CH636206A5 - Process for the simultaneous determination of folic acid and/or tetrahydrofolic acid and also vitamin B12 by radioactivity measurement - Google Patents

Description

According to the present invention, a sample containing a folic acid and / or tetrahydrofolic acid and vitamin B12 is (30) with (1) a binder for folic acid and / or tetrahydrofolic acid, (2) a binder for vitamin B12, (3) a labeling substance, which consists of Folic acid, 5-methyltetrahydrofolic acid and analogs of folic acid is selected and which is labeled with a first radioactive isotope 35, and (4) brought into contact with a labeling vitamin B12, which is labeled with a second radioactive isotope different from the first , which results in a competitive bond between labeled and unlabeled folic acid and / or tetrahydrofolic acid and labeled and unlabeled vitamin B12 in relation to their respective binding sites. The bound proportions of marked and unmarked folic acid and / or tetrahydrofolic acid and the bound proportions of marked and unmarked vitamin B12 are simultaneously from the unbound 45 parts of marked and unmarked folic acid and / or tetrahydrofolic acid and from the unbound parts of marked and unlabeled vitamin B12, and then the radioactivity of at least one of the bound and unbound portions is measured, whereby the amounts of folic acid and / or tetrahydrofolic acid and vitamin B12 are determined, for example from reference curves.

For the purposes of the invention, folic acid and vitamin B12 can be released from binding to endogenous carriers 55 by heating a sample containing folic acid and vitamin B12, for example a serum or plasma sample, thereby destroying the endogenous carriers. The heating can be carried out in a wide range of pH values, the pH 60 being generally 5 or above 5. The pH is usually at least 7.0 (i.e. neutral or alkaline pH); the preferred pH is at least 9.0 and preferably not above 9.6. The most preferred pH is 9.2 to 9.4 (generally 9.3), since this value enables the release to be carried out at the same pH as the following determination, with folic acid instead of the reduced methyl derivative of folic acid as the reference substance is used. Heating to release the vitamin

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Bu and the folic acid and / or tetrahydrofolic acid from their binding to the binders is generally carried out at a temperature of 95 to 110 ° C., preferably 98 to 105 ° C.

The release of folic acid and vitamin B12 from their binding to endogenous carriers can also be carried out in the presence of a suitable reducing agent, as a result of which the endogenous folic acid present in the serum is kept in a reduced form. The reducing agent added during the heating is one which keeps the reduced folic acid in a reduced form without damaging the vitamin B12 and which is stable under the conditions of determination. Representative examples of suitable reducing agents include ascorbates, the reagent from Cleland (Dithiothreit), monothioglycol, thiodiglycol, thio-glycolic acid, cysteine, homocysteine, glutathione, mercapto-ethanol, reducing agents with a sulfhydryl group and inorganic reducing agents such as sodium sulfite sulfite Sodium sulfide and sodium metabi sulfite; the organic reducing agents are preferred.

After the folic acid and vitamin B12 have been released from their binding to endogenous carriers and the destruction of these endogenous carriers, the sample is treated with two different labeling agents, i. H. a marker for folic acid and / or tetrahydrofolic acid and a marker for vitamin B12, and with two different binders, i.e. H. a binder for folic acid and / or tetrahydrofolic acid and a binder for vitamin B12. The receptors for folic acid, both those that occur naturally and the antibodies, are well known to those skilled in the art, and each of these receptors can be used as a binder in the method of the present invention. Representative examples of such receptors include receptors or binders extracted from various animal organs, in particular the kidneys and pancreas, preparations containing β-lactoglobulin, cow's milk, dolphin serum, etc .; binders based on milk are preferred. Binders or receptors for vitamin B12, for example saliva, chicken serum, the intrinsic factor, etc., are also well known to the person skilled in the art; the intrinsic factor is preferred as the binder.

The labeling agent for folic acid and / or tetrahydrofolic acid is either folic acid (pteroylmonoglutaminic acid; PG) or the reduced 5-methyl derivative of folic acid (5-methyl-tetrahydrofolic acid; MTFA) or suitable analogues thereof, which contain a radioactive isotope, preferably one radioactive isotope of iodine. The term "marking agent" for folic acid and /

or tetrahydrofolic acid generally refers to the radiolabeled folic acid, the radiolabeled reduced methyl derivative thereof, and the radiolabeled analogues thereof. Because of its instability, the 5-methyl derivative is generally not used as a marking agent. The radiolabelled folic acid used as the labeling agent is preferably the radiolabelled folic acid, and the term "radiolabelled folic acid" includes the radiolabelled analogs thereof, i.e. H. folic acids substituted with a radiolabelled residue. The preferred radioactive isotope is a radioactive isotope of iodine, mostly J125, while the labeling agent is a folic acid that contains a substituent containing a radioiodine-labeled phenol or imidazole group, such as histidine, histamine, tyrosine or tyramine, with a radioactive one Isotope of iodine is substituted. A particularly preferred labeling agent is one in which the cx-carboxyl group of the glutamyl radical is substituted by tyrosyl or histidyl labeled with radioiodine; however, it is clear that the labeling agent can also be one in which the y-carboxyl group is substituted as described in US Patent No. 3989812.

The marker for vitamin B12 is a radioactively labeled 5 vitamin B12; the vitamin B12 is preferably marked with "Co".

According to the preferred embodiment of the method, the determination is carried out at an alkaline pH; thus the determination and release of folic acid and vitamin B12 from their binding are carried out at an alkaline pH, the release taking place at a temperature customary in this field; generally the temperature is 98 to 105 ° C. Of course, the release and the subsequent determination can be carried out at different pH values; however, the same pH values are used with advantage, because this eliminates the need to readjust the pH.

According to the particularly preferred embodiment of the determination according to the invention, the folic acid and the vitamin B12 are released from their binding to endogenous carriers at a pH in the range from 9.2 to 9.4, and the determination is then carried out at a pH of 9 , 2 to 9.4, the marker for the folic acid and / or tetrahydrofolic acid being in the form of a folic acid labeled with radioiodine and the folic acid labeled with radioiodine preferably being an analog of folic acid labeled with radioiodine, for example histidyl, histamyl substituted with radioiodine , Tyrosyl or Tyramyl, preferably Tyrosyl. The marker for vitamin B12 is a 30 "Co labeled vitamin B12. The use of the radiolabeled folic acid and the determination of the endogenous folic acid at the pH mentioned makes it possible to use folic acid instead of MTFA as a reference substance - as in US Pat 3988431. 35 It goes without saying that, according to the preferred embodiment, using the folic acid as reference substance, the release from the binding to the carrier substances can be carried out at a different pH, even if the determination is carried out at a pH of 9 , 2 to 9.4. ”Likewise, the determination and / or the release, if MTFA is used as the reference substance for the determination, can be carried out at pH values other than 9.2 to 9.4.

The bound and unbound portions can be separated by methods well known to those skilled in the art; in general, these portions are separated from one another using an adsorbent in the form of fine particles. The preferred adsorbent is activated carbon coated with dextran; however, it is clear that a wide variety of other adsorbents, such as ion exchange resins, inorganic adsorbents, etc., can be used to separate the bound and free portions. In addition, the determination can be carried out using the so-called «determination in solid phase» method; 55 the receptors for folic acid and / or tetrahydrofolic acid and vitamin B12 are previously applied or bound to a solid support, such as a test tube, or to an insoluble polymer as a coating, whereby the bound and free portions are easily separated 60 can. The methods for separating the bound and free portions, whether by adding an adsorbent present as fine particles or by using a receptor bound to a solid phase, are not the subject of the present invention.

65 After the bound and free fractions have been separated, the radioactivity of either the bound or the free fractions or the two fractions is determined, and the radioactivity determined is known to the person skilled in the art

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Methods, preferably compared with a reference curve. Since different radioactive isotopes are used to label the marker for folic acid and / or tetrahydrofolic acid and the marker for vitamin B12, it is clear that the respective marker can be measured in different channels of a counter or if the counter has only one channel , it can be set so that different counting settings only register one isotope at a time.

The invention is described in more detail by the following examples.

example

The following reagents are used in the dual determination:

1. Double marker

1.5 (J.CÌ a- (pteroylglutamyl) l25J-L-tyrosine (made as described in U.S. Patent Application No. 727,408) and 0.75 nCi Vitamin Bi2 [S7Co], human serum albumin, sodium borate, dextran and preservatives.

2. Double binder

Binder for the folic acid and / or tetrahydrofolic acid from cow's milk and pork intrinsic factor, both formulated to bind 55 + 15% marker (Bo), albumin from human serum, dextran and preservative.

3. Double reference substance, which contains albumin from human serum, sodium borate, sodium chloride and preservative.

3A double ref. Substance A zero value

3B double ref. Substance B 1.0 ng / ml folic acid; 100 pg / ml B12 3C double reference substance C 2.0 ng / ml folic acid; 200 pg / ml B12 3D double reference substance D 4.0 ng / ml folic acid; 400 pg / ml B12 3E double reference substance E 10 ng / ml folic acid; 1000 pg / ml B12 3F double reference substance F 20 ng / ml folic acid; 2000 pg / ml B12

4. pH 9.3 buffer consisting of 0.05 mol / liter sodium borate and 6.25 jig potassium cyanide / ml.

5. 5% solution of Cleland's reagent (dithiothreitol).

5A. Buffer for the determination

A mixture of 1 ml of the reagent mentioned under 5 (Cleland's reagent) and 50 ml of reagent 4.

6. Suspension of activated carbon coated with dextran. 4.4 ± 0.1 g dry mixture of dextran activated carbon (1:10), wetting agent and sodium chloride in 100 ml sterile distilled water.

Description of how it works

10th

Tube no.

Reference sub

Folic acid

Vitamin B12

punch

in ng / înl in pg / ml

3, 6

100 (j.1 A

0

0

7, 8

100 [Û B

1.0

100

9.10

100 never

2.0

200

11.12

100 m. 1 D

4.0

400

13.14

100 (j.1 E

10th

1000

15.16

100 nlF

20th

2000

4. Except for tubes 1 and 2, all tubes are covered loosely with a plastic stopper.

15 5. In addition to tubes 1 and 2, all tubes are heated in a glycerol or water bath at 100 ° C for 45 minutes.

6. The tubes are removed from the glycerol or water bath and cooled in running water to a temperature of 20-25 ° C; the determination must not be made until the tubes are at the temperature mentioned. All tubes are covered.

7. Add 100 µL of the double marker (reagent 1) to each tube and gently shake the contents of the tube by hand.

8. Add 100 [al of the double binder (Reagent 2) to each tube and gently shake the contents of the tube by hand.

Creating a reference curve 1.16 polypropylene tubes are numbered from 1 to 16.

2. The buffer for the determination (Reagent 5A) is added as follows:

Tubes 1, 2 3-16

3. The double reference substances (reagents 3A-3F) are added as follows and the tube contents are shaken gently for mixing.

Clinical regulations

1. For each clinical sample, two tubes made of polypropylene are numbered consecutively from number 17.

2. 1000 μl of the buffer for the determination (reagent 5A) are added to each tube.

Buffer 1600 M.1 1000 [X \

3. 100 | ml of sample from a patient is added to each tube of a pair of tubes, and the contents of the tube are gently shaken to mix.

7. Each tube will be 100 times twice

25 markers

(Reagent 1) is added and the tube contents are shaken gently by hand.

Tubes 1 and 2

30 are set aside until level 15 and kept at room temperature.

8. Tubes 5—16 become 100 | _ü des

35 double binder (reagent 2) are added, and the tube contents are shaken gently by hand.

From this point on, all tubes are

40 masses treated:

9. Incubate at room temperature for 45 minutes from the last time the binder was added. In order to avoid any influence of light, the frame containing the tubes is covered with an aluminum film.

45 lie covered or kept in the dark.

10. Add to tubes 3-16 and all tubes containing a sample from a patient (tubes # 17, 18, etc.) 0.4 ml of activated carbon coated with dextran. Nothing is added to tubes 1 and 2. The so-called reagent is “injected” into each tube in order to obtain a uniform suspension of the reaction mixture.

11. During 10 minutes from the time of the last addition according to step 10, the tubes are

55 temperature maintained.

12. The tubes are centrifuged for 15 minutes, preferably in the cold, with an acceleration of at least 1240 g. If a higher centrifugal force is used, shorter times may suffice.

60 13. Starting with number 3, a set of clean tubes is numbered consecutively.

14. The clear supernatant liquid from each tube is carefully decanted into the tube provided according to level 13 with the same number. You get one

65 maximum transfer by placing the tube edges together. Any slipping of the activated carbon into the tube intended for counting should be avoided. The residues consisting of activated carbon are discarded.

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15. The radioactivity in the supernatants and tubes 1 and 2 is measured for one or more minutes for each tube using a scintillation counter (gamma counter).

If the counter has 2 or more channels, it should be set to 5 so that the "25J" is counted in one channel and the "Co" in another channel. If the counter has only one channel, it should be set so that different Register one isotope at a time, in the latter case the tubes should be counted twice, once when the counter is set to l25J, the count values of the reference curve for folic acid and the count values of the samples are obtained, then when the counter is set to "Co, whereby the counts of the reference curve for vitamin Bn and the counts of the samples are obtained. If, when registering one isotope, the values obtained are overshadowed by the other isotope, the setting of the counter should be corrected accordingly. By reducing the channel width, such overshadowing by the other isotope can be reduced to a minimum or excluded.

The reference curve and the content of folic acid are calculated from the values obtained by counting 125J; the reference curve and the content information for vitamin B12 are obtained by counting the "Co. 25

Because the radioactivity of the labeling agent decreases over time, an increase in the counting time may be necessary; when using a sufficiently effective

However, the extension is not required. The volume of the marking agent used should correspond to that described in the present description of the method of operation; the volume of the marker must not be increased to compensate for the decrease in radioactivity. If the volume of the marking agent specified above is used, the binding of vitamin B12 to the respective binding agent remains essentially unchanged in the sample during the period of the measurement. If the described procedure is strictly applied, reproducible values are obtained.

The reference curve covers the range from 1.0 to 20 ng / ml for folic acid and the range from 100 to 2000 pg / ml for vitamin B12. A zero value (“blank”) consisting of tubes 3 and 4 is used in the calculation in order to take into account the correction caused by the count values of the background and the radioactive marking agent not adsorbed on the activated carbon.

The method according to the present invention offers a particular advantage in that it enables a simultaneous determination of folic acid and / or tetrahydrofolic acid and vitamin B12. In addition, it was unexpectedly found that, in accordance with the particularly preferred embodiments of the invention, both vitamin B12 and folic acid and / or tetrahydrofolic acid can be effectively released from their binding to endogenous carriers and can be effectively determined at an alkaline pH.

G

Claims (14)

636 206 1. A method for the simultaneous determination of folic acid and / or tetrahydrofolic acid and vitamin B12, characterized in that a sample containing (1) a binder containing a folic acid and / or tetrahydrofolic acid and vitamin B12 which have been released from their binding to endogenous carriers for folic acid and / or tetrahydrofolic acid, (2) a binder for vitamin B12, (3) a labeling substance which is selected from folic acid, 5-methyl-tetrahydrofolic acid and analogs of folic acid and which is labeled with a first radioactive isotope, and (4) a labeling vitamin B12, labeled with a second radioactive isotope different from the first, contacts the bound portions of folic acid and / or tetrahydrofolic acid and vitamin B12 from the unbound portions of folic acid and / or separates tetrahydrofolic acid and vitamin B12 and the radioactivity of at least one of the bound and unbound Measures proportions, whereby the content of both labeled folic acid and / or tetrahydrofolic acid and labeled vitamin B12 is determined. 2. The method according to claim 1, characterized in that the folic acid and / or tetrahydrofolic acid and the vitamin B12 are released from their binding to endogenous carriers by heating before contacting. 2nd PATENT CLAIMS 3. The method according to claim 2, characterized in that the release is carried out by heating at a pH of at least 7.0. 4. The method according to claim 3, characterized in that the release is carried out by heating at a pH of at least 9.0, but not higher than 9.6. 5. The method according to claim 4, characterized in that the release is carried out by heating at a pH of 9.2 to 9.4. 6. The method according to any one of the preceding claims, characterized in that the contacting is carried out at a pH of at least 7.0. 7. The method according to any one of the preceding claims, characterized in that the vitamin B12 is marked with 57Co. 8. The method according to any one of the preceding claims, characterized in that the substance used for labeling is a folic acid labeled with radioiodine. 9. The method according to any one of the preceding claims, characterized in that the substance used for labeling is a folic acid in which a carboxyl group of the glutamyl part is substituted by a substituent which contains a phenol or imidazole labeled with radioiodine. 10. The method according to any one of the preceding claims, characterized in that the substance used for labeling is a folic acid in which a carboxyl group of the glutamyl part is substituted by histidyl, tyra-myl, histamyl or tyrosyl labeled with radioiodine. 11. The method according to claim 10, characterized in that the carboxyl group of the glutamyl part is substituted by 125J-L-tyrosyl. 12. The method according to any one of the preceding claims, characterized in that the heating and the subsequent contacting are carried out at the same pH. 13. The method according to any one of the preceding claims, characterized in that the heating and the contacting are carried out at a pH of 9.2 to 9.4. 14. The method according to any one of the preceding claims, characterized in that the binder for vitamin B12 is a naturally occurring receptor thereof and the binder for folic acid and / or tetrahydrofolic acid is a naturally occurring receptor thereof. Endogenous folic acid is usually determined by a method based on competitive binding to proteins. In a nutshell, competitive binding to proteins (CPB) for the determination of folic acid presupposes the ability of unlabelled folic acid in blood serum or in other media with labeled folic acid in competition for a binder specific for folic acid that is found in sources such as cow's milk, Pig kidneys, etc., is present in usable concentrations, thereby inhibiting the binding of the 10 labeled folic acid. As a result of this competitive inhibition, the ratio of bound labeled folic acid to free labeled folic acid decreases as the concentration of unlabeled folic acid increases. Accordingly, the concentration of folic acid in an unknown sample, for example in a patient's serum, is obtained by comparing the measured inhibition with that caused by known amounts of folic acid and shown in a reference curve. Endogenous vitamin B12 is also determined by a similar method based on competitive binding to proteins; Pig intrinsic factor is generally used as a binder for vitamin B12. The subject of the present invention is therefore an improved method by radioactivity measurement, in which the folic acid and / or tetrahydrofolic acid and the vitamin B12, which are present in a sample, can be determined simultaneously.