react in 
aqueous solution with nucleophiles stronger than water and which are not 
attacked, or are attacked only in small degree, by water in the 
physiologically meaningful pH-region, i.e. particularly in the region from 
6.0 to 9.0, particularly from 6.5 to 8.0. 
The choice of the functional groups takes into consideration that the 
material to be fixed, particularly material of a biological origin, 
generally contains the (free) amino group as the nucleophilic group, but 
possibly also phenolic, hydroxy, or thiol groups in addition. 
The polymer of said shell comprises 
(a) from 4.9 to 99.9 percent, by total weight of the polymer material of 
said shell, of a combination of at least one monomer having a functional 
group and at least one hydrophilic monomer, but the content of said 
monomer having a functional group being at least 0.1 percent; 
(b) from 0 to 95 percent by weight of at least one non-hydrophilic monomer; 
and 
(c) from 0.1 to 20 percent by weight of at least one crosslinking monomer. 
The construction of the shell portion of the latex according to the present 
invention in its reactive form can thus be represented as follows in a 
highly schematized manner: 
##STR1## 
wherein X represents the functional groups to be covalently bonded, 
preferably those which fulfill the conditions described earlier; R 
represents a spacer between the functional and the polymerizable groups, 
with the size and type of the spacer being comparatively uncritical; Z 
represents the polymerized form of a polymerizable group Z', present in a 
polymerizable monomer of the type Z'--(R).sub.n --X, discussed more in 
detail hereinafter; B represents a hydrophilic component of the shell 
derived from one or more hydrophilic monomers described more in detail 
hereinafter; A represents a component imparting hardness or rigidity of 
form to the shell and is also described more in detail below; and n has 
the value 0 to 1, respectively defining embodiments in which a spacer, R, 
is absent or present between groups Z and X. In a number of examples, the 
group R can be entirely absent, i.e. n can have the value 0. 
As a rule, X signifies a group which can react with one of the nucleophiles 
in question, i.e. is an activated group. Preferably, it signifies a 
sulfonic acid halide group, a thioisocyanate group, an activated ester, or 
a thiocarbonyldioxy-, carbonylimidoyldioxy-, haloethoxy-, haloacetoxy-, 
oxirane-, aziridine-, formyl-, keto-, acryloyl-, or anhydride-group. 
As sulfonic acid halides, the chloride and bromide can be used. The fluoro, 
chloro, and bromo compounds can be used as haloacetoxy compounds. As ester 
components, the activated esters of hydroxylamine compounds (such as those 
of N-hydroxysuccinimide or of N-hydroxyphthalimide), of phenols activated 
with electron-attracting groups (such as halophenols like trichlorophenol, 
or of nitrophenols), or of heterocyclic lactams such as pyridone can be 
used. 
Oxirane, keto, formyl, sulfonic acid chloride, thioisocyanate, activated 
carboxylic acid ester, and carboxylic acid anhydride groups are 
particularly preferred. Among the monomers of the type Z'--(R)n--X, Z' 
represents a (free radically) polymerizable unit and n is 0 or 1. 
Such free radically polymerizable units are, for example, vinyl groups 
wherein Z' for example represents 
##STR2## 
wherein R.sub.1 is hydrogen or methyl or is --CH.sub.2 --COOR.sub.2, 
CH.sub.2 --CONHR.sub.2, or is --CH.sub.2 --CON R.sub.2).sub.2, wherein 
R.sub.2 is alkyl having 1 to 4 carbon atoms. 
Further, Z' can be derived from maleic acid 
##STR3## 
As groups which are both polymerizable and capable of reaction, maleic acid 
anhydride and itaconic acid anhydride, as well as acrolein, methacrolein, 
methylvinylketone, and activated esters will serve. Derivatives of 
(meth)acrylic acid and of maleiimide, as well as maleic acid anhydride and 
itaconic acid anhydride, are particularly preferred. 
The following examples are given for elucidation of the formula Z'--R--X: 
##STR4## 
(condensation product of methacrylic acid and 1,4-butanedioldiglycidyl 
ether) 
CH.sub.2 =CH--COO--CH.sub.2 --CH.sub.2 --O--CSNH--(CH.sub.2).sub.6 --N=C=S 
(condensation product of acrylic acid-2-hydroxyethyl ester with 
1,6-hexanediisothiocyanate) 
##STR5## 
As to the remaining units contributing to the structure of the shell (A and 
B in the schematic representation), these are by definition such as impart 
the required properties, namely hydrophilicity and hardness, to the shell. 
A T.sub..lambda.max between 20.degree. and 250.degree. C., particularly 
between 50.degree. C. and 200.degree. C. (as determined by DIN 53445) can 
serve as an indication of the desired hardness in an anhydrous condition. 
On the other hand, the monomers involved in the construction of the shell 
should themselves suitably contain no strongly nucleophilic groups (such 
as --NH.sub.2, --SH). Further, the shell should preferably not contain any 
aromatic groups. Further, the components of the shell must in some way be 
crosslinked. Y serves as a symbol for this crosslinking or for the bonding 
with the core. 
Referring further to the schematic representation, the component primarily 
responsible for the hydrophilicity of the shell portion is designated as 
B. Further components, the choice of which must be primarily coordinated 
with the resulting hardness of the overall polymer, are designated as A. 
The conditions described for the shell construction of the latex to be used 
according to the present invention are met, for example, by copolymers of 
the methacrylate and/or acrylate type, wherein the qualitative and 
quantitative amount of component B is so measured that the criteria given 
earlier herein for the shell of the polymer latex are met. 
For example, as the hydrophilic component B, substituted methacrylamides 
and acrylamides of the general formula 
##STR6## 
can be used, wherein R.sub.1 is hydrogen or methyl and R.sub.3 and 
R.sub.4, independently of each other, can be hydrogen and/or alkyl having 
1 to 4 carbon atoms, that is unsubstituted amides as well as amides formed 
with primary and secondary amines. (Meth)acrylamide, N-methyl- or 
N-isopropyl- or N-butyl-(meth)acrylamide, and 
N,N-dimethyl-(meth)acrylamide should be especially mentioned, as well as 
(meth)acrylic acid morpholide (a particular case in which R.sub.3 and 
R.sub.4, together with the nitrogen atom to which they are attached, form 
a ring), and N-vinyl-pyrrolidone-2. 
Other hydrophilic components B include acrylate or methacrylate monomers 
containing hydroxy groups, particularly esters or amides of acrylic acid 
or of methacrylic acid containing hydroxy groups, as well as alkoxyalkyl 
esters and/or alkoxyalkyl amides of acrylic acid and of methacrylic acid, 
e.g. representatives of the general formula 
##STR7## 
wherein R'.sub.1 is hydrogen or methyl, R'.sub.2 is hydrogen or alkyl 
having 1 to 4 carbon atoms, Q is oxygen or --N(R.sub.3 ')--, wherein 
R.sub.3 ' is hydrogen or alkyl having 1 to 4 carbon atoms, p is an integer 
from 1 to 3, preferably 2, and m is an integer from 1 to 25. However, if Q 
is oxygen, then p is not equal to 1. Hydroxyethyl acrylate, hydroxyethyl 
methacrylate, 2-hydroxyethyl(meth)acrylamide, 
2-hydroxypropyl(meth)acrylamide, and monoesters of (meth)acrylic acid with 
glycerine and other polyols are especially mentioned. 
Further, 2-(methylsulfinyl)ethyl- acrylate and -methacrylate, as well as 
N-[2(methylsulfinyl)ethyl]acrylamide and -methacrylamide, are included 
within monomer type B. Polymerizable acids such as (meth)acrylic acid, 
itaconic acid, or maleic acid can also be incorporated as hydrophilic 
groups in the shell of the latex, as can polymerizable tertiary amines 
like 2-N,N-dimethylaminoethyl-(meth)acrylamide or -(meth)acrylic acid 
esters or 3-N,N-dimethylaminopropyl-(meth)acrylamide or -(meth)acrylic 
acid esters. To avoid imparting a net electrical charge to the latex 
particles, these acid or basic groups should always be simultaneously 
present in a particle (e.g. methacrylic acid and 2-N,N-dimethylaminoethyl 
methacrylate), so that the particles are substantially electrically 
neutral. 
As monomers of type A, those monomers are employed which are insoluble in 
water or have at most limited water solubility, whereby the qualitative 
and quanitative amount is so measured that the hardness criterion for the 
resulting polymer mentioned earlier is satisfied. 
Typical of these monomers are: 
(a) esters of acrylic acid and/or of methacrylic acid with alcohols having 
from 1 to 20 carbon atoms, particularly the methyl, ethyl, propyl, and 
butyl esters of methacrylic acid, as well as the methyl, ethyl, propyl, 
butyl, and 2-ethylhexyl esters of acrylic acid, and 
(b) polymerizable monomers of the vinyl acetate type, particularly vinyl 
acetate, vinyl propionate, vinyl butyrate, and vinyl isobutyrate. 
It is understood that the so-called "soft" monomers of type A can only be 
present in subordinate amounts, generally less than 50 percent by weight 
of the polymer of the shell. 
The hardness or other relevant properties of the polymer films formed from 
specific monomers is known, as is also the contribution of such monomers 
to the properties of copolymers in which they are present. [See U.S. Pat. 
No. 2,795,564; Rauch-Puntigam et al., "Acryl- und Methacryl-verbindungen" 
("Acrylic and Methacrylic Compounds"), Springer-Verlag, Berlin, 1967, 
pages 303-304; T. G. Fox, Bull. Am. Phys. Soc. 1, 123 (1956)]. 
The amount of the crosslinking agent Y is so measured that a washing away 
of the latex shell is no longer possible: as a rule at least 0.1 percent 
by weight of the material is necessary for this purpose. Larger amounts of 
crosslinking agent are in no way interfering, so that as a rule amounts 
from 0.1 to 20 percent, particularly from 1 to 10 percent, by weight are 
used. 
From a chemical viewpoint, Y can be any multi-functional acrylate or 
methacrylate, e.g. glycol dimethacrylate, butanediol diacrylate, 
triethyleneglycol dimethacrylate, tetraethyleneglycol diacrylate, and 
pentaerythritol tetraacrylate, inter alia. Not all functional OH groups of 
the polyol which is used as a basis for the crosslinking agent need be 
esterified with polymerizable acids (e.g. pentaerythritol dimethacrylate 
has two free OH groups), so that these crosslinking agents also can 
exhibit a thoroughly hydrophilic character. A further example for a 
hydrophilic crosslinking agent, Y, is N,N-methylene-bis-(methacrylamide). 
In addition, naturally, such monomers which contain easily graftable units 
in addition to an easily polymerizable group, e.g. allyl methacrylate, are 
also useable as crosslinking agents. 
The core of the core-shell particles according to the present invention is 
not fundamentally critical, as long as the condition is met that the 
resulting latex particles have a stable form, i.e. exhibit sufficient 
rigidity. From the point of view of technology, the redispersibility of 
the core-shell latex system should be guaranteed. The polymer material of 
the core can, for example, meet these requirements as well if it is a 
material which is soft per se but is a strongly crosslinked polymer as if 
it is per se a hard polymer (whether crosslinked or not crosslinked). It 
is in the nature of the core-shell construction that disturbing 
interactions which could be caused by the core material are less to be 
feared, so that also from this aspect a relatively free choice of 
materials exists. Thus, the core can be the carrier of a property suitable 
for physical separation or identification, e.g. the carrier of a marking 
which is detectable in a physical manner. For example, one can think of 
the use of dyes or fluorescent dyes, or of radioactive marking of the 
core, inter alia. Further, the core can make a physical separation 
possible as a result of a difference between its density and that of the 
surrounding medium. 
Accordingly, it is possible to construct the core material from such 
monomers or comonomers as are compatible with the requirement for a 
redispersibility of the latex, e.g. all copolymer compositions from 
derivatives of methacrylic acid and of acrylic acid as well as various 
vinyl esters which impart to the copolymer a T.sub..lambda.max (according 
to DIN 53445) of at least 0.degree. C. These "hard" copolymers are, for 
example compolymers comprising methyl methacrylate, butyl methacrylate, 
and methyl acrylate, inter alia, and need not be crosslinked. Whereas care 
must be taken to avoid the presence of aromatic groups in the region of 
the shell material if the particles are to be used for diagnostic reagents 
involving antigen-antibody reactions (because of potential hapten 
properties), monomers of the styrene type can also be employed in the core 
of the latex, e.g. styrene, vinyl toluene, divinylbenzene, and, therewith, 
also copolymers of styrene and maleic acid esters or fumaric acid esters. 
If the glass transition temperature, T.sub..lambda.max, of the core polymer 
is clearly below 0.degree. C., i.e. if the polymer is instrinsically 
"soft", then the use of at least 1 percent of a crosslinking agent is 
recommended, e.g. glycol dimethacrylate, divinylbenzene, etc. 
In connection with the requirement for a density of the core particles 
which deviates from the density of the carrier medium or the continuous 
phase, such monomers which impart an increased density to the latex 
acquire a particular significance. 
In particular, "heavy" monomers offer themselves, especially those having 
one or more halogen atoms, and particularly chlorinated or brominated 
monomers. Mentioned as exemplary are vinyl compounds such as vinyl 
chloride, styrene derivatives such as chlorostyrene or bromostyrene, as 
well as derivatives of (meth)acrylic acid which carry these heavy groups 
in a side chain, e.g. 2,4,6-tribromophenoxyethyl methacrylate. 
Alternatively, the core can also be constructed of monomers whose density, 
as a polymer, distinguishes itself less strongly from the density of the 
carrier medium or of the continuous phase. In such a case, the size of the 
core is to be increased to such an extent that nevertheless good 
separability is guaranteed. 
The preparation of latices of core-shell particles of the kind of interest 
to the present invention can take place following techniques known per se 
(cf. German Auslegeschrift No. 27 22 752). As exemplary of a preferred 
embodiment, methods for the preparation of a core-shell latex material 
having coarse particles and of one having fine particles are given further 
below. 
Whether a latex is to comprise coarse or fine particles is suitably 
determined by the nature of the core material. The preparation of a 
coarse-particle polymer core can result, for example, from a 
polymerization which is completely free of emulsifier. 
An advantageous embodiment involves dropping the monomer or a monomer 
mixture over a period of one-half to four hours into water pre-heated to 
about 50.degree. to about 100.degree. C. which contains a sufficient 
amount of a water soluble initiator, such as potassium- or 
ammonium-peroxydisulfate, hydrogen peroxide, or salts of 
4,4'-azobis(cyanovalerianic acid). Instead of a thermal polymerization in 
the region of about 50.degree. C. to 100.degree. C., however, the reaction 
can also be initiated at lower temperatures with the aid of a redox 
initiator system. Oil soluble initiators, too, for example dibenzoyl 
peroxide or azo-bis-isobutyronitrile, are suitable as polymerization 
initiators. In this case, the use of at least small amounts of an 
emulsifier is advantageous, or may even be necessary. Another way for 
achieving large latex particles involves a multi-stage process with the 
aid of a seed latex. In this case, the desired monomer or monomer mixture 
is polymerized in a second or even subsequent stage onto a seed latex 
which has been priorly prepared in a desired manner. Procedurally, batch 
processes, multiple-batch processes, and also, and better, monomer-feed or 
emulsion-feed processes are suitable. It is essential for these 
embodiments that the total emulsifier concentration in the stages 
following the seed latex is kept so low that all of the monomer 
polymerizes on the seed latex particles and there is no new particle 
formation. Particularly large polymer cores are obtained if the 
aforementioned latices having coarse particles and prepared without an 
emulsifier are employed as the seed latex (cf. European patent publication 
No. 79101398.0 or German Offenlegungsschrift No. 28 33 601). 
Systems having coarse particles are also obtained if a seed latex is 
prepared in a first stage to contain a polymer of very low molecular 
weight. These latex particles can be swollen with monomer or a monomer 
mixture and polymerized to form large latex particles. The additional use 
of subordinate amounts of a completely water insoluble substance with the 
monomer or monomer mixture can have the same effect as the low molecular 
weight polymer (cf. German Offenlegungsschrift No. 27 51 867 or European 
Pat. No. 0 003 905). Coarse particle cores, for example having a diameter 
from about 0.5 to more than 2 microns, are advantageous if the density of 
the core polymer does not deviate significantly from the density of the 
carrier medium or of the continuous phase. 
The preparation of a polymer core comprising fine particles involves, in 
principle, the synthesis of a latex according to the known criteria for an 
emulsion polymerization, wherein the desired size of the core latex 
particles is controlled at the beginning of the polymerization by the 
emulsifier concentration. As a result of the fact that the composition of 
the core material is relatively uncritical, it is in principle here 
possible also to use any desired latex as the core material, to the extent 
that the particles therein fulfill the aforementioned requirements such as 
form stability and high density, for example. For the preparation of a 
fine-particle core material, one stage or multi-stage batch processes, 
monomer-feed or emulsion-feed processes, or continuous modes of operation 
are suitable as methods. As initiators, those water soluble or oil soluble 
starters mentioned in a preceding paragraph for the preparation of a 
coarse-particle core latex can be used. As there taught, the 
polymerization can be purely thermal or may occur with the aid of redox 
system. 
In principle, all anionic, cationic, non-ionic, or amphoteric surface 
active agents, alone or in combination, are suitable as emulsifers. 
However, anionic and/or non-ionic emulsifers are preferred. A particularly 
advantageous embodiment for the preparation of fine-particle core latices 
involves heating a solution of a suitable buffer (about pH 7) containing 
an emulsifier to the desired polymerization temperature, adding a water 
soluble initiator in a certain amount, and then adding a monomer emulsion 
(including a crosslinking agent) dropwise over a period of 0.5 to 6 hours. 
Fine particle cores, for example from about 0.1 to 0.5 micron in diameter, 
can be used if the density of the core polymer differs sufficiently 
strongly from the density of the carrier medium or of the continuous 
phase. 
The polymerization of the shell onto the latex core can follow directly 
after the polymerization of the core material. The method of procedure 
resembles in principle that described earlier above for the seed latex. 
The monomer mixture of the shell composition, in which suitably monomers 
of the Z'RX-type are present, is added per se or as an emulsion in water 
or a buffer solution over a period of time from 0.5 to 4 hours to the core 
latex. In this, attention must again be paid that the total emulsifier 
concentration remains so low that the formation of new particles is 
avoided. In certain cases, it can be necessary to add two different 
monomer feeds simultaneously, one of which may possibly contain water. 
Such a procedure is always necessary if the monomers do not dissolve in 
one another, or if a part of the monomers is only soluble in water but the 
other part is not water soluble. 
Following these prescriptions, the shell monomer polymerizes on the polymer 
core which is present. It may prove suitable additionally to add initiator 
or buffer solution before the addition of the shell monomers, particularly 
if the polymerization of the latex core does not occur in a buffer 
solution and if the shell monomers are added in a monomer-addition 
technique. 
The addition of a buffer is above all of extraordinary importance if the 
functional monomers, Z'--(R).sub.n --X, are highly reactive compounds. 
Then, the buffer mixture is naturally so adjusted that any disturbance of 
these reactive groups (e.g. by hydrolysis) is kept as minimal as possible 
during the course of synthesis of the latex particles. 
The polymerization conditions are, with the exception of a limited 
emulsifier concentration, similar to those which have been described for 
the preparation of the core. A single batch or multiple batch method is 
possible; however, a monomer-feed or emulsion-feed method is preferred. 
The polymerization can occur thermally in the region of about 50.degree. 
C.-100.degree. C. or it can take place also at lower temperatures with the 
aid of a redox initiator system. As polymerization initiators, preferably 
those water-soluble starters which are conventionally used in emulsion 
polymerization are preferred. In principle, however, oil soluble 
initiators can also be used providing their decomposition temperature lies 
in the aforementioned temperature range. 
An appropriate ratio of the shell thickness to the size of the core is 
attained, for example, if the weight of the core material to that of the 
shell material is in a ratio from 1:3 to 5:1. However, also more extreme 
core-shell ratios are possible in principle, for example 10:1. It is 
understood that the shell portion should be chosen to be larger the 
smaller the latex core is. 
The particles are obtained in the form of aqueous dispersions (latices) of 
a relatively low viscosity. The polymer content can--as a guideline--be in 
the region from 15-30 percent by weight, for example, in such a 
dispersion. In principle, however, a solids content from a few percent by 
weight up to about 70 percent by weight is possible. 
When dispersed, the polymer particles generally have a size between about 
and about 0.05 micron and about 5 microns, and more often between about 
0,5 micron and about 2 microns. 
The particles can be recovered as a powder by a variety of techniques such 
as spray drying, freeze drying, precipitation, etc. In this powdered 
condition, the primary particles may aggregate loosely to form 
"superstructures" of varying sizes, but wherein the individual primary 
particles retain their original size. On redispersion in a liquid, usually 
an aqueous medium, these "superstructures" swell and revert to the 
original primary particles. 
The dry polymer particles are preferably stored under anhydrous conditions, 
protected from any reaction with nucleophilic reagents, and suitably below 
about 50.degree. C. 
Redispersion is simply effected by combining the dry particles with a 
liquid and stirring. The dispersing phase is usually water or an aqueous 
medium, suitably a medium such as a buffer solution in which a desired 
further reaction with a biologically active substance can be effected. 
The core-shell latices are used for the preparation of diagnostic reagents 
as follows. 
The novel reagents according to the invention can be prepared by a reaction 
of a latex of the novel core-shell particles with a biologically active 
substance or structure. The biologically active substances or structures 
can be, for example, "immunologically active" materials. As 
"immunologically active" materials, for example, the components of 
physiological liquids, cell extracts, and tissue extracts can be 
mentioned, providing that an immunologic anti-reactant is available or can 
be prepared. 
As representative of immunologically active materials, amino acids, 
peptides, proteins, enzymes, lipoproteins, glycoproteins, lipoids, nucleic 
acids, polysaccharides, primary amines, alkaloids, hormones, vitamins, 
sterols, and steroids can be mentioned. 
As immunologically active structures, microorganisms such as gram-positive 
and gram-negative bacteria, spirochetes, mycoplasmas, mycobacteria, 
vibrionaceae, actinomyces, protozoa such as intestinal protozoa, amebas, 
flagellates, spores, intestinal nematodes and tissue nematodes (worms), 
trematodes (schistosomes, leeches), cestodes, and toxoplasmas can be 
mentioned. Also, fungi such as sporotrichum, cryptococcus, blastomyces, 
histoplasma, coccidioides, and candida, viruses and rickettsia such as 
canine hepatitis, Shope-papilloma, influenza A and B, chicken pox, herpes 
simplex, adenoviruses, polyomas, Rous-sarcoma, smallpox, polio virus, 
measles, canine distemper, leukemia, mumps, Newcastle-disease, sendai, 
echovirus, hoof-and-mouth disease, psittacosis, rabies, extromelia, and 
tree viruses. Further, tissue antigens, hormones such as the hypophysis 
hormone, insulin, glucagon, thyroid hormone, chorionic gondatropin, 
chorionic growth hormone-prolactin, and human placental lactogen are 
considered, as are enzymes such as pancreatic chymotrypsinogen, 
procarboxypeptidase, glucose-oxidase, lactate dehydrogenase, uricase, 
amino acid-oxidase, urease, asparaginase, and proteases. Further, blood 
cell antigens, blood group substances and other isoantigens such as blood 
platelets, leucocytes, plasma proteins, milk proteins, saliva proteins, 
urine proteins, and antibodies, including auto-antibodies, can be 
mentioned. 
The use of the core-shell latices for the immobilization of enzymes is 
discussed more in detail below. 
For reactions of the polymer particles according to the invention with 
enzymes, the enzyme can simply be incubated in an aqueous medium with an 
adequate amount of the particles. The aqueous medium should preferably 
approach physiological conditions, for example a buffer adjusted to be 
suitable to the type of enzyme employed. The materials are incubated 
preferably not significantly above room temperature and with moderate 
stirring. If the epoxy group is used as the functional group, one can work 
in the pH region between 6 and 9, for example, without limitation. In 
general, a time period from one to several days, for example three days, 
is appropriate for the reaction. The non-covalently bound enzyme can be 
separated by repeated centrifugation (at about 5000 rpm) and redispersion 
in buffer solution. A determination of the activity can follow patterned 
on the known enzyme-specific determination methods. A particular advantage 
of the present invention is that even loaded polymer particles can be 
redispersed--for example in the form of a freeze dried powder--and may 
optionally be stored over a long period of time. The limiting factor is in 
any case the stability of the bound biological material. 
The polymer particles according to the present invention can also be used 
as a carrier for other--for instance industrially useful--enzymes in a 
suitable form. Acylases, penicillinase, glucose-isomerase, and peroxidases 
should be mentioned, for example, inter alia. 
For various reasons, for example for following immunoagglutination, it can 
be advantageous--as already mentioned--to provide the particles with a 
marker, for example a fluorescent dyestuff. 
The polymer latices of the present invention are suitable for the 
immobilization of microorganisms in general, for which the reaction 
conditions are similar to those for the immobilization of proteins. In 
contrast to the state of the art, the present method offers a better 
accessability of the immobilized microorganisms for substrate molecules. 
The small cytotoxicity characteristic of the present immobilization method 
should be emphasized. 
The comments above are valid also for the immobilization of viruses and of 
eukariotic cells. The polyfunctional nature of the polymer particles also 
in general permits their use for the crosslinking of biologically active 
substances. From this viewpoint, those latex particles of small diameter 
(500 Angstrom units is a guide value) are of particular significance. 
The polymer particles of the invention can also be used to advantage in 
preparative organic syntheses. In this case it is not necessary to operate 
in an aqueous medium: rather, also organic reaction media can be employed 
or co-employed. 
For example, protective groups can be introduced in this manner. A 
particularly interesting aspect lies in the use of the materials according 
to a peptide synthesis according to Merrifield [cf. Merrifield, Adv. 
Enzymol. 32, 221-296 (1969)]. 
A better understanding of the present invention and of its many advantages 
will be had by referring to the following specific Examples, given by way 
of illustration.

Example 1-Preparation of Latex No. 1 
(Exemplary of a coarse-particle latex) 
(a) Synthesis of a parent dispersion 
1600 g of water are introduced into a polymerization vessel equipped with a 
reflux condenser, stirrer, and thermometer, and are heated to 80.degree. 
C. After the addition of a monomer mixture comprising 
3 g of isobutyl methacrylate, 
3 g of methyl methacrylate, and 
0.3 g of ethyleneglycol dimethacrylate. 
4 g of ammonium persulfate, dissolved in 36 g of water, are added. 
Then, at 80.degree. C., a mixture of 
200 g of isobutyl methacrylate, 
200 g of methyl methacrylate, and 
20 g of ethyleneglycol dimethacrylate 
is added thereto within a period of two hours. After the end of the monomer 
addition, the mixture is maintained for one further hour at 80.degree. C. 
A coagulate-free, readily filterable, dispersion containing about 20 
percent solids and of low viscosity is obtained. 
(b) Synthesis of a dispersion containing oxirane groups 
350 ml of water are introduced into a polymerization vessel equipped with a 
reflux condenser, stirrer, and thermometer. 10 ml of a phosphate buffer 
solution (pH=7, "TITRISOL") and 80 g of the parent dispersion are added 
thereto. After heating to 80.degree. C. 0.4 g of the sodium salt of 
4,4'-azobis-(4-cyanovalerianic acid) in 4 ml of water is added. 
Thereafter is added, at 80.degree. C. over a period of three hours, an 
emulsion comprising 
1000 g of water, 
1 g of sodium lauryl sulfate, 
1 g of the sodium salt of 4,4'-azobis(cyanovaleric acid), 
150 g of methyl methacrylate, 
150 g of isobutyl methacrylate, and 
15 g of ethylene glycol dimethacrylate. 
Subsequently, over a period of 60 minutes, the following are added 
simultaneously: a solution of 20 g of methacrylamide and 0.6 g of the 
sodium salt of 4,4'-azobis-(4-cyanovalerianic acid) in 300 g of water, as 
well as a monomer mixture comprising 35 g of methyl methacrylate, 40 g of 
glycidyl methacrylate, and 4 g of ethylene glycol dimethacrylate. 
Thereafter, the mixture is stirred for a further 60 minutes at 80.degree. 
C. 
A dispersion free of coagulate and of low viscosity having a solids content 
of about 20.degree. C. is obtained. The particle size is about 2 microns. 
Example 2--Preparation of Latex No. 2 
(Exemplary of a coarse-particle latex) 
(a) Synthesis of a parent dispersion 
1600 g of water are introduced into a polymerization vessel like that of 
Example 1 and warmed to 80.degree. C. After the addition of a monomer 
mixture comprising 
6.24 g of styrene and 
0.06 g of allyl methacrylate, 
4 g of ammonium persulfate, dissolved in 36 g of water, are added. To this 
mixture, again at 80.degree. C., a mixture of 
415 g of styrene and 
5 g of allyl methacrylate 
is added dropwise over a period of 2 hours. 
After the end of the monomer addition, the mixture is kept for a further 2 
hours at 80.degree. C. A viscous dispersion containing about 20 percent 
solids which is free of coagulates and can be coarsely filtered is 
obtained. 
(b) Synthesis of a dispersion containing oxirane groups 
The procedure of Example 1 is followed, but heating is at 85.degree. C. and 
1.0 g of the sodium salt of 4,4'-azobis-(4-cyanovaleric acid) in 10 ml of 
water is 
To this mixture, an emulsion is added, over a period of 3 hours at 
85.degree. C., consisting of: 
1000 g of water, 
1 g of sodium lauryl sulfate, 
4 g of the sodium salt of 4,4'-azobis-(cyanovalerianic acid), 
312 g of styrene, and 
4 g of allyl methacrylate. 
Subsequently, over a period of 90 minutes, the following are added 
simultaneously a solution of 20 g of methacrylamide and 0.6 g of the 
sodium salt of 4,4'-azobis-(4-cyanovalerianic acid) in 300 g of water, as 
well as a monomer mixture comprising 35 g of methyl methacrylate, 40 g of 
glycidyl methacrylate, and 4 g of ethylene glycol dimethacrylate. The 
mixture is then stirred for a further 60 minutes at 80.degree. C. 
A readily filterable dispersion of low viscosity, free of coagulate and 
having a solids content of about 20 percent, is obtained. The particle 
size is about 2 microns. 
Example 3--Preparation of Latex No. 3 
(Exemplary of a fine particle latex) 
5 ml of a phosphate buffer solution (pH=7, "TITRISOL"), 0.03 g of sodium 
lauryl sulfate, and 0.2 g of the sodium salt of 
4,4'-azobis-(4-cyanovalerianic acid) are dissolved in 100 ml of water 
present in a polymerization vessel equipped as described earlier. The 
mixture is heated to 80.degree. C. and, over a period of three hours, an 
emulsion is added comprising 
0.1 g of sodium lauryl sulfate, 
0.5 g of the sodium salt of 4,4'-azobis(4-cyanovalerianic acid), 
80 g of methyl methacrylate, 
15 g of [2-(2,4,6-tribromophenoxy) 
ethyl]-methacrylate, 
5 g of ethylene glycol dimethacrylate, and 
200 g of water. Subsequently, a solution of 5 g of methacrylamide in 75 g 
of water and a monomer mixture comprising 
10 g of glycidyl methacrylate, 
1 g of ethylenglycol dimethacrylate, and 
9 g of methyl methacrylate 
are added simultaneously over a period of 90 minutes. The mixture is held 
for a further 60 minutes at 80.degree. C. 
A dispersion of low viscosity having a solids content of about 25 percent 
is obtained. The particle size is 0.3 micron. The content of oxirane 
groups is 31 percent, based on the glycidyl methacrylate introduced 
(determined by titration with sodium thiosulfate). 
Example 4--Preparation of Latex No. 4 
(a) Preparation of a core dispersion 
0.3 g of sodium tetradecyl sulfonate, 
0.6 g of ammonium sulfate, and 
500 g of distilled water 
are introduced into a polymerization vessel equipped as in Example 1 and 
are warmed to 80.degree. C. 
An emulsion comprising 
500 g of p-bromostyrene, 
300 g of fumaric acid diethyl ester, 
4 g of sodium tetradecyl sulfonate, 
4 g of sodium persulfate, and 
710 g of distilled water 
is introduced into this mixture dropwise at 80.degree. C. over a period of 
6 hours. After the introduction, the mixture is stirred for a further two 
hours at 80.degree. C., then cooled to room temperature and filtered. The 
dispersion obtained is of low viscosity and has a solids content of about 
40 percent. 
(b) Preparation of a core-shell dispersion 
500 g of the 40 percent dispersion of Example 4 a) are adjusted with 
phosphate buffer to a pH of 7.0 and are diluted to a total volume of 1000 
ml with a solution comprising 1 g of the sodium salt of 
4,4'-azobis-(cyanovalerianic acid) and 0.5 g of sodium tetradecyl 
sulfonate in 1000 ml of distilled water. [This is equivalent to a 20 
percent dispersion as in Example 4 a) at a pH of 7.0]. 
This mixture is heated to 80.degree. C. in a polymerization vessel, held 
for 15 minutes at this temperature, and then the following two solutions 
are introduced dropwise at 80.degree. C., simultaneously 
Solution A: 
20 g of 2-bromoethyl methacrylate, 
2.5 g of glycol dimethacrylate, 
17.5 g of N-t.-butyl methacrylamide, and 
10 g of methyl methacrylate; 
Solution B: 
1 g of the sodium salt of 
4,4'-azobis-(cyanovalerianic acid) in 
50 g of distilled water. 
The total time for dropwise addition is about two hours. For both 
additions, the rate of addition should be as closely the same as possible. 
After the end of the addition, the mixture is kept for one further hour at 
80.degree. C. Thereafter, it is cooled and filtered. A dispersion of low 
viscosity containing fine particles and having a solids content of about 
23 percent is obtained. 
(c) Preparation of the core-shell dispersion 
Example 4(b) is followed (dilution of dispersion 4a) neutralization, etc.) 
but the following solutions are added. 
Solution A: 
10 g of vinyl acetate, 
30 g of chloroacetic acid vinyl ester, 
2.5 g of methylene-bis (acrylamide), and 
7.5 g of acrylamide. 
Solution B: 
2 g of the sodium salt of 4,4'-azobis-(cyanovaleric acid) in 
50 g of distilled water. 
The total time of dropwise addition is about 3 hours. After addition is 
concluded, the mixture is kept for a further 2 hours at 80.degree. C. 
After cooling and filtration, a low viscosity dispersion containing fine 
particles is obtained. 
Example 5: Synthesis of Latex No.5 cl Stage I 
The following components are introduced into a polymerization vessel as in 
Example 1: 
1550 g of distilled water, 
0.8 g of sodium lauryl sulfate, 
3.2 g of methyl methacrylate, and 
3.2 g of isobutyl methacrylate 
and are then warmed to 80.degree. C. with stirring. Subsequently, a 
solution of 4 g of ammonium persulfate in 40 ml of water is added. 
Thereafter, the following monomer mixture is added at 80.degree. C.: 
190 g of methyl methacrylate, 
190 g of isobutyl methacrylate, and 
20 g of glycol-bis(methacrylate). 
The time for addition of the monomer is two hours. After addition, the 
mixture is kept for a further two hours at 50.degree. C. After cooling, a 
readily filterable dispersion free of coagulate is obtained having a 
solids content of 19 percent, a pH of 2.2, and viscosity of 4 mPa.sec. 
Stage II 
160 g of the dispersion of stage I are introduced into a polymerization 
vessel as in Example I and the following are added: 
10 g of phosphate buffer (pH=7, "TITRISOL"), 
0.4 g of the sodium salt of 4,4'-azobis-(cyanovalerianic acid), and 
310 g of distilled water. 
This mixture is warmed to 80.degree. C. and the following emulsion is added 
over a period of 3 hours: 
143 g of methyl methacrylate, 
143 g of isobutyl methacrylate, 
15 g of ethylene glycol-bis (methacrylate), 
1 g of sodium lauryl sulfate, 
1.8 g of the sodium salt of 
4,4'-azobis(cyanovalerianic acid), and 
970 g of distilled water. 
Immediately thereafter, the following two mixtures are added simultaneously 
over a period of one hour: 
Mixture A: 
44 of methyl methacrylate, 
4 g of ethylene glycol-bis (methacrylate), and 
42 g of glycidyl methacrylate; 
Mixture B: 
0.6 g of the sodium salt of 4,4'-azobis-(cyanovalerianic acid), 
10 g of methacrylamide, and 
320 g of distilled water. 
After the addition, the mixture is kept for a further hour at 80.degree. C. 
After cooling, a coagulate-free dispersion having a solids content of 
about 19 percent is obtained. The particle size is about 0.4 micron. 
Example 6--Purification of the latex according to Example 1 
(Removal of the auxiliaries, emulsifiers, initiators, etc. required for the 
synthesis) 
10 ml of the dispersion of Example 1 are centrifuged for 15 minutes at 5000 
rpm. The remaining serum is poured off and the particles are subsequently 
redispersed in 1 N NaCl (equivalent to 1 g of polymer solids in about 50 
ml of 1 N NaCl). These are then again centrifuged for 10 minutes at 5000 
rpm and decanted. Redispersion in 1 N NaCl and centrifugation are repeated 
two more times. 
The particles are then redispersed in 0.05 M phosphate buffer at pH 7.5 
(equivalent to 1 g of polymer solids in 50 ml of 0.05 M phosphate buffer 
at pH 7.5). This is then centrifuged for 10 minutes at 5000 rpm and the 
residue is poured off. 
This procedure is repeated once more. The latex so obtained is then stored 
in a refrigerator at +5.degree. C. 
Example 7--Purification of the latex according to Example 3 
The procedure of Example 6 is followed, but the duration of centrifugation 
(5000 rpm) is raised to 30 minutes in each case. 
Example 8--Reaction for the immobilization of Trypsin 
15 ml of the dispersion of Example 1 (containing about 3 g of solid 
polymer) are combined with 300 mg of trypsin (dissolved in 6 ml of 1 M 
phosphate buffer at pH 7.5). The mixture is then stirred at 23.degree. C. 
for 72 hours. 
Non-covalently bonded enzyme is then removed by a three-fold centrifugation 
and redispersion in 0.05 M phosphate buffer (procedure as in Example 6). 
Example 9--Measurement of the activity of the immobilized enzyme 
(a) Hydrolysis of N-benzoyl-arginine-ethyl ester (BAEE) at 37.degree. C. 
and at an automatically maintained constant pH of 7.5; 
1 g of the dry substance of the latex purified by centrifugation according 
to Example 8 (and introduced as about 2 g of moist substance with about 1 
g of water) is dispersed in 20 ml of a 2 percent BAEE-solution. 
______________________________________ 
Cycle Activity (U/g) 
______________________________________ 
1. 14.2 
2. 12.1 
3. 11.8 
4. 11.8 
______________________________________ 
The activity is in each case referred to 1 g of carrier material. 1 U 
corresponds to 1 micromol/minute based on the initial rate of reaction. 
For each sample, 3 to 4 subsequent determinations (cycles) are carried out 
to discriminate between bound and unbound trypsin. 
(b) Hydrolysis of casein (37.degree. C., pH8.0) 
1 g of the dry latex purified by centrifugation according to Example 8 
(introduced as about 2 g of moist substance with about 1 g of water) is 
dispersed in 20 ml of a 4 percent casein solution. 
______________________________________ 
Cycle Activity (U/g) 
______________________________________ 
1. 3.2 
2. 2.6 
3. 2.6 
4. 2.6 
______________________________________ 
Example 10--Freeze drying of a reactive latex 
15 ml of the dispersion of Example 1 are purified as in Example 6. A 
polymer containing about 50 percent of residual moisture is obtained. 
This centrifuged latex is freeze dried and is subsequently stored for 6 
months at -20.degree. C. 
Redispersion of the freeze dried latex 
Redispersion follows in 0.05 M phosphate buffer at pH 7.5. The mixture must 
be vigorously stirred for about 5 minutes. Alternatively, the sample 
suspended in the buffer solution can be treated briefly with ultrasound. 
Subsequently, the material is reacted with an enzyme as in Example 8 (10 
percent of trypsin, calculated on the latex introduced). 
The activity of the freeze dried latex, stored for six months at 
-20.degree. C., redispersed, and reacted with 10 percent of trypsin is 
reported below: 
______________________________________ 
Activity (U/g) 
Activity (U/g) 
Cycle (Substrate: BAEE) 
(Substrate: Casein) 
______________________________________ 
1. 13.3 2.9 
2. 10.6 2.2 
3. 10.6 2.2 
______________________________________ 
Example 11--Freeze drying of a latex having trypsin immobilized thereon 
1 g of the latex of Example 8, reacted with trypsin, is freeze dried and 
stored thereafter for 6 months at -20.degree. C. Redispersion follows as 
described in Example 10, with 0.05 M phosphate buffer. 
The activity toward casein (1 g of redispersed latex solids in 20 ml of 4 
percent casein solution) at 37.degree. C. and pH 8.0 is reported below: 
______________________________________ 
Cycle Activity (U/g) 
______________________________________ 
1. 3.6 
2. 2.4 
3. 2.4 
______________________________________ 
Example 12--The immobilization of trypsin The procedure of Example 8 is 
followed but 15 ml of the dispersion of Example 3 (centrifugation for 30 
minutes at 5000 rpm) are used for the immobilization of trypsin. The 
activity toward casein as a substrate (pH 8.0, 37.degree. C.) is reported 
below: 
______________________________________ 
Cycle Activity (U/g) 
______________________________________ 
1. 5.5 
2. 4.2 
3. 4.0 
______________________________________ 
Example 13 --Synthesis of a fluorescently marked latex 
40 g of the parent dispersion of Example 1 a) are introduced into a 
polymerization vessel according to Example 1.5 ml of phosphate buffer 
(pH=7, "TITRISOL"), 0.2 g of the sodium salt of 
4,4'-azobis-(cyanovalerianic acid), and 180 g of distilled water are added 
thereto. 
After warming of the mixture to 80.degree. C., an emulsion is added over a 
period of 3 hours--also at 80.degree. C.--comprising the following: 
127 g of methyl methacrylate, 
15 g of isobutyl methacrylate, 
7.5 g of ethylene glycol-bis (dimethacrylate), 
0.6 g of flurol-green-gold [=solvent green 5 (Colour Index Part I, No. 
79075)], 
1.0 g of the sodium salt of 4,4'-azobis-(cyanovalerianic acid), 
0.5 g of sodium lauryl sulfate, and 
450 g of distilled water. 
After conclusion of the addition (=latex core), the following two mixtures 
are added at 80.degree. C. simultaneously over a period of one hour: 
Mixture A: 
24 g of methyl methacrylate, 
2 g of ethylene glycol-bis (methacrylate), and 
21 g of glycidyl methacrylate; 
Mixture B: 
3 g of methacrylamide, 
0.3 g of the sodium salt of 4,4'-azobis-(cyanovalerianic acid), and 
155 g of distilled water. 
After conclusion of the addition, the mixture is held for a further 60 
minutes at 80.degree. C. and is then cooled. 
A readily filterable dispersion, free of coagulate and having a solids 
content of 19 percent, a pH of 7.7, and a viscosity of 10 mPa.sec. is 
obtained. Particle size is about 2 microns. 
The fluorescence under UV stimulation is clearly visible macroscopically as 
well as in a fluorescence microscope. 
Example 14--Immobilization of anti-albumin 
10 ml of the dispersion according to Example 5 are diluted to 100 ml with 
0.05 M of phosphate buffer at pH 7.5. (Suitably, 0.05 percent of sodium 
azide is added to the phosphate buffer.) A dispersion containing about 2 
percent of polymer solids results. 
The anti-serum identified as Cat. No. 61-015,6389 (goat) in the catalog of 
Miles-Yeda, Ltd., (Kiryat Weizmann, Rehovoth, Israel) is diluted with 
buffer to the following concentrations: 
(a) 1000 micrograms of antibody/ml 
(b) 200 micrograms of antibody/ml 
(c) 40 micrograms of antibody/ml 
(d) 8 micrograms of antibody/ml 
(e) 0 micrograms of antibody/ml 
The bonding of the anti-albumin onto the latex particles occurs by 
reaction, in each case, of 1 ml of the 2 percent dispersion with 1 ml of 
the dilution series (a)-(e). The mixtures are stirred for 5 days at room 
temperature and the latex particles are purified by centrifugation as 
described in Example 6. 
The product can be used directly for the detection of human albumin using 
the latex agglutination technique. 
Example 15--Synthesis of a compound containing dueterium as a marker 
In 2 ml of purified methylene chloride as a solvent, 0.146 g (1 m mols) of 
deuterated benzoyl chloride-d.sub.5 is reacted with 0.18 g (3 m mols) of 
ethanol amine during 1 day at 0.degree. C. and 1 day at ambient 
temperature. Thereafter, the methylene chloride is vaporized and the 
residue is mixed with an amount of the polymeric material of Example 10 
containing 5 m mol equivalents of oxirane groups and the mixture is 
adjusted to pH 8 by the addition of aqueous phosphate buffer solution. 
After 3 days at 37.degree. C. the mixture is diluted with distilled water 
and the latex containing the excess of ethanol amine in covalently bound 
form is centrifuged. The supernatant aqueous solution contains the desired 
end product N-2-hydroxyethyl benzoyl amide-d.sub.5, which is ready for use 
in biological test.