US 3574818 A
Description (OCR text may contain errors)
United States Patent Int. Cl. G01n 33/16 US. Cl. 424-9 3 Claims ABSTRACT OF THE DISCLOSURE A diagnostic for rheumatism, adaptable to topical application to the skin, consisting essentially of an aqueous solution of a nucleoproteide. A method for preparing the diagnostic by isolation from aqueous homogenizates of a streptococcus of Group A or C.
It is known that in the case of rheumatism, which is prevailingly caused by streptococcal infection, antibodies against streptococcal substances can be identified in the serum. In clinical diagnosis only the determination of the anti-streptolysin titer has had good results. The antibodies thereby identified have no connection with Rheumatismus verus. They merely show an immunisation against the streptococcal streptolysin. Consequently, the reaction is unspecific and, being effected in vitro, requires an expensive and wearisome technique.
'Now, a diagnostic has been found which can be used, especially as a skin test, to detect rheumatic fever.
The diagnostic according to the present invention consists essentially of a nucleoproteide obtained from streptococci which has a UVmaximum of approximately 259 mg and a migration velocity of 4.6 to 6 cm./2 hrs. in paper electrophoresis (Veronal buffer 0.1 M; pH 8.6; 11 v./cm.; 22 C.). The diagnostic consists preferably of a solution of the nucleoproteide obtained from streptococci in water or water-containing solvents, for instance physiological NaCl solution, and other solvents or mixtures of solvents tolerated by the skin without or with only a very reduced reaction. Such solutions can be applied in form of suitable dispersions, for example oil-inwater or water-in-oil emulsions, to which further substances usual in such preparations may be added. Such include nonsoluble substances, for instance fats and oils, as well as emulsifying or dispersing agents like protective colloids of organic or inorganic origin. Further adjuvants conventionally used in such preparations such, for example, as buffers, stabilizers and the like, may be added as well.
The content of nucleoproteide of these preparations usually ranges between 10- and 10 g. percent, referred to the total amount of the preparation. In special cases, however, said content may be above or below the indicated limits. If the degree of immunisation is particularly high, a higher concentration will be necessary. If the degree of immunisation is relatively low, a lower concentration will be sufficient. Particularly adequate concentrations are those of 1(l g. percent to g. percent.
The diagnostic according to the present invention permits the identificationtRheumatismus verus by a very simple intracutaneous test. According to the type of preparation the diagnostic may be used intracutaneously or percutaneously.
In the intracutaneous test, for example, 0.1 cc. of the test solution is injected intracutaneously. A positive result of the test is indicated by a reddening and infiltration at the point of injection. The diagnostic leads to a skin reaction of the type known from tuberculin. A time in- 3,574,818 Patented Apr. 13, 1971 terval of 48 hours after the application of the test has proved favorable for examining the result of the reaction. The test may also be carried out as a percutaneous test by rubbing in a pea-sized amount of an ointment preparation of the nucleoproteide or in the form of a plastersample, similar to variations of the tuberculin test. In the serum of the treated patients, humoral antibodies against the above-described nucleoproteide are detectable by various immunologic reactions such as by the Ouchterlony test, by immunoelectrophoresis and the like. The antibody content in the serum is, however, in most cases so slight that the gamma globuline fraction must be concentrated.
The intracutaneous test is to be considered superior in being easy to manage and capable of being carried out without any laboratory equipment.
The nucleoproteide used as active substance in the diagnostic according to the present invention is obtained from streptococci, preferably from those of the groups A and C.
The separation of the nucleoproteide from the extract can be effected by combining centrifugation and a treatment with protein-precipitating salts or salt solutions of different concentrations, or by extraction with appropriate solutions, or by electrophoresis, counter-current distribution, chromatography and so on, as well as by combinations of these processes.
A very suitable method of isolating the nucleoproteide consists in homogenising streptococci with a two-fold amount of water or Water-containing solvents (referred to the weight of the streptococci) and isolating the extract by the usual methods of separation, such as centrifugation. Subsequently, the homogenate is adjusted to a high content of salt, e.g. approximately 40% of ammonium sulfate. Thus a pre-fraction is separated which is discarded. After isolating the pre-fraction, the content of salt is increased, for instance to approximately 70% of ammonium sulfate. During this, a precipitate results which is separated, for example by centrifuging, and dialysed in aqueous or water-containing solution against water or a water-containing solvent. The dialysed solution is passed, either as such or after drying, into a buffer of a pH-value of about 7 and the solution is added to a basic ion-exchanger, for exmple DEAE-cellulose. The column is washed with buffer of pH 3 until the pH of the extract is 3. Then a salt, for instance NaCl in a concentration of approximately 3%, is added to the buffer. A pre-fraction is discarded and the subsequent main fraction is collected. The extracted material is dialysed, for example against distilled water, in order to eliminate the NaCl and the buffer substances. Then the solution can be dried over silica gel.
The nucleoproteide used according to the present invention is distinguished by a UV-maximum of approximately 259 me and shows a migration velocity of 4.66 cm./ 2 hrs. in paper electrophoresis (Veronal buffer 0.1 M; pH 8.6; 11 v./cm.). Further reactions are blackening with amido-black 10B, phosphate coloring according to Waid and Morgan, slight coloring on calcium according to Pollard and McOmie, lipid coloring according to Swalm and slight coloring on purine according to Michel and Harberler. On alkaline and acid hydrolysis, adenine, cytosine, guanine and uracil are identified. Ribose can also be identified according to Partridge. Aspartic acid, glutamic acid, glycine, alanine, serine, threonine, valine and leucine have been detected according to Dose and Caputo.
The addition of the nucleoproteide, in a higher degree of dilution, to tropocollagen solutions results in the forma tion of collagenous fibrils. The nucleoproteide binds excellently to human serum protein. This can be demonstrated in the Ouchterlong-test as well as by immunoelectrophoresis. 'Furthermore, it reacts with highly purified 3 human gamma-globulin. This reaction can equally well be demonstrated by immunoelectrophoresis and by the Ouchterlony-test. The nucleoproteide is easily Water-soluble, but insoluble in alcohol, ether and chloroform.
The new diagnostic obtained according to the present invention constitutes a valuable hitherto un'kown expedient in medical diagnosis. Surprisingly, it makes it possible to identify the existence of a rheumatic disease by a most simple method, which means considerable progress. It was not foreseeable that the nucleoproteide used according to the present invention would lead to such a test reaction.
EXAMPLE 50 grams of dried streptococci are homogenised with 100 m1. of distilled water in a Potter homogenizer. Subsequently, the homogenised product is agitated mechanically for three hours at room temperature, and then centrifuged for 15 minutes at 5,000 revolutions. The separated solution is heated on a water bath to 80 C., and, after cooling, a pre-fraction of unspecific material is precipitated by adding 40% (g./volume) ammonium sulfate. The mixture is kept for 12 hours in a refrigerator at +4 C. and then centrifuged for 15 minutes at 5,000 revolutions. In the resulting solution the content of ammonium sulfate is increased to 70%, the precipitate is once again kept in the refrigerator for 12 hours at +4 C. and centrifuged for 15 minutes at 5,000 revolutions. The precipitate is then dissolved in water and dialysed twice for 24 hours each against distilled water. After this time no sulfate ions can be detected with barium chloride. Then the dialysed solution is dried over silica gel.
For further purification DEAE cellulose is used. For this purpose 12 g. of DEA-E-cellulose are suspended in 500 cc. of Michaelis butfer of pH 7.0. After 45 minutes the solution is decanted and the suspension of the ion-exchanger is filled into a glass column of 30 volume. 0.5 gram of the streptococcal substance are dissolved in ml. of buffer of pH 7.0, the solution is cleared by centrifuging for minutes at 20,000 revolutions and then introduced on top of the exchanger column. The column is washed with buffer of pH 3.0 till the eluate has a pH- value of 3.0. Then, the adsorbate is desorbed with the same bulfer to which 3% of NaCl have been added. The first ml. of the extract are discarded. The following 80 ml. are collected and dialysed for 24 hours at 4 C. against distilled water. After this time no chlorine ions can be detected. The suspension is then dried over silica gel.
1. A process for making a diagnostic for rheumatism which comprises preparing an aqueous homogenizate of a streptococcus of Group A or C; centrifuge to concentrate the bacterial extract; adding a salt to the extract to precipitate a discardable first fraction therefrom; adding further quantities of a salt to precipitate a desired main fraction therefrom; dissolving said main fraction in water; dialysing said solution to remove salt therefrom, and then drying the resulting dialysed solution; dissolving the dried solution in a neutral buffer; adsorbing the buffered solution on a basic ion exchanger; washing the ion exchanger with a buffer at pH 3; washing the ion exchanger with a salt solution buffer at pH 3 to desorb a discardable first fraction therefrom and then eluting a desired main fraction by further washing with said buffered salt solution; dialysing the eluate to remove salt therefrom; and drying said dialysed solution to obtain said diagnostic.
2. A process according to claim 1 wherein the salt used for the separation of said discardable first fraction from said desired main fraction is ammonium sulfate.
3. A diagnostic for rheumatism prepared according to claim 1 comprising an aqueous solution of a nucleoproteide having a UV-maximum of approximately 259 millimicrons and a migration velocity of 4.5-6 cm./2 hours in paper electrophoresis (Veronal buffer 0.1 M; pH 8.6; 11 v./cm.); said nucleoproteide being readily soluble in water but insoluble in alcohol, ether, and chloroform; said nucleoproteide buackening with amido black 10B, phosphate coloring according to Waid and Morgan, coloring slightly on calcium according to Pollard and McOrnie, lipid coloring according to Swalm, and coloring slightly on purine according to Michel and Harberler; adenine,
cytosine, guanine, and uracil being identifiable on alkaline and acid hydrolysis, ribose being identifiable according to Partridge, and aspartic acid, glutamic acid, glycine, alanine, serine, threonine, valine, and leucine being detectable according to Dose and Caputo.
References Cited Stedmans Medical Dictionary, 17th ed. (1949), pp. 1138-1139.
STANLEY J. FRLEDMAN, Primary Examiner US. Cl. X.R.