US3658982A - Stable latex reagent for the detection of rheumatoid arthritis - Google Patents
Stable latex reagent for the detection of rheumatoid arthritis Download PDFInfo
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- US3658982A US3658982A US824314A US3658982DA US3658982A US 3658982 A US3658982 A US 3658982A US 824314 A US824314 A US 824314A US 3658982D A US3658982D A US 3658982DA US 3658982 A US3658982 A US 3658982A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/815—Carrier is a synthetic polymer
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/866—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/868—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, or immunotolerance
Definitions
- a stable reagent having avidity for the agglutination reaction in the serological determination of rheumatoid factors is composed of rabbit gamma globulin which is degraded with a proteolytic enzyme, coated on latex particles, heat treated and stored at low temperatures u nt1l used. This reagent is capable of identifying true positives when the sodium chloride of the serum diluent is main tained within certain critical limits.
- the present invention relates to a method for the serological determination of agglutinating factors, commonly called rheumatoid factors, that are found in the sera of patients suffering from rheumatoid arthritis. More particularly, the invention relates to a stable reagent for the quantitation of the rheumatoid factors and to the method for its preparation, and particularly the conditions under which it is used.
- agglutinating factors commonly called rheumatoid factors
- rheumatoid arthritis The sera of many patients with rheumatoid arthritis contain high molecular weight gamma globulins possessing a sedimentation constant of 19 S. These protein moie-' ties have been termed rheumatoid factors and the detection of rheumatoid factors in sera is a convenient means for the early diagnosis of rheumatoid arthritis.
- Nonbiological particles such as polystyrene latex, acrylic resins and bentonite that have been sensitized with human gamma globulin have been used to detect rheumatoid factor.
- US. Pat. 3,088,875 teaches one such diagnostic reagent. Although these tests are used widely, they can best be classified as general screening tests in that they are extremely sensitive, but at the same time yield a considerable number of false positive results as has been described by Heiner and Rudd, Arth. & Rheumat, :110 (1962).
- Biological particles are also used as carriers for the apparent antigens of the rheumatoid factor.
- human red blood cells either selected human gamma globulin containing specific nonagglutinating anti- D (Rh is used for the sensitization, or the red blood cells are first treated with a reagent such as tannic acid which alters the surface properties so that protein is readily but nonspecifically adsorbed.
- a reagent such as tannic acid which alters the surface properties so that protein is readily but nonspecifically adsorbed.
- the reagents consist of latex sensitized with rabbit gamma globulin and a buffer system.
- the test is made by preparing doubling dilutions in a series of test tubes of the blood serum using the buffer with a critical concentration of sodium chloride as diluent and adding an equal volume of latex reagent.
- the tubes containing the serum-latex mixture are then placed at 56 C. for 2 hours and at '+4 C. overnight. If rheumatoid factors are present in the test serum, a direct reaction is evidenced by agglutination of the particles of the latex reagent. If there is no rheumatoid factor present, the latex reagent will remain smoothly suspended throughout the fluid medium. Reaction can be graded with respect to the degree of agglutination present.
- Polystyrene particles having an average diameter of from 0.15 micron to 14 microns, but preferably 0.81 micron are used to prepare the latex system. Acrylic particles having an average within the same range may also be used.
- the test will not function properly with ordinary rabbit gamma globulin .Treatment of the rabbit gamma globulin to alter its antigenic properties is essential to the test.
- the alteration found most effective is accomplished by enzyme treatment of the rabbit gamma globulin prior to sensitization of the latex particles.
- the latex reagent is then heat treated to complete the alteration of the rabbit gamma globulin. Heat treatment at 50 C. for a period of 7 to 10 days has been found to be elfective. Higher temperatures for shorter periods of time are also effective. For example, heat treatment of the latex reagent at C. (boiling) for 2 to 5 minutes was found to be effective. In the alternative heating at 70 C. for approximately 5 days can be practiced. However, heating of the latex reagent in the absence of prior enzyme treatment does not result in a suitable reagent.
- the whole product of fragmentation may be used for the preparation of the test reagent.
- papain is employed as the enzyme, isolation of the relatively insoluble portion of the fragmented rabbit gamma globulin and selection of the more nearly soluble part of this fraction is desirable.
- the yields of treated rabbit globulin on latex particles is relatively low compared to the trypsin or chymotrypsin treated material. Heating of the fragmented material can be expected to result in further collapse of the native structure which exposes the desired reactive site areas.
- human gamma globulin treated in the manner described becomes grossly nonspecific and unlfit for reagent purposes.
- Pretreatment of human gamma globulin prior to sensitization of the latex has not been found to enhance the reactivity while still maintaining the specificity of the reaction.
- a further difference in the reagents consisting of human and rabbit gamma globulin can be seen in the butfer systems required.
- sodium chloride in a borate butter system enhances the speed of reaction of human globulin sensitized latex
- a concentration beyond 0.1 M sodium chloride in borate bufier negates the reaction of the rabbit gamma globulin.
- a concentration above 0.05 M sodium chloride in borate buifer results in a substantial increase in the number of false positives.
- a salt concentration of 0.025 M in borate buifer yields optimum reactions. Concentrations of sodium chloride below 0.025 M result in insolubility of serum proteins, and consequent precipitation of these proteins.
- the effective critical concentration of sodium chloride is the concentration of that salt in the serum diluent.
- Normal human serum has a concentration of 0.15 M sodium chloride.
- the first dilution in a tube test involves 0.1 ml. of serum with 1.9 ml. of 0.025 M borate buffer containing 0.025 M sodium chloride. This 1:20 dilution results in an efiective sodium chloride concentration close to 0.025 M. Further dilutions (1:40, 1:80, 1:160, etc.) bring the concentration even closer to 0.025 M sodium chloride.
- EXAMPLE I 100 milligrams of rabbit gamma globulin are dissolved in milliliters of 0.1 M borate buffer, pH 8.6. Crude trypsin equal to one-tenth the weight of rabbit gamma globulin is added to the globulimbuiier solution and brought to a temperature of 37 C. and maintained at that temperature for one hour. The enzyme digested gamma globulin is then diluted to 50 ml. with 0.1 M borate buffer, pH 8.6 and is added slowly with constant stirring to 50 milliliters of a two percent by weight suspension of polystyrene particles in which the particles have an average diameter of 0.81 micron. Thimerosal is added in a final concentration of 115000. The resulting material is brought slowly to a temperature of 50 C. and maintained at that temperature for days. Salt free bovine serum albumin is added to a final concentration of 0.1% for stabilizing purposes and the material thus prepared is stored at +4 C.
- EXAMPLE II 100 milligrams of rabbit gamma globulin are disoslved in 5 milliliters of 0.1 M borate buffer, pH 8.6. Crude trypsin equal to one-tenth the weight of rabbit gamma globulin is added to the globulinabuffer solution and brought to a temperature of 37 C. and maintained at that temperature for one hour. The enzyme digested gamma globulin is then diluted to 50 ml. with 0.1 M borate buffer, pH 8.6 and is added slowly with constant stirring to 50 milliliters of a two percent by weight suspension of polystyrene particles in which the particles have an average diameter of 0.81 micron. Thimerosal is added in a final concentration of 1:5000. The resulting material is brought to a temperature of 93 C., and maintained at that temperature for 10 minutes. The material thus prepared is stored at +4 C.
- EXAMPLE III milligrams of rabbit globulin is dissolved in 5 milliliters of 0.05 M phosphate bufier pH 7.1. Papain equal to one-fiftieth of the weight of rabbit gamma globulin is added to the globulin-buffer solution and brought to a temperature of 37 C. and maintained at that temperature for one hour. The preparation is then dialysed against borate buffer pH 8.6, diluted to fifty milliliters with the same buffer and added slowly with constant stirring to 50 milliliters of a two percent by weight suspen sion of polystyrene particles in which the particles have an average diameter of 0.81 micron. Thimerosal is added in a final concentration of 1 :5000. The resulting material is brought slowly to a temperature of 50 C., and maintained at that temperature for 10 days. The material thus prepared is stored at +4 C.
- the reagent of this invention may be employed as the initial test for rheumatoid arthritis or as the confirming test.
- the faster, but less accurate slide test based on human gamma globulin may be used for the initial screening test when large numbers of tests are involved.
- the following example will serve to illustrate the employment of the reagent in testing for rheumatoid arthritis.
- Tube #10 is the control.
- the tubes may be centrifuged and thereafter shaken gently before observing the agglutination.
- centrifuging requires substantial skill for determination of the approximate centrifugal force and time.
- the preferred practice is to maintain the tubes overnight at 4 C. instead of centrifuging.
- the following tables show the results of confirmation tests where a positive response was obtained in a latex slide screening test.
- the highest doubling dilution at which agglutination occurred with the reagent of the invention is compared with the highest doubling dilution of the Waaler-Rose sheep cell test.
- the reliable Waaler-Rose test data indicated true positive responses and the latex slide test data and the latex tube test data (i.e., the reagent of this invention) also showed positive responses.
- the latex slide test employed is substantially that disclosed in US. Pat. 3,088,875.
- a reagent for determining rheumatoid factor in 160 human serum comprising latex particles, having an average diameter of from about 0.15 micron to about 14 TABLE 3.PATIENTS WHOSE SERA WERE SENT FOR microns, coated with a proteolytic enzyme-treated rabbit SYPHILIS SEROLOGY gamma globulin, said globulin having been prepared by ClinicalData Unknown adding to rabbit gamma globulin in a buttered solution, Waaler- Latex, Latex, pH 7.1-8.6, a proteolytic enzyme selected from the group 1 Slide consisting of trypsin, chymotrypsin and papain and heatest test test ing at 37 C.
- said coated latex particles g g having been treated for 5-10 days at temperatures from Nd Nd 50-70 C. or for 2-5 minutes at temperatures from g I 93-100 C.
- Nd Nd 2.
- the method of determining the presence of rheumagg I 3% toid factor in human serum which comprises preparing Nd Nd doubling dilutions of human serum with a buffer solution gg fig containing sodium chloride in the range of 0.025 M to 1 3% fig 0.05 M, adding to said dilutions a buffered suspension of Nd I Nd the reagent of claim 1, incubating said serum-suspension gg fig mixtures at about 56 C., reducing said temperature to Nd Nd about 4 C., and observing said mixtures for agglutina- N N tion. 3 3 5.
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Abstract
A STABLE REAGENT HAVING AVIDITY FOR THE AGGLUTINATION REACTION IN THE SEROLOGICAL DETERMINATION OF RHEUMATOID FACTORS IS COMPOSED OF RABBIT GAMMA GLOBULIN WHICH IS DEGRADED WITH A PROTEOLYTIC ENZYME, COATED ON LATEX PARTICLES, HEAT TREATED AND STORED AT LOW TEMPERATURES UNTIL USED. THIS REAGENT IS CAPABLE OF IDENTIFYING TRUE POSITIVES WHEN THE SODIUM CHLORIDE OF THE SERUM DILUENT IS MAINTAINED WITHIN CERTAIN CRITICAL LIMITS.
Description
United States Patent 3,658,982 STABLE LATEX REAGENT FOR THE DETECTION OF RHEUMATOID ARTHRITIS Alice M. Reiss, Somerville, and Rosemary K. Chachowski,
Manville, N.J., assignors to Ortho Pharmaceutical Corporation No Drawing. Continuation-impart of application Ser. No. 558,255, June 17, 1966. This application May 13, 1969, Ser. No. 824,314
Int. Cl. G01n 31/00, 33/16 US. Cl. 424-12 6 Claims ABSTRACT OF THE DISCLOSURE A stable reagent having avidity for the agglutination reaction in the serological determination of rheumatoid factors is composed of rabbit gamma globulin which is degraded with a proteolytic enzyme, coated on latex particles, heat treated and stored at low temperatures u nt1l used. This reagent is capable of identifying true positives when the sodium chloride of the serum diluent is main tained within certain critical limits.
The present application is a continuation-in-part of our copending application, Ser. No. 558,255, filed June 17, 1966 now abandoned.
The present invention relates to a method for the serological determination of agglutinating factors, commonly called rheumatoid factors, that are found in the sera of patients suffering from rheumatoid arthritis. More particularly, the invention relates to a stable reagent for the quantitation of the rheumatoid factors and to the method for its preparation, and particularly the conditions under which it is used.
The sera of many patients with rheumatoid arthritis contain high molecular weight gamma globulins possessing a sedimentation constant of 19 S. These protein moie-' ties have been termed rheumatoid factors and the detection of rheumatoid factors in sera is a convenient means for the early diagnosis of rheumatoid arthritis.
Nonbiological particles such as polystyrene latex, acrylic resins and bentonite that have been sensitized with human gamma globulin have been used to detect rheumatoid factor. US. Pat. 3,088,875 teaches one such diagnostic reagent. Although these tests are used widely, they can best be classified as general screening tests in that they are extremely sensitive, but at the same time yield a considerable number of false positive results as has been described by Heiner and Rudd, Arth. & Rheumat, :110 (1962).
Attempts to quantitate the level of rheumatoid factor in the serum of patients by tube test procedures employing these sensitized particles frequently result in astronomically high titers that bear less relation to the clinical condition of the patient than to the particular preparation of human gamma globulin used for sensitization.
Biological particles are also used as carriers for the apparent antigens of the rheumatoid factor. When human red blood cells are used, either selected human gamma globulin containing specific nonagglutinating anti- D (Rh is used for the sensitization, or the red blood cells are first treated with a reagent such as tannic acid which alters the surface properties so that protein is readily but nonspecifically adsorbed. Neither of these two procedures has received the widespread acceptance accorded to the sheep red blood cell-rabbit anti-sheep cell test first described by Waaler [Acta Path. Microbiol. Scand., 17:172 (1940)], and subsequently by Rose et al. [Proc. Soc. Exper. Biol. & Med., 68:1 (1948)].
.Although the reasons for the specificity of the sheep cell test are not known, its specificity has been amply demonstrated by clinical follow up of patients whose sera has revealed positive test reactions. Tube test titers seldom extend beyond 1:5000 and stand in contrast to titers obtained with either tanned and sensitized red blood cells or sensitized latex which may extend beyond 1: 100,000.
Despite the reliability of the Waaler-Rose sheep cell test, its use has been somewhat curtailed due to the instability of the red blood cell carrier. Standardization and sensitization of red blood cells are time consuming procedures that must be performed at frequent intervals.
It is the primary object of the present invention to provide a stable nonbiological particle sensitized with rabbit gamma globulin to obtain a sensitive, specific reagent for rheumatoid factor.
It is another object of the invention to provide a tube test confirming reagent to be used whenever positive reactions are obtained with a rapid screening type of test procedure.
It is still another object of the invention to provide a test reagent which mimics the titers obtained with the sheep red cell test.
It is a further object to provide test conditions that will render the confirming agent capable of identifying true positives.
In the test of the present invention, the reagents consist of latex sensitized with rabbit gamma globulin and a buffer system. The test is made by preparing doubling dilutions in a series of test tubes of the blood serum using the buffer with a critical concentration of sodium chloride as diluent and adding an equal volume of latex reagent. The tubes containing the serum-latex mixture are then placed at 56 C. for 2 hours and at '+4 C. overnight. If rheumatoid factors are present in the test serum, a direct reaction is evidenced by agglutination of the particles of the latex reagent. If there is no rheumatoid factor present, the latex reagent will remain smoothly suspended throughout the fluid medium. Reaction can be graded with respect to the degree of agglutination present.
Polystyrene particles having an average diameter of from 0.15 micron to 14 microns, but preferably 0.81 micron are used to prepare the latex system. Acrylic particles having an average within the same range may also be used.
The test will not function properly with ordinary rabbit gamma globulin .Treatment of the rabbit gamma globulin to alter its antigenic properties is essential to the test. The alteration found most effective is accomplished by enzyme treatment of the rabbit gamma globulin prior to sensitization of the latex particles. The latex reagent is then heat treated to complete the alteration of the rabbit gamma globulin. Heat treatment at 50 C. for a period of 7 to 10 days has been found to be elfective. Higher temperatures for shorter periods of time are also effective. For example, heat treatment of the latex reagent at C. (boiling) for 2 to 5 minutes was found to be effective. In the alternative heating at 70 C. for approximately 5 days can be practiced. However, heating of the latex reagent in the absence of prior enzyme treatment does not result in a suitable reagent.
Several enzymes have been tested and found satisfactory for alteration of the rabbit gamma globulin, namely crude and crystalline trypsin, chymotrypsin and papain. Fungal proteases and pepsin have been found only partially eifective. While the nature of the alteration of the rabbit gamma globulin is not fully understood, presumably the reactivity of rheumatoid factor with rabbit globulin depends upon sites that are unavailable after adsorption of the rabbit globulin onto latex. Fragmentation of the globulin molecules by various proteolytic enzymes provides a means by which vital portions of the structure become available. In the case of trypsin or chymotrypsin treated rabbit gamma globulin, the whole product of fragmentation may be used for the preparation of the test reagent. When papain is employed as the enzyme, isolation of the relatively insoluble portion of the fragmented rabbit gamma globulin and selection of the more nearly soluble part of this fraction is desirable. In any event the yields of treated rabbit globulin on latex particles is relatively low compared to the trypsin or chymotrypsin treated material. Heating of the fragmented material can be expected to result in further collapse of the native structure which exposes the desired reactive site areas.
In contrast, human gamma globulin treated in the manner described becomes grossly nonspecific and unlfit for reagent purposes. Pretreatment of human gamma globulin prior to sensitization of the latex has not been found to enhance the reactivity while still maintaining the specificity of the reaction.
A further difference in the reagents consisting of human and rabbit gamma globulin can be seen in the butfer systems required. Whereas sodium chloride in a borate butter system enhances the speed of reaction of human globulin sensitized latex, a concentration beyond 0.1 M sodium chloride in borate bufier negates the reaction of the rabbit gamma globulin. A concentration above 0.05 M sodium chloride in borate buifer results in a substantial increase in the number of false positives. A salt concentration of 0.025 M in borate buifer yields optimum reactions. Concentrations of sodium chloride below 0.025 M result in insolubility of serum proteins, and consequent precipitation of these proteins.
The effective critical concentration of sodium chloride is the concentration of that salt in the serum diluent. Normal human serum has a concentration of 0.15 M sodium chloride. However, the first dilution in a tube test involves 0.1 ml. of serum with 1.9 ml. of 0.025 M borate buffer containing 0.025 M sodium chloride. This 1:20 dilution results in an efiective sodium chloride concentration close to 0.025 M. Further dilutions (1:40, 1:80, 1:160, etc.) bring the concentration even closer to 0.025 M sodium chloride.
The following examples illustrate the preparation of the reagent.
EXAMPLE I 100 milligrams of rabbit gamma globulin are dissolved in milliliters of 0.1 M borate buffer, pH 8.6. Crude trypsin equal to one-tenth the weight of rabbit gamma globulin is added to the globulimbuiier solution and brought to a temperature of 37 C. and maintained at that temperature for one hour. The enzyme digested gamma globulin is then diluted to 50 ml. with 0.1 M borate buffer, pH 8.6 and is added slowly with constant stirring to 50 milliliters of a two percent by weight suspension of polystyrene particles in which the particles have an average diameter of 0.81 micron. Thimerosal is added in a final concentration of 115000. The resulting material is brought slowly to a temperature of 50 C. and maintained at that temperature for days. Salt free bovine serum albumin is added to a final concentration of 0.1% for stabilizing purposes and the material thus prepared is stored at +4 C.
EXAMPLE II 100 milligrams of rabbit gamma globulin are disoslved in 5 milliliters of 0.1 M borate buffer, pH 8.6. Crude trypsin equal to one-tenth the weight of rabbit gamma globulin is added to the globulinabuffer solution and brought to a temperature of 37 C. and maintained at that temperature for one hour. The enzyme digested gamma globulin is then diluted to 50 ml. with 0.1 M borate buffer, pH 8.6 and is added slowly with constant stirring to 50 milliliters of a two percent by weight suspension of polystyrene particles in which the particles have an average diameter of 0.81 micron. Thimerosal is added in a final concentration of 1:5000. The resulting material is brought to a temperature of 93 C., and maintained at that temperature for 10 minutes. The material thus prepared is stored at +4 C.
EXAMPLE III milligrams of rabbit globulin is dissolved in 5 milliliters of 0.05 M phosphate bufier pH 7.1. Papain equal to one-fiftieth of the weight of rabbit gamma globulin is added to the globulin-buffer solution and brought to a temperature of 37 C. and maintained at that temperature for one hour. The preparation is then dialysed against borate buffer pH 8.6, diluted to fifty milliliters with the same buffer and added slowly with constant stirring to 50 milliliters of a two percent by weight suspen sion of polystyrene particles in which the particles have an average diameter of 0.81 micron. Thimerosal is added in a final concentration of 1 :5000. The resulting material is brought slowly to a temperature of 50 C., and maintained at that temperature for 10 days. The material thus prepared is stored at +4 C.
The reagent of this invention may be employed as the initial test for rheumatoid arthritis or as the confirming test. The faster, but less accurate slide test based on human gamma globulin may be used for the initial screening test when large numbers of tests are involved. The following example will serve to illustrate the employment of the reagent in testing for rheumatoid arthritis.
EXAMPLE IV In conducting the confirming test for rheumatoid factor utilizing the reagent of the invention, blood serum from a patient which has given a positive response in screening test for rheumatoid factor is used. The reagent as prepared in any one of Examples I to III is diluted 1:20 with 0.025 M borate butter. Ten 12 x 75 mm. glass test tubes are placed in a rack. Doubling dilutions of the serum are prepared as follows:
0.1 ml. of the patients serum is pipetted into tube #1. 1.9 ml. of 0.025 M borate buifer containing 0.025 M sodium chloride is added to tube #1 and 1.0 ml. of the same buffer is added to the remaining nine tubes. The contents of tube #1 are mixed and 1.0 ml. of the contents of tube #1 are transferred to tube #2. The contents of tube #2 are mixed and 1.0 ml. thereof is transferred to tube #3. The transfer from one tube to the next progresses through tube #9 and 1.0 ml. of the final contents of tube #9 is discarded. Tube #10 is the control.
1.0 ml. of the diluted reagent is then added to each of the ten tubes. The contents of the tubes are mixed well and incubated for 2 hours at 56 C. The tubes are then maintained at 4 C. overnight. The reactions are read for agglutination of particles and clarity of supernate. The results are recorded as negative (an even suspension comparable to the control), 1+, 2+, 3+, or 4+.
The tubes may be centrifuged and thereafter shaken gently before observing the agglutination. However, centrifuging requires substantial skill for determination of the approximate centrifugal force and time. Hence, the preferred practice is to maintain the tubes overnight at 4 C. instead of centrifuging.
The following tables show the results of confirmation tests where a positive response was obtained in a latex slide screening test. The highest doubling dilution at which agglutination occurred with the reagent of the invention is compared with the highest doubling dilution of the Waaler-Rose sheep cell test. In each of these tables, the reliable Waaler-Rose test data indicated true positive responses and the latex slide test data and the latex tube test data (i.e., the reagent of this invention) also showed positive responses. The latex slide test employed is substantially that disclosed in US. Pat. 3,088,875.
TABLE 1 PATIENTS UNDER CLINICAL OBSERVATION reaCtiOn mixture donsistislg of Seruln (1 WITH A? IE STABLISHED DIAGNOSIS OF HEUMATO D ml., using a buffer solution containing sodium chloride ARTHR T within the critical range described hereinbefore) and s w e h latex reagent (0.1 ml.). The following tables illustrate the f 2? i 5 ffiiticzalitty of the sodium chloride concentration during e es +H+ 040 I Nd l 3% TABLE 4.-TUBE TEsT USING LATEX REAGENT 0F 40 EXAMPLE I 2 500 10 Dilutions of a patient's 1 230 serum known to contain 560 rheumatoid factors in low titer 20 Composition of the serum diluent I; 1 280 b er 1:20 1:40 1:80 1:160
640 j 0. a 1 0 0 160 0 3 1 0 0 160 0.025 M borate plus 0.025 NaCl a 2 1 o 160 0.025 M borate plus 0.05 NaCl 3 2 :i; 0 H 320 0.025 M borate plus 0.10 NaCl 3 1 0 0 160 0.025 M borate plus 0.15 NaCl 1 0 0 0 20 0.025 M borate plus 0.20 NaCl 0 0 0 0 +1+1+ 160 The results are recorded as negative (an even suspension comparable Nd means not done. No confirmation test is run where the slide to the control) or test shows a negative response.
TABLE 2.-PATIENTS WHOSE SERA WERE SENT FOR RHEUMATOID ARTHRITIS SEROLOGY TABLE 5.-'lUBE TEST USING LATEX REAGENT OF EXAMPLE I Clinical Data Uncertain Dilutions of a patient's serum known to contain Waaler- Latex, Latex, rheumatoid factors in high titer Serum Rose slide tube Composition of the tes test e serum diluent buffer 1:20 1:40 1=s0 1=100 1:320 1:640 1=1,2s0
320 0.025 M borate plus 80 0.025MNaCl 3 4 4 a 2 2 320 0.025 M borate plus 0.05MNaCl 1 1 2 2 2 2 $0 160 312% It can be seen that salt affects the two serum titration 33 reactions differently. However, unless the optimum con- 20 80 centration is used, the reaction is affected adversely. $33 Optimum sensitivity is requisite for the test as is avoidance of prozoning of high titered sera.
:1" S8 What 1s claimed 1s: 150 1. A reagent for determining rheumatoid factor in 160 human serum comprising latex particles, having an average diameter of from about 0.15 micron to about 14 TABLE 3.PATIENTS WHOSE SERA WERE SENT FOR microns, coated with a proteolytic enzyme-treated rabbit SYPHILIS SEROLOGY gamma globulin, said globulin having been prepared by ClinicalData Unknown adding to rabbit gamma globulin in a buttered solution, Waaler- Latex, Latex, pH 7.1-8.6, a proteolytic enzyme selected from the group 1 Slide consisting of trypsin, chymotrypsin and papain and heatest test test ing at 37 C. for one hour, said coated latex particles g g having been treated for 5-10 days at temperatures from Nd Nd 50-70 C. or for 2-5 minutes at temperatures from g I 93-100 C. Nd Nd 2. The reagent of claim 1, wherein said latex particles I fig are polystyrene particles having an average diameter of Nd Nd 0.81 micron. gg R; 3. The reagent of claim 1, wherein said enzyme is 320 320 added to said rabbit gamma globulin dissolved in 0.1 M 1 3 fig borate buffer having a pH of 8.6. Nd Nd 4. The method of determining the presence of rheumagg I 3% toid factor in human serum which comprises preparing Nd Nd doubling dilutions of human serum with a buffer solution gg fig containing sodium chloride in the range of 0.025 M to 1 3% fig 0.05 M, adding to said dilutions a buffered suspension of Nd I Nd the reagent of claim 1, incubating said serum-suspension gg fig mixtures at about 56 C., reducing said temperature to Nd Nd about 4 C., and observing said mixtures for agglutina- N N tion. 3 3 5. The method of claim 4 wherein said serum-suspen- Nd Nd sion mixtures are observed for agglutination after maintaining them at 4 C. overnight. Not y 18 necessary to enzyme treat the Tabblt 6. The method of claim 4 wherein said serum-suspengamma gloiflllin P to adsorption to and F sion mixtures are observed for agglutination after centrifploy a heating step on the latex reagent itself, but it is ugatiom also critical to control the salt concentration of the final (References on following page) Referenzas Cited UNITED STATES PATENTS 5/1963 Fisk 424-12 3/1967 Treacy 42412 OTHER REFERENCES Ling, J. Med. Lab. Tech., vol. 18, 1961, pp. 94401. Rheins, PSEBM, vol. 96, October 1957, pp. 67-71. Edelman, J. Exptl. Med., Vol. 108, July 1958, pp.
Christian, I. Exptl. Med., vol. 1 08, July 1958, pp.
Porter, Biochern. 1., vol. 73, September 1959, pp.
US. Cl. X.R.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US55825566A | 1966-06-17 | 1966-06-17 | |
US82431469A | 1969-05-13 | 1969-05-13 |
Publications (1)
Publication Number | Publication Date |
---|---|
US3658982A true US3658982A (en) | 1972-04-25 |
Family
ID=27071674
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US824314A Expired - Lifetime US3658982A (en) | 1966-06-17 | 1969-05-13 | Stable latex reagent for the detection of rheumatoid arthritis |
Country Status (6)
Country | Link |
---|---|
US (1) | US3658982A (en) |
BE (1) | BE700023A (en) |
CH (1) | CH485460A (en) |
DE (1) | DE1598898A1 (en) |
GB (1) | GB1179435A (en) |
NL (1) | NL6708441A (en) |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2522086A1 (en) * | 1974-05-20 | 1975-12-11 | Technicon Instr | ANALYSIS OF BIOLOGICAL FLUIDS |
US4054646A (en) * | 1973-07-30 | 1977-10-18 | General Electric | Method and apparatus for detection of antibodies and antigens |
US4138213A (en) * | 1974-05-20 | 1979-02-06 | Technicon Instruments Corporation | Agglutination immunoassay of immune complex with RF or Clq |
US4153417A (en) * | 1976-09-08 | 1979-05-08 | Pharmacia Diagnostics Ab | Method of indicating rheumatoid factors |
US4162895A (en) * | 1976-12-10 | 1979-07-31 | Technicon Instruments Corporation | Mouse serum |
US4172827A (en) * | 1972-06-26 | 1979-10-30 | General Electric Company | Method for concentration and purification of antigens and antibodies |
US4184847A (en) * | 1976-09-08 | 1980-01-22 | Pharmacia Diagnostics Ab | Method of indicating rheumatoid factors |
US4189466A (en) * | 1975-09-19 | 1980-02-19 | Technical Research Affiliates, Inc. | Detection of rheumatoid factor by antibody sensitized microbial particles |
FR2439402A1 (en) * | 1977-10-17 | 1980-05-16 | Gen Electric | METHOD FOR DETECTING IMMUNOLOGICAL REACTIONS USING A VOLTAGE BEAT MEASUREMENT TECHNIQUE |
EP0019741A1 (en) * | 1979-05-07 | 1980-12-10 | BEHRINGWERKE Aktiengesellschaft | Latex reagent, process for its preparation, its application and an agent containing this reagent |
WO1982002773A1 (en) * | 1981-02-05 | 1982-08-19 | Icl Scient | Polystyrene latex reagents and related methods for use in immunological diagnostic procedures |
EP0107861A1 (en) * | 1982-10-29 | 1984-05-09 | Research Corporation | Diagnostic test for rheumatological diseases |
US5026512A (en) * | 1975-08-30 | 1991-06-25 | Chang Shao C | Method for manufacturing molded products of thermoplastic and inorganic materials |
US5827668A (en) * | 1990-05-25 | 1998-10-27 | Peptide Therapeutics Limited | Immunodiagnostic assay for rheumatoid arthritis |
US6159748A (en) * | 1995-03-13 | 2000-12-12 | Affinitech, Ltd | Evaluation of autoimmune diseases using a multiple parameter latex bead suspension and flow cytometry |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4434227A (en) * | 1982-02-08 | 1984-02-28 | Abbott Laboratories | Immunoassay for class specific immunoglobulin antibodies |
-
1967
- 1967-05-18 GB GB23157/67A patent/GB1179435A/en not_active Expired
- 1967-06-07 DE DE19671598898 patent/DE1598898A1/en active Pending
- 1967-06-16 NL NL6708441A patent/NL6708441A/xx unknown
- 1967-06-16 BE BE700023D patent/BE700023A/xx unknown
- 1967-06-16 CH CH859067A patent/CH485460A/en not_active IP Right Cessation
-
1969
- 1969-05-13 US US824314A patent/US3658982A/en not_active Expired - Lifetime
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4172827A (en) * | 1972-06-26 | 1979-10-30 | General Electric Company | Method for concentration and purification of antigens and antibodies |
US4054646A (en) * | 1973-07-30 | 1977-10-18 | General Electric | Method and apparatus for detection of antibodies and antigens |
US4062935A (en) * | 1974-05-20 | 1977-12-13 | Technicon Instruments Corporation | Immunoassay involving the binding of RF to the antigen-antibody complex |
US4138213A (en) * | 1974-05-20 | 1979-02-06 | Technicon Instruments Corporation | Agglutination immunoassay of immune complex with RF or Clq |
DE2522086A1 (en) * | 1974-05-20 | 1975-12-11 | Technicon Instr | ANALYSIS OF BIOLOGICAL FLUIDS |
US5026512A (en) * | 1975-08-30 | 1991-06-25 | Chang Shao C | Method for manufacturing molded products of thermoplastic and inorganic materials |
US4189466A (en) * | 1975-09-19 | 1980-02-19 | Technical Research Affiliates, Inc. | Detection of rheumatoid factor by antibody sensitized microbial particles |
US4153417A (en) * | 1976-09-08 | 1979-05-08 | Pharmacia Diagnostics Ab | Method of indicating rheumatoid factors |
US4184847A (en) * | 1976-09-08 | 1980-01-22 | Pharmacia Diagnostics Ab | Method of indicating rheumatoid factors |
US4162895A (en) * | 1976-12-10 | 1979-07-31 | Technicon Instruments Corporation | Mouse serum |
FR2439402A1 (en) * | 1977-10-17 | 1980-05-16 | Gen Electric | METHOD FOR DETECTING IMMUNOLOGICAL REACTIONS USING A VOLTAGE BEAT MEASUREMENT TECHNIQUE |
EP0019741A1 (en) * | 1979-05-07 | 1980-12-10 | BEHRINGWERKE Aktiengesellschaft | Latex reagent, process for its preparation, its application and an agent containing this reagent |
WO1982002773A1 (en) * | 1981-02-05 | 1982-08-19 | Icl Scient | Polystyrene latex reagents and related methods for use in immunological diagnostic procedures |
US4351824A (en) * | 1981-02-05 | 1982-09-28 | Icl Scientific | Polystyrene latex reagents, methods of preparation, and use in immunological procedures |
EP0107861A1 (en) * | 1982-10-29 | 1984-05-09 | Research Corporation | Diagnostic test for rheumatological diseases |
US5827668A (en) * | 1990-05-25 | 1998-10-27 | Peptide Therapeutics Limited | Immunodiagnostic assay for rheumatoid arthritis |
US6159748A (en) * | 1995-03-13 | 2000-12-12 | Affinitech, Ltd | Evaluation of autoimmune diseases using a multiple parameter latex bead suspension and flow cytometry |
Also Published As
Publication number | Publication date |
---|---|
GB1179435A (en) | 1970-01-28 |
NL6708441A (en) | 1967-12-18 |
DE1598898A1 (en) | 1971-04-01 |
BE700023A (en) | 1967-12-18 |
CH485460A (en) | 1970-02-15 |
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