Stable diagnostic reagent and method for qualitative determinations of streptococci infections

A method and a stable reagent for the detection of pathogenic bacterial infections in humans, and in particular, a stable diagnostic composition comprising non-degraded streptolysin-O protein directly adsorbed onto polystyrene latex particles and method of use are disclosed. The reagent may be prepared by adsorbing one or more layers of the streptolysin-O toxin onto the surface of the latex particle, effecting an intermolecular cross-linkage of the streptolysin-O, and further stabilizing the reagent by the addition of a bacteriostatic agent. Accordingly, the present invention provides a significant advance regarding the qualitative determinations of streptococci infections by providing a stable diagnostic reagent and method for such determinations.

BACKGROUND OF THE INVENTION 
The present invention relates to a method and a stable reagent for the 
chemical diagnosis of a pathogenic bacterial infection in humans. More 
particularly, the present invention relates to the production of a stable 
reagent which permits qualitative determination of streptococci infections 
by unaided visual determination. 
When a human or other animal is exposed to foreign proteins of antigenic 
character, an immunologic response will often occur. One aspect of this 
response is the presence in the animal's blood of water-soluble antibodies 
capable of specifically recognizing and binding the foreign protein. 
During a bacterial infection in an animal, the invading organisms may 
excrete a great number of these foreign proteins. Streptococci bacteria 
have been shown to excrete over 20 exocellular toxins, one of which is a 
cytolytic protein known as streptolysin-O. 
Streptolysin-O is an extremely destructive protein, one of its prime 
functions being the hemolysis of red blood cells. Fortunately, 
streptolysin-O is also antigenic and the animal can respond 
immunologically with the appropriate antibodies (anti-streptolysin) which 
can inhibit lysogenesis by binding onto the streptolysin-O. 
The amount of anti-streptolysin present in serum, called the "titer" is an 
important tool to clinicians, not only for the diagnosis of streptococci 
infection, but also for its relationship with rheumatic fever, acute 
gloumerulonephritis, rheumatoid arthritis and erythema nodosum. To 
determine this titer, some technicians have utilized the inhibitory 
properties of anti-streptolysin in conjunction with the lytic properties 
of streptolysin-O on red blood cells. In this respect, the study done by 
Rammelkamp [Am. J. Med. 10:673, 1951] teaches one such method. 
The most commonly used method for determining the concentration of 
streptolysin-O antibodies is to perform a hemolytic inhibition test. One 
of the activities of the streptolysin-O is its ability to hemolyze human 
red blood cells and blood cells of other animal species. In the presence 
of anti-streptolysin in the patient's serum, the hemolysis is inhibited. 
Thus, an increased concentration of anti-streptolysin present in the serum 
will be reflected in greater inhibition. Quantitative expression is 
measured by an elaborate dilution scheme of the patient's serum. 
The isolation of streptolysin-O from the culture medium is a long and 
tedious process. In addition, after several days of handling in an aerobic 
atmosphere, the toxin is in an oxidized form. For hemolytic activity, 
streptolysin-O must be in a reduced state. This is usually accomplished by 
the addition of cysteine, 2-mercaptoethanol or other thiols as reducing 
agents. Even with the addition of any of these compounds, the hemolytic 
potency of the toxin is short lived. Commercially available streptolysin-O 
reagents are usually reduced, freeze dried, and sealed under an inert gas 
atmosphere. Under reconstitution, the reduced material must be used within 
thirty minutes. 
Although this test procedure is widely used, it is tedious and necessitates 
maintaining a constant supply of viable red blood cells. Also, because 
streptolysin-O loses potency when oxidized, the procedure must be done 
quickly or in the presence of reducing agents which may adversely affect 
the analysis. 
From the literature, it is apparent that streptolysin-O and its activity 
will form precipitins without the necessity of activating the oxidized 
toxin. Thus, the indication is that while the hemolytic activity is 
dependent on the toxin being in a reduced form, the precipitin formation 
is independent of its redox state. 
Antigen-antibody reactions may manifest themselves under various 
conditions. In soluble forms, the antigen and antibodies will combine to 
form loose aggregates, which continue to a lattice build-up to become 
visible precipitates. If this soluble antigen-antibody reaction should 
take place in a semi-solid medium such as agar, a macroscopically visible 
precipitin line will result. 
Attempts to simplify the foregoing test procedure have been based on the 
well-known antibody agglutination process and the fact that 
anti-streptolysin recognition of streptolysin-O is independent of the 
toxin's redox state. In addition, due to the importance of qualitative 
determination of anti-streptolysin titers, much research has been directed 
towards rendering the agglutination product macroscopically visible. 
The conversion of a "precipitin" reaction to an "agglutination" reaction 
has occupied the time and efforts of clinical immunochemists throughout 
the world for the past three decades. Adsorption of a reactant to a 
carrier particle serves to demonstrate the presence of either the antigen 
or antibody within a relatively short length of time, and secondly, the 
macroscopic visualization of the antigen-antibody reaction does not 
require the need for elaborate or sophisticated equipment. For these 
reasons, simple agglutination reactions have enjoyed wide popularity in 
laboratory use of immunodiagnosis of disease states. Various carrier 
particles such as erythrocytes, bentonite, collodium, quartz, synthetic 
resins and latex particles have been employed as the serologic carrier of 
one of the reactants. 
In instances of antigen-antibody reaction, where the reactant is in a 
particulate form, visible agglutination of the particles will demonstrate 
the antibody-antigen reaction. 
Technical difficulties, availability, variation in sizes and compositions 
have eliminated many of the aforementioned carriers as reactant particles. 
However, commercially available uniform size polystyrene latex has enjoyed 
immense popularity as a serologic carrier. 
Various techniques for forming visible agglutination products in general 
are known in the art and good results have been obtained by adsorbing 
either the antigen or the antibody to a carrier. In this regard, U.S. Pat. 
No. 3,088,875 teaches the use of polymerized styrene latex particles as 
carriers. 
Polystyrene latex particles are usually hydrophobic and negatively charged, 
and will non-specifically adsorb many proteinaceous materials. However, 
because the adsorption is often not permanent, a number of procedures have 
been developed to stabilize the protein-latex complex. These methods 
usually require some modification of the protein and the addition of 
extraneous high molecular weight materials. A representative sampling of 
these procedures can be found in U.S. Pat. Nos. 3,658,982 and 3,992,517, 
and German Pat. No. 1,914,081. 
These polystyrene particles are usually prepared by initiating the 
polymerization of styrene monomers with potassium persulfate in the 
presence of the emulsifier sodium laurel sulfate. In preparation of 
particles through emulsion polymerization the stability of the polystyrene 
particles is due in part to the charged surface groups originated from the 
initiator through surface sulfate groups. However, the emulsifier also 
contributes to the stability of the particles by imparting an electrical 
negative charge. The greater the quantity of emulsifier the greater the 
negative charge on the latex particle surface. A double layer of ions and 
counter ions is usually responsible for the stability of the particles. 
Divalent and trivalent cations as well as high ionic strength buffers may 
cause destabilization of the polystyrene latex colloid. 
A review of the art reveals that IgG molecules containing antibody activity 
are usually non-specifically adsorbed onto the polystyrene latex particles 
which then serve as a reagent. Heat and other methods of denaturation will 
tend to "stabilize" the reagent. These antibodies, usually composed of 
negatively charged IgG molecules, will be adsorbed onto the negatively 
charged latex particles due to the influence of the non-ionic van der 
Waals forces and the hydrophobic bond effects. 
When streptolysin-O is combined with polystyrene latex particles by 
alteration of the protein, either through partial degradation or by the 
addition of stabilizing materials, the modification may cause a loss of 
some antigenic determinants and a decrease in assay sensitivity. 
Furthermore, the extraneous materials utilized may interfere with the 
antibody-antigen interaction or the agglutination reaction, resulting in 
false results and incorrect clinical diagnosis. 
Thus, those skilled in the art have recognized a significant need for a 
stable streptolysin-O-latex complex in which the protein retains 
significant preadsorption characteristics and wherein the entire complex 
is free from potentially contaminating intermediaries. The present 
invention fulfills this need. 
SUMMARY OF THE INVENTION 
The present invention relates to a method and a stable reagent for the 
detection of pathogenic bacterial infections in humans, and in particular, 
provides a stable diagnostic composition consisting of non-degraded 
streptolysin-O protein directly adsorbed onto polystyrene latex particles. 
The reagent may be prepared by adsorbing one or more layers of the 
streptolysin-O toxin onto the surface of the latex particle, effecting an 
intermolecular cross-linkage of the streptolysin-O, and further 
stabilizing the reagent by the addition of a bacteriostatic agent. 
More specifically, one significant feature of the invention relates to the 
non-covalent coating of a polystyrene latex particle with streptolysin-O 
protein by utilizing an alkaline pH to properly orient the determinant 
groups of the protein, in conjunction with a carbodiimide to effect 
intermolecular cross-linking. An additional feature of the invention is 
that it provides for the maintenane of the stability of the streptolysin-O 
latex complex by utilizing sodium azide, or other suitable bacteriostatic 
agents such as thimerosal. 
One presently preferred embodiment for coating the material onto 
polystyrene utilizes an inorganic buffer at pH 10.0. The preferred buffer 
is prepared with Boric Acid-KCl-NaOH at 0.5 M, although freshly prepared 
carbonate-bicarbonate buffer at pH 10.0, among others, is also suitable. 
The final latex streptolysin-O reagent is suspended in a 0.05 M Boric 
Acid-HCl-NaOH buffer, pH 8.2 with 0.1% NaN.sub.3, added. 
Accordingly, the present invention significantly advances the state of the 
art regarding the qualitative determinations of streptococci infections by 
providing a stable diagnostic reagent and method for such determinations. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A significant feature of the present invention is embodied in a complex 
consisting of polystyrene latex particles adsorbed with streptolysin-O 
antigen in an alkaline environment, the adsorption occurring in the 
presence of carbodiimide. This complex has been shown to be particularly 
useful for the qualitative determination of streptococci infections in 
animals because it enhances formation of a visible agglutination product. 
While those skilled in the art have not completely understood the 
interaction between polystyrene particles and proteins, it is believed 
that absent any covalent coupling between the two materials, the 
adsorption occurs in the form of an equilibrium reaction, and therefore is 
susceptible to small changes in pH, temperature, ionic strength or other 
environmental factors. A change in any one of these factors could cause 
disassociation of the latex-protein complex and streptolysin-O adsorbed 
onto polystyrene particles has proven to be partially susceptible to this 
problem. The addition of other materials to the complex may tend to lessen 
the chance of disassociation, but also, the addition of other materials to 
the complex tends to complicate the determination of optimal conditions 
for assay procedures. 
It has been found that if the adsorption is carried out at a relatively 
high alkaline pH in the presence of carbodiimide, the resulting product is 
stable and has a minimal number of critical components. The precise 
structure of the product formed during the reaction is not fully 
understood; however, it is believed that at the pH utilized, the 
determinant groups of the streptolysin-O are beneficially arranged to 
retain a sufficient amount of antigenicity even while being cross-linked 
by a carbodiimide. It should be noted that carbodiimides are capable of 
forming covalent bonds between many types of functional groups, including 
carboxylic acids, amines, alcohols, and thiols. The actual cross-links 
formed in the present invention could involve one, some, or all of these 
groups. 
Thus, in accordance with the present invention, streptolysin-O protein is 
diluted in a buffered solution with a pH range of from about 8.5 to about 
11.9, but preferably a pH of about 10.0 is utilized. In this respect, it 
is noted, however, that a relatively small amount of streptolysin-O 
protein is adsorbed at about pH 8.2 to about pH 8.5. Below about pH 8.2, 
possibly due to the orientation of the streptolysin-O molecules, an 
unstable reagent may result, and activity may disappear within a few days. 
Further, a similar situation was encountered at pH levels above about pH 
11.5. One buffer is a freshly prepared carbonate-bicarbonate solution, and 
preferred buffers are inorganic buffers, such as 0.5 molar Boric 
Acid-KCl-NaOH. The buffers may be adjusted to the proper pH by any one of 
well known methods. 
The preparation of the streptolysin-O toxin may suitably be prepared in 
accordance with the following procedure. Streptococcus pyrogenes, group A. 
American Type Culture collection #12383, is cultivated in a commercially 
prepared broth medium known as Todd-Hewitt broth. The culture medium is 
enhanced with dextrose to promote the production of the streptolysin-O. 
Following over night growth, the pH of the culture is adjusted with sodium 
hydroxide to neutralize the acid formed. Additional dextrose is added and 
the incubation continued for an additional 6-8 hours. The culture is then 
frozen at -40.degree. C. for 24 or more hours, then thawed at +5.degree.. 
The top one-third of the thawed culture is collected and centrifuged to 
remove the bacteria. Solid ammonium sulfate is added to the centrifuged 
concentrate to a final concentration of 75%. The streptolysin-O is 
precipitated by the ammonium sulfate. The precipitate is suspended in 
water, and dialyzed continuously to remove the precipitant. 
The isolated streptolysin-O is assayed by preparing two-fold dilutions of 
the streptolysin-O in a 0.01 M cysteine-HCl solution. To each tube is 
added 0.2 ml of a 5% suspension of thrice-washed human or sheep 
erythrocytes suspended in phosphate buffer saline, pH 6.6. The tubes are 
incubated in a 37.degree. C. water bath for two hours, and the tubes are 
centrifuged. The end point is the greatest dilution displaying hemolysis 
of the cells. This is usually 1:2048 dilution, the range being 1:512 to 
1:4096. 
In this regard, streptolysin-O protein may be prepared according to the 
general method of J. E. Alouf and M. Raynaud, Biochimie, 55,1187, (1973), 
which method is incorporated herein by reference, or may be purchased 
commercially. Alternatively, the streptolysin-O protein may be prepared 
from standard reagents which are available for hemolysis-inhibition tests. 
These reagents require concentration before use, for instance, about one 
thousand fold. The crude streptolysin material may be filtered through a 
0.22.mu. porosity cellulose membrane filter as a sterilizing procedure. 
When a new lot of streptolysin-O protein has been prepared for coating the 
polystyrene latex particles, a suitable formulation procedure is used to 
prepare reagents containing varying amounts of streptolysin-O attached to 
the particles. Each reagent may thus be tested with a panel of positive 
and negative serums, the serums having been tested by hemolysis inhibition 
and assigned values in Todd or International units. The formulation of 
streptolysin-O protein chosen may vary, and the preferred formulation 
giving proper reactivity with the panels may then be used to prepare large 
amounts of reagents. 
In a presently preferred embodiment, the suitably diluted protein is mixed 
with a 10% (w/v) solution of uniform sized polystyrene latex particles 
dispersed in deionized water, and an 0.2% (w/v) aqueous solution of water 
soluble carbodiimide. In this regard, the carbodiimide solution must be 
freshly prepared and used after solubilization. After mixing from about 12 
to about 20 hours, the sensitized particles are washed with a suitable 
buffer, such as 0.05 M Boric Acid-KCl-NaOH buffer at pH 8.2, and then 
centrifuged down at 10,000 rcf. This washing procedure is repeated as 
required, for instance, three times, whereafter the particles are finally 
re-suspended in the same buffer. A suitable bacteriostatic agent, 
preferably sodium azide, is then added to achieve an 0.1% (w/v) 
concentration although a useful range is from about 0.05% (w/v) to 2.0% 
(w/v). 
Another suitable bacteriostatic agent which can be utilized in the 
preparation of the latex-streptolysin-O reagent is thimerosal. In this 
respect, it was determined that in terms of being a bacteriostat, 
thimerosal was found to be effective at 4.degree. C. and 32.degree. C. in 
the concentration range of from about 0.001% to about 0.00001%. Malachite 
green dye (C. I. No. 42000) may be added, if desired, to a final 
concentration of 0.001% (w/v) to enhance the stability and facilitate 
visual determination of agglutination. For further disclosure of this 
particular dye and other suitable dyes, reference may be had to the 
treatise entitled "Conn's Biological Stains", including pages 168, et seq. 
The size of the particles, available commercially from Dow Chemical 
Company, Midland, Mich., may vary from about 0.5 microns to about 7.0 
microns, but a uniform size of about 0.62 microns is optimal because of 
reduced centrifugation time, better separation of the latex-protein 
complex from other materials during the washing procedure and 
reproducibility of results. 
Suitable water soluble carbodiimides are 1-ethyl-3-(3-dimethylaminopropyl) 
hydrochloride carbodiimide, available from Ott Chemical Company, Muskegon, 
Mich., or 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide 
morpho-p-toluenesulfonate, available from Aldrich Chemical Company, 
Milwaukee, Wis., the latter being preferable. The concentration of the 
carbodiimide added may vary from about 0.1% (v/w) to about 0.6% (v/w).