A suspension of diagnostic particles comprising antibody molecules attached to a carboxylate derivatized polymer core is provided for agglutination tests. The antibody is linked to the core through an avidin-biotin bridge. Avidin is joined by an amide bond to carboxyl groups on the core, and biotin is linked by an amide bond to amino groups on the antibody molecule. The core-bound antibody is exposed to a mixture of free biotin and biotinylated antibody to attach a controlled amount of antibody that is consistent with suspension stability prior to its use in a test and rapid cross-linking of suspended particles in the presence of antigen.

The present invention relates to tests which employ antibodies to detect 
the presence of particular antigenic substances and more particularly to 
improved suspensions of antibody molecules bound to solid support 
particles which agglutinate in the presence of the particular antigen to 
which the antibody is reactive. 
BACKGROUND OF THE INVENTION 
Antibodies are large proteinaceous molecules that are produced by animals 
in response to the presence of a foreign substance for the purpose of 
neutralizing that substance. An antibody molecule may be highly specific, 
recognizing only a certain site of a particular molecule which is the 
antigen to that antibody. Because of their high specificities, antibodies 
are very useful in ascertaining the presence or absence of various 
antigenic substances, and a number of test procedures, such as 
radioimmunoassays, have been developed to take advantage of the 
specificity of the antibodies. Relatively recently, monoclonal antibodies 
have been developed, providing a practical method for assuring that an 
antibody fraction contains only a single type of antibody molecule. 
Monoclonal antibodies may be substituted for conventional antibody 
fractions in most diagnostic tests, providing greater accuracy and 
reliability than tests which utilize conventional antibody fractions. 
One particular type of test which has been developed that is particularly 
useful for detecting the presence of large antigens having multiple 
antibody-recognition sites (antigenic determinants), is an agglutination 
test. Antibody molecules are bound to minute particles formed of a 
polymer, and the particle-bound antibody is suspended in a liquid medium. 
In the presence of the antigen, the particle-bound antibody attaches to 
the recognition sites on the antigen. If antibody molecules on more than 
one particle attach to the same antigen molecule, the particles become 
cross-linked, and as multiple particles cross-link, they agglutinate and 
precipitate from the solution. Agglutination and precipitation of the 
suspended particles is readily observable by the naked eye, providing a 
very simple and very certain test that a particular antigen is present. 
One currently used method of attaching antibodies to polymer particles is 
described in Molday, R. S., W. J. Dreyer, A. Rembaum, and S. P. S. Yen; 
"New Immunolatex Spheres: Visual Makers of Antigens on Lymphocytes for 
Scanning Electron Microscopy", J. Cell Biol (1975) 64, pp. 75-88. 
Carboxylate derivatized latex particles are reacted with the antibody in 
the presence of a carbodiimide, coupling amino groups on the antibody to 
the carboxyl groups on the peptide. This procedure is useful for binding 
relatively small antibody or immunoglobulin molecules, such as IgG, but 
does not work in acceptable fashion for binding larger antibody molecules 
such as IgM. Furthermore, this method provides no method for controlling 
the amount of antibody that binds to the particle. If excessive amounts of 
antibody bind to the particles, the particles may tend to agglutinate 
prematurely, i.e., before indroduction of the antigen. 
Another method of binding antibodies to latex particles is through 
adsorption of antibodies to the surface of latex particles, as is 
described in Carel J. Van Oss and J. M. Singer; "The Binding of Immune 
Globulins and Other proteins by Polystyrene Latex Particles", J. 
Reticuloendothelial Soc. (1966) 3, pp. 29-40. The success of this 
procedure depends to a great extent on the exact lot of the latex, 
different lots having vastly different adsorptive properties and 
stabilities. Because of the unpredictability of results, this procedure is 
used largely for larger antibody molecules, such as IgM. 
It would be desirable to provide latex particles having bound antibody 
which can be more reproducibly manufactured, irrespective of the type of 
antibody and irrespective of the adsorptive properties of the particular 
lot of latex particles. It would be further desirable to control the 
number of antibody molecules binding to the particles to assure that 
premature agglutination will not occur due to the particles having 
excessive bound antibody molecules and yet assure that there are 
sufficient bound antibody molecules to agglutinate the particles rapidly 
in the presence of the antigen. 
SUMMARY OF THE INVENTION 
The invention provides a diagnostic composition comprising a suspension of 
carboxylate-derivatized latex particles bound to antibody molecules 
through avidin-biotin bridges. The latex particles, having multiple free 
carboxyl groups on their surfaces, are bound to avidin using a 
carbodiimide intermediate. Biotin is covalently bound to the antibody. A 
mixture of biotin and biotinylated antibody in a predetermined molar ratio 
are reacted with the latex-bound avidin, linking antibody moieties to a 
selected portion of the avidin binding sites. The number of antibody 
molecules attached through avidin-biotin bridges to the latex is 
controlled to assure that the particles remain in suspension until they 
cross-link and agglutinate in the presence of an antigen to the antibody. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the invention, diagnostic particles are provided in 
which antibodies are attached to carboxylate-derivatized latex particles 
through avidin-biotin bridges. The diagnostic particles comprise 
carboxylate-derivitized polymeric core particles, avidin moieties linked 
through amide bonds to the core particles, biotin moieties complexed to 
the avidin moities and antibody (immunoglobulin) molecules linked to the 
biotin moieties through amide bonds. The diagnostic particles are 
suspended in a liquid medium to form a latex composition that is useful 
for diagnosing the presence of an antigen to which the bound antibody is 
specific. The diagnostic particles suspended in the liquid medium 
agglutinate by cross-linking through antigen molecules that have multiple 
antigenic determinants that are recognized by the antibody, and the 
cross-linked diagnostic particles precipitate from the liquid medium. 
To form the diagnostic particles, carboxylate-derivatized latex core 
particles are attached to avidin through an amide bond formed between the 
surface carboxyl groups of the polymeric core particle and primary amino 
groups on the avidin. An antibody of interest is attached to biotin 
through an amide bond formed by the carboxyl group of biotinic acid and an 
amine group of the antibody. Avidin has a very strong affinity for biotin, 
and the antibody-bound biotin moiety readily complexes to an avidin moiety 
linked to the core particle. To assure that the diagnostic particles will 
be stable as a latex suspension until agglutination testing, the number of 
antibody moieties linked to each core particle is controlled. To prevent 
excessive biotinylated antibody from complexing to the avidin moities 
linked to the core particles, a mixture of free biotinic acid and 
biotinylated antibody of a predetermined molar ratio is reacted with the 
core particle-bound avidin so that biotinic acid occupies a portion of the 
binding sites on the avidin which might otherwise be occupied by the 
biotinylated antibody. 
The term "latex" herein is used broadly to include stable dispersions of 
particles of polymeric material. Suitable latexes include suspensions of 
minute polystyrene and polyacrylamide particles. To provide that a stable 
suspension of the diagnostic particle can be formed which will precipitate 
within a reasonable time upon exposure to the antigen, the starting 
polymeric core particles should be between about 0.2 and about 1.0 micron 
in diameter. 
The polymeric core particles are carboxylate-derivatized to provide exposed 
carboxyl groups at their surfaces for attachment of avidin. Polyacrylamide 
particles may be derivatized by the method of John K. Inman, "Covalent 
Linkage of Functional Groups, Ligands, and Proteins to Polyacrylamide 
Beads", in Methods in Enzymology, Vol XXXIV, (ed. William B. Jakoby and 
Meir Wilchek, Academic Press, N.Y., 1974) pp. 30-58. Suitable 
carboxylate-derivatized latex particles are commercially available; for 
example, carboxylate-latex sold by Polysciences. It is found that 
particles carboxylated to between 0.1 and about 0.5 milliequivalents per 
gram are most suitable. This degree of carboxylation provides more surface 
carboxyl groups than are eventually used to bind avidin and, subsequently, 
biotinylated antibody. However, less successful results are achieved with 
particles carboxylated to a lesser degree. Therefore, it is not considered 
desirable to limit the amount of antibody bound on each particle through 
the number of carboxyl moieties on the polymeric core particles. 
The high affinity of avidin for biotinic acid is well known, and the 
combination of avidin and biotin are found to provide a very effective 
means for linking controlled amounts of antibody to the latex. Biotin 
(hexahydro-2-oxo-1H-thieno [3,4] imidazole-4-pentanoic acid) is a growth 
factor present in very minute amounts of every living cell and is found 
mainly bound to proteins or polypeptides. Avidin is a glycoprotein 
containing four essentially identical peptide subunits, each having an 
attached carbohydrate moiety. Each subunit of avidin has a single biotin 
binding site. The combined molecular weight of the subunits is about 
66,000. Avidin is most commonly isolated from raw egg whites but is 
probably found in the genital tract of all animals. Avidin is also 
produced by certain bacteria, such as Streptomyces avidinii, and avidin 
used herein is to be understood to refer to animal avidin as well as 
bacterial avidin, such as streptavidin. The high affinity of avidin for 
biotin has been demonstrated by the ability of large amounts of avidin to 
produce biotin deficiency in rats and chicks. 
Because the core particles have a greater number of surface carboxyl groups 
than are to be eventually linked to antibody molecules, avidin is reacted 
with the core particles in amounts less than the stoichiometric 
concentration which would link to all core surface carboxyl groups. It is 
not preferred, however, to control the number of biotinylated antibody 
molecules that are subsequently linked to the cores by limiting the number 
of avidin molecules that are bound to the cores to the minimum that would 
be required to complex stoichiometrically to the desired number of 
biotinylated antibody molecules. It is found that best results are 
achieved when avidin is reacted with the latex particles to provide 
between about 10.sup.10 and about 10.sup.13 molecules of avidin per 
cm.sup.2 of estimated core particle surface area, and to this end, between 
about 1.2.times.10.sup.-3 and about 1.2.times.10.sup.-2 gm of avidin are 
reacted per gm of styrene core particles, and between about 
1.2.times.10.sup.-3 and about 1.2.times.10.sup.-2 gm of avidin are reacted 
per gm of polyacrylamide core particles. 
The antibody is selected according to the antigen to be detected. Any of 
the known types of immunoglobulins can be linked to latex core particles 
by the method of the present invention, including IgG, IgA and IgM. A 
general requirement is that the antibody be specific for an antigen having 
at least two antigenic determinants so that the antigen can bind to 
antibodies on different diagnostic particles and thereby cross-link the 
particles. Many large antigens of interest, such as the group carbohydrate 
antigen of Group A Streptococcus, have multiple, substantially identical, 
antigenic determinants. If the particular antigen does not have duplicate 
antigenic determinants, it may have spaced-apart distinct antigenic 
determinants, in which case a mixture of two or more antibodies, each 
reactive with one of the determinants, might be linked to the latex core 
particles to allow cross-linking between diagnostic particles to take 
place through the unique antigenic determinants. 
It is preferred that, if available, monoclonal antibodies be used to detect 
antigens. Monoclonal antibodies, consisting of identical antibody 
molecules, are much more specific than conventionally obtained antibody 
fractions and provide for much greater reproducability between lots of 
diagnostic particles. "Monoclonal antibodies" is used herein to refer to 
antibodies generated by hybridomas, as well as antibodies produced by 
other cell immortalization techniques, e.g., by infection with certain 
viruses. However, the invention is intended to encompass diagnostic 
particles incorporating conventional antibody fractions, particularly to 
detect antigens for which no monoclonal antibody is presently available. 
The formation of the amide bond between the carboxyl groups on the latex 
core particles and an amine group of the avidin is preferably facilitated 
through an intermediate formed by reaction of a crabodiimide, such as 
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, with the carboxyl groups 
on the latex particles. After the carboxyl groups have been activated 
through reaction with the carbodiimide, the avidin is introduced, 
whereupon primary amino groups on the avidin replace the carbodiimide 
linked to the carbonyl. These reactions are represented in equation 1 
below: 
##STR1## 
A preferred method of forming the amide bond between the carboxyl group of 
biotin and an amino group of the antibody molecule is by initially forming 
an ester between an N-hydroxy imide, such as N-hydroxy-succinimide and 
biotin and then reacting the N-hydroxy imide-biotin with the 
immunoglobulin, whereupon an amino group of the immunoglobulin replaces 
the N-hydroxyimide linked to the carbonyl. This reaction is carried out at 
slightly alkaline conditions, preferably at a pH of between about 7.5 and 
about 9.0. These reactions are represented in equation 2 below: 
##STR2## 
Because avidin is reacted in less than stoichiometric amounts, unbound 
surface carboxyl groups remain on the latex core particles. These unbound 
carboxyl groups are preferably neutralized, e.g., with an amine, such as 
ethanol amine. 
It has been found that diagnostic particles have greater stability if, 
subsequent to neutralization, non-immunogenic proteinaceous material, such 
as bovine serum albumin (BSA), is adsorbed onto the surfaces of the 
avidin-bound latex core particle. 
Control of the amount of antibody on the surface of the diagnostic 
particle, which is accomplished by mixing free biotinic acid with 
biotinylated antibody so that they compete for the excess avidin binding 
sites, is considered an important aspect of the invention. Too few 
antibody molecules may not afford agglutination at a suitable rate, 
whereas too many antibody molecules may result in premature precipitation. 
Generally, it is preferred that between 2.times.10.sup.11 and about 
2.times.10.sup.12 antibody molecules be bound per cm.sup.2 of estimated 
surface area of the latex core particles, although this may vary somewhat 
depending upon whether the antibody is small, e.g., IgG, or large, e.g., 
IgM. The molar ratio of biotinic acid to biotinylated antibody ratio is 
dependent on the number of avidin binding sites. It must also be taken 
into account that free biotinic acid reacts somewhat more rapidly with the 
avidin than does the antibody-bound avidin. Providing a desired amount of 
antibody on the diagnotic particles generally requires that a mixture of 
biotinic acid and biotinylated antibody in a molar ratio of between about 
1:1 and about 10:1 be reacted with particles having avidin, in the 
above-mentioned preferred amounts, bound to the core particles. The 
reaction between a mixture of biotinylated antibody and biotinic acid with 
core particle-linked avidin is represented in equation 3 below: 
##STR3## 
The liquid medium in which the diagnostic particles are suspended is 
generally aqueous as is consistent with the natural environment of 
antibody molecules. A slightly basic pH, e.g., between about 7.5 and about 
8.5, contributes to stability of the diagnostic particles. Antimicrobial 
agents, such as NaN.sub.3 may also be added to the medium. Generally latex 
suspensions for use in agglutination tests contain between about 5 and 
about 10 gm. of particles per liter of suspension. 
The invention will now be described in greater detail by way of example.

EXAMPLE 
4.0 ml of carboxylate-latex at 2.5 weight percent solids, obtained from 
Polysciences Inc. Warrington, Pa., is washed three times with distilled 
water, and after the final wash, the particles are resuspended in 4.0 ml 
of distilled water. 1.0 ml of 0.05 M KH.sub.2 PO.sub.4, pH 4.5 is added. 
The suspension is placed on a magnetic stirrer and maintained at 
22.degree. C., and 5 ml. of a solution of 2 weight percent 
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (obtained from Sigma Chem. 
Co., St. Louis, Mo.) is added and allowed to react with the latex for 3.5 
hours. The carbodiimide-activated carboxylate-latex is then washed once in 
saline and resuspended in 5 ml of saline. 
1.2 mg of avidin (all procedures are performed in duplicate, once using egg 
white avidin and once using streptavidin) is dissolved in 5 ml of 0.2 M 
borate, pH 8.5, and the 5 ml. latex suspension is added. The 
carbodiimide-activated carboxylate-latex and avidin are allowed to react 
for 20 hrs. at 22.degree. C. To neutralize surface carboxyl groups that 
are not bound to avidin, 5 mM ethanolamine is added, and then BSA is added 
to a concentration of 2 weight percent. The avidin-latex is washed and 
taken up in 0.1 M glycine-saline, pH 8.2 containing 0.2% NaN.sub.3, 0.2% 
BSA and 0.05% Tween-20 and stored at 4.degree. C. 
Antibodies are bound to biotin as follows: 
(A) 1 mg of polyvalent rabbit IgG (anti-group A streptococcal antigen) in 
1.0 ml. of 0.2 M NaHCO.sub.3 is reacted with 50 .mu.l of a solution of 
d-biotin-N-hydroxysuccinimide ester in DMSO (1.0 mg/ml). The reaction is 
allowed to proceed for 4 hours at 22.degree. C., and the reaction mixture 
is then dialized at 4.degree. C. for 18 hours against a 500-fold excess of 
0.91 M tris-saline buffer, pH 8.0 containing 0.2% NaN.sub.3. 
(B) 1.0 mg of mouse monoclonal IgG.sub.3 (anti-group A streptococcal 
antigen) in 1.0 ml of 0.2 M NaHCO.sub.3 is reacted with 8 .mu.l of a 
solution of d-biotin-N-hydroxysuccinimide ester in DMSO (1.0 mg/ml). The 
reaction is allowed to proceed for 4 hours at 22.degree. C., and the 
reaction mixture is then dialyzed at 4.degree. C. for 18 hours against a 
500-fold excess of 0.01 M tris-saline buffer, pH 8.0 containing 0.2% 
NaN.sub.3. 
(C) 1.0 mg of mouse monoclonal IgM (anti-N meningitis B capsular 
polysaccharide) in 1.0 ml of 0.2 M NaHCO.sub.3 is reacted with 8 to 10 
.mu.l of a solution of d-biotin-N-hydroxysuccinimide ester in DMSO (1.0 
mg/ml) for 4 hours at 22.degree. C., and the reaction mixture is then 
dialyzed against a 500-fold excess of 0.01 M tris-saline buffer, pH 8.0 
containing 0.2% NaN.sub.3. 
Each of the biotinylated antibodies, formed above, is attached to the 
avidin-latex particles. 1.0 ml. of avidin latex is pelleted and the 
supernatant removed. The pellet is taken up in 1.0 ml of biotinylated 
antibody containing 5 .mu.l of 10.sup.-3 M biotinic acid, and the mixture 
is stirred for 1 hour at 22.degree. C. to react the biotin and 
biotinylated antibody. After 1 hour, a second 5 .mu.l aliquot of 10.sup.-3 
M biotinic acid is added to block potentially unoccupied biotin binding 
sites on the avidin moieties. 
The diagnostic particles are centrifuged, washed and suspended at 0.6% 
solids in 0.1 M glycine-saline buffer, pH 8.2 containing 0.2% NaN.sub.3, 
0.2% BSA and 0.05% Tween-20. 
To 15 ml of the diagnostic particle suspension formed from the polyvalent 
rabbit IgG, 50 ml containing 0.1 .mu.g of Group A Streptoccous antigen are 
added. Agglutination of the suspension is noted after 4 minutes. 
To 15 ml of the diagnostic particle suspension formed from the mouse 
monoclonal IgG.sub.3 antibody, 50 ml containing 0.1 .mu.g of Group A 
Streptococcus antigen are added. Agglutination of the suspension is noted 
after 10 minutes. 
To 15 ml of the diagnostic particle suspension formed from the mouse 
monoclonal IgM antibody, 50 ml containing 0.1 .mu.g of N mening B 
polysaccharide antigen are added. Agglutination of the suspension is noted 
after 10 minutes. 
Suspensions of diagnostic, particles prepared according to the present 
invention, are stable if stored under refrigeration for periods of several 
months. The diagnostic particles are extremely sensitive, and using 
microtechniques, the suspensions can be used to detect as little as 
nanogram quantities of antigen. 
Several advantages of the present invention can now be more fully 
appreciated. The invention provides for attachment of all types of 
immunoglobulins, including IgM. The amount of antibody attached to the 
latex is not dependent upon the adsorption characteristics of a particular 
lot of latex. Similarly, although the degree of carboxylation is 
preferably within a certain range, the amount of antibody attached is 
determined independently of the precise degree of carboxylation of the 
core particles, i.e., by the less than stoichiometric amount of avidin 
bound to the carboxyl groups and then by the selected molar ratios of 
biotinic acid and biotinylated antibody. Thus reproducability of 
manufacture is significantly enhanced relative to prior latex 
particle-to-antibody linking procedures, particularly with respect to IgM, 
which previously had to be bound to the latex by the highly variable 
adsorption technique. 
While the invention has been described in terms of certain preferred 
embodiments, modifications obvious to one with ordinary skill in the art 
may be made without departing from the scope of the present invention. For 
example, while it is not preferred, the amount of antibody bound to the 
particles could be made through control of the number of avidin molecules 
bound to the latex core particles followed by saturation of the biotin 
binding sites with biotinylated antibody molecules. While the diagnostic 
particle suspensions generally require that the antigen be large, having 
at least two antigenic determinant sites, it is also contemplated that a 
small antigenically active molecule or hapten could agglutinate the 
particles if the particles were suspended in a liquid medium that 
contained a chemical which would link two or more of the hapten molecules. 
Various features of the present invention are set forth in the following 
claims: