CA1098015A - Simultaneous radioassay of folate and vitamin b in12 xx - Google Patents
Simultaneous radioassay of folate and vitamin b in12 xxInfo
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- CA1098015A CA1098015A CA295,736A CA295736A CA1098015A CA 1098015 A CA1098015 A CA 1098015A CA 295736 A CA295736 A CA 295736A CA 1098015 A CA1098015 A CA 1098015A
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- folate
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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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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Abstract
Abstract of the Disclosure A serum sample is heated at an alkaline pH to release folate and vitamin B12 from endogenous binders. A
simultaneous radioassay for folate and vitamin B12 is effected by contacting the sample with binder for folate, binder for vitamin B12, folate labeled with one radioactive isotope and vitamin B12 labeled with another radioactive isotope, followed by separation of bound and free portions, and determination of the radioactivity of at least one of the portions. The amounts of folate and vitamin B12 present in the sample may be determined from standard curves.
simultaneous radioassay for folate and vitamin B12 is effected by contacting the sample with binder for folate, binder for vitamin B12, folate labeled with one radioactive isotope and vitamin B12 labeled with another radioactive isotope, followed by separation of bound and free portions, and determination of the radioactivity of at least one of the portions. The amounts of folate and vitamin B12 present in the sample may be determined from standard curves.
Description
This invention relates to radioassay, and more particularly, to a radioassay for folate and vitamin Bl2.
Currently, endogenous folate is measured by a competitive protein-binding technique. In brief, competitive protein-binding (CPB) for the assay of folate involves the ability of unlabeled folate in serum or other media to compete with labeled folic acid for a specific folate binder, present in usable concentrations in such sources as cow's milk, hog kidney, etc., and thereby inhibit the binding of labeled folic acid. As a result of the competitive inhibition, the ratio of bound labeled folic acid to free labeled folic acid diminishes as the concentration of unlabeled folate is increased. Accordingly, the concentration of folate in an unknown sample; e.g., a patient's serum, is obtained by comparing the inhibition with that produced by known amounts of folate, as presented in a standard curve.
Endogenous vitamin Bl2 is also determined by a similar competitive protein-binding technique, with the vitamin Bl2 binder generally being hog intrinsic factor.
~ The present invention is direcked to an improved radioassay and reagents therefor whereby fola-te and vitamin B12 present in a sample may be determined simultaneously.
In accordance with one aspect of the present invention, a sample containing folate and vitamin Bl2 is contacted with a binder for the folate, a binder for the vitamin Bl2, a folate tracer labeled with a first radioactive isotope and a vitamin Bl2 tracer labeled with a second radioactive isotope different from the first radioactive isotope, resulting in competitive binding between the labeled and unlabeled folate and the labeled and unlabeled vitamin B12 for their respective binder sites. The bound portions of labeled and unlabeled folate and bound portions of labeled and ~mlabeled vitamin B,l2 are " ~.
Currently, endogenous folate is measured by a competitive protein-binding technique. In brief, competitive protein-binding (CPB) for the assay of folate involves the ability of unlabeled folate in serum or other media to compete with labeled folic acid for a specific folate binder, present in usable concentrations in such sources as cow's milk, hog kidney, etc., and thereby inhibit the binding of labeled folic acid. As a result of the competitive inhibition, the ratio of bound labeled folic acid to free labeled folic acid diminishes as the concentration of unlabeled folate is increased. Accordingly, the concentration of folate in an unknown sample; e.g., a patient's serum, is obtained by comparing the inhibition with that produced by known amounts of folate, as presented in a standard curve.
Endogenous vitamin Bl2 is also determined by a similar competitive protein-binding technique, with the vitamin Bl2 binder generally being hog intrinsic factor.
~ The present invention is direcked to an improved radioassay and reagents therefor whereby fola-te and vitamin B12 present in a sample may be determined simultaneously.
In accordance with one aspect of the present invention, a sample containing folate and vitamin Bl2 is contacted with a binder for the folate, a binder for the vitamin Bl2, a folate tracer labeled with a first radioactive isotope and a vitamin Bl2 tracer labeled with a second radioactive isotope different from the first radioactive isotope, resulting in competitive binding between the labeled and unlabeled folate and the labeled and unlabeled vitamin B12 for their respective binder sites. The bound portions of labeled and unlabeled folate and bound portions of labeled and ~mlabeled vitamin B,l2 are " ~.
-2~ i simultaneously separated from the unbound portions of labeled and unlabeled folate and unbound portions of labeled and unlabeled vitamin B12, followed by counting the radioactivity of at least one of the bound or unbound portions, with the amounts of folate and vitamin B12 then being determined from standard curves.
In accordance with another aspect of the present invention, there is provided reagent for use in effecting a simultaneous assay for folate and vitamin B12 which includes a binder ~or the folate, a binder for the vitamin B12, a folate tracer labeled with a first radioactive isotope and a vitamin sl2 tracer labeled with a second radioactive isotope -different from the first radioactive isotope, whereby a simultaneous assay for folate and vitamin B12 can be ef~ected by us~e of the reagent in a sample containing both folate and vitamin B12.
In accordance with the present invention, folate and vitamin B12 are released from t:heir endogenous binders by heating a sample, containing folate and vitamin B12, e~.g., a serum or plasma sample, with the endogenous binders being destroyed by such heating. The heating can be effected at a wide variety of pH values, with the pH generally being 5 or greater. The pH is most generally at least 7.0 (neutral or al~aline pH), with the pH preferably being at least 9.0 and preferably no greater than 9.~. The most preferred p~I is from 9.2 to g.4 ~generally 9.3) in that this permits the release to ~ be effected at the same pH as the subse~uent assay in which ; folic acid is employed as a standard, instead of the reduced methyl derivative of folic acid. The heating to release vitamin ~12 and folate from their binders is generally effected at a temperature of from 95C to 110C, and preferably of from 98C to 105C.
.~
The release of folic acid and vitamin B12 from their endogenous binders is also effected in the presence of a suitable reducing agent in order to preserve -the endogenous folate present in the serum in reduced form. The reducing agent which is included during the heating step is a reducing agent which maintains the reduced folate without adversely affecting the vitamin B12 and which is stable under the assay conditions. As representative examples of suitable reducing agents there may be mentioned: ascorbate, Cleland's reagent ~dithiothreitol); dithioerythritol, monothioglycol; thiodigly-col; thioglycollic acid; cysteine; homocysteine; glythathione, ~ ~ .
-3a-mercaptoethanol; sulfhydryl sodium dithionite; sodium sulfide, sodium metablsulfite, with the organic reducing agents being preferred.
After release of folic acid and vitamin 312 from their endogenous binders and destruction of such endogenous binders, the sample is contacted with a dual tracer; i.e., a tracer for folate and a tracer for vitamin B12, and a dual - binder; i.e., a binder for folate and a binder for vitamin B12.
The xeceptors, both naturally occurring and antibodies, for folate are well known in the art, and any one of such receptors may be employed in the assay of the present invention. As representative examples of such receptors, there may be mentioned: receptors or binders extracted from various animal organs, particularly kidneys and pancreas; ~-lactoglobulin preparations; cow's milk, dolphin serum and the like, with milk binder being preferred. Similarly, binders or receptors for vitamin B12 are known in the art, e.g., saliva, chicken serum, intrinsic factor, with the preferred binder being intrinsic factor.
;20 The folate tracer is either folic acid ~pteroylmon3-ylutamic acid) (PG[or the reduced 5-methyl derivative of folic acid, 5-methyl-tetrahydrofolic acid (MTFA)] or appropriate analogs thereo, laheled with a radioactive isotope, which is preferably a radioactive isotope of iodine. The term folate tracer generically refers to radiolabeled folic acid, the radiolabeled reduced methyl derivative and the radiolabeled analogs thereof. The 5-methyl derivative is generally not employed as a tracer as a result of the instability thereo.
The radiolabeled olate employed as a tracer is preferably radiolabeled folic acid, and the term radiolabeled folic acid includes the radiolabeled analogs thereof i.e., folic acid substituted with a radiolabeled radical. The preferred radioactive isotope is a radioactive isotope of iodine and most preferably I125 with the tracer being a folic acid including a substituent which includes a radioiodinated phenol or imidazole group such as histidine, histamine, tyrosine or tyramine, which is substituted with a radioactive isotope of iodine.
A particularly preferred tracer is one in which the ~-carboxyl group of the glutamyl moiety is substituted with radioiodinated tyrosyl or histidyl; however, it is to be understood that the tracer can also be one in which the y-carboxyl group is so substituted, as disclosed in U.S.
Patent No. 3,989,812.
The vitamin B12 tracer is preferably a radio-labeled vitamin B12, with the vitamin B12 preferably being labeled with 57Co.
In accordance with the preferred procedure, the assay is effected at an alkaline pH; i.e., the assay and release of folate and vitamin B12 from binders are both effected at alkaline p~, with the release being effected at temperatures known in the art; i.e., generally in the order or from 98C to 105C. It is to be understood that the release ana subse~uent assay could be effected at diferent pH values; however, identical pH values are preferably employed in that this eliminates the necessity for a pH adjustment.
In accordance with the most preferred procedure for effecting the assay, folate and vitamin B12 are released from their endogenous binders at an alkaline pH in the order of from 9.2 to 9.4 followed b~r effecting the assay at a pH of from 9.2 to 9.4 with the folate tracer being in the form of a radioiodinated folic acid, with the radioiodinated folic acid preferably being a radioiodinated analog of folic acid; e.g., radioiodo-substituted histidyl, histamyl, tyrosyl or tyramyl, preferably tyrosyl. The tracer for vitamin B12 is 7Co.
labelled vitamin B12. The use of radiolabeled folic acid and ~,' .
`'~. ib, ' ' ~'- ' ~ ' , assay of endogenous folates at such a pH permits the use of folic acid as a standard, instead of MTFA, as described in U.S~ Patent No. 3,988,431.
It is to be understood that in accordance with the preferred procedure wherein folic acid is employed as a standard, even though the assay should be effected at pH 9.2-9.4, the release from binder can be effected at another pH value.
Similarly, if the standard for the assay is MTFA, the assay and/or release can be effected at pH values other than 9.2-9.4.
The bound and unbound portions are separated by procedures known in the art, with such portions generally being separated by the use of a particulate adsorbent. The preferred adsorbent is dextran-coated charcoal; however, it is to be understood that any one of a wide variety oE other adsorbents, such as ion exchange resins, inorganic adsorbents, etc., may be employed for separating the bound and free portions. In addition, the assay may be effected by a so-called solid phase assay technique, wherein the receptors or folate and ~itamin sl? are previously coated on or bound to a solid O support, such as a test tube, or insoluble polymer, whereby the bound and free portions may be readily separated from each other. The techniques for effecting separation of bound and free portions, whether by the addition of a particulate adsorbent, or by the use of a receptor bound to a solid phase, forms no part of the present invention.
After separation of the bound and free portions, the radioactivity of either the bound or free portion or both portions is determined, and the determined radioactivity is compared with a standard curve, by procedures known in the art.
As should be apparent, since different radioactive isotopes are employed for labeling the folate tracer and vitamin B12 tracer, the respective tracers may be counted in different channels of a ~ 6 --counter or if the counter has one channel, it can be calibrated so that different counter settings will count one isotope at a time.
The invention will be further described with respect to the following example; however, the scope of the invention is not to be limited thereby:
EX~MPLE
The following reagents are used in the dual assay:
1. Dual Tracer 1.5 ~ Ci ~-(pteroylglutamyl)125 I-L-tyrosine and 0.75 ~ Ci vitamin B12 [57Co.~, human serum albumin, sodium borate, dextran and preservatives.
2. Dual Binder ~ Folate binder from bovine milk and hog intrinsic - factor, both formulated for a trace binding (Bo) of 55 + 15%, h~an serum albumin, dextran and preservatives.
In accordance with another aspect of the present invention, there is provided reagent for use in effecting a simultaneous assay for folate and vitamin B12 which includes a binder ~or the folate, a binder for the vitamin B12, a folate tracer labeled with a first radioactive isotope and a vitamin sl2 tracer labeled with a second radioactive isotope -different from the first radioactive isotope, whereby a simultaneous assay for folate and vitamin B12 can be ef~ected by us~e of the reagent in a sample containing both folate and vitamin B12.
In accordance with the present invention, folate and vitamin B12 are released from t:heir endogenous binders by heating a sample, containing folate and vitamin B12, e~.g., a serum or plasma sample, with the endogenous binders being destroyed by such heating. The heating can be effected at a wide variety of pH values, with the pH generally being 5 or greater. The pH is most generally at least 7.0 (neutral or al~aline pH), with the pH preferably being at least 9.0 and preferably no greater than 9.~. The most preferred p~I is from 9.2 to g.4 ~generally 9.3) in that this permits the release to ~ be effected at the same pH as the subse~uent assay in which ; folic acid is employed as a standard, instead of the reduced methyl derivative of folic acid. The heating to release vitamin ~12 and folate from their binders is generally effected at a temperature of from 95C to 110C, and preferably of from 98C to 105C.
.~
The release of folic acid and vitamin B12 from their endogenous binders is also effected in the presence of a suitable reducing agent in order to preserve -the endogenous folate present in the serum in reduced form. The reducing agent which is included during the heating step is a reducing agent which maintains the reduced folate without adversely affecting the vitamin B12 and which is stable under the assay conditions. As representative examples of suitable reducing agents there may be mentioned: ascorbate, Cleland's reagent ~dithiothreitol); dithioerythritol, monothioglycol; thiodigly-col; thioglycollic acid; cysteine; homocysteine; glythathione, ~ ~ .
-3a-mercaptoethanol; sulfhydryl sodium dithionite; sodium sulfide, sodium metablsulfite, with the organic reducing agents being preferred.
After release of folic acid and vitamin 312 from their endogenous binders and destruction of such endogenous binders, the sample is contacted with a dual tracer; i.e., a tracer for folate and a tracer for vitamin B12, and a dual - binder; i.e., a binder for folate and a binder for vitamin B12.
The xeceptors, both naturally occurring and antibodies, for folate are well known in the art, and any one of such receptors may be employed in the assay of the present invention. As representative examples of such receptors, there may be mentioned: receptors or binders extracted from various animal organs, particularly kidneys and pancreas; ~-lactoglobulin preparations; cow's milk, dolphin serum and the like, with milk binder being preferred. Similarly, binders or receptors for vitamin B12 are known in the art, e.g., saliva, chicken serum, intrinsic factor, with the preferred binder being intrinsic factor.
;20 The folate tracer is either folic acid ~pteroylmon3-ylutamic acid) (PG[or the reduced 5-methyl derivative of folic acid, 5-methyl-tetrahydrofolic acid (MTFA)] or appropriate analogs thereo, laheled with a radioactive isotope, which is preferably a radioactive isotope of iodine. The term folate tracer generically refers to radiolabeled folic acid, the radiolabeled reduced methyl derivative and the radiolabeled analogs thereof. The 5-methyl derivative is generally not employed as a tracer as a result of the instability thereo.
The radiolabeled olate employed as a tracer is preferably radiolabeled folic acid, and the term radiolabeled folic acid includes the radiolabeled analogs thereof i.e., folic acid substituted with a radiolabeled radical. The preferred radioactive isotope is a radioactive isotope of iodine and most preferably I125 with the tracer being a folic acid including a substituent which includes a radioiodinated phenol or imidazole group such as histidine, histamine, tyrosine or tyramine, which is substituted with a radioactive isotope of iodine.
A particularly preferred tracer is one in which the ~-carboxyl group of the glutamyl moiety is substituted with radioiodinated tyrosyl or histidyl; however, it is to be understood that the tracer can also be one in which the y-carboxyl group is so substituted, as disclosed in U.S.
Patent No. 3,989,812.
The vitamin B12 tracer is preferably a radio-labeled vitamin B12, with the vitamin B12 preferably being labeled with 57Co.
In accordance with the preferred procedure, the assay is effected at an alkaline pH; i.e., the assay and release of folate and vitamin B12 from binders are both effected at alkaline p~, with the release being effected at temperatures known in the art; i.e., generally in the order or from 98C to 105C. It is to be understood that the release ana subse~uent assay could be effected at diferent pH values; however, identical pH values are preferably employed in that this eliminates the necessity for a pH adjustment.
In accordance with the most preferred procedure for effecting the assay, folate and vitamin B12 are released from their endogenous binders at an alkaline pH in the order of from 9.2 to 9.4 followed b~r effecting the assay at a pH of from 9.2 to 9.4 with the folate tracer being in the form of a radioiodinated folic acid, with the radioiodinated folic acid preferably being a radioiodinated analog of folic acid; e.g., radioiodo-substituted histidyl, histamyl, tyrosyl or tyramyl, preferably tyrosyl. The tracer for vitamin B12 is 7Co.
labelled vitamin B12. The use of radiolabeled folic acid and ~,' .
`'~. ib, ' ' ~'- ' ~ ' , assay of endogenous folates at such a pH permits the use of folic acid as a standard, instead of MTFA, as described in U.S~ Patent No. 3,988,431.
It is to be understood that in accordance with the preferred procedure wherein folic acid is employed as a standard, even though the assay should be effected at pH 9.2-9.4, the release from binder can be effected at another pH value.
Similarly, if the standard for the assay is MTFA, the assay and/or release can be effected at pH values other than 9.2-9.4.
The bound and unbound portions are separated by procedures known in the art, with such portions generally being separated by the use of a particulate adsorbent. The preferred adsorbent is dextran-coated charcoal; however, it is to be understood that any one of a wide variety oE other adsorbents, such as ion exchange resins, inorganic adsorbents, etc., may be employed for separating the bound and free portions. In addition, the assay may be effected by a so-called solid phase assay technique, wherein the receptors or folate and ~itamin sl? are previously coated on or bound to a solid O support, such as a test tube, or insoluble polymer, whereby the bound and free portions may be readily separated from each other. The techniques for effecting separation of bound and free portions, whether by the addition of a particulate adsorbent, or by the use of a receptor bound to a solid phase, forms no part of the present invention.
After separation of the bound and free portions, the radioactivity of either the bound or free portion or both portions is determined, and the determined radioactivity is compared with a standard curve, by procedures known in the art.
As should be apparent, since different radioactive isotopes are employed for labeling the folate tracer and vitamin B12 tracer, the respective tracers may be counted in different channels of a ~ 6 --counter or if the counter has one channel, it can be calibrated so that different counter settings will count one isotope at a time.
The invention will be further described with respect to the following example; however, the scope of the invention is not to be limited thereby:
EX~MPLE
The following reagents are used in the dual assay:
1. Dual Tracer 1.5 ~ Ci ~-(pteroylglutamyl)125 I-L-tyrosine and 0.75 ~ Ci vitamin B12 [57Co.~, human serum albumin, sodium borate, dextran and preservatives.
2. Dual Binder ~ Folate binder from bovine milk and hog intrinsic - factor, both formulated for a trace binding (Bo) of 55 + 15%, h~an serum albumin, dextran and preservatives.
3. Dual Standards containing human serum albumin, sodium borate, sodium chloride and preservatives.
3A Dual Standard A Zero level 3B Dual Standard s 1.0 ng/ml Folic acid; 100 pg/ml sl2 2Q 3C Dual Standard C 2.0 ng/ml Folic acid; 200 pg/ml s 3D Dual Standard D 4.0 ng/ml Folic acid; 400 pg/ml B12 3E Dual Standard E 10 ng/ml Folic acid; 1000 pg/ml B
3F Dual Standard F 20 ng/ml Folic acid; 2000 pg/ml B12
3A Dual Standard A Zero level 3B Dual Standard s 1.0 ng/ml Folic acid; 100 pg/ml sl2 2Q 3C Dual Standard C 2.0 ng/ml Folic acid; 200 pg/ml s 3D Dual Standard D 4.0 ng/ml Folic acid; 400 pg/ml B12 3E Dual Standard E 10 ng/ml Folic acid; 1000 pg/ml B
3F Dual Standard F 20 ng/ml Folic acid; 2000 pg/ml B12
4. Buffer p~ 9.3, 0O05M sodium borate with 6.25~g potassium cyanide/ml.
5. Cleland's Reagent(dithiothreitol~ solution, 5%.
5A. Assay Buffer A mixture of 1 ml of Reagent 5 to 50 ml of Reagent :
4.
5A. Assay Buffer A mixture of 1 ml of Reagent 5 to 50 ml of Reagent :
4.
6. Dextran - coated charcoal suspension, 4.4 + 0.1 g dextran-charcoal dry mix (1:10) suspending agent and sodium chloride in 100 ml sterile distilled water. :
7 --.
~9~
PROTOCOL
Preparation of a Standard Curve Clinical Determinations 1. Number 16 polypropylene tubes 1. Starting with 17, con-sequentially from 1 - 16. secutively number two polypropylene tubes for each clinical sample.
2. Add assay suffer (Reagent 5A) 2. Add 1000~1 Assay Buffer as follows: (Reagent 5A) to each tube.
Tubes Buf~er 1,-2- 1600~1 3-16 1000~1 3. Add Dual Standards (Reagents) 3. Add 100~1 patient sample 3A 3F) as follows: to each of two tubes.
Mix gently.
Tube No. Standard Folic Acid Vitamin B
as nq/ml _ as pq/ml_l2 3,6 100~;ZA 0 0 7,8 100~1B 1.0 100 9,10 100~1C 2.0 200 11,12 100~1D 4.0 400 13,14 100~1E 10 1000 15,16 100~1F 20 2000 Mix gently.
4. Cover all tubes loosely with plastic caps (except 1 and 2).
5. Heat all tubes (except 1 and 2) in a glycerin or water bath at 100C for 45 minutes~
6. Remove all tubes from 100C bath. Cool to 20-25C in a running water bath~ Do not continue assay until the tubes are within this range. Uncap all tubes. .
7. Add 100~1 Dual Tracer 7. Add 100~1 Dual Tracer (Reagent 1) to all tubes. Mix (Reagent 1) to each gently by hand. Set tubes 1 tube. Mix gently and 2 aside at room temperature by hand.
until Step 15.
~9~
PROTOCOL
Preparation of a Standard Curve Clinical Determinations 1. Number 16 polypropylene tubes 1. Starting with 17, con-sequentially from 1 - 16. secutively number two polypropylene tubes for each clinical sample.
2. Add assay suffer (Reagent 5A) 2. Add 1000~1 Assay Buffer as follows: (Reagent 5A) to each tube.
Tubes Buf~er 1,-2- 1600~1 3-16 1000~1 3. Add Dual Standards (Reagents) 3. Add 100~1 patient sample 3A 3F) as follows: to each of two tubes.
Mix gently.
Tube No. Standard Folic Acid Vitamin B
as nq/ml _ as pq/ml_l2 3,6 100~;ZA 0 0 7,8 100~1B 1.0 100 9,10 100~1C 2.0 200 11,12 100~1D 4.0 400 13,14 100~1E 10 1000 15,16 100~1F 20 2000 Mix gently.
4. Cover all tubes loosely with plastic caps (except 1 and 2).
5. Heat all tubes (except 1 and 2) in a glycerin or water bath at 100C for 45 minutes~
6. Remove all tubes from 100C bath. Cool to 20-25C in a running water bath~ Do not continue assay until the tubes are within this range. Uncap all tubes. .
7. Add 100~1 Dual Tracer 7. Add 100~1 Dual Tracer (Reagent 1) to all tubes. Mix (Reagent 1) to each gently by hand. Set tubes 1 tube. Mix gently and 2 aside at room temperature by hand.
until Step 15.
8. Add 100~1 Dual Binder 8. Add 100~1 Dual Binder (Reagent 2) to tubes 5-16. (Reagent 2) to each Mix gently by hand. tube. Mix gently by hand.
From this point, all tubes are treated as follows:
From this point, all tubes are treated as follows:
9. Incubate at room temperature for 45 minutes from the time of the last addition of the binder. Cover the rack of tubes with aluminum foil to exclude light or keep in the dark.
10. Add 0.4 ml dextran-coated charcoal to tubes 3-16 and to all patient sample tubes (17, 18, etc.). Do not add to tubes 1 and 2. This reagent is "squirted" into each tube to obtain a uniform suspension in the reaction mixture.
11. Keep at room temperature for 10 minutes from the time of last addition in Step 10.
12. Centrifuge at a minimum of 1240 x g for 15 minutes, pre-ferably in the cold. Shorter times may be sufficient in equipment of higher centrifugal force.
13. Consecutively number a set of clean tubes, beginning with 3.
14. Gently decant each clear supernatent into the similarly numbered tube prepared in Step 13. Maximal transfer is obtained by hitting the rim together. Avoid decanting over any charcoal to the counting tube. Discard the charcoal residues.
15. Count the radioac~ivity in the supernatants and tubes 1 and 2 in sequence for one or more minutes with a scintillation (gamma) counter.
If the counter has two or more channels it should be calibrated to count [125I] in one channel and [57Co] in another channel. If the counter has one channel it should be calibrated so that different counter settings will count one isotope at a time. In the case of the latter, it will be necessary to count the tubes twice, once setting the counter for [125I] and obtaining the folate curve counts and sample counts, then setting the counter for [57Co] to obtain the Vitamin ~12 curve counter and sample counts. If in counting one isotope there is spillover from the other isotope, counter adjustments must be made. Decreasing channel wid-ths can minimize or eliminate such 9 _ . ~, ~
.
s spillover.
The folate curve and values are calculated from the counts obtained by counting ~125I]; the vitamin B12 curve and values are obtained by counting [57Co.].
As the radioactive tracer decays with age, increased counting times may be required. ~This will be unnecessary with efficient equipment.) The volume of tracer used must be that specified in this protocol; tracer volume should not be increased to compensate for decay. Binding of each vitamin to its respective binder remains essentially unchanged with time using the volume of tracer specified. Reproducible results will be obtained by following the protocol which has been described.
The Standard Curve covers the range of 1.0 to 20 ng/ml folate and 100 to 2000 pg/ml of Vitamin B12. A "Blank"
(tubes 3 and 4) is used to correct ~or background counts and radioactive tracer which is not adsorbed onto the charcoal.
The present invention is particularly advantageous in that it is possible to effect a simultaneous assay for folate and vitamin B12. Moreover, in accordance with the particularly preferred embodiments, Applicant unexpectedly found that vitamin B12 and folate can both be effectively released from their endogenous binders and effectivaly assayed at an alkaline pH.
~' .
:
_ ~Q _
If the counter has two or more channels it should be calibrated to count [125I] in one channel and [57Co] in another channel. If the counter has one channel it should be calibrated so that different counter settings will count one isotope at a time. In the case of the latter, it will be necessary to count the tubes twice, once setting the counter for [125I] and obtaining the folate curve counts and sample counts, then setting the counter for [57Co] to obtain the Vitamin ~12 curve counter and sample counts. If in counting one isotope there is spillover from the other isotope, counter adjustments must be made. Decreasing channel wid-ths can minimize or eliminate such 9 _ . ~, ~
.
s spillover.
The folate curve and values are calculated from the counts obtained by counting ~125I]; the vitamin B12 curve and values are obtained by counting [57Co.].
As the radioactive tracer decays with age, increased counting times may be required. ~This will be unnecessary with efficient equipment.) The volume of tracer used must be that specified in this protocol; tracer volume should not be increased to compensate for decay. Binding of each vitamin to its respective binder remains essentially unchanged with time using the volume of tracer specified. Reproducible results will be obtained by following the protocol which has been described.
The Standard Curve covers the range of 1.0 to 20 ng/ml folate and 100 to 2000 pg/ml of Vitamin B12. A "Blank"
(tubes 3 and 4) is used to correct ~or background counts and radioactive tracer which is not adsorbed onto the charcoal.
The present invention is particularly advantageous in that it is possible to effect a simultaneous assay for folate and vitamin B12. Moreover, in accordance with the particularly preferred embodiments, Applicant unexpectedly found that vitamin B12 and folate can both be effectively released from their endogenous binders and effectivaly assayed at an alkaline pH.
~' .
:
_ ~Q _
Claims (31)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An improved simultaneous assay for folate and vitamin B12 comprising:
contacting a sample containing folate and vitamin B12 released from endogenous binders therefor with a binder for the vitamin B12, a binder for folate, a folate tracer labeled with a first radioactive isotope and vitamin B12 tracer labeled with a second radioactive isotope different from the first radioactive isotope;
separating bound portions of folate and vitamin B12 from unbound portions of folate and vitamin B12; and counting the radioactivity of at least one of the bound and unbound portions to determine both folate tracer and vitamin B12 tracer.
contacting a sample containing folate and vitamin B12 released from endogenous binders therefor with a binder for the vitamin B12, a binder for folate, a folate tracer labeled with a first radioactive isotope and vitamin B12 tracer labeled with a second radioactive isotope different from the first radioactive isotope;
separating bound portions of folate and vitamin B12 from unbound portions of folate and vitamin B12; and counting the radioactivity of at least one of the bound and unbound portions to determine both folate tracer and vitamin B12 tracer.
2. A process according to claim 1 wherein, prior to the contacting, folate and vitamin B12 are heat released from endogenous binders therefor.
3. A process according to claim 2 wherein said heat release is effected at a pH of at least 7Ø
4. A process according to claim 3 wherein the vitamin B12 is labeled with 57Co.
5. A process according to claim 4 wherein the folate tracer is a radioiodinated folic acid.
6. A process according to claim 5 wherein the contacting is effected at a pH of at least 7Ø
7. A process according to any one of claims 2, 3 or 5 wherein the heat release and contacting are each effected at a pH of at least 9.0 and no greater than 9.6.
8. A process according to any one of claims 2, 3 or 5 wherein the heat release and contacting are each effected at a pH of from 9.2 to 9.4.
9. A process according to any one of claims 1, 3 or 6 wherein the folate tracer is folic acid in which a carboxyl group of the glutamyl moiety is substituted with a substituent which includes a radioiodinated phenol or imidazole.
10. A process according to any one of claims 1, 3 or 6 wherein the folate tracer is folic acid in which a carboxyl group of the glutamyl moiety is substituted with a member selected from the group consisting of radioiodinated histidyl, radioiodinated tyramyl, radioiodinated histamyl and radio-iodinated tyrosyl.
11. A process according to any one of claims 1, 3 or 6 wherein the folate tracer is folic acid in which a carboxyl group of the glutamyl moiety is substituted with 125I-L-tyrosyl.
12. A process according to any one of claims 2, 3 or 6 wherein the heat release and subsequent contacting are effected at the same pH.
13. A process according to any one of claims 1, 3 or 6 wherein the binder for vitamin B12 is a naturally occurring receptor and the binder for folate is a naturally occurring receptor.
14. An improved simultaneous assay for folate and vitamin B12 comprising:
heating a sample containing folate and vitamin B12 associated with endogenous binders therefor in the presence of a reducing agent which maintains reduced folate without adversely affecting vitamin B12 at a pH of at least 7.0, said heating being effected to a temperature to heat release folate and vitamin B12 from their endogenous binders;
contacting the sample at a pH of at least 7.0 with a binder for the folate, a binder for the vitamin B12, folate tracer labeled with a first radioactive isotope and vitamin B12 tracer labeled with a second radioactive isotope different from the first radioactive isotope;
separating portions of folate and vitamin B12 bound to the binders from unbound portions of folate and vitamin B12; and counting the radioactivity of at least one of the bound and unbound portions to determine both folate tracer and vitamin B12 tracer.
heating a sample containing folate and vitamin B12 associated with endogenous binders therefor in the presence of a reducing agent which maintains reduced folate without adversely affecting vitamin B12 at a pH of at least 7.0, said heating being effected to a temperature to heat release folate and vitamin B12 from their endogenous binders;
contacting the sample at a pH of at least 7.0 with a binder for the folate, a binder for the vitamin B12, folate tracer labeled with a first radioactive isotope and vitamin B12 tracer labeled with a second radioactive isotope different from the first radioactive isotope;
separating portions of folate and vitamin B12 bound to the binders from unbound portions of folate and vitamin B12; and counting the radioactivity of at least one of the bound and unbound portions to determine both folate tracer and vitamin B12 tracer.
15. The process of claim 14 wherein the folate tracer is radioiodine labeled folic acid and vitamin B12 tracer is radiocobalt labeled vitamin B12.
16. The process of claim 15 wherein the heating is effected at a pH of at least 9.0 and no greater than 9.6, and said contacting is effected at a pH of at least 9.0 and no greater than 9.6.
17. The assay of claims 14, 15 or 16 wherein the folic acid is labeled with a member selected from the group consisting of radioiodinated tyrosyl, radioiodinated tyramyl, radioiodinated histamyl and radioiodinated histidyl, said member being substituted on a carboxyl group of the glutamyl moiety.
18. The process of claims 14, 15 or 16 wherein the heating and subsequent contacting are effected at the same pH.
19. The process of claims 14, 15 or 16 wherein said heating and said contacting are effected at a pH of from 9.2 to 9.4.
20. The process of claim 16 wherein the folic acid is labeled with a member selected from the group consisting of radioiodinated tyrosyl, radioiodinated tyramyl, radioiodinated histamyl and radioiodinated histidyl, said member being substituted on a carboxyl group of the glutamyl moiety.
21. The process of claim 20 wherein said member is 125I-L tyrosyl.
22. Reagent for use in effecting a simultaneous assay for folate and vitamin B12, comprising:
a binder for the folate and a binder for the vitamin B12; and a folate tracer labeled with a first radioactive isotope and a vitamin B12 tracer labeled with a second radioactive isotope different from the first radioactive isotope, whereby a simultaneous assay for folate and vitamin B12 can be effected by use of said reagents in a sample containing both folate and vitamin B12.
a binder for the folate and a binder for the vitamin B12; and a folate tracer labeled with a first radioactive isotope and a vitamin B12 tracer labeled with a second radioactive isotope different from the first radioactive isotope, whereby a simultaneous assay for folate and vitamin B12 can be effected by use of said reagents in a sample containing both folate and vitamin B12.
23. The reagents of claim 22 wherein the folate and vitamin B12 binders are in admixture with each other to provide a dual binder separate from the folate tracer and the vitamin B12 tracer.
24. The reagents of claim 22 wherein the folate and vitamin B12 tracers are in admixture with each other to provide a dual tracer separate from the folate binder and the B12 binder.
25. The reagents of any one of claims 22, 23 or 24 and further including a buffer for maintaining a sample contain-ing both folate and vitamin B12 to be simultaneously assayed at a pH of at least 7.0 during said assay.
26. The reagents of any one of claims 22, 23 or 24 and further comprising a plurality of dual standards, each com-prising a premeasured standard for folate and a premeasured standard for vitamin B12.
27. The reagents of any one of claims 22, 23 or 24 and further comprising a reducing agent capable of maintaining reduced folate in a sample without adversely affecting vitamin B12 in the sample.
28. The reagents of any one of claims 22, 23 or 24 and further comprising a reducing agent capable of maintaining reduced folate without adversely affecting vitamin B12 in the sample and buffer means for maintaining a sample at a pH of at least 7Ø
29. The reagents of any one of claims 22, 23 or 24 wherein the folate binder is bovine milk binder and the vitamin B12 binder is hog intrinsic factor.
30. The reagents of any one of claims 22, 23 or 24 wherein the folate tracer is a radioiodinated folic acid and the vitamin B12 tracer is vitamin B12 labeled with 57Co.
31. The reagents of any one of claims 22, 23 or 24 wherein the folate tracer is (pteroylglutamyl)-125I-L-tyrosine, and the vitamin B12 tracer is vitamin B12 [57Co].
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US76299277A | 1977-01-27 | 1977-01-27 | |
| US762,992 | 1977-01-27 | ||
| US817,563 | 1977-07-21 | ||
| US05/817,563 US4146602A (en) | 1977-01-27 | 1977-07-21 | Simultaneous radioassay of folate and vitamin B12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1098015A true CA1098015A (en) | 1981-03-24 |
Family
ID=27117207
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA295,736A Expired CA1098015A (en) | 1977-01-27 | 1978-01-26 | Simultaneous radioassay of folate and vitamin b in12 xx |
Country Status (8)
| Country | Link |
|---|---|
| BR (1) | BR7800477A (en) |
| CA (1) | CA1098015A (en) |
| CH (1) | CH636206A5 (en) |
| DE (1) | DE2803154C2 (en) |
| FR (1) | FR2379068A1 (en) |
| GB (1) | GB1601094A (en) |
| IT (1) | IT1153998B (en) |
| SE (1) | SE7800974L (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL55816A (en) * | 1978-10-30 | 1982-04-30 | Ames Yissum Ltd | Method for simultaneous immunoassay of several different antibodies and a kit therefor |
| WO1980002076A1 (en) * | 1979-03-19 | 1980-10-02 | Diagnostic Techn Int Inc | Double tagged immunoassay |
| CA1148859A (en) * | 1979-06-14 | 1983-06-28 | Lacy R. Overby | Simultaneous assay of two hepatitis viruses using a solid phase |
| US4399228A (en) * | 1981-07-30 | 1983-08-16 | Corning Glass Works | Polate competitive protein binding assay |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3577511A (en) * | 1968-02-21 | 1971-05-04 | Adrian Leonard Luhby | Process and compositions for differential diagnosis of the megaloblastic anemia syndromes |
| US3952091A (en) * | 1974-10-23 | 1976-04-20 | Hoffmann-La Roche Inc. | Simultaneous multiple radioimmunoassay |
| US3988431A (en) * | 1974-12-12 | 1976-10-26 | Becton, Dickinson And Company | Radioassay of folates |
| US3981863A (en) * | 1975-02-25 | 1976-09-21 | Micromedic Diagonistics, Inc. | Cyanocobalamin derivatives |
-
1978
- 1978-01-25 DE DE2803154A patent/DE2803154C2/en not_active Expired
- 1978-01-26 SE SE7800974A patent/SE7800974L/en unknown
- 1978-01-26 CH CH88778A patent/CH636206A5/en not_active IP Right Cessation
- 1978-01-26 CA CA295,736A patent/CA1098015A/en not_active Expired
- 1978-01-26 GB GB3284/78A patent/GB1601094A/en not_active Expired
- 1978-01-26 IT IT19687/78A patent/IT1153998B/en active
- 1978-01-26 BR BR7800477A patent/BR7800477A/en unknown
- 1978-01-27 FR FR7802421A patent/FR2379068A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| IT1153998B (en) | 1987-01-21 |
| DE2803154C2 (en) | 1986-07-24 |
| DE2803154A1 (en) | 1978-08-17 |
| FR2379068A1 (en) | 1978-08-25 |
| SE7800974L (en) | 1978-07-28 |
| IT7819687A0 (en) | 1978-01-26 |
| BR7800477A (en) | 1978-08-29 |
| CH636206A5 (en) | 1983-05-13 |
| FR2379068B1 (en) | 1983-11-25 |
| GB1601094A (en) | 1981-10-21 |
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