US3852415A - Compositions for use in radioimmunoassay as a substitute for blood plasma extract in determination of carcinoembryonic antigen - Google Patents

Compositions for use in radioimmunoassay as a substitute for blood plasma extract in determination of carcinoembryonic antigen Download PDF

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US3852415A
US3852415A US00297565A US29756572A US3852415A US 3852415 A US3852415 A US 3852415A US 00297565 A US00297565 A US 00297565A US 29756572 A US29756572 A US 29756572A US 3852415 A US3852415 A US 3852415A
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cea
diluent
composition
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J Vandervoorde
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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Priority to CH1403573A priority patent/CH584409A5/xx
Priority to CA182,470A priority patent/CA1012458A/en
Priority to DE2349738A priority patent/DE2349738C3/en
Priority to IL43378A priority patent/IL43378A/en
Priority to IT29875/73A priority patent/IT1001584B/en
Priority to AU61242/73A priority patent/AU467511B2/en
Priority to FR7336328A priority patent/FR2203525A5/fr
Priority to NL7314093.A priority patent/NL161884C/en
Priority to JP11463773A priority patent/JPS5315130B2/ja
Priority to GB4780473A priority patent/GB1423244A/en
Priority to SE7313919A priority patent/SE417460B/en
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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
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y10S436/813—Cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/106664—Blood serum or blood plasma standard or control
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/108331—Preservative, buffer, anticoagulant or diluent

Definitions

  • FIG. 1 ANTIBODY TITRATION CURVES I l I I AA PLASMA EXTRACT I50 PLASMA EXTRACT SUBSTITUTE I30 W 1/0 cpm x IO' 90 0 50 I00 200 500 ,u! Of ANT/SERUM (l' 2500 dill?) FIG. 1
  • a diluent medium a material which will permit consistent, reliable results.
  • plasma extracted by glycoprotein solvents and dialysis from normal blood group Type A+ blood was used as the diluent medium.
  • Such as extraction process is disclosed, for example, in US. Pat. No. 3,663,684, Example 5. While the results using this diluent are satisfactory, there is difficulty in obtaining sufficient quantities of normal plasma extract without an interfering amount of CEA.
  • FIG. 1 shows antibody titration curves.
  • FIG. 2 shows standard inhibition curves.
  • This substitute is a buffer composition containing as the buffer, a salt of an organic acid and a strong inorganic base, a preservative, a protein to coat glass tubes, a base and water.
  • the concentrations and amounts of ingredients as well as pH must be adjusted so the curve is substantially identical to that resulting when plasma extract from blood group Type A+ is used.
  • the substitute plasma extract composition when used to make a standard inhibition curve must produce a curve which is substantially identical to that resulting when plasma extract from blood group Type A+ is used.
  • this range includes the pKs of polybasic acids. For purposes of this invention, however, only the pK and pK are significant.
  • a preferred pK is in the range of about 2 to 3.
  • Other criteria for choosing a suitable organic acid are that it must form water soluble salts with alkali metals, and it must cause the pH of the composition to be near neutral on the acid side.
  • Typical, suitable, organic acids are the tetraacetic acid compounds, particularly ethylenediamine tetraacetic acid (EDTA) which has a pK of 2.0 and a pK of 2.7.
  • EDTA ethylenediamine tetraacetic acid
  • Other similar organic acids are suitable, for example, diethylenetriamine pentacetic acid.
  • Preferred alkali metal salts are the disodium and dipotassium salts.
  • the concentration of the organic acid should be equivalent in function to 1.28-1.32 gm./liter of disodium EDTA. Preferably, 1.3 gm./liter is required. In the event a tenfold concentration increase of the plasma extract substitute per liter is made, then the equivalent to 13.0 gm./l. of disodium EDTA is used.
  • disodium EDTA means the disodium salt of ethylenediamine tetraacetic acid with two molecules of water of hydration, i.e., disodium EDTA dihydrate.
  • CEA has a tendency to adhere to glass, it has been found necessary to use a small amount of proteinaceous material to coat the glass receptacles containing the diluent medium and prevent CEA from adhering. Such proteinaceous material must be inert with respect to the antibody-antigen reactions present when the curves are developed.
  • a typical suitable material is bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the BSA is commercially available as a 30 percent aqueous solution and is suitable in this form for use in the invention, also BSA in the form of a dry powder is suitable.
  • the amount used in forming the compositions of this invention is u liters/liter or 21 mg./ liter when dry powder is used. In the event an increase in concentration of the buffer is desired then 700 uL/l. or 210 mg./liter is used.
  • the receptables are a material other than glass or glass coated with a non-reactive coating, e.g., Teflon, then there is no need for the proteinaceous material.
  • a non-reactive coating e.g., Teflon
  • a small amount of a preservative should be used to prevent growth or microorganisms, particularly fungi. It has been found that either 0.17 gm./l. of sodium azide or a functionally equivalent amount of other preservatives, e.g., potassium sorbate, is suitable for the compositions of this invention. In the event the more concentrated plasma extract substitute is used, then 1.7 gm./l. of preservative is required.
  • the invention is not limited to the use of the specific preservatives named herein since other functionally equivalent materials or 3 mixtures thereof are suitable and would be obvious to the skilled artisan.
  • the base used should be a base of the same metal forming the salt with the acid.
  • 1M sodium hydroxide is used when disodium EDTA is the organic acid salt.
  • the pH is adjusted similarly to about 6.25 since upon dilution it then approaches the preferred 6.5.
  • the amount of base used varies depending upon the pH of the composition prior to its addition. Generally, however, approximately ml. of l M base are added.
  • the composition is prepared by mixing all the ingredients except the base in about 800 ml. deionized or distilled water. Following this, the solution is brought to the preferred pH, 6.5 with the base and the solution is made to 1,000 ml. with deionized or distilled water. (In the case of a tenfold concentration increase, the pH is brought to 6.25.) The resulting solution is stable at room temperature (25C.) or 4C. for several weeks.
  • carcinoembryonic antigen includes all antigenic material which is specific to antibodies of carcinoembryonic antigen.
  • Typical CEA antigen materials are disclosed in US. Pat. No. 3,663,684 and US. Pat. Application Ser. No. 133,404, filed Apr. 2, 1971 now US. Pat. No. 3,697,638.
  • EXAMPLE 1 Preparation of Plasma Substitute 1.3 Grams of EDTA-Na -2H O (3.7 mM) is dissolved in about 800 ml. of water. 0.17 Grams of sodium azide and 70 ul. of 30 percent aqueous BSA are added to the solution. The pH is adjusted to 6.5 with 1M sodium hydroxide and the volume made up to 1,000 with deionized or distilled water. This solution is stable at 4C. or at room temperature for several weeks.
  • EXAMPLE 2 Preparation of Concentrated Plasma Substitute 13.0 Grams of EDTA-Na -2H O (37 mM) is dissolved in about 800 m1. of water. 1.7 Grams of sodium azide and 700 pl. of 30 percent aqueous BSA are added to the solution. The pH is adjusted to 6.25 with 1M sodium hydroxide and the volume made up to 1,000 ml. with deionized or distilled water. The pH of this stock solution, upon diluting tenfold to make the working solution, approaches 6.5. This stock solution is stable for several weeks at 4C. or at room temperature.
  • EXAMPLE 3 Preparation of Plasma Extract 1 MI. of normal blood group Type A+ plasma is diluted with 4 ml. of saline solution in a test tube. 5 M1. of 1.2 M perchloric acid is added to each tube. The mixtures are centrifuged for 20 minutes at 1,000 X g. Thesupemates are decanted in dialysis bags which are then sealed. The bags are placed in a dialysis bath and dialyzed for 18 hours against 60 volumes of deionized or distilled water which is changed several times during the dialysis. A final 3 hour dialysis against 60 volumes of 0.01 M ammonium acetate buffer, pH 6.8 is performed. The extracts are then transferred to disposable test tubes.
  • EXAMPLE 4 Antiserum Titration Curve with Plasma Extract Graded amounts, i.e., 50, 100, 200 and 500 pl of antiserum to CEA which is diluted 1 to 2,500 in a buffer comprising 9 volumes of borate buffer (pH 8.4) and 1 volume of blood group Type A+ plasma are added to four test tubes, each containing 10 ml. of plasma extract prepared as in Example 3. Only water was added to one of the test tubes for a zero measurement. The mixtures were incubated for 0.5 hour at 45C. about 3 nanograms of I CEA having 150,000-200,000 cpm. were then added to each tube and the mixture incubated for 0.5 hour at 45C. 5 M1.
  • the assay indicates the activity of the antiserum, knowledge of which is needed for use in the radioimmunoassay for CEA.
  • Example 5 Antiserum Titration Curve with Plasma Extract Substitute The identical procedure of Example 4 was followed except that 10 ml. of the composition of Example 1 was used in place of the 10 ml. of plasma extract. The results are shown in FIG. 1.
  • EXAMPLE 6 Antiserum Titration Curve with Plasma Extract Substitute 5 ml. of the product of Example 2 were diluted to 50 ml. with deionized or distilled water and utilized as the diluent following the identical procedure of Example 5. The resulting curve is identical to that of Example 5 and shown in FIG. 1.
  • EXAMPLE 7 Standard Inhibition Curve with Plasma Extract pl. of antiserum of CEA was added to each of five test tubes containing 10 ml. of the plasma extract of Example 3.
  • Graded amounts of CEA i.e., 0, 2.5, 6.25, 12.5 and 25 mg, were added to the tubes and the mixture incubated for 0.5 hour at 45C.
  • about 3 ng. of I -CEA with 150,000-200,000 cpm were added to each tube and the mixture incubated for 0.5 hour at 45C. 5 M].
  • zirconyl phosphate gel (pH 6.25) was then added to each tube. The tubes were centrifuged at 1,000 X g for 5 minutes at room temperature.
  • Example 8 Standard Inhibition Curve with Plasma Extract Substitute
  • the identical procedure of Example 7 was followed except that 10 ml. of the composition of Example 1 was EXAMPLE 9 Standard Inhibition'Curve with Plasma Extract Substitute From Example 2 5 ml. of the product of Example 2 were diluted to 50 ml. with deionized or distilled water and utilized as diluent following the identical procedure of Example 8. The resulting curve is identical to that of Example 8 and shown in FIG. 2.
  • the slightly higher level of radioactivity of the curve using the plasma extract at the zero value indicates the presence of a very small but measurable amount of CEA in the plasma extract.
  • composition of claim 2 wherein the preservative is sodium azide.
  • a diluent composition suitable for forming an antibody to CEA titration curve or a CEA standard inhibition curve substantially identical to that resulting when blood plasma is used as the diluent said composition having a pH 6.25 and comprising per liter; 13.0 grams of. disodium EDTA dihydrate, 1.7 grams of an antimi crobial preservative, 700 pl. of 30 percent w/w aqueous BSA, sodium hydroxide, and sufficient water to make 1 liter.
  • composition of claim 4 wherein the preservative is sodium azide.

Abstract

A composition suitable for use as a substitute for blood plasma extract in producing antiserum titration curves and standard antigen inhibition curves for the determination of carcinoembryonic antigen in human plasma is disclosed.

Description

nited States Patent 1 1 3,852,415 Vandervoorde Dec. 3, 1974 1 COMPOSITIONS FOR USE IN 3,720,760 3/1973 Bennich 424/1 RADIOIMMUNOASSAY AS A SUBSTITUTE F OR BLOOD PLASMA EXTRACT IN DETERMINATION OF CARCINOEMBRYONIC ANTIGEN Inventor: Jacques Pierre Vandervoorde, West Caldwell, NJ.
Assignee: Hoffman-La Roche, Inc., Nutley,
Filed: Oct. 13, 1972 Appl. No.: 297,565
11.8. CI. 424/1, 23/230 B, 424/2,
424/12, 250/303 Int. CL... A61k 27/04 Field of Search 424/1, 2, 359, 177, 12;
References Cited UNlTED STATES PATENTS l0/l972 Hansen 424/1 OTHER PUBLICATIONS Aach et al., Proc. Nat. Acad. Sci., U.S.A., Vol. 68, No.5, p. 1056, May 1971.
Salmon et al., The Journal of Immunology, Vol. 104, No. 3, March 1970, pp. 665-667.
Salmon et al., The Journal of Immunology, Vol. 103, No. 1, July 1969, pp. 129-131.
Primary Examiner-Benjamin R. Padgett Attorney, Agent, or Firm samuel L. Welt; Jon S. Saxe; Gerald S. Rosen 5 7 ABSTRACT 7 Claims, 2 Drawing Figures PATENTEL 31974 3. 852.41 5
ANTIBODY TITRATION CURVES I l I I AA PLASMA EXTRACT I50 PLASMA EXTRACT SUBSTITUTE I30 W 1/0 cpm x IO' 90 0 50 I00 200 500 ,u! Of ANT/SERUM (l' 2500 dill?) FIG. 1
CEA STANDARD INHIBIT/ON CURVES H5 1 PLASMA EXTRACT I05 PLASMA EXTRACT SUBSTITUTE 95 H*\\ 85 cPm x IO' ng CEA FIG. 2
COMPOSITIONS FOR USE IN RADIOIMMUNOASSAY AS A SUBSTITUTE FOR BLOOD PLASMA EXTRACT IN DETERMINATION OF CARCINOEMBRYONIC ANTIGEN BACKGROUND OF THE INVENTION In order to accurately assay circulating carcinoembryonic antigen (CEA) in human blood, it is necessary to run antiserum titration curves and standard antigen inhibition curves for use in comparing against the test sample. The test sample is usually either blood serum or blood plasma. The antiserum titration curve is used to tell the activity of the antiserum and results from plotting the microliters of antiserum containing a known activity versus the countsper minute of radioiodinated CEA. The standard antigen inhibition curve is used to find the amount of CEA in a test sample and results from plotting nanograms of CEA versus counts per minute of radioiodinated CEA.
In performing the measurements required to develop the curves, it is necessary to use as a diluent medium a material which will permit consistent, reliable results. Prior to this invention, plasma extracted by glycoprotein solvents and dialysis from normal blood group Type A+ blood was used as the diluent medium. Such as extraction process is disclosed, for example, in US. Pat. No. 3,663,684, Example 5. While the results using this diluent are satisfactory, there is difficulty in obtaining sufficient quantities of normal plasma extract without an interfering amount of CEA.
There is therefore a need for a diluent medium which is economical and easy to obtain, permits consistent, reliable results and which further results in standard curves which are substantially identical to those resulting from the use of plasma extract from blood group Type A+ blood.
This latter property is important since it eliminates the need for developing new data which will be acceptable to the scientific community and governmental agencies. Such development is very time consuming and costly.
DRAWINGS FIG. 1 shows antibody titration curves. FIG. 2 shows standard inhibition curves.
DESCRIPTION OF THE INVENTION According to this invention, a substitute for the plasma extract of blood type A+ blood has been developed. This substitute is a buffer composition containing as the buffer, a salt of an organic acid and a strong inorganic base, a preservative, a protein to coat glass tubes, a base and water.
In order to insure that the buffer is of proper composition to make it suitable for use in forming the standard antiserum titration curve the concentrations and amounts of ingredients as well as pH must be adjusted so the curve is substantially identical to that resulting when plasma extract from blood group Type A+ is used.
The substitute plasma extract composition, when used to make a standard inhibition curve must produce a curve which is substantially identical to that resulting when plasma extract from blood group Type A+ is used.
Surprisingly, it has been found that the same composition can be used as a plasma extract substitute when running the titrations for both of the standard curves discussed.
' about 5 to 6 is operable, this range includes the pKs of polybasic acids. For purposes of this invention, however, only the pK and pK are significant. A preferred pK is in the range of about 2 to 3. Other criteria for choosing a suitable organic acid are that it must form water soluble salts with alkali metals, and it must cause the pH of the composition to be near neutral on the acid side.
Typical, suitable, organic acids are the tetraacetic acid compounds, particularly ethylenediamine tetraacetic acid (EDTA) which has a pK of 2.0 and a pK of 2.7. Other similar organic acids are suitable, for example, diethylenetriamine pentacetic acid.
Preferred alkali metal salts are the disodium and dipotassium salts.
The concentration of the organic acid should be equivalent in function to 1.28-1.32 gm./liter of disodium EDTA. Preferably, 1.3 gm./liter is required. In the event a tenfold concentration increase of the plasma extract substitute per liter is made, then the equivalent to 13.0 gm./l. of disodium EDTA is used. As used herein disodium EDTA means the disodium salt of ethylenediamine tetraacetic acid with two molecules of water of hydration, i.e., disodium EDTA dihydrate.
Other increases or decreases in concentration of the plasma extract substitute can be used but are not sufficiently accurate for practical purposes when making the standard curves.
Since CEA has a tendency to adhere to glass, it has been found necessary to use a small amount of proteinaceous material to coat the glass receptacles containing the diluent medium and prevent CEA from adhering. Such proteinaceous material must be inert with respect to the antibody-antigen reactions present when the curves are developed. A typical suitable material is bovine serum albumin (BSA). The BSA is commercially available as a 30 percent aqueous solution and is suitable in this form for use in the invention, also BSA in the form of a dry powder is suitable. The amount used in forming the compositions of this invention is u liters/liter or 21 mg./ liter when dry powder is used. In the event an increase in concentration of the buffer is desired then 700 uL/l. or 210 mg./liter is used.
If the receptables are a material other than glass or glass coated with a non-reactive coating, e.g., Teflon, then there is no need for the proteinaceous material.
A small amount of a preservative should be used to prevent growth or microorganisms, particularly fungi. It has been found that either 0.17 gm./l. of sodium azide or a functionally equivalent amount of other preservatives, e.g., potassium sorbate, is suitable for the compositions of this invention. In the event the more concentrated plasma extract substitute is used, then 1.7 gm./l. of preservative is required. The invention is not limited to the use of the specific preservatives named herein since other functionally equivalent materials or 3 mixtures thereof are suitable and would be obvious to the skilled artisan.
Finally, sufficient base is used to adjust the pH of the plasma extract substitute to 6.45-6.55, preferably 6.5. The base used should be a base of the same metal forming the salt with the acid. For example, 1M sodium hydroxide is used when disodium EDTA is the organic acid salt. When the more concentrated medium is used, the pH is adjusted similarly to about 6.25 since upon dilution it then approaches the preferred 6.5. The amount of base used varies depending upon the pH of the composition prior to its addition. Generally, however, approximately ml. of l M base are added.
The composition is prepared by mixing all the ingredients except the base in about 800 ml. deionized or distilled water. Following this, the solution is brought to the preferred pH, 6.5 with the base and the solution is made to 1,000 ml. with deionized or distilled water. (In the case of a tenfold concentration increase, the pH is brought to 6.25.) The resulting solution is stable at room temperature (25C.) or 4C. for several weeks.
As used herein carcinoembryonic antigen (CEA) includes all antigenic material which is specific to antibodies of carcinoembryonic antigen. Typical CEA antigen materials are disclosed in US. Pat. No. 3,663,684 and US. Pat. Application Ser. No. 133,404, filed Apr. 2, 1971 now US. Pat. No. 3,697,638.
The following Examples illustrate the invention.
EXAMPLE 1 Preparation of Plasma Substitute 1.3 Grams of EDTA-Na -2H O (3.7 mM) is dissolved in about 800 ml. of water. 0.17 Grams of sodium azide and 70 ul. of 30 percent aqueous BSA are added to the solution. The pH is adjusted to 6.5 with 1M sodium hydroxide and the volume made up to 1,000 with deionized or distilled water. This solution is stable at 4C. or at room temperature for several weeks.
EXAMPLE 2 Preparation of Concentrated Plasma Substitute 13.0 Grams of EDTA-Na -2H O (37 mM) is dissolved in about 800 m1. of water. 1.7 Grams of sodium azide and 700 pl. of 30 percent aqueous BSA are added to the solution. The pH is adjusted to 6.25 with 1M sodium hydroxide and the volume made up to 1,000 ml. with deionized or distilled water. The pH of this stock solution, upon diluting tenfold to make the working solution, approaches 6.5. This stock solution is stable for several weeks at 4C. or at room temperature.
EXAMPLE 3 Preparation of Plasma Extract 1 MI. of normal blood group Type A+ plasma is diluted with 4 ml. of saline solution in a test tube. 5 M1. of 1.2 M perchloric acid is added to each tube. The mixtures are centrifuged for 20 minutes at 1,000 X g. Thesupemates are decanted in dialysis bags which are then sealed. The bags are placed in a dialysis bath and dialyzed for 18 hours against 60 volumes of deionized or distilled water which is changed several times during the dialysis. A final 3 hour dialysis against 60 volumes of 0.01 M ammonium acetate buffer, pH 6.8 is performed. The extracts are then transferred to disposable test tubes.
EXAMPLE 4 Antiserum Titration Curve with Plasma Extract Graded amounts, i.e., 50, 100, 200 and 500 pl of antiserum to CEA which is diluted 1 to 2,500 in a buffer comprising 9 volumes of borate buffer (pH 8.4) and 1 volume of blood group Type A+ plasma are added to four test tubes, each containing 10 ml. of plasma extract prepared as in Example 3. Only water was added to one of the test tubes for a zero measurement. The mixtures were incubated for 0.5 hour at 45C. about 3 nanograms of I CEA having 150,000-200,000 cpm. were then added to each tube and the mixture incubated for 0.5 hour at 45C. 5 M1. of zirconyl phosphate gel (pH 6.25) was then added to each tube. The tubes were centrifuged at 1,000 X g for 5 minutes at room temperature. The supernatant was then discarded and the gel precipitate was resuspended in 10 ml. of ammonium acetate solution 0.1 M, pH 6.25). The gel was then separated by centrifugation at 1,000 X g for 5 minutes and assayed for gel bound I The results are shown in FIG. 1. The assay indicates the activity of the antiserum, knowledge of which is needed for use in the radioimmunoassay for CEA.
EXAMPLE 5 Antiserum Titration Curve with Plasma Extract Substitute The identical procedure of Example 4 was followed except that 10 ml. of the composition of Example 1 was used in place of the 10 ml. of plasma extract. The results are shown in FIG. 1.
EXAMPLE 6 Antiserum Titration Curve with Plasma Extract Substitute 5 ml. of the product of Example 2 were diluted to 50 ml. with deionized or distilled water and utilized as the diluent following the identical procedure of Example 5. The resulting curve is identical to that of Example 5 and shown in FIG. 1.
EXAMPLE 7 Standard Inhibition Curve with Plasma Extract pl. of antiserum of CEA was added to each of five test tubes containing 10 ml. of the plasma extract of Example 3. Graded amounts of CEA, i.e., 0, 2.5, 6.25, 12.5 and 25 mg, were added to the tubes and the mixture incubated for 0.5 hour at 45C. about 3 ng. of I -CEA with 150,000-200,000 cpm were added to each tube and the mixture incubated for 0.5 hour at 45C. 5 M]. of zirconyl phosphate gel (pH 6.25) was then added to each tube. The tubes were centrifuged at 1,000 X g for 5 minutes at room temperature. The supernatant was then discarded and the gel precipitate was resuspended in 10 ml. of ammonium acetate solution (0.1 M, pH 6.25 The gel was then separated by centrifugation at 1,000 X g for 5 minutes and assayed for gel bound I The results are shown in FIG. 2. The curve is used to find the amount of CEA in a plasma extract sample.
EXAMPLE 8 Standard Inhibition Curve with Plasma Extract Substitute The identical procedure of Example 7 was followed except that 10 ml. of the composition of Example 1 was EXAMPLE 9 Standard Inhibition'Curve with Plasma Extract Substitute From Example 2 5 ml. of the product of Example 2 were diluted to 50 ml. with deionized or distilled water and utilized as diluent following the identical procedure of Example 8. The resulting curve is identical to that of Example 8 and shown in FIG. 2.
The slightly higher level of radioactivity of the curve using the plasma extract at the zero value indicates the presence of a very small but measurable amount of CEA in the plasma extract.
I claim:
11. A diluent composition suitable for forming an antibody to CEA titration curve or a CEA standard inhibition curve substantially identical to that resulting when blood plasma is used as the diluent, said composition having a pH 6.45-6.55 and comprising per liter; an amount of a salt of an organic carboxylic acid with an alkali metal which is equivalent to 1.3 grams of disodium ethylenediamine tetraacetic acid dihydrate, 0.17 grams of an antimicrobial preservative, 70 ul. of a 30 percent w/w aqueous solution of bovine serum albumin, sodium hydroxide, and sufficient water to make 1 liter.
2. A diluent composition suitable for forming an antibody to CEA titration curve or a CEA standard inhibition curve substantially identical to that resulting when blood plasma is used as the diluent, said composition having a pH 6.456.55 and comprising per liter; 1.3 grams of disodium ethylenediamine tetraacetic acid dihydrate, 0.17 grams of an antimicrobial preservative, 70 ul. of a 30 percent w/w aqueous solution of bovine serum albumin, sodium hydroxide, and sufficient water to make 1 liter.
3. The composition of claim 2 wherein the preservative is sodium azide.
4. A diluent composition suitable for forming an antibody to CEA titration curve or a CEA standard inhibition curve substantially identical to that resulting when blood plasma is used as the diluent, said composition having a pH 6.25 and comprising per liter; 13.0 grams of. disodium EDTA dihydrate, 1.7 grams of an antimi crobial preservative, 700 pl. of 30 percent w/w aqueous BSA, sodium hydroxide, and sufficient water to make 1 liter.
5. The composition of claim 4 wherein the preservative is sodium azide.
6. In a method of making an antibody to CEA titration curve by forming graded amounts of antiserum to CEA by dilution with a buffer, incubating in a diluent, adding-radiolabelled CEA, incubating, adding zirconyl phosphate gel, separating the resulting precipitated gel bound with radiolabelled material, assaying it for radio activity and recording the results on a graph as counts per minute vs. p. liters of antiserum, the improvement which comprises utilizing as the diluent, the composition of claim 1.
7. In a method of making a CEA standard inhibition curve by adding antiserum to CEA to graded amounts of CEA in a diluent, incubating the mixture, adding radiolabelled CEA, incubating, adding zirconyl phosphate gel, recovering the resulting gel precipitate bound with radiolabelled material, assaying it for radioactivity and recording the results on a graph as counts per minute vs. nanograms of CEA, the improvement which comprises utilizing as the diluent, the composi-

Claims (7)

1. A DILUENT COMPOSITION SUITABLE FOR FORMING AN ANTIBODY TO CEA TITRATION CURVE OR A CEA STANDARD INHIBITION CURVE SUBSTANTIALLY IDENTICAL TO THAT RESULTING WHEN BLOOD PLASMA IS USED AS THE DILUENT, SAID COMPOSITION HAVING A PH 6.45-6.55 AND COMPRISING PER LITER, AN AMOUNT OF A SALT OF AN ORGANIC CARBOXYLIC ACID WITH AN ALKALI METAL WHICH IS EQUIVALENT TO 1.3 GRAMS OF DISODIUM ETHYLENEDIAMINE TETRAACETIC AND DIHYDRATE, 0.17 GRAMS OF AN ANTIMICROBIAL PRESERVATIVE, 70 UL. OF A 30 PERCENT W/W AQUEOUS SOLUTION OF BOVINE SERUM ALBUMIN, SODIUM HYDROXIDE, AND SUFFICIENT WATER TO MAKE 1 LITER.
2. A diluent composition suitable for forming an antibody to CEA titration curve or a CEA standard inhibition curve substantially identical to that resulting when blood plasma is used as the diluent, said composition having a pH 6.45-6.55 and comprising per liter; 1.3 grams of disodium ethylenediamine tetraacetic acid dihydrate, 0.17 grams of an antimicrobial preservative, 70 Mu l. of a 30 percent w/w aqueous solution of bovine serum albumin, sodium hydroxide, and sufficient water to make 1 liter.
3. The composition of claim 2 wherein the preservative is sodium azide.
4. A diluent composition suitable for forming an antibody to CEA titration curve or a CEA standard inhibition curve substantially identical to that resulting when blood plasma is used as the diluent, said composition having a pH 6.25 and comprising per liter; 13.0 grams of disodium EDTA dihydrate, 1.7 grams of an antimicrobial preservative, 700 Mu l. of 30 percent w/w aqueous BSA, sodium hydroxide, and sufficient water to make 1 liter.
5. The composition of claim 4 wherein the preservative is sodium azide.
6. In a method of making an antibody to CEA titration curve by forming graded amounts of antiserum to CEA by dilution with a buffer, incubating in a diluent, adding radiolabelled CEA, incubating, adding zirconyl phosphate gel, separating the resulting precipitated gel bound with radiolabelled material, assaying it for radioactivity and recording the results on a graph as counts per minute vs. Mu liters of antiserum, the improvement which comprises utilizing as the diluent, the composition of claim 1.
7. In a method of making a CEA standard inhibition curve by adding antiserum to CEA to graded amounts of CEA in a diluent, incubating the mixture, adding radiolabelled CEA, incubating, adding zirconyl phosphate gel, recovering the resulting gel precipitate bound with radiolabelled material, assaying it for radioactivity and recording the results on a graph as counts per minute vs. nanograms of CEA, the improvement which comprises utilizing as the diluent, the composition of claim 1.
US00297565A 1972-10-13 1972-10-13 Compositions for use in radioimmunoassay as a substitute for blood plasma extract in determination of carcinoembryonic antigen Expired - Lifetime US3852415A (en)

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ZA723264A ZA723264B (en) 1972-10-13 1972-05-15 Improvements in toys
US00297565A US3852415A (en) 1972-10-13 1972-10-13 Compositions for use in radioimmunoassay as a substitute for blood plasma extract in determination of carcinoembryonic antigen
ZA737693A ZA737693B (en) 1972-10-13 1973-10-01 Dilution agent
CH1403573A CH584409A5 (en) 1972-10-13 1973-10-01
CA182,470A CA1012458A (en) 1972-10-13 1973-10-02 Physiological buffer system
DE2349738A DE2349738C3 (en) 1972-10-13 1973-10-03 Aqueous plasma substitute
IL43378A IL43378A (en) 1972-10-13 1973-10-04 Aqueous buffer solution for use as a plasma substitute
IT29875/73A IT1001584B (en) 1972-10-13 1973-10-08 AGENT FOR DILUTION
AU61242/73A AU467511B2 (en) 1972-10-13 1973-10-10 Dilution agent
FR7336328A FR2203525A5 (en) 1972-10-13 1973-10-11
NL7314093.A NL161884C (en) 1972-10-13 1973-10-12 METHOD FOR DETERMINING CARCINO EMBRYONIC ANTIGENS (CEA) IN A SAMPLE
JP11463773A JPS5315130B2 (en) 1972-10-13 1973-10-12
GB4780473A GB1423244A (en) 1972-10-13 1973-10-12 Dilution agent
SE7313919A SE417460B (en) 1972-10-13 1973-10-12 SET IN TESTING CARCINO MEMBRANAL ANTIGEN USING A NEW DILUTER
BE136609A BE805991A (en) 1972-10-13 1973-10-12 DILUENT
DD174034A DD108151A5 (en) 1972-10-13 1973-10-12

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US3901870A (en) * 1974-03-12 1975-08-26 Behringwerke Ag Derivative of alpha' 1'-fetospecific serum protein and process for its manufacture
USB554039I5 (en) * 1975-02-28 1976-02-24
US4043757A (en) * 1975-05-20 1977-08-23 Ortho Pharmaceutical Corporation Method for detection of human mammary carcinoma
US4132769A (en) * 1974-10-30 1979-01-02 Osther Kurt B Cancer antigen, cancer therapy, and cancer diagnosis
US4152410A (en) * 1975-09-03 1979-05-01 Eisai Co., Ltd. Diagnosis reagent for neoplasm and method for diagnosis of neoplasm
WO1981001469A1 (en) * 1979-11-21 1981-05-28 Wistar Inst Monoclonal hybridoma antibody specific for high molecular weight carcinoembryonic antigen
US4379839A (en) * 1977-05-23 1983-04-12 The Trustees Of Columbia University In The City Of New York Method for detecting cancer
US4624916A (en) * 1984-04-06 1986-11-25 International Immunoassay Laboratories, Inc. Process and composition for the rapid quantitation of small levels of creative kinase-MB isoenzyme
US4631254A (en) * 1982-12-06 1986-12-23 Hoffmann-La Roche Inc. Carcinoembryonic antigen determination
US5296377A (en) * 1992-12-15 1994-03-22 Boehringer Mannheim Corporation Control reagent containing a hydroxylamine or an antioxidant
US6013772A (en) * 1986-08-13 2000-01-11 Bayer Corporation Antibody preparations specifically binding to unique determinants of CEA antigens or fragments thereof and use of the antibody preparations in immunoassays
US10563153B2 (en) 2010-05-20 2020-02-18 Ecolab Usa Inc. Rheology modified low foaming liquid antimicrobial compositions and methods of use thereof
US11474114B2 (en) * 2020-06-19 2022-10-18 Republic of Korea (National Forensic Service Director Ministry of the Interior and Safety) Artificial blood for bloodstain pattern analysis

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JPS57208459A (en) * 1981-06-19 1982-12-21 Eisai Co Ltd Measuring method using enzyme-labelled antibody and reagent
JPS61280566A (en) * 1984-09-03 1986-12-11 Hiroaki Sawai Direct immunoassay of 2'-5'-oligoadenylic acid
US4690772A (en) * 1985-06-03 1987-09-01 National Medical Care Sterilant compositions
CA1296253C (en) * 1986-10-20 1992-02-25 Praveen Tyle Stabilized growth hormone compositions

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3901870A (en) * 1974-03-12 1975-08-26 Behringwerke Ag Derivative of alpha' 1'-fetospecific serum protein and process for its manufacture
US4132769A (en) * 1974-10-30 1979-01-02 Osther Kurt B Cancer antigen, cancer therapy, and cancer diagnosis
USB554039I5 (en) * 1975-02-28 1976-02-24
US3999944A (en) * 1975-02-28 1976-12-28 Hoffmann-La Roche Inc. Detection of breast cancer
US4043757A (en) * 1975-05-20 1977-08-23 Ortho Pharmaceutical Corporation Method for detection of human mammary carcinoma
US4152410A (en) * 1975-09-03 1979-05-01 Eisai Co., Ltd. Diagnosis reagent for neoplasm and method for diagnosis of neoplasm
US4379839A (en) * 1977-05-23 1983-04-12 The Trustees Of Columbia University In The City Of New York Method for detecting cancer
US4349528A (en) * 1979-11-21 1982-09-14 The Wistar Institute Monocolonal hybridoma antibody specific for high molecular weight carcinoembryonic antigen
WO1981001469A1 (en) * 1979-11-21 1981-05-28 Wistar Inst Monoclonal hybridoma antibody specific for high molecular weight carcinoembryonic antigen
US4631254A (en) * 1982-12-06 1986-12-23 Hoffmann-La Roche Inc. Carcinoembryonic antigen determination
US4624916A (en) * 1984-04-06 1986-11-25 International Immunoassay Laboratories, Inc. Process and composition for the rapid quantitation of small levels of creative kinase-MB isoenzyme
US6013772A (en) * 1986-08-13 2000-01-11 Bayer Corporation Antibody preparations specifically binding to unique determinants of CEA antigens or fragments thereof and use of the antibody preparations in immunoassays
US6022958A (en) * 1986-08-13 2000-02-08 Bayer Corporation cDNAs coding for members of the carcinoembryonic antigen family
US5296377A (en) * 1992-12-15 1994-03-22 Boehringer Mannheim Corporation Control reagent containing a hydroxylamine or an antioxidant
US10563153B2 (en) 2010-05-20 2020-02-18 Ecolab Usa Inc. Rheology modified low foaming liquid antimicrobial compositions and methods of use thereof
US11268049B2 (en) 2010-05-20 2022-03-08 Ecolab Usa Inc. Rheology modified low foaming liquid antimicrobial compositions and methods of use thereof
US11474114B2 (en) * 2020-06-19 2022-10-18 Republic of Korea (National Forensic Service Director Ministry of the Interior and Safety) Artificial blood for bloodstain pattern analysis

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GB1423244A (en) 1976-02-04
CH584409A5 (en) 1977-01-31
IL43378A0 (en) 1974-01-14
NL161884C (en) 1980-03-17
JPS5315130B2 (en) 1978-05-23
DD108151A5 (en) 1974-09-05
CA1012458A (en) 1977-06-21
NL7314093A (en) 1974-04-16
JPS4971133A (en) 1974-07-10
ZA737693B (en) 1974-08-28
DE2349738A1 (en) 1974-04-18
NL161884B (en) 1979-10-15
FR2203525A5 (en) 1974-05-10
AU6124273A (en) 1975-05-01
IT1001584B (en) 1976-04-30
ZA723264B (en) 1974-08-28
BE805991A (en) 1974-04-12
DE2349738B2 (en) 1978-01-19
DE2349738C3 (en) 1981-06-19
AU467511B2 (en) 1975-12-04
IL43378A (en) 1976-04-30
SE417460B (en) 1981-03-16

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