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Publication numberUS3852415 A
Publication typeGrant
Publication dateDec 3, 1974
Filing dateOct 13, 1972
Priority dateOct 13, 1972
Also published asCA1012458A1, DE2349738A1, DE2349738B2, DE2349738C3
Publication numberUS 3852415 A, US 3852415A, US-A-3852415, US3852415 A, US3852415A
InventorsJ Vandervoorde
Original AssigneeHoffmann La Roche
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Compositions for use in radioimmunoassay as a substitute for blood plasma extract in determination of carcinoembryonic antigen
US 3852415 A
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.
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Description  (OCR text may contain errors)

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-

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3697638 *Apr 12, 1971Oct 10, 1972Hoffmann La RocheAntigens
US3720760 *Sep 6, 1968Feb 7, 1984Pharmacia AbTitle not available
Non-Patent Citations
Reference
1 *Aach et al., Proc. Nat. Acad. Sci., U.S.A., Vol. 68, No. 5, p. 1056, May 1971.
2 *Salmon et al., The Journal of Immunology, Vol. 103, No. 1, July 1969, pp. 129 131.
3 *Salmon et al., The Journal of Immunology, Vol. 104, No. 3, March 1970, pp. 665 667.
Referenced by
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US3901870 *Mar 12, 1974Aug 26, 1975Behringwerke AgDerivative of alpha' 1'-fetospecific serum protein and process for its manufacture
US3999944 *Feb 28, 1975Dec 28, 1976Hoffmann-La Roche Inc.Detection of breast cancer
US4043757 *May 20, 1975Aug 23, 1977Ortho Pharmaceutical CorporationMethod for detection of human mammary carcinoma
US4132769 *Oct 28, 1975Jan 2, 1979Osther Kurt BAntisera and antibodies specific against a human cancer associated protein, immunology
US4152410 *Sep 1, 1976May 1, 1979Eisai Co., Ltd.Diagnosis reagent for neoplasm and method for diagnosis of neoplasm
US4349528 *Nov 21, 1979Sep 14, 1982The Wistar InstituteMonocolonal hybridoma antibody specific for high molecular weight carcinoembryonic antigen
US4379839 *Feb 29, 1980Apr 12, 1983The Trustees Of Columbia University In The City Of New YorkMethod for detecting cancer
US4624916 *Apr 6, 1984Nov 25, 1986International Immunoassay Laboratories, Inc.Process and composition for the rapid quantitation of small levels of creative kinase-MB isoenzyme
US4631254 *Sep 6, 1985Dec 23, 1986Hoffmann-La Roche Inc.Carcinoembryonic antigen determination
US5296377 *Dec 15, 1992Mar 22, 1994Boehringer Mannheim CorporationControl reagent containing a hydroxylamine or an antioxidant
US6013772 *Jun 6, 1995Jan 11, 2000Bayer CorporationAntibody preparations specifically binding to unique determinants of CEA antigens or fragments thereof and use of the antibody preparations in immunoassays
US6022958 *Jun 6, 1995Feb 8, 2000Bayer CorporationcDNAs coding for members of the carcinoembryonic antigen family
USB554039 *Feb 28, 1975Feb 24, 1976 Title not available
WO1981001469A1 *Nov 21, 1980May 28, 1981Wistar InstMonoclonal hybridoma antibody specific for high molecular weight carcinoembryonic antigen
Classifications
U.S. Classification436/539, 250/303, 435/967, 436/17, 436/18, 436/542, 436/543, 436/16, 436/813
International ClassificationG01N33/96, G01N33/574, G01N33/531
Cooperative ClassificationY10S436/813, Y10S435/967, G01N33/96
European ClassificationG01N33/96