WO1993019369A1 - Technique de prevention de fausses reactions positives dans les essais immunologiques - Google Patents

Technique de prevention de fausses reactions positives dans les essais immunologiques Download PDF

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Publication number
WO1993019369A1
WO1993019369A1 PCT/US1993/002901 US9302901W WO9319369A1 WO 1993019369 A1 WO1993019369 A1 WO 1993019369A1 US 9302901 W US9302901 W US 9302901W WO 9319369 A1 WO9319369 A1 WO 9319369A1
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Prior art keywords
body fluid
sample
chemical compound
temperature
antigen
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PCT/US1993/002901
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English (en)
Inventor
Jacques Singer
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Montefiore Medical Center
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Priority to JP5516856A priority Critical patent/JPH07500190A/ja
Publication of WO1993019369A1 publication Critical patent/WO1993019369A1/fr

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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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • 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

Definitions

  • a ligand binding assay which employs a specific antigen or antibody capable of binding to the analyte.
  • Analytes are the substance, set of substances or factors to be assayed. Radioimmunoassay
  • ELISA enzyme-linked adsorbent when one im unoreagent can be immobilized on the carrier surface while retaining its activity and the reciprocal immunoreagent can be linked to an enzyme in such a manner that both the enzymatic reactivity and the immunoreactivity of this conjugate are retained.
  • Capillary Enzyme Immunoassay Immunoassay reaction occur within glass capillary tubes, polyvinylchloride tubes and between small glass or clear plastic plates.
  • Latex particles are captured on filters, either by physical entrapment or by chemical adhesion to the filter fibers or capturing latex particles in suspension.
  • the capture reagent is bound to a microporous membrane and the specimen containing the analyte is allowed to flow over and around the capture reagent conjugate binding and color development in the
  • DIB assay occurs as described for particle capture assay (immunofiltration assay) .
  • Protein A rich cells of Staphylococus aureus bind immunoglobulins from many animal cells, and these antibody coated cell are use in agglutination reactions.
  • Complement Fixation A standard serologic assay used for the detection of an antigen-antibody reaction in which complement is fixed as a result of the formation of an immune complex.
  • IgG is the predominant immunoglobulin class present in serum.
  • I M is the predominant immunoglobulin class present in serum.
  • a pentameric immunoglobulin comprising approximately 10% of normal human serum immunoglobulin with a molecular weight of 900,000 and a sedimentation coefficient of 19S(Svedberg units).
  • Fc Fragment A fragment obtained by pepsin digestion of immunoglobulin molecule. It contains antigen antibody activity.
  • An F(ab ) 2 fragment and an Fc fragment comprise an active monomeric immunoglobulin molecule.
  • a crystalizable fragment obtained by papain digestion of IgG molecule it contains antigen binding capacity. Attenuation
  • antibody-antigen reaction takes place. Specificity refers to the degree to which the antibody recognition of its antigen is unique. Many body fluids contain factors, largely undefined, which gave false positive or false negative results in an antigen-antibody test system.
  • the term antibody is commonly used to describe the substance gamma globulin which is present in serum, spinal fluid and other fluids.
  • Antibodies identified as IgG, IgA, IgM, IgD and IgE are complex proteins or glycoprotein molecules. They are elaborated by the immune system in response to antigenic stimulation. Most known antigens are multivalent in that they have more than one combining site (or antigenic determinants) to which antibody may be bound.
  • the precipitin reaction is basic to immunology and immunochemistry because it permits the detection and quantitation of antigen and antibody in solution, in tissues, in individual cells or in region of cells.
  • Radioimmunoassay enzyme linked immunosorbent assay (ELISA)
  • ELISA enzyme linked immunosorbent assay
  • precipitin in tubes or by turbidimetry light scattering
  • fluorescence immunoassay precipitation on nylon or on paper membranes dot or blot assay
  • chromatography neutrophile and platelet assays
  • acquired inhibition to clotting proteins detection of antibody to tissue specific antigen and HLA typing, antiglobulin tests, i mmuno d i f f u s i o n (double Ouchterlony, count rimmunoelectrophoresis, immunoelectrophoresis) , complement fixation, DNA Proithermunology and the like.
  • Agglutination techniques are based on the use • of particles (latex, charcoal, bentonite, sheep cell, etc.); on slides, in test tubes, and/or on paper or utilizing instrument particle enhanced immunoassay; light scattering assay, particle counting assay, turbidometric assay and spectroscopic assay, Petinia assay, PACIA assay, rids instrumentation assay, and the like.
  • False positive results may be caused by the presence in all sera of C ⁇ which is a subcomponent of the C 1 complex. False positive results may also be caused by the presence in a large number of sera of rheumatoid factors (RF) . False negative results may be caused by the presence of the undissociated C ⁇ complex.
  • RF rheumatoid factors
  • the first component of the complement C ⁇ is largely dissociated under physiological conditions.
  • the C ⁇ complement is composed of three subcomponents known as C ; C lr ; and C la which are held together in a calcium dependent complex.
  • C when diluted with glycine buffer, pH 8.2 and/or when treated with EDTA-Na will dissociate into the three components (C ⁇ , C ⁇ r and C ls ) .
  • the C component was considered to be a thermolabile substance that could be inactivated by heat at 56 ⁇ >C for 30 minutes.
  • the present inventor has discovered that C and C j are only attenuated and not inactivated by heating a serum.sample to 56 C for 30 minutes.
  • C ⁇ will react with gamma globulin in solution to agglutinate uncoated latex particles or latex particles coated with gamma globulin.
  • C ⁇ has binding sites for IgG and IgM and initiates the activation of the classical pathway cascade.
  • the binding sites for C are located on the Fc portion of IgG and IgM. These binding sites are made up of known sequences of amino acids.and the binding sites are sensitive to high ionic strength salts " which is suggestive of electrostatic interaction.
  • the binding between C ⁇ and immunoglobulin is known to be inhibited by inorganic ions and organic ions in concentrations of 100-300mM.
  • C 1 forms an ionic bond and links many antibodies to their antigenic surface.
  • C lq in human sera is capable of binding to ' antigen antibody complexes and to immunoglobulins such a IgG and IgM.
  • the normal concentration of C ⁇ is 70 meg./ml. It will also bind to aggregated gamma globulin, will precipitate gamma globulin from solution as well as in agar gel systems.
  • C ⁇ binds to bacteria, bacterial lipopolysaccharide, dextran, heparin, polyinosinic acid carrageenan, DNA, C reactive protein, fibronectin, mitochondria, platelets, lymphocytes, monocytes, null cells, monosodium urate crystals, cytoskeletal filaments hybrid monoclonal mouse antibodies. C will bind or adsorb on glass, paper, plastic, etc.
  • particles which will bind C ⁇ include synthetic polymeric particles of various chemical, composition such as polystyrene, polyvinyl toluene, styrenebutadiene, styrene-acrylic acid and the like.
  • the particles will react with C ⁇ alone or when coated with anyone of the following: immunoglobulin, aggregated immunoglobulin, antigens, specific antibodies, sheep cell sensitized with coated anti-rabbit sheep cell antiserum.
  • C ⁇ is used as an exogenous reagent, to detect and quantitate antigen-antibody complexes which are present in certain diseases such as lupus erythematosus, rheuma*.
  • C lq examples of the tests in which C lq is added are C, binding assay, C ⁇ solid phase assay, C inhibitory assay, C ⁇ enzyme linked immunosorbent assay (ELISA), C ⁇ AgAb assay (ELISA), C ⁇ cocktail assay, (C ⁇ bovine conglutin in and low affinity rabbit IgM antihuman Fc fragments) radioimmunoassay,
  • C, 1 and C,lq under certain conditions can cause disaggregation or solubilization of AgAb of heat aggregation of gamma globulin.
  • the classic methodology for inactivation of complement involves heating samples to 56°C for 30 minutes. Repeated freezing and thawing of sera may cause aggregation of gamma globulin. The aggregated gamma globulin will bind C ⁇ thereby decreasing the activity of the complement. This property of the aggregated gamma globulin is called the anticomplementary property. Heating serum at 56oC for 30 minutes has been shown to be ineffective as a procedure for the inactivation of ⁇ . The present inventor has discovered that only when the temperature is raised to 59-64 ⁇ >C for about 3 to 30 minutes in the presence of organic or inorganic ions, the C ⁇ and C ⁇ is inactivated. The higher the temperature the shorter will be the required heating time.
  • a further example of complement interference is when normal sera are added to a solid state immunoassay as a blocking agent.
  • the normal sera contains C ⁇ and C ⁇ which will bind non-specifically to AgAb complexes or it may dissociate and solubilize AgAb complexes in the test sample.
  • the gamma globulin to be adsorbed on the surface of latex is replaced with a fragment of the same antibody F(ab ) 2 fragment for coating latex particle.
  • C ⁇ binds to Fc fragment of immunoglobulin but not to the F(ab ) 2 fragment.
  • CSF is boiled at 100°C for five minutes or urine is heated at 100°C for 3-5 minutes. Following heat treatment urine or CSF are centrifuged and the supernatants are tested.
  • a euglobulin from fraction is used which is obtained from ascitic mouse fluid.
  • IgM consists of approximately 10% of normal immunoglobulin of about 900,000 and a sedimentation coefficient in the ultracentrifuge of 19S (Svedberg) units, while those of IgG has a sedimentation coefficient of 7S and 160,000 molecular weight. In normal human adults IgG constitutes approximately 75% of the total serum immunoglobulin. The concentration range in normal serum for IgG is 8-16 mg/ml and IgM is 0.5-2 mg/ml.
  • the major immunoglobulin expressed on the surface of B cells is formed as a receptor in small T lymphocytes, as monoclonal cryoglobulin, or as mixed polyclonal cryoglobulins IgM- IgG, IgM-IgG-IgA, on the circulating immune complexes IgG-IgM, IgA-IgG-IgM.
  • Rheumatoid factors present in Rheumatoid arthritis sera represent only 1-2% of the IgM molecule.
  • These antibodies react with IgG immunoglobulin or aggregated gamma globulins are called RF. They primarily react with the Fc portion of the IgG but some of them also react with the Fab fragment of these immunoglobulins.
  • Some other classes of immunoglobulin IgA, IgE and IgD have shown to be RF because of their reactivity with IgG. They also react with the Fc portion.
  • IgM rheumatoid factor antibodies have been found to be present in 70-80% of patients with rheumatoid arthritis; in a 0.5-2% of normal population healthy control; and in 20-30% of sera of people over 65 years of age.
  • Rheumatoid factor is also present in percentage varying from 1 to 30% in sera of patients with liver disease, subacute bacterial endocarditis, viral disease, leprosy, tuberculosis, syphilis, cirrhosis of the liver, hepatitis, sarcoidosis, mixed cryoglobulinemia, malignant tumor, trypanosomiasis, in heroin addiction, and in other connective tissue diseases such as lupus erythematosus, Sjogren's syndrome, scleroderma and polymyositis.
  • Rheumatoid factor also makes a transient appearance in patient after vaccination against various diseases.
  • RF represents normal components of the immune network.
  • the synthesis of RF regularly accompanies all primary immune responses and is usually transient.
  • Rheumatoid factor is recognized to interfere in all immunoassay procedures using particulate carriers, such as: latex, sheep cells, bentonite, and charcoal particles.
  • particulate carriers such as: latex, sheep cells, bentonite, and charcoal particles.
  • particles are coated physically or chemically with human and/or animal gamma globulin (immunoglobulins).
  • gamma globulins human and/or animal gamma globulins
  • the gamma globulin in these tests are obtained commercially by chemical fractionation of human or animal sera.
  • polyethylene glycol is used to increase the sensitivity of the tests and can lead to sslf-aggregation of immunoglobulins.
  • Complexes may be formed between RF and these immunoglobulins producing false positive results.
  • the antibody used in general immunoassay procedures for the detection or quantitation of analytes are either polyclonal or monoclonal.
  • the polyclonal are prepared by in vivo immunization while the monoclonal antibody can be prepared in tissue cultures or by ascites formation in syngeneic mice.
  • Monoclonal antibodies appear to have excellent affinity and specificity for antigens. They are commonly used in many immunoassay proce ⁇ ures such as in immunology, chemistry, hematology and in microbiology.
  • Polyclonal antibody which are produced in animals in response to bacteria, fungi, parasites and viruses may react with RF.
  • Rheumatoid factor is known to react with gamma globulins of rabbits, sheep, gcrts, horses, cows, as well as mice and produce false positive reactions.
  • Monoclonal antibodies produced in mice have been shown to react with rheumatoid factors. Patients receiving mouse antibody therapy mount an immune response against the Fab and Fc region of murine IgG. Preexisting antimouse immunoglobulin antibodies have been detected in sera of healthy individuals and patients with various disease.
  • the antibody which is added for the detection of the ligand may crosslink with RF and produce a false positive result because of an elevated response signal. Similar false positive results are obtained in most other immunoassays included radioimmunoassays.
  • RF can erroneously enhance immune complex formation; it is known that the complement can solubilize antigen antibody complexes by preventing the formation of large, insoluble lattices, or it may enhance the immune complex formation.
  • RF has multiple binding activity for the Fc portion of immunoglobulin and the complement may bind also to the same Fc fragment. Through steric hindrance RF may interfere with the binding of complement in these assays.
  • an autoantibody in a patients serum binds to the cell surface molecule and RF is present, it can cross-link the auto antibody with a labeled detection antibody.
  • the non competitive, two site, solid phase immunoassay appears to be most susceptible to RF interference because it uses two antibodies, especially when human immunoglobulin are being measured.
  • RF interference is especially problematic in assays used to assess human sera for the presence of autoantibodies specific to selective tissue (e.g. skin, thyroid) when these patients also have circulating RF.
  • RF can form stable complexes with IgG if either reactant is in a high enough concentration, or if these reactants produce sufficient multiple sites of reactivity which shift the equilibrium from unbound reactant toward a stable product (e.g. due to IgG aggregation and/or immune complex formation and IgM RF multivalency.
  • the subcomponent Cl of the immunomolecular complex of the complement Cl binds to IgG, IgM and to antigen antibody complexes.
  • Rheumatoid factor binds also to the same immune globulin and complexes. Binding sites are located on the same Fc portion of the immunoglobulin.
  • Latex particles sensitized with human gamma globulin are used for adsorbing serum specimens for RF. This procedure may also inhibit the AgAb reaction, IgG added to the latex particles may elute from the particles and inhibit the AgAb reaction (crossreacting or sterically hindrance) . IgG may be eluted from the surface of latex particles which have gamma globulins covalently bound to the surface.
  • Antibodies are produced for use in the immunoassay procedures which are obtained from a species that is immunologically distinct from human species (for example, chicken gamma globulin does not react with RF) .
  • Heating sera at 56°C or 62°C may produce
  • Blocking tests may be used to detect nonspecific positive reactions.
  • the positive test sample is retested after incubation with specific and nonspecific sera or immunoglobulin fractions.
  • a large number of blocking substances have been used in the tests for detection of antigen by an immunoassay procedure.
  • the purpose of the blocker step is to fill any protein adsorbing sites on the solid phase which are not already occupied so as to prevent nonspecific adsorption of the solid phase and preventing false positive results.
  • bovine albumin, or normal human serum, or normal rabbit serum, or fetal calf serum are added to bring the C ⁇ and C to the solid phase component.
  • C r and C x crosslink immunoglobulins, producing AgAb complexes aggregating AgAb or solubilizing AgAb complex, and thus produce false positive reactions.
  • RF containing immuno ⁇ globulin fraction of serum can be separated from the remainder of the serum to permit their analysis in assays free from RF interference.
  • IgM is separated from IgG to allow the separate analysis in an IgG or IgM assay.
  • Mini exchange e.g. di and quaternary aminoethyl Sephadex
  • This approach may not be successful at all times.
  • a single step absorption with Protein A Sepharose and IgG saturated protein A-Sepharose may be used. In this procedure absorption would remove 50-90% of both IgM RF and total IgG.
  • RF are present in all five class of immunoglobulin IgM, IgA, IgG, IgD and IgE, but 90-98% in the IgM class.
  • IgM antibodies are split into smaller units by breaking their disulfide bonds with sulphydryl reagents such as mercaptoethanol, dithioerythrol or cysteine.
  • sulphydryl reagents such as mercaptoethanol, dithioerythrol or cysteine.
  • the disulfide bonds joining the two heavy chains of the IgG or of the IgM are reduced to - SH groups and these two halves of the IgG are. no longer covalently bond.
  • This procedure in immunology is also associated with heating sera at 56°C or 60°c It is well recognized that heating will produce aggregation of immunoglobulin.
  • RF binds strongly to aggregated gamma globulin and in the same time the C ⁇ component of the human serum will also bind to aggregate gamma globulin.
  • the invention provides a method for the prevention of the interfering effects of C in a body fluid.
  • the invention in its broadest sense comprises a method for the prevention of the interfering effect of C ⁇ and C ⁇ from a body fluid in immunoassays.
  • the method comprises the steps of:
  • the following steps comprise a preferred method:
  • a 0.25ml of the sample which is to be tested in an immunoassays procedure is diluted 1:10 in glycine buffer, pH 7.8-8.2, ionic strength 0.025-0.0125. Then, 20 ⁇ l of a solution of a neutral salt (2M NaCl or 2M KC1 or 0.1M EDTA-Na) is added to the diluted body fluid is incubated at 63°C for 3 minutes. If the sample is to assayed for RF, the preferred temperature is 60°C for 30 minutes.
  • a neutral salt 2M NaCl or 2M KC1 or 0.1M EDTA-Na
  • the invention also includes the screening method which uses latex particles to determine if a sample of a body fluid, i.e. sera, synovial fluid, pleural fluid or ascitic fluid contains C ⁇ or RF using a sequence of steps which are based on the reactivity of C ⁇ and RF with latex particles and the use of heat in the presence of a suitable chemical compound, as herein defined, to inactivate C ⁇ .
  • a screening method has also been devised for the demonstration of the presence of by the Ouchterlony technique has been devised.
  • the invention further includes the method for the detection and semiquantitive determination of RF in human sera and fluid by the use of heat and a suitable chemical compound which is used to prevent interference caused by C ⁇ to make the latex test specific for RF.
  • the invention also provides a novel apparatus for automatically carrying out latex agglutination tests without interference from C,iq or RF.
  • FIG. 1 is a graph which illustrates a plot from an X-Y recorder of a photometer which shows both a baseline (negative curve) of a negative turbidometric test and a positive curve (slope >0) of a positive turbidometric test.
  • the dashed line marked "tangent to maximum” is the line drawn with a tangent meter to measure the maximum slope as shown by the superimposed right triangle marked "measurement of maximum slope”.
  • FIG. 2 is a flow chart which shows the sequence of steps for the screening method of the invention for determining if a body fluid is RF positive or contains
  • FIG. 3 is a schematic of an apparatus according to the invention.
  • FIG. 4 is a graphical representative of the test results of Example 12B.
  • body fluid includes cerebrospinal fluid, synovial, ascitic fluid, pleural fluid, blood and the like, urine and serum.
  • body fluids When body fluids are tested, the interferring effect of C ⁇ and C ⁇ in an immunoassay may be prevented by the methods set forth herein.
  • the preferred detection method for use in demonstrating the operability of the invention is the photometric latex agglutination technique.
  • the photometric latex agglutination technique is a simple, rapid, qualitative and quantitative immunological assay. It can be performed in minutes with small quantities of reagents. The sensitivity can be adjusted over a wide range and it can quantitate as little as 10 nanograms of antigen and antibody.
  • a photometer which may be used for latex agglutination is a Model 500 (Chronolog Corp.) used for blood Aggregometry. The instrument is provided with temperature controls and the heater block is controlled to 37°C. It has a stirring motor with a stirring speed at 1200 rpm. The reaction between the latex antigen and antibody mixture is performed in a single tube with continuous stirring and then measured by monochromatic light transmitted through the suspension which is undergoing continual flocculation.
  • Flocculation is measured by monitoring the changes in transmission as a function of time using a single channel recorder connected with the aggregometer.
  • the difference in light transmission is expressed as a voltage and recorded as in function of time.
  • the ensuing flocculation is evidenced by a change in transmission (T) with time (t) .
  • the variation of % transmission with time is a measure of the rate of changes of particle size during flocculation.
  • the increase in transmission is a result of the 0.8 ⁇ size latex particle colliding during AgAg reaction and forming larger and fewer aggregates of latex particle up to 5-6 ⁇ in diameters. When it reaches the maximum size, the sigmoidal flocculation curve become asymptotic.
  • the point in flocculation when the particle size changes most rapidly is represented by the slope of the percent transmission curve. This slope is measured with a tangent meter within a few minutes after the latex suspension is added. In the use of negative fluid where no antigen antibody reaction takes place, the transmission versus the time will result in a straight line. The maximum slope value is detected in less than 3 minutes.
  • the sera are diluted and each aliquot is run in the photometer. A family of curves for serum samples of different dilutions is then obtained and the slopes are calculated.
  • a standard curve is generated by plotting the maximum slope response of the various dilutions of the fluid containing a certain amount of antigen or antibody. The curves . for the unknown samples are compared to the standard curve and the q_ -itive estimation is based on the comparison.
  • a typical base line or negative curve which is actually a substantially straight line is shown by the horizontal dotted line and a positive "curve" is shown in FIG. 1.
  • the dotted line is a line which is drawn on a tangent to the sigmoid shaped curve at a point ,n the curve where the slope is at a maximum as recorded by the X-Y plotter.
  • the maximum slope is the region where the rate of change of the particle size of the flocculating particles is the greatest. It is possible to predict from the standard curve the results for any dilution of a body fluid without testing the unknown body fluid at several dilutions.
  • the preferred chemical compounds for use in the practice of the invention include the neutral salts of the Hofmeister series, organic acid salts or diamino compounds. Any of these compounds may be used in the practice of the invention provided that when they are added to a 1:10 dilution of a sample of a body fluid in a glycine buffer pH 7.0-8.5, pH 8.2, and a ionic strength of 0.0125-0.025 ⁇ at a concentration of 40mM to 200mM, and the sample is heated to a temperature of 59-64°C for a sufficient period time, e.g. from 3 to 30 minutes, the interference of C ⁇ or C ⁇ is prevented without affecting the reactivity of the antigen or antibody in said body fluid.
  • neutral salts of the Hofmeister series which inactivate the C ⁇ or C ⁇ when heated to a temperature of 59-64°C for a sufficient period of time may also be utilized.
  • the neutral salts are strong electrolytes serving as a source of ionic strength.
  • reaction with charge residues the reaction with dipolar groups such as pept-ide bond, amino, carboxyl, hydroxyl, primary and tertiary amide, it will alter the free energy difference between folded (associated) and unfolded (dissociated) form of the macromolecule neutral salt exercising striking effects on association and dissociation equilibria.
  • dipolar groups such as pept-ide bond, amino, carboxyl, hydroxyl, primary and tertiary amide
  • Protein interaction is a complex process in which shape, electrostatic potential and hydrophobicity and hydrophilicity of the interacting molecules plays a role. Stability of IgG as well as of all proteins depends greatly on factors such as hydrogen bonding, hydrophobic bonding, van der Waals interaction, electrostatic forces of attraction and repulsion among charged surface and bound water. Therefore, the effect of salts on C ⁇ and immunoglobulins or on any protein will depend on the salt effect on these stabilizing factors.
  • the inorganic and organic salts exert a structural perturbance on protein. They modify the environment of a macromolecule and thus affect the way they operate in aqueous solution through temperature, pH, compete hydrogen bonding, hydrophobic bond effectors.
  • Neutral salts are defined as strong electrolytes which are significantly soluble in water without imparting major charge to in solution pH. The mechanism by which neutral salts or aliphatic acids act on proteins, in association and dissociation of large protein has been postulated. The first mechanism is through reaction with charged residue. Since the charges of the proteins are mostly located at the surface of the macromolecule they should not have a major effect on the residue located inside the macromolecule.
  • the second mechanism is the reaction with dipolar groups in the macromolecule such as the peptide bond, amino, carboxyl and hydroxyl groups, primary, secondary and tertiary amino groups, etc.
  • the third mechanism is the non-polar interaction. These are compounds which are partially by non-polar or hydrophobic in character.
  • the fourth mechanism is organic salts are added to the protein, both intra and intermolecular bonds are broken, the side chains of the molecule are exposed and the water (or the solvent) alters its structure to make it thermodynamically hospitable for the newly exposed non-polar group.
  • the present invention provides the novel methodology which is based on the use of heat and an inorganic or organic anion to produce a change in the configuration of immunoglobulin and bind C without producing a degree of aggregation which will irreversibly bind the C ⁇ to the aggregated immunoglobulin.
  • Ci,q is released from the C,i macromolecular complex; released from the binding site of C with IgG, IgM, IgGIgM and IgGIgG complexes or from the C Jn inhibitor and other binding sites by: a. dilution of serum with low ionic strength buffers such as glycine, borate and phosphate buffers; b. addition of inorganic or organic neutral salts which affect the binding sites of C ⁇ with immunoglobulins or antigen-antibody complex or C ⁇ inhibitor; c. use of heat to produce an unfolding or a conformational change in the molecule of immunoglobulin for a greater capacity of fixing C 1 ; and d.
  • a sample For the detection or quantitation of rheumatoid factor in the latex photometric technique, it is preferred to prepare a sample by adding a chemical compound as hereinabove described to a diluted body fluid and apply heat at a temperature of 60°C for a period f about 30 minutes although higher temperatures, i.e., 63°C for ⁇ :: shorter period of time, i.e., 3 minutes could be utilized. Thereafter, the prepared sample is tested for the presence of RF by contacting the sample with coated or uncoated latex particles.
  • Any agglutination of the latex particle may be rapidly detected visually or in a spectrophotometer.
  • the optical density of the sample increases in direct relation to the amount of RF in the sample.
  • the sample is placed in the spectrophotometer and if a flocculaton curve is observed within three minutes, the sample is positive for RF.
  • the sample which has been qualitatively tested is serially diluted. Each serially diluted sample is tested in the spectrophotometer to obtain a curve until the point when a serially diluted sample does not give a curve.
  • the titer is the dilution of the most dilute sample which yields a curve.
  • a series of reference standard curves in a photometer are prepared using known amounts of purified RF or standardized rheumatoid factor serum which is free of Ci,q and coated latex particles.
  • the reference standard curves are compared to the curve for the test sample to determine the quantity of RF present in the sample.
  • the following latex immunoassays are examples of the tests which may utilize the present invention:
  • Neisseria meningitidis (dif. groups)
  • Amoebiasis Entamoeba histolytica
  • Filariasis (elephantiasis) Hydatid disease (Echinococcus granulosus)
  • Trichinella spiralis Trichomonas vaginalis
  • CMV Cytomegalovirus
  • HCG Human Chorionic Gonadotropin
  • HPL Human Placental Lactogen
  • CEA Carcino-Embryonic Antigen
  • CRP C-reactive Protein
  • MTA Multi-Tumor Antibody
  • Ciq and RF are inactivated.
  • the following technique is recommended for inactivation of RF as well of C,iq in sera or other fluids
  • Fluid is diluted 1/10 in a low ionic strength glycine buffer 0.02 ⁇ (ionic strength) pH 8.2; to 0.25 ml serum dilution is added 10 to 20 ⁇ l microliter 0.1M solution of EDTA-tetra Na, and 2 microliters of a freshly prepared 5M mercaptoethanol solution which fluid is immediately heated at 60°C for 30 minutes.
  • This procedure can be utilized in all tests for the detection of bacteria, viruses or parasites, which do not contain SS bonds. It can be used for tests or kits for:
  • Drug assay amikacin, barbiturate, gentamycin, morphine,
  • the preferred chemical compounds for use in the practice of the invention include the neutral salts of the Hofmeister series, organic acid salts or diamino compounds. Any of these compounds may be used in the practice of the invention provided that when they are added to a 1:10 dilution of a sample of a body fluid in a 1/8 diluted glycine buffer pH 8.2, at a concentration of 40mM to 200mM, and the sample is heated to a temperature of 59-62°C for a sufficient period time, e.g. 30 minutes, the C 1 or C ⁇ are inactivated without affecting the reactivity of the antigens or antibody in said body fluid.
  • neutral salts which are used in this invention function as an activator to unmask and release the Ci,q component of the C ⁇ complex (the main nonspecific component agglutinator in latex system) .
  • the RF is to be inactivated, it is necessary to use the particular compounds of the formula R-SH include mercaptoethanol, cysteine, dithioerythritol thioglycolic acid and thioalcohols, lower alkyl (C ⁇ _ 5 ) mercaptans and the like. These compounds will react with proteins according to the following reaction mechanism:
  • RSH compounds may be used which also inactivate RF using the procedures described herein.
  • the amounts of RSH compounds are not critical and from 25mM to 50mM and preferably about 30mM (in the final dilution) may be used.
  • the sample is heated to a temperature of about 59-62°C for a sufficient period of time to inactivate the C ⁇ and RF. Usually a period of 30 minutes is effective.
  • Latex photometric rate reaction as a representative of particulate immunoassay techniques.
  • Immunoprecipitation is the simplest and most direct means of demonstrating antigen antibody reaction in the laboratory and can be applied to all other immunoassay procedures. Since the present invention was developed using latex photometry, this procedure will be described first: A. LATEX PHOTOMETRIC RATE REACTION TECHNIQUES
  • Reagents Glycine saline buffer. To 975 ml of 0.1M glycine add 2.5 ml of IN NaOH made up to 1000 ml with distilled water and the pH adjusted to pH 8.2 Ten gram of sodium chloride are then added to each 1000 ml buffer.
  • Latex particles Seragen Diagnostic, Bang Laboratories, Indianapolis, IN., Polystyrene latex 10 percent solid particle size 0.777 u diameter.
  • Human gamma globulin PENTEX 0.5 gr percent solution in glycine saline buffer #1.
  • Latex particles suspension is prepared by adding 25 ⁇ l of latex 10% particles size O . lll ⁇ diameter to 10 ml of glycine buffer #1.
  • Latex human gamma globulin suspension To 10 ml glycine saline buffer #1 add 25 ⁇ l of latex 10% and 50 microliter of human gamma globulin.
  • EDTA tetrasodium 0.1M In the general procedure of the invention, sera or fluids to be tested are diluted in 1:10 glycine buffer # 2, to 0.25 ml of the diluted serum is added 10 ⁇ l of 0.1M EDTA tetrasodium dihydrate and incubated in a temperature controlled bath, at 59°-64°C. for 3-30 minutes. Thereafter, latex particle, coated with IgG or uncoated, are added and tested for the presence of flocculation or aggregation of the latex particles.
  • Example 1 This example describes a screening method to determine if a sample of a body fluid is RF positive or if it contains Ci.q.
  • Sera is diluted to a dilution of 1:10 with glycine buffer #2.
  • the diluted sample is placed in the Chronolog photometer with continuous stirring.
  • To this sample is added 0.25 ml of a suspension of uncoated latex particles. If the recorder of the photometer shows a straight line it indicates that there is no detectable C ⁇ and RF. If a sigmoidal curve is obtained, this indicates the the sample contains C ⁇ or rheumatoid factor.
  • a neutral salt (10 ⁇ l 2M NaCl) is added to the mixture of sera and latex. If a flocculation curve is then obtained in the photometer. this indicates the presence of masked C ⁇ or RF or a mixture of C,iq and RF. If no flocculation curve is obtained, an aliquot of the sera, buffer, and 2M NaCl is prepared as described above. That mixture is heated at 56°C for 30 minutes and uncoated latex is added. A flocculation curve should be observed, which indicates the presence of C ⁇ and RF.
  • Example 2 This example demonstrates that the C ⁇ subcomponent of the C will agglutinate sera and is inactivated by 63°C for 3 minutes in the presence of salt.
  • a commercial preparation of a C ⁇ depleted human serum is tested with a latex IgG preparation in the photometer. No agglutination of latex particles take place when 0.25 ml of serum diluted 1:10 with glycine buffer #2 pH 8.2 is added to the latex IgG suspension. If 0.25 ml serum which is depleted of C ⁇ , is combined with 15 ⁇ l of purified commercial C ⁇ preparation, the serum gives strong positive latex agglutination. Diluted sera was then heated at 63°C, for 3 minutes in the presence of 15 ⁇ l of 2M NaCl and when tested with latex coated with IgG, no agglutination was detected.
  • COMPARATIVE EXAMPLE 1 This example shows that IgG agglutinate with various purified complement components and with Rheumatoid factor with added C ⁇ or added negative serum when the sample is not treated with heat at 59-64°C for a sufficient period of time.
  • Latex particles coated with HgG or uncoated can be used to detect the presence of Ci n q.
  • Latex particles uncoated or latex particles coated with human gamma globulin were added to purified C ⁇ from Diamedix Corp., Sigma Lab, Behring, Atlantic Lab each in concentrations of 0.5 mg of C ⁇ /ml. All of these preparations agglutinated latex particles that were coated or uncoated with gamma globulin.
  • Example 4 This Example demonstrates the effect of various concentrations of the neutral salts on normal sera hich is heated at 60°C for 30 minutes.
  • Example 5 This example reports the testing of 100 normal donor sera by latex photometry.
  • the sera should not be treated at a temperature of 63°+l°C without added salt because if a sample has a high level of gamma globulin i.e., more than 18 mg/ml, the high level of gamma globulin may cause aggregation.
  • Example 6 This example shows the effect of salt and heat in RF containing sera.
  • Fifty rheumatoid sera were diluted in glycine buffer #1 to a dilution of 1:10 and tested unheated, heated at 56°C for 30 minutes and with 15 ⁇ l EDTA-Na added and heated at 60°C for 30 minutes with EDTA-Na added. All these sera were positive by the slide test as well as by the latex IgG tube technique.
  • temperatures of 60°C for 30 minutes should be used for preventing the agglutination of C ⁇ in latex IgG particles.
  • Example 7 Serum was obtained from a patient suffering from a Cryptococcus neoformans infection.
  • the blood culture was positive for Cryptococcus neoformans.
  • the serum had a titer of 1:60 when tested by the slide latex agglutination test (Meridian Diagnostics).
  • the patients serum was serially diluted with glycine buffer #2 and a titer of 1:120 was detected using the Meridian Diagnostics latex antibody test.
  • Example 8 A 30 ⁇ l sample of serum depleted of C is modified by adding 30 ⁇ l of purified C lq and Cryptococcus polysaccharide (0.1 mg/ml) to form a test mixture. One aliquot of the mixture is tested on uncoated latex and latex coated with Cryptococcus antibody. A second aliquot is heated at 63°C for 3 minutes prior to testing on the uncoated latex and the Cryptococcus latex. The results are as follows:
  • Example 9 The invention also includes a novel apparatus for automatically carrying out immunoassays in which it is desired to inactivate Ci, or C,iq in a sample of body fluid.
  • the apparatus may be used for: 1. Screening methodology for confirming that C ⁇ has been inactivated.
  • FIG. 3 shows a schematic diagram of an apparatus of the invention.
  • the basic structure of the invention comprises an irradiation unit comprising a light source 1; a filter or prism 2; a sample cell 3 for holding a sample of body fluid; an electronically controlled variable electric heater block 4 in proximity to cell 4 which is adapted to hold a test sample tube.
  • the electronic control on the heater block 4 is capable of providing temperatures which will heat the sample in sample cell 3 to a temperature in the range of about 59-64°C for periods of time ranging from 3 minutes to 30 minutes or longer.
  • the sample cell 3 has a number of associated reagent feed lines 6, 7 and 8 which are provided with automatic metering valves which are electronically controlled to deliver the required amounts of dispersed latex particles, chemical solution, and/or antigen or antibody solution, and/or mercaptoethanol solution.
  • line 6 is provided for delivering the dispersion of latex particles
  • line 7 is provided for the delivery of a neutral salt solution such as 2M NaCl
  • line 8 is provided for delivery of mercaptoethanol solution for immunoassays in which it desired to eliminate RF from the sample of body fluid.
  • Reference cell 3A with light source IA is provided for holding a control sample for compensation.
  • Heater 4A and lines 6A, 7A and 8A are provided in the event it is desired to run a parallel control.
  • Photocells 12 and 14 are placed in such a way that they will sense the light transmitted through the cells 3 and 3A.
  • An amplifier 16 is used to amplify the signals from the photocells 12 and 14.
  • the signals are sent to interface 18 and are processed in a computer for display on a CRT and/or printer which are not shown in FIG. 3.
  • Cells 3 and 3A are preferably equipped with conventional magnetic stirrers which are utilized to keep the components of the test cell in suspension.
  • the novel apparatus of the invention may be operated as follows: an analyte such as sera (diluted with glycine buffer #2-1:10), which is to be tested for Cryptococcus neoformans is placed in the cell 3 and lOul of a 2M solution of KC1 in water is metered into cell 3 through line 6. Heater block 4 is energized and the temperature of the cell is raised to 63°C for three minutes to inactivate the Ci, and C,iq.
  • an analyte such as sera (diluted with glycine buffer #2-1:10), which is to be tested for Cryptococcus neoformans is placed in the cell 3 and lOul of a 2M solution of KC1 in water is metered into cell 3 through line 6.
  • Heater block 4 is energized and the temperature of the cell is raised to 63°C for three minutes to inactivate the Ci, and C,iq.
  • the apparatus of the invention may possess the basic structure of prior art photometric devices such as the Chronolog Model 570VS for performing optical aggregation tests.
  • This apparatus may be modified to include a variable heater block means and associated timer means as well as reagent feed means which have been described herein. Suitable photometers which may be modified to embody the invention are described in United States Letters Patent No. 4,118,192, which is incorporated herein by reference or photometers which are commercially available such as * the LA-2000 which is manufactured by Eiken Chemical Co.
  • the antibody or the antigen are applied to the center of the well and antigen or antibody are applied to 5 wells situated at the periphery of the plate.
  • To each well is applied lO ⁇ l of the material to be tested and lO ⁇ l of antisera.
  • the presence or absence of a precipitin line between antigen and antibody is read after the plates are kept at room temperature or in a refrigerator for 24 to 48 hours.
  • Example 10 This examples is a method for the demonstration of the presence of C ⁇ in sera and the use. The Methodology utilized for preparing the latex particles has been described in PROCEDURE A. Material: Ouchterlony diffusion on plate. In each of 6 tubes was:
  • the Cl,r inhibitor had 10 International Units
  • the invention is illustrated by Run No. 3 which .shows that in the presence of a neutral salt, the C in serum is thermolabile at a temperature of 63°C for 3 minutes while it is not thermolabile at a temperature of 56°C for 30 minutes in the presence of a neutral salt.
  • Example 12A To a 0.75 ml of a solution of human gamma globulin, 0.50 percent solution of 0.25 ml of glycine buffer No. 2 is added to six test tubes. To each of the tubes are added:
  • Control + 25 ⁇ l of buffer no. 2;
  • Example 13 The procedure of run No. 2 of Example 12A was repeated using various salts and the turbidometric readings are reported in FIG. 4. The results show that these salts substantially prevented the formation of aggregates by heat Example 13
  • each containing 1 ml of glycine buffer (pH 8.2) are added 20 ⁇ l of two different preparations of purified IgM RF (obtained from Dr. Blass Frangione, NYU Medical School and Dr. Ralph Heimer, Jefferson Medical College) .
  • the samples were serially diluted up to 1:8000 with glycine buffer and tested by the latex fixation tube method of Singer and Plotz for detection of RF. Titers of 1:2000 and 1:4000 were obtained respectively.
  • this experiment was repeated with the modification that 2 ⁇ l of a 5M 2-mercaptoethanol solution were added to each test tube and heat was applied (60°C for 30 minutes), both preparations yielded negative results. This shows that the combination of mercaptoethanol and heat at 60°C for 30 minutes destroys the RF.
  • Example 14 Two sera from patients with Cryptococcal meningitis were tested by the latex agglutination test of (CALAS) Meridian Diagnostic Inc. Latex particles in these kits are coated with an anticryptococcal globulin. One sera was negative for RF and the other sera had a titer of 1:60 for RF in the latex gamma globulin test of Singer and Plotz. The technique utilized in the kit should eliminate from the fluid tested the presence of nonspecific agglutinator, the RF.
  • One control is a 25 ⁇ l of a 1 mg C (purified C ⁇ ) which is added to a 0.25 ⁇ l of a Glycine buffer solution; and a second control is a dilution of 1:50 of a RF serum obtained from patient suffering from active rheumatoid arthritis.
  • Sera is serially diluted with 0.1M glycine buffer pH 8.2 and tested with Cryptococcal antibody, (latex kit of the Meridian Diagnostic Inc.). Both sera, as well the as the control were tested with the same reagents. Both sera gave agglutination titers of 1:256 and 1:512 respectively. The titers were similar with that obtained by the original procedure of Meridian Diagnostic; both controls were negative demonstrating that C ⁇ and RF are eliminated from this sera using the technique of the present invention.
  • Patient sera are diluted 1:10 with a glycine buffer diluted 1:8 with distilled water.
  • a glycine buffer diluted 1:8 with distilled water.
  • the controls were a 1/50 dilution of serum containing RF obtained, from sera of patients suffering from RA and glycire buffer (pH 8.2) containing lmg of a C purified preparation (Sigma Co.).
  • the invention also includes kits for the carrying out of an immunoassay.
  • kits of the invention may comprise the components of any conventionally available kit and (a) a chemical agent which when added to a sample of body fluid is capable of inactivating the C ⁇ or C ⁇ in said body fluid when a mixture of said body fluid and said chemical compound are heated to a temperature of 59- 62°C for a sufficient period of time to inactivate said C. and C without affecting the reactivity of an antigen or antibody in said body fluid and (b) a solution containing C ⁇ .
  • kits of the invention may comprise the components of any conventionally available kit and (a) a chemical agent which when added to a sample of body fluid is capable of inactivating the C ⁇ or C ⁇ in said body fluid when a mixture of said body fluid and said chemical compound are heated to a temperature of 59- 64°C for a sufficient period of time to inactivate said C ⁇ and C ⁇ without affecting the reactivity of an antigen or antibody in said body fluid and (b) a solution containing C ⁇ .
  • kits will contain reagents used in the respective immunoassay procedures:
  • the buffer to contain 0.1% sodium azide as a preservative.

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Abstract

L'invention concerne une nouvelle technique de prévention de fausses réactions positives dans les essais immunologiques dues à l'interférence de C1 et C1q. Ce procédé consiste à chauffer un échantillon de fluide du corps à une température de 59-64 °C en présence d'un sel neutre particulier. Un procédé de dépistage et d'inactivation du facteur rhumatoïde est également décrit.
PCT/US1993/002901 1992-03-26 1993-03-26 Technique de prevention de fausses reactions positives dans les essais immunologiques WO1993019369A1 (fr)

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JP5516856A JPH07500190A (ja) 1992-03-26 1993-03-26 免疫試験中の誤反応を防止する技術

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US1454993A 1993-02-08 1993-02-08
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4062935A (en) * 1974-05-20 1977-12-13 Technicon Instruments Corporation Immunoassay involving the binding of RF to the antigen-antibody complex
US4379085A (en) * 1982-05-14 1983-04-05 American National Red Cross Heat stabilization of plasma proteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4062935A (en) * 1974-05-20 1977-12-13 Technicon Instruments Corporation Immunoassay involving the binding of RF to the antigen-antibody complex
US4379085A (en) * 1982-05-14 1983-04-05 American National Red Cross Heat stabilization of plasma proteins

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Journal of Immunological Methods, Volume 121, issued 1989, O.P. BORMER, "Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal antibodies", pages 85-93, see especially pages 87, 89, 92. *
Journal of Immunological Methods, Volume 73, issued 1984, G. VANHAM et al., "Influence of serum complement and rheumatoid factor on detection of immune complexes by the clq and monoclonal rheumatoid factor solid-phase assay", pages 301-311, see especially pages 301-303, 310. *
Journal of Laboratory Clinical Medicine, Volume 92, Number 4, issued October 1978, J.E. RUTSTEIN et al., "Rheumatoid factor interference with the latex agglutination test for fibrin degradation products", pages 529-535, see especially pages 530-531. *
N.R. ROSE et al., "Manual of Clinical Laboratory Immunology", published 1986 by American Society for Microbiology (Washington, D.C.), pages 204-210, see especially page 208. *
See also references of EP0586693A4 *

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CA2110159A1 (fr) 1993-09-30

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