Classifications

• • 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
• • 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
• • 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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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Hematology (AREA)
• Engineering & Computer Science (AREA)
• Urology & Nephrology (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Rehabilitation Therapy (AREA)
• Biotechnology (AREA)
• Rheumatology (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

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Cited By (15)

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• 1967
  • 1967-05-18 GB GB23157/67A patent/GB1179435A/en not_active Expired
  • 1967-06-07 DE DE19671598898 patent/DE1598898A1/de 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/de not_active IP Right Cessation
• 1969
  • 1969-05-13 US US824314A patent/US3658982A/en not_active Expired - Lifetime

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