CA1202235A - Immunoassay for complement fragments - Google Patents
Immunoassay for complement fragmentsInfo
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- CA1202235A CA1202235A CA000435635A CA435635A CA1202235A CA 1202235 A CA1202235 A CA 1202235A CA 000435635 A CA000435635 A CA 000435635A CA 435635 A CA435635 A CA 435635A CA 1202235 A CA1202235 A CA 1202235A
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Abstract
ABSTRACT
A method for removing complement components C3, C4 and C5 from samples of biological fluids and recovering from the fluids complement fragments C3a, C4a and C5a or the des-Arg derivatives thereof, which com-prises: combining equal volumes of the biological fluid sample and a buffered solution of 0.8 to 1.6% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflavine hydrochloride and aminacrine; incubating the mixture for about one minute to 2 hours at about 25°C; and recovering the supernatant, containing the complement fragments, from the resultant precipitate. Also a method for assaying the complement fragments, which comprises: incubating the supernatant with a known amount of a labeled complement fragment C3a, C4a or C5a, or the des-Arg derivative thereof and a known amount of antibody which recognizes the complement fragment or the des-Arg derivative thereof; separating the free labeled complement fragment from the bound labeled complement fragment; measuring the amount of labeled complement fragment in either the free or antibody bound complement component; and determining the concen-tration of complement fragment or the des-Arg derivative thereof in the biological sample by comparison to a standard curve. A kit for effecting the above processes is also disclosed.
A method for removing complement components C3, C4 and C5 from samples of biological fluids and recovering from the fluids complement fragments C3a, C4a and C5a or the des-Arg derivatives thereof, which com-prises: combining equal volumes of the biological fluid sample and a buffered solution of 0.8 to 1.6% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflavine hydrochloride and aminacrine; incubating the mixture for about one minute to 2 hours at about 25°C; and recovering the supernatant, containing the complement fragments, from the resultant precipitate. Also a method for assaying the complement fragments, which comprises: incubating the supernatant with a known amount of a labeled complement fragment C3a, C4a or C5a, or the des-Arg derivative thereof and a known amount of antibody which recognizes the complement fragment or the des-Arg derivative thereof; separating the free labeled complement fragment from the bound labeled complement fragment; measuring the amount of labeled complement fragment in either the free or antibody bound complement component; and determining the concen-tration of complement fragment or the des-Arg derivative thereof in the biological sample by comparison to a standard curve. A kit for effecting the above processes is also disclosed.
Description
-1- 4022.1 FIELD OF INYENTION
_ ~ The present invention provides an improved method for assaying complement fragments C3a, C4a and C5a or the des-Arg derivatives thereof and a mercantile kit useful in performing said immunoassay.
The complement system of humans and other mammals involves more than 20 components which participate in an orderly sequence of reac tions resulting in complement acti~ation, Numerous studies indicate that the complement system is a fundamental element of normal host defense mechanisms. As a consequence, complement activation is com-monly associated with a variety of pathological states such as certain malignancies, myocardial infarction, systemic lupus erythematosis, and adult respiratory distress syndrome. Because of these correlations clinical laboratory methods that detect complement activation are use-ful in diagnosing certain disease conditions.
Complement activation can occur by either of two primary modes known as the "classical" pathway and the "alternative" pa~hway, respectively. These different pathways are generally distinguished according to the process which inîtiates complement activation. Acti-vation v~a the classical pathway is usually associated with an immuno-logic stimulus whereas activation via the alternative pathway is mo~t commonly associated with non-immunoloyic stimuli. Regardless of the initiating stimulus both pathways converge, followed by the conversion of the C3 component of complement into its C3a and C3b fragments.
This cleavage of C3 into its subcomponents is considered to be one of the significant events signalling activation of the alternate comple-ment cascade. Following the conversion of C3, a Cs convertase enzyme complex is formed.- This enzyme cleaves the Cs component to yield the fragments Csa and Csb. Complement activation by the classical pathway mechanism îs uniquely characterized by the fact that this route leads to the conversion of the C4 component to its fragments C4a and C4b.
The physicochemical and physiological properties of the cleavage products C3a, C4a and C5a, termed anaphylatoxins are well known. Each is a potent bioactive polypeptlde and plays a key role as a mediaeor ~2~9~2~
-Z- 4n~20i of acute inflammatory processes~ hmong these three anaphylatoxins Csa alone is uniquely characterized by its abil~ty to interact with white blood cells. Both C3a and C4a are rendered inactive in vivo by con-version to their respective des arginine derivatives (C3ades Arg or C3aj, C4adeS Arg or C4aj) by a serum carboxypeptidase. Human Csa, on the other hand, is converted to C5ades Arg by this serum carboxypepti-dase only after all available white blood cell binding sites for C5a have been saturated.
Conversion of the human complement components C3 and C5 to yield their respective anaphy1atoxin products has been implicated in certain naturally occurring pathologic statles including: autoimmune disorders such as systemic lupus erythematosis, rheumatoid arthritis, malig-nancy, myocardial in~arction, Purtscher's rekinopathy, and adult respiratory distress syndrome. In addition, increased circulating 7evels of C3a and C5a have been detected in certain conditions associ-ated with iatrogenic complement activation such as: cardiopulmonary bypass surgery, renal dialysis, and nylon Fiber leukaphoresis. Ele-vated levels of C4a anaphylatoxin are commonly associated with the autoimmune disorders mentioned above. Therefore9 the ability to quan-titatively measure the circulating levels of these anaphylatoxins ortheir des-Arg derivatives is of great utility in diagnosing a variety of important pathological conditions. Additionally, the ability to measure levels of C4a or C~ades Ar9 enables one to determine the path-way by which complement activation occursO This facility enables one not only to determine the precise mechanism of complement activation but also whether a patient's natural immunological defense mechanisms are functional~
Until the development of the radioimmunoassay (RIA) method of Tony E. Hugli and Dennis E. Chenoweth reported in ''Immunoassays: Clin-ical Laboratory Techniques for the 1980s," 443-460, Alan R. Liss, lnc., New York, NY (198D), measurement of the anaphylatoxins C3a, C4a and C5a or their des-Arg derivatives had only been achieved when the levels of these factors were relatively elevated, for example, when the disease process had reached an advanced stageO The RI~ techniques of Hugli and Chenoweth permit quantitative measurement of trace amounts of the anaphylatoxins or their des-Arg derivatives and hence provide a 5ensitive diagnostic tool. However, the means known hereto-fore for measuring these factors have been frought with a significant 1%~2;23~
~ 4022.1 problem associated with the requirement that the C3, C4 and C5 plasma precursors of the anaphylatoxins must be removed from the biological fluid to be tested. This stringent requirement is predicated on the observation that the anti~odies raised to the anaphylatoxins possess a significant cross-reactivity with their respective plasnla precursor as C33, C4a or C5a is a part of the parent molecule C3, C4 and C5, respectively. Because of this unavoidable cross-reactivity it is imperative that the precursor which exists in relatively high concen-trations in serum and plasma be completely removed to avoid detecting artifactually elevated levels of the anaphylatoxins which are normally present in only trace amounts. Prior known methods of separating the anaphylatoxins from their plasma precursors involves diluting the plasma with sodium chloride and acidifying with hydrochloric acid and subsequently neutralizing the recovered serurn sample. See Hugli and Chenoweth cited above. The present invention prov~des a novel and simplified means of quantitatively removing the plasma precursor of the anaphylatoxin from the biological samples yet simultaneously permitting a quantitative recovery of the low molecular weight anà~
phylatoxins, C3a, C4a and C5a or the des-Arg derivat~ves thereof.
SUMMARY OF INVENTION
~ . .
The present invention provides a novel method for removing sub-stantially all traces of complement precursors or components 03, C4 and C~ from samples o~ biological fluids and recovering from said fluids complement fragments C3a, C4a and C5a or the des-Arg derivci-tives thereof without interfering with the immunogenicity of saidfragments which comprises combining equal volumes of the biological fluid sample and a ouffered solution of 0.8 to 1.6~ of an acridine derivative selected from the group consisting of acrinol 9 acriflavine, acriflavine hydrochloride and aminacrine, incubating the mixture for from one minute to 2 hvurs at about 25C and recovering the super-natant from the resultant- precipitate.
~ he present invention also provides a novel and improved method for quantitatively measuring complement fragments C3a, C4a or Csa or the des-Arg derivatives thereof in a biological sample which comprises combining equal volumes of the biological sample and 0.8 to 106% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflavine hydrochloride and aminacrine, incubating ~he mixture for from one minute to 2 hours at about 25~C, recovering the _4_ 4022~1 supernatant from the resultant precipitate~ and incubating the supernatant with a known amount of a labeled complement fragment selected from C3a, C4a or Csa or the des-Arg derivative thereof and a known amount of an antibody which recognizes sald labeled complement fragment or des-Arg derivative thereof, separating the free labeled complement fragment from the bound labeled complement fragment~
measuring the amount of labeled complement fragment in either the free or antibody bound complement component, and determining the concentration of complement fragment in the biological sample by reference to a standard curve.
The present invention also provides a novel kit wherein the com-ponent parts are assembled for use in assaying samples of biological fluids for complement fragments C3a, C4a and Csa or the des-Arg deriv-atives thereof which comprises a ~irst container having therein a buffered solution of 0.8 to 1.6% solution of an acridine derivative selected from the group consisting of acrinol~ acriflavine, acriflav-ine hydrochloride and aminacrine, a second container having therein.a labeled reagent selected from labeled complement fragments C3a, ~4a or Csa or the des-Arg derivatives thereof; and a third container having therein an antibody reagent selected from antibody which recognizes complement fragments C3a, C4a or C5a or the des-Arg derivatives thereof.
DETAILED DESCRIPTION OF INVENTION
.
As used herein acrinol is taken to mean 2-ethoxy-699-diamino-
_ ~ The present invention provides an improved method for assaying complement fragments C3a, C4a and C5a or the des-Arg derivatives thereof and a mercantile kit useful in performing said immunoassay.
The complement system of humans and other mammals involves more than 20 components which participate in an orderly sequence of reac tions resulting in complement acti~ation, Numerous studies indicate that the complement system is a fundamental element of normal host defense mechanisms. As a consequence, complement activation is com-monly associated with a variety of pathological states such as certain malignancies, myocardial infarction, systemic lupus erythematosis, and adult respiratory distress syndrome. Because of these correlations clinical laboratory methods that detect complement activation are use-ful in diagnosing certain disease conditions.
Complement activation can occur by either of two primary modes known as the "classical" pathway and the "alternative" pa~hway, respectively. These different pathways are generally distinguished according to the process which inîtiates complement activation. Acti-vation v~a the classical pathway is usually associated with an immuno-logic stimulus whereas activation via the alternative pathway is mo~t commonly associated with non-immunoloyic stimuli. Regardless of the initiating stimulus both pathways converge, followed by the conversion of the C3 component of complement into its C3a and C3b fragments.
This cleavage of C3 into its subcomponents is considered to be one of the significant events signalling activation of the alternate comple-ment cascade. Following the conversion of C3, a Cs convertase enzyme complex is formed.- This enzyme cleaves the Cs component to yield the fragments Csa and Csb. Complement activation by the classical pathway mechanism îs uniquely characterized by the fact that this route leads to the conversion of the C4 component to its fragments C4a and C4b.
The physicochemical and physiological properties of the cleavage products C3a, C4a and C5a, termed anaphylatoxins are well known. Each is a potent bioactive polypeptlde and plays a key role as a mediaeor ~2~9~2~
-Z- 4n~20i of acute inflammatory processes~ hmong these three anaphylatoxins Csa alone is uniquely characterized by its abil~ty to interact with white blood cells. Both C3a and C4a are rendered inactive in vivo by con-version to their respective des arginine derivatives (C3ades Arg or C3aj, C4adeS Arg or C4aj) by a serum carboxypeptidase. Human Csa, on the other hand, is converted to C5ades Arg by this serum carboxypepti-dase only after all available white blood cell binding sites for C5a have been saturated.
Conversion of the human complement components C3 and C5 to yield their respective anaphy1atoxin products has been implicated in certain naturally occurring pathologic statles including: autoimmune disorders such as systemic lupus erythematosis, rheumatoid arthritis, malig-nancy, myocardial in~arction, Purtscher's rekinopathy, and adult respiratory distress syndrome. In addition, increased circulating 7evels of C3a and C5a have been detected in certain conditions associ-ated with iatrogenic complement activation such as: cardiopulmonary bypass surgery, renal dialysis, and nylon Fiber leukaphoresis. Ele-vated levels of C4a anaphylatoxin are commonly associated with the autoimmune disorders mentioned above. Therefore9 the ability to quan-titatively measure the circulating levels of these anaphylatoxins ortheir des-Arg derivatives is of great utility in diagnosing a variety of important pathological conditions. Additionally, the ability to measure levels of C4a or C~ades Ar9 enables one to determine the path-way by which complement activation occursO This facility enables one not only to determine the precise mechanism of complement activation but also whether a patient's natural immunological defense mechanisms are functional~
Until the development of the radioimmunoassay (RIA) method of Tony E. Hugli and Dennis E. Chenoweth reported in ''Immunoassays: Clin-ical Laboratory Techniques for the 1980s," 443-460, Alan R. Liss, lnc., New York, NY (198D), measurement of the anaphylatoxins C3a, C4a and C5a or their des-Arg derivatives had only been achieved when the levels of these factors were relatively elevated, for example, when the disease process had reached an advanced stageO The RI~ techniques of Hugli and Chenoweth permit quantitative measurement of trace amounts of the anaphylatoxins or their des-Arg derivatives and hence provide a 5ensitive diagnostic tool. However, the means known hereto-fore for measuring these factors have been frought with a significant 1%~2;23~
~ 4022.1 problem associated with the requirement that the C3, C4 and C5 plasma precursors of the anaphylatoxins must be removed from the biological fluid to be tested. This stringent requirement is predicated on the observation that the anti~odies raised to the anaphylatoxins possess a significant cross-reactivity with their respective plasnla precursor as C33, C4a or C5a is a part of the parent molecule C3, C4 and C5, respectively. Because of this unavoidable cross-reactivity it is imperative that the precursor which exists in relatively high concen-trations in serum and plasma be completely removed to avoid detecting artifactually elevated levels of the anaphylatoxins which are normally present in only trace amounts. Prior known methods of separating the anaphylatoxins from their plasma precursors involves diluting the plasma with sodium chloride and acidifying with hydrochloric acid and subsequently neutralizing the recovered serurn sample. See Hugli and Chenoweth cited above. The present invention prov~des a novel and simplified means of quantitatively removing the plasma precursor of the anaphylatoxin from the biological samples yet simultaneously permitting a quantitative recovery of the low molecular weight anà~
phylatoxins, C3a, C4a and C5a or the des-Arg derivat~ves thereof.
SUMMARY OF INVENTION
~ . .
The present invention provides a novel method for removing sub-stantially all traces of complement precursors or components 03, C4 and C~ from samples o~ biological fluids and recovering from said fluids complement fragments C3a, C4a and C5a or the des-Arg derivci-tives thereof without interfering with the immunogenicity of saidfragments which comprises combining equal volumes of the biological fluid sample and a ouffered solution of 0.8 to 1.6~ of an acridine derivative selected from the group consisting of acrinol 9 acriflavine, acriflavine hydrochloride and aminacrine, incubating the mixture for from one minute to 2 hvurs at about 25C and recovering the super-natant from the resultant- precipitate.
~ he present invention also provides a novel and improved method for quantitatively measuring complement fragments C3a, C4a or Csa or the des-Arg derivatives thereof in a biological sample which comprises combining equal volumes of the biological sample and 0.8 to 106% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflavine hydrochloride and aminacrine, incubating ~he mixture for from one minute to 2 hours at about 25~C, recovering the _4_ 4022~1 supernatant from the resultant precipitate~ and incubating the supernatant with a known amount of a labeled complement fragment selected from C3a, C4a or Csa or the des-Arg derivative thereof and a known amount of an antibody which recognizes sald labeled complement fragment or des-Arg derivative thereof, separating the free labeled complement fragment from the bound labeled complement fragment~
measuring the amount of labeled complement fragment in either the free or antibody bound complement component, and determining the concentration of complement fragment in the biological sample by reference to a standard curve.
The present invention also provides a novel kit wherein the com-ponent parts are assembled for use in assaying samples of biological fluids for complement fragments C3a, C4a and Csa or the des-Arg deriv-atives thereof which comprises a ~irst container having therein a buffered solution of 0.8 to 1.6% solution of an acridine derivative selected from the group consisting of acrinol~ acriflavine, acriflav-ine hydrochloride and aminacrine, a second container having therein.a labeled reagent selected from labeled complement fragments C3a, ~4a or Csa or the des-Arg derivatives thereof; and a third container having therein an antibody reagent selected from antibody which recognizes complement fragments C3a, C4a or C5a or the des-Arg derivatives thereof.
DETAILED DESCRIPTION OF INVENTION
.
As used herein acrinol is taken to mean 2-ethoxy-699-diamino-
2~ acridine or the lactate monohydrade salt thereof; aeriflavine means amixture of 3,6-diamino-10-methylacridinium chloride and 3,6-diamino-acridine contairing when dried at 105C for ~wo hnurs not less than 13.3% and not more than 15.8~ of Cl; acriflavine hydrochloride means a mixture of the hydrochlorides of 3,6-diamino-lO-methylacridinium chloride and 3,6-diaminoacridine containing when dried for one hour at 105C not less than 23X and not more than 23.5% of Cl; and aminacrine means 5-aminoacridine .
In practicing the present invention the sample of biological fluid can be any bodily fluid such as, ~or example, serum, plasma, urine or cerebrospinal fluid. When the biological fluid employed is plasma or cerebrospinal fluid EDTA is added thereto.
Equal volumes of the biological fluid and 0.8 to 1.6% of the acri-d;ne derivative are conbined for a final concentration of 0.4 to 0.8~
Z~ 3~i _5_ 4022.1 of said acrid;ne derivative. The preferred flnal concentration of acridine derivative ;s 0.6%. The acrid;ne derivative-protein complex is buffered to a pH of about 7.4. Any buffer which does not contain chloride ;ons is suitab1e, for example~ carbonate buffers, phosphate buffers, or HEPES (N-2-hydroxyethylpiperazineethane sulfonic acid) may be e~ployed. Preferably the molarity of the buffer is between 0.05 to O.lM with a pH of from 6.8 to 7.8. The preferred buffer for use in practicing the present invention is 0.05M phosphate buffer, pH 7.4.
Once the biological fluid and acridine derivative are combined a precipitate forms almost immediately. However, the complex preferably should be permitted to incubate for at least about 20 minutes to effect maximum separatlon of the complement precursors C3, C4 and C5 from their activated fragments. We have found that precipitation of the complement component is complete within about one minute to 2 hours. The complex may be permitted to incubate longer than one hour if necessary, however, no beneficial additional separation of protein is expected to occur beyond one hour. Incubation is carried out àt room temperature, that is, about 25C.
Removal of the precipitate is conveniently accomplished by cen-trifuging the acridine derivative-protein complex dt about 3~000 to 7,000 x 9 for about 1~ tù 20 minutes then decanting the supernatant which contains the complement fragments C3a, C4a and C5a or the des-Arg derivatives thereof. As indicated hereinabvve9 complement fragments C3a, C4a and C5a are rendered inactive in vivo in serum and plasma by carboxypeptidases by conversion to their respective inactive des-Arg derivatives. Swch conversion also occurs in vivo in urine.
Hence, the supernatant recovered from the novel acridine derivative-precipitation method of the present invention will contain primarily the des-Arg derivative of the complement fragments`C3a, C~a and C5a rather than the activated form of said ~ragments when the biological fluid is plasma, serum or urine. For diagnostic purposes measurement of the inactive des-Arg derivative is just as meaningful as the direct measurement of the active forrn of the complement fragments.
The recovered supernatant is used without further treatment or processing in the immunoassay procedure. We have found that the presence of the acridine derivative in the test samples does not interfere with the antigenicity of the complement fragments or the des-Arg derivatives thereof, thus providing a unique and greatly %;i~;~5 -6- ~022.1 simplified means of assaying for sa1d fragments.
The immunoassay procedure is not substantially different from known immunoassays for C3a, C.,a and Csa or the des-Arg derivatives thereof. Equal volumes -of the test sample to be assayed, labeled complement fragment G3a, C4a or Csa or the des-Arg deri~ative thereof, and antibody which recognizes said complement fragment are combined and incubated after which the antibody bound and unbound, or free, labeled complement fragment are separa~ed and measured to determine the amount of complement fragment in the test sample by comparison to standard curves. An assay buffer is generally added to the incubate.
We have found a buffer comprising HEPES, protamine sul~ate, thimerosol and gelatin to be particularly useful.
Purification of the complement fragment C3al C4a or Csa or the des-Arg derivative thereof for use in raising antibody thereto, and for use in generating standard curves as well as for preparing labeled complement fragment can be achieved by the general method described in J. Biol. Chem. 256, 8685-8~9~ (1981). We have found that this proce-dure can be modi~ied an~ improved by substituting the acridine deriva-tive-precipitation technique described hereinabove for the hydrochlor 2C ic acid precipitation technique.
Antibody to complement fragments ~3a, C4a, C5a or the des-Arg derivatives thereof is r~ised as generally described by Hugli, et al., J. Biol. Chem. 250, 1472-1478 (1975;, and in J. Biol. Chem~ 25Ç, 8685-86~2 (1981~.
The label utilized in the labeled complement fragment or the des-Arg derivative thereof can be any substance capable of detection by physical or chemieal means. Radioisotopes such as tritium iodine-131 and iodine-125 are useful with l25I being preferred. The 125I
labeled material can be prepared by various means generally known in the art, such as, the solid-phase lactoperoxidase method. A preferred method employs the use of TCDG (1,3,4,6-tetrachloro-3a,6a-diphenyl-glycouril) in phosphate buffered saline.
The antibody bound complement fragment can be separated from the free or unbound complement fragment by various means commonly employed in immunoassay procedures. For example, this separation can be achieved by treatment with polyethylene glycol ~Desbuquois, 8. and Aurback, G.D., J. Clin. Endocrinol. Metab. 33, 732 (1971)3 or IgG Sorb or by contacting the incubate with a second antibody. The second 2~
_7 4022.1 antibody, which is prepared by standard procedures~ for example, as described in Daughaday, et al~, "Principle of Competitive Protein Binding Assay", J.B~ Llppincott~ Philadelphia (1971~ recognizes the complement fragment-antibody complex contained in the incubate. Use of the second antibody technique is particularly preferred~
Standard curves are derived essentially by performing the above-described assay procedure using known quantities of complement frag-ment C3a, C4a or C5a or the des-Arg ~derivative thereof in plaçe of the test sample. The assay procedure described herein is highly sensi-tive, and we have found it particularly useful to develop standard curves for concentrations of the complement fragments ranging from 1.0 ng to 25 ng.
The following examples further illustrate the invention~ In Examples 3-5 acrinol is used as the precipitating agent and is the preferred acridine derivative of the present invention.
~%~%~
-8- 402~.1 Example 1 Purification o~ complement Fragments or khe des-Arg derivative From 2 to 4 liters of serum was activated at 37DC by addition of boiled yeast (20 mg/ml serum) and allowing the mixture to stand ~or 45 minutes to one hourn Equal volumes of ac-ti~ated serum and 0.8 to 1.6%
acrinol, bu~fered to pH 7.4 with 0.05M phosphate buf~er, were combined and let stand for 30 minutes at 25C a~ter which the mixture was centrifuged and the supernatant was decanted. The supernatant was dialyzed against running water overnight at 5~-8~C then gel filtered 1~ on a p-60 column ~quilibrated with O.lM ammonium formate, pH 500~ lhe fractions containing complernent fragments C3a, C4a or C5a or the des-Arg derivatives thereof were pooled and applied to an SP-Sephadex*col-umn equilibrated with O~lM ammonium formate~ pH 5.0, rhe column was eluted by a linear gradient of 0~1M to 0.8M ammonium ~ormate, pH 7Ø
The fractions of C3a, C4a and 05? or the des Ar~ derivatives thereof were collected and appl-ied individually to separate CM-cellulose col-umns and eluted by a gradient of 0.15M to 0.43M ammonium formate, pH
7.0, at a flow rate of 75 ml/hour. Each of the complement components C3a, C4~ or C5a was pooled with its respective des-Arg deriva~ive recovered ~rom the column and lyophilized then redissolved in water and dialyzed against 1% acetic acid. A final lyophilization and resuspension ir water of about 10 mg/ml provided each fragment ready for use~
Example_2 1251 Complement C3a or the des-Arg deriYative thereof To an incubation tube was added 50 ~g of complement fragment C3a or the des-Arg deri~ative thereof, 150 ~1 of phosphate bu~fered saline, 100 ~9 of TCDG and lmCi (10 ~1~ 125~ sodium. The phosphate buffered saline was prepared as a stock solution comprising 81 my sodium chloride, 39 mg anhydrous sodium biphosphate, 199 mg sodium phosphate dibasic heptahydrate reagent and 10~ ml glass distilled - deionized water. 7he mixture was incubated for 20 minutes at about 25C then transferred to a chromatography column equilibrated with phosphate buffered saline with gelatin which was prepared as a stock solution comprising 143 9~ sodium chloride, 200 mg thimerosol NF? 6 g anhydrous sodium biphosphate~ 34 g sodium phosphate dibasic hepta-hydrate reagent, 17 ~ gelatin and glass distilled deionized water to a volume o~ 17~5 liters. Radioactive fractions of 0~ mt each were collected and diluted to give 20,000 to 60,000 cpmlO.05ml in phosphate * trade mark ~9~ 4022.1 buffered saline with gelatin.
~e~
(a) A mixture of 0.45 ml act1vated plasma, 0.45 ml of 0.8% acri-nol in 0.05M phosphate buffer, pH 7,4, and Ool ml distilled water was reacted at about 25C for 20 minutes then centrifuged at 1700 x 9 for lO minutes. The supernatant was collected. A mixture of 50 ~l of assay buffer prepared as described hereinabove9 50 ~l of supernatant obtained above, ~0 ~l of l25I complement fragment C3a or the des-Arg deriYatiYe ther~of and 50 ~l of complement fragment C3a rabbit anti~
sera was incubated for 30 minutes at about 25C after which 50 ~l of goat anti-rabbit antisera was added and mixed well. The mixture was incubated for an additional 30 minutes at about 25C after which 2 ml of isotonic saline was added. Ihe mixture was cen~rifuged at 2noo g for 10 minutes and the supernatant decanted. The radioactivity of the l~ pellet was 4555 cpm.
(b) The foregoing procedure was performed only the acid-precipi-tation technique of Hugli and Chenoweth described in Immunoassays:
Clinical Laboratory Techniques for the 1980s: 443-460 was substituted fo~ the acrinol precipitation step. The radioactivity of the result-ant pellet was 4521-4757 cpm demonstrating that the acrinol precipita-tion technique removes intact complement protein precursor as effect-ively as treatment with hydrochloric acid followed by base neutraliza-tion providing a greatly improved simplified assay procedure~ Also the results of the two experiments indicate that the presence of acrinol does not affect either the first or the second immunological reactions.
In generating standard curves for complement fragment C3a or the des-Arg derivative thereof using 1.0 ng9 2.5 ng, 5.0 ng~ 10 ng, 25 ng and 50 ng quantities of C3a we found that use of thè acrinol precipi-tation step in place of the acid precipitation-base neutralization step of Hugli and Chenoweth increased the sensitivity of the immuno-assay by a factor of 12 as evidenced by the fact that the range of detection became shorter and the slope %B/Bo (Standard Counts/Bound Standard) was increased from -2.3 to -4Ø
Example 4 The assay as described in 3(a) above was performed using acivated plasma samples to which known quantities of complement factor C3a or the des-Arg derivative thereof ranging from 2 to 20 ng was added. The ~2~
-10- 4022.1 results indicate that there was 100~ of the known C3a concentrations in each of the "spiked" normal sampies assayed.
Example 5 The essential reagents for the performance of the immunoassay of the present invention are assembled into a mercantile unit as a kit.
The kit comprises multiple containers7 such as, bottles or other suit-able containers as follows:
(a) a first container having therein a buffered solution of 0.8 to 1.6% acrinol;
(b3 a second container having therein a labeled reagent selected from I25I labeled complement ~ragments C3a, Clla or Csa or the ~es-Arg deri~atives thereof;
(c) a third container having therein rabbit antisera to the par ticular complement fragment, and which optionally contains the following additional reagents:
(d) a container having therein assay buffer, prepared as described hereinabove;
(e) a container having therein second antibody which binds thP
product of the initial complement fragment-antibody reaction, preferably goat anti-rabbit antisera;
~f) a oontainer having therein complement fragment standard, 25 ng;
(g) a container having therein complement fragment standard, 10 ng;
(h) a container having therein complement fragment standard, 5 ng;
(i) a container having therein complement fragment standard, ?.5 ng;
(j) a container having therein complement fragment standard, 1.0 ng.
In each of (f) thrQugh (j) the complement fragment is either C3a9 C4a or C5a or the des-Arg derivative thereof depending on whioh oom-plement fragment is being assayed.
Although the foregoing examples are limited to the separation and assay of complement fragment C3a or the des-Arg derivative thereof the novel separation and assay procedure of the present invention is equally appliciable to complement fragments C4a and Csa or the des-Arg derivatives thereof.
,,
In practicing the present invention the sample of biological fluid can be any bodily fluid such as, ~or example, serum, plasma, urine or cerebrospinal fluid. When the biological fluid employed is plasma or cerebrospinal fluid EDTA is added thereto.
Equal volumes of the biological fluid and 0.8 to 1.6% of the acri-d;ne derivative are conbined for a final concentration of 0.4 to 0.8~
Z~ 3~i _5_ 4022.1 of said acrid;ne derivative. The preferred flnal concentration of acridine derivative ;s 0.6%. The acrid;ne derivative-protein complex is buffered to a pH of about 7.4. Any buffer which does not contain chloride ;ons is suitab1e, for example~ carbonate buffers, phosphate buffers, or HEPES (N-2-hydroxyethylpiperazineethane sulfonic acid) may be e~ployed. Preferably the molarity of the buffer is between 0.05 to O.lM with a pH of from 6.8 to 7.8. The preferred buffer for use in practicing the present invention is 0.05M phosphate buffer, pH 7.4.
Once the biological fluid and acridine derivative are combined a precipitate forms almost immediately. However, the complex preferably should be permitted to incubate for at least about 20 minutes to effect maximum separatlon of the complement precursors C3, C4 and C5 from their activated fragments. We have found that precipitation of the complement component is complete within about one minute to 2 hours. The complex may be permitted to incubate longer than one hour if necessary, however, no beneficial additional separation of protein is expected to occur beyond one hour. Incubation is carried out àt room temperature, that is, about 25C.
Removal of the precipitate is conveniently accomplished by cen-trifuging the acridine derivative-protein complex dt about 3~000 to 7,000 x 9 for about 1~ tù 20 minutes then decanting the supernatant which contains the complement fragments C3a, C4a and C5a or the des-Arg derivatives thereof. As indicated hereinabvve9 complement fragments C3a, C4a and C5a are rendered inactive in vivo in serum and plasma by carboxypeptidases by conversion to their respective inactive des-Arg derivatives. Swch conversion also occurs in vivo in urine.
Hence, the supernatant recovered from the novel acridine derivative-precipitation method of the present invention will contain primarily the des-Arg derivative of the complement fragments`C3a, C~a and C5a rather than the activated form of said ~ragments when the biological fluid is plasma, serum or urine. For diagnostic purposes measurement of the inactive des-Arg derivative is just as meaningful as the direct measurement of the active forrn of the complement fragments.
The recovered supernatant is used without further treatment or processing in the immunoassay procedure. We have found that the presence of the acridine derivative in the test samples does not interfere with the antigenicity of the complement fragments or the des-Arg derivatives thereof, thus providing a unique and greatly %;i~;~5 -6- ~022.1 simplified means of assaying for sa1d fragments.
The immunoassay procedure is not substantially different from known immunoassays for C3a, C.,a and Csa or the des-Arg derivatives thereof. Equal volumes -of the test sample to be assayed, labeled complement fragment G3a, C4a or Csa or the des-Arg deri~ative thereof, and antibody which recognizes said complement fragment are combined and incubated after which the antibody bound and unbound, or free, labeled complement fragment are separa~ed and measured to determine the amount of complement fragment in the test sample by comparison to standard curves. An assay buffer is generally added to the incubate.
We have found a buffer comprising HEPES, protamine sul~ate, thimerosol and gelatin to be particularly useful.
Purification of the complement fragment C3al C4a or Csa or the des-Arg derivative thereof for use in raising antibody thereto, and for use in generating standard curves as well as for preparing labeled complement fragment can be achieved by the general method described in J. Biol. Chem. 256, 8685-8~9~ (1981). We have found that this proce-dure can be modi~ied an~ improved by substituting the acridine deriva-tive-precipitation technique described hereinabove for the hydrochlor 2C ic acid precipitation technique.
Antibody to complement fragments ~3a, C4a, C5a or the des-Arg derivatives thereof is r~ised as generally described by Hugli, et al., J. Biol. Chem. 250, 1472-1478 (1975;, and in J. Biol. Chem~ 25Ç, 8685-86~2 (1981~.
The label utilized in the labeled complement fragment or the des-Arg derivative thereof can be any substance capable of detection by physical or chemieal means. Radioisotopes such as tritium iodine-131 and iodine-125 are useful with l25I being preferred. The 125I
labeled material can be prepared by various means generally known in the art, such as, the solid-phase lactoperoxidase method. A preferred method employs the use of TCDG (1,3,4,6-tetrachloro-3a,6a-diphenyl-glycouril) in phosphate buffered saline.
The antibody bound complement fragment can be separated from the free or unbound complement fragment by various means commonly employed in immunoassay procedures. For example, this separation can be achieved by treatment with polyethylene glycol ~Desbuquois, 8. and Aurback, G.D., J. Clin. Endocrinol. Metab. 33, 732 (1971)3 or IgG Sorb or by contacting the incubate with a second antibody. The second 2~
_7 4022.1 antibody, which is prepared by standard procedures~ for example, as described in Daughaday, et al~, "Principle of Competitive Protein Binding Assay", J.B~ Llppincott~ Philadelphia (1971~ recognizes the complement fragment-antibody complex contained in the incubate. Use of the second antibody technique is particularly preferred~
Standard curves are derived essentially by performing the above-described assay procedure using known quantities of complement frag-ment C3a, C4a or C5a or the des-Arg ~derivative thereof in plaçe of the test sample. The assay procedure described herein is highly sensi-tive, and we have found it particularly useful to develop standard curves for concentrations of the complement fragments ranging from 1.0 ng to 25 ng.
The following examples further illustrate the invention~ In Examples 3-5 acrinol is used as the precipitating agent and is the preferred acridine derivative of the present invention.
~%~%~
-8- 402~.1 Example 1 Purification o~ complement Fragments or khe des-Arg derivative From 2 to 4 liters of serum was activated at 37DC by addition of boiled yeast (20 mg/ml serum) and allowing the mixture to stand ~or 45 minutes to one hourn Equal volumes of ac-ti~ated serum and 0.8 to 1.6%
acrinol, bu~fered to pH 7.4 with 0.05M phosphate buf~er, were combined and let stand for 30 minutes at 25C a~ter which the mixture was centrifuged and the supernatant was decanted. The supernatant was dialyzed against running water overnight at 5~-8~C then gel filtered 1~ on a p-60 column ~quilibrated with O.lM ammonium formate, pH 500~ lhe fractions containing complernent fragments C3a, C4a or C5a or the des-Arg derivatives thereof were pooled and applied to an SP-Sephadex*col-umn equilibrated with O~lM ammonium formate~ pH 5.0, rhe column was eluted by a linear gradient of 0~1M to 0.8M ammonium ~ormate, pH 7Ø
The fractions of C3a, C4a and 05? or the des Ar~ derivatives thereof were collected and appl-ied individually to separate CM-cellulose col-umns and eluted by a gradient of 0.15M to 0.43M ammonium formate, pH
7.0, at a flow rate of 75 ml/hour. Each of the complement components C3a, C4~ or C5a was pooled with its respective des-Arg deriva~ive recovered ~rom the column and lyophilized then redissolved in water and dialyzed against 1% acetic acid. A final lyophilization and resuspension ir water of about 10 mg/ml provided each fragment ready for use~
Example_2 1251 Complement C3a or the des-Arg deriYative thereof To an incubation tube was added 50 ~g of complement fragment C3a or the des-Arg deri~ative thereof, 150 ~1 of phosphate bu~fered saline, 100 ~9 of TCDG and lmCi (10 ~1~ 125~ sodium. The phosphate buffered saline was prepared as a stock solution comprising 81 my sodium chloride, 39 mg anhydrous sodium biphosphate, 199 mg sodium phosphate dibasic heptahydrate reagent and 10~ ml glass distilled - deionized water. 7he mixture was incubated for 20 minutes at about 25C then transferred to a chromatography column equilibrated with phosphate buffered saline with gelatin which was prepared as a stock solution comprising 143 9~ sodium chloride, 200 mg thimerosol NF? 6 g anhydrous sodium biphosphate~ 34 g sodium phosphate dibasic hepta-hydrate reagent, 17 ~ gelatin and glass distilled deionized water to a volume o~ 17~5 liters. Radioactive fractions of 0~ mt each were collected and diluted to give 20,000 to 60,000 cpmlO.05ml in phosphate * trade mark ~9~ 4022.1 buffered saline with gelatin.
~e~
(a) A mixture of 0.45 ml act1vated plasma, 0.45 ml of 0.8% acri-nol in 0.05M phosphate buffer, pH 7,4, and Ool ml distilled water was reacted at about 25C for 20 minutes then centrifuged at 1700 x 9 for lO minutes. The supernatant was collected. A mixture of 50 ~l of assay buffer prepared as described hereinabove9 50 ~l of supernatant obtained above, ~0 ~l of l25I complement fragment C3a or the des-Arg deriYatiYe ther~of and 50 ~l of complement fragment C3a rabbit anti~
sera was incubated for 30 minutes at about 25C after which 50 ~l of goat anti-rabbit antisera was added and mixed well. The mixture was incubated for an additional 30 minutes at about 25C after which 2 ml of isotonic saline was added. Ihe mixture was cen~rifuged at 2noo g for 10 minutes and the supernatant decanted. The radioactivity of the l~ pellet was 4555 cpm.
(b) The foregoing procedure was performed only the acid-precipi-tation technique of Hugli and Chenoweth described in Immunoassays:
Clinical Laboratory Techniques for the 1980s: 443-460 was substituted fo~ the acrinol precipitation step. The radioactivity of the result-ant pellet was 4521-4757 cpm demonstrating that the acrinol precipita-tion technique removes intact complement protein precursor as effect-ively as treatment with hydrochloric acid followed by base neutraliza-tion providing a greatly improved simplified assay procedure~ Also the results of the two experiments indicate that the presence of acrinol does not affect either the first or the second immunological reactions.
In generating standard curves for complement fragment C3a or the des-Arg derivative thereof using 1.0 ng9 2.5 ng, 5.0 ng~ 10 ng, 25 ng and 50 ng quantities of C3a we found that use of thè acrinol precipi-tation step in place of the acid precipitation-base neutralization step of Hugli and Chenoweth increased the sensitivity of the immuno-assay by a factor of 12 as evidenced by the fact that the range of detection became shorter and the slope %B/Bo (Standard Counts/Bound Standard) was increased from -2.3 to -4Ø
Example 4 The assay as described in 3(a) above was performed using acivated plasma samples to which known quantities of complement factor C3a or the des-Arg derivative thereof ranging from 2 to 20 ng was added. The ~2~
-10- 4022.1 results indicate that there was 100~ of the known C3a concentrations in each of the "spiked" normal sampies assayed.
Example 5 The essential reagents for the performance of the immunoassay of the present invention are assembled into a mercantile unit as a kit.
The kit comprises multiple containers7 such as, bottles or other suit-able containers as follows:
(a) a first container having therein a buffered solution of 0.8 to 1.6% acrinol;
(b3 a second container having therein a labeled reagent selected from I25I labeled complement ~ragments C3a, Clla or Csa or the ~es-Arg deri~atives thereof;
(c) a third container having therein rabbit antisera to the par ticular complement fragment, and which optionally contains the following additional reagents:
(d) a container having therein assay buffer, prepared as described hereinabove;
(e) a container having therein second antibody which binds thP
product of the initial complement fragment-antibody reaction, preferably goat anti-rabbit antisera;
~f) a oontainer having therein complement fragment standard, 25 ng;
(g) a container having therein complement fragment standard, 10 ng;
(h) a container having therein complement fragment standard, 5 ng;
(i) a container having therein complement fragment standard, ?.5 ng;
(j) a container having therein complement fragment standard, 1.0 ng.
In each of (f) thrQugh (j) the complement fragment is either C3a9 C4a or C5a or the des-Arg derivative thereof depending on whioh oom-plement fragment is being assayed.
Although the foregoing examples are limited to the separation and assay of complement fragment C3a or the des-Arg derivative thereof the novel separation and assay procedure of the present invention is equally appliciable to complement fragments C4a and Csa or the des-Arg derivatives thereof.
,,
Claims (15)
1. A method for assaying complement fragment C3a, C4a or C5a or the des-Arg derivative thereof in a biological sample which comprises com-bining equal volumes of the biological sample and a solution of 0.8 to 1.6% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflavine hydrochloride, and aminacrine, incu-bating the mixture for about one minute to 2 hour at about 25°C, recovering the supernatant from the resultant precipitate, incubating the supernatant with a known amount of a labeled complement fragment selected from C3a, C4a or C5a or the des-Arg derivative thereof and a known amount of antibody which recognizes said complement fragment or des-Arg derivative thereof, separating the free labeled complement fragment from the bound labeled complement fragment, measuring the amount of labeled complement fragment in either the free or antibody bound complement component, and determining the concentration of com-plement fragment or the des-Arg derivative thereof in the biological sample by comparison to a standard curve.
2. The method of claim 1 wherein the biological sample and the acri-dine derivative are incubated for about 20 minutes to one hour.
3. The method of claim 1 wherein the solution of the acridine deriva-tive is 2-ethoxy-6,9-diaminoacridine lactate monohydrate buffered with 0.05M phosphate buffer, pH 7.4.
4. The method of claim 3 wherein the label is 125I.
5. The method of claim 4 wherein the free labeled complement fragment or des-Arg derivative thereof is separated from the bound labeled com-plement fragment by the addition of a second antibody.
6. The method of claim 5 wherein complement fragment being assayed is C3a or the des-Arg derivative thereof.
7. The method of claim 5 wherein the complement fragment being assayed is C4a or the des-Arg derivative thereof.
8. The method of claim 5 wherein the complement fragment being assayed is C5a or the des-Arg derivative thereof.
9. A method for removing complement components C3, C4 and C5 from samples of biological fluids and recovering from said fluids comple-ment fragments C3a, C4a and C5a or the des-Arg derivatives thereof which comprises combining equal volumes of the biological fluid sample and a buffered solution of 0.8 to 1.6% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflav-ine hydrochloride, and aminacrine, incubating the mixture for about one minute to 2 hours at about 25°C and recovering the supernatant, containing said complement fragments, from the resultant precipitate.
10. The method of claim 9 wherein the acridine derivative is 2-eth-oxy-6,9-diaminoacridine lactate monohydrate and the buffer is 0.05M
phosphate buffer, pH 7.4.
phosphate buffer, pH 7.4.
11. The method of claim 9 wherein the mixture is incubated for about 20 minutes to one hour.
12. A mercantile kit wherein the component parts are assembled for use in assaying biological fluids for complement fragments C3a, C4a or C5a or the des-Arg derivatives thereof which comprises (a) a first container having therein a buffered solution of 0.8 to 1.6% of an acridine derivative selected from the group consisting of acrinol, acriflavine, acriflavine hydrochloride, and aminacrine;
(b) a second container having therein a labeled reagent selected from labeled complement fragments C3a, C4a or C5a or the des-Arg derivatives thereof; and (c) a third container having therein an antibody reagent selected from antibody which recognizes complement fragment C3a, C4a or C5a or the des-Arg derivative thereof.
(b) a second container having therein a labeled reagent selected from labeled complement fragments C3a, C4a or C5a or the des-Arg derivatives thereof; and (c) a third container having therein an antibody reagent selected from antibody which recognizes complement fragment C3a, C4a or C5a or the des-Arg derivative thereof.
13. The mercantile kit of claim 12 wherein the acridine derivative is 2-ethoxy-6,9-diaminoacridine lactate monohydrate.
14. The mercantile kit of claim 12 which additionally comprises (a) a fourth container having therein assay buffer;
(b) a fifty container having therein a second antibody;
(c) a sixth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 25 ng;
(d) a seventh container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 10 ng;
(e) an eighth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 5.0 ng;
(f) a ninth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 2.5 ng; and (g) a tenth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 1.0 ng.
(b) a fifty container having therein a second antibody;
(c) a sixth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 25 ng;
(d) a seventh container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 10 ng;
(e) an eighth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 5.0 ng;
(f) a ninth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 2.5 ng; and (g) a tenth container having therein complement fragment C3a, C4a or C5a or the des-Arg derivative thereof standard, 1.0 ng.
15. The mercantile kit of claim 14 wherein the acridine derivative is 2-ethoxy-6,9-diaminoacridine lactate monohydrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000435635A CA1202235A (en) | 1983-08-30 | 1983-08-30 | Immunoassay for complement fragments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000435635A CA1202235A (en) | 1983-08-30 | 1983-08-30 | Immunoassay for complement fragments |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1202235A true CA1202235A (en) | 1986-03-25 |
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ID=4125972
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000435635A Expired CA1202235A (en) | 1983-08-30 | 1983-08-30 | Immunoassay for complement fragments |
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| Country | Link |
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| CA (1) | CA1202235A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2490025A3 (en) * | 2007-03-27 | 2012-11-28 | Immunovia AB | Method, array and use thereof |
| US11320436B2 (en) | 2020-07-16 | 2022-05-03 | Immunovia Ab | Methods, arrays and uses thereof |
-
1983
- 1983-08-30 CA CA000435635A patent/CA1202235A/en not_active Expired
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2490025A3 (en) * | 2007-03-27 | 2012-11-28 | Immunovia AB | Method, array and use thereof |
| US11525832B2 (en) | 2007-03-27 | 2022-12-13 | Immunovia Ab | Protein signature/markers for the detection of adenocarcinoma |
| US11320436B2 (en) | 2020-07-16 | 2022-05-03 | Immunovia Ab | Methods, arrays and uses thereof |
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