Serum Vitamin B.sub.12 Assay and Kit Therefor

This invention relates to the release of serum vitamin B.sub.12 from endogenous binders therefor, and more particularly, to an improved assay and kit for vitamin B.sub.12.

Currently, vitamin B.sub.12 is measured by a competitive protein binding technique. In brief, competitive protein binding for the assay of vitamin B.sub.12 involves the ability of unlabeled vitamin B.sub.12 in serum or other media to compete with labeled vitamin B.sub.12 for a specific vitamin B.sub.12 binder, and thereby inhibit the binding of labeled vitamin B.sub.12. As a result of the competitive inhibition, the ratio of bound labeled vitamin B.sub.12 to free labeled vitamin B.sub.12 diminishes as the concentration of unlabeled vitamin B.sub.12 is increased. Accordingly, the concentration of vitamin B.sub.12 in an unknown sample; e.g., a patient's serum, is obtained by comparing the inhibition observed with that produced by known amounts of vitamin B.sub.12, as presented in a standard curve. In determining vitamin B.sub.12 in a serum, it is necessary to initially release the vitamin B.sub.12 from endogenous binders therefor. Currently, such a release is effected by heating the serum to elevated temperatures for a period of time; e.g., temperature in the order of about 100.degree. C. for about 15 minutes or longer. Such heating increases the length of the assay, necessitates the use of a water bath, and in addition requires cooling before the competitive protein binding technique can be initiated.

In accordance with one aspect of the present invention, serum vitamin B.sub.12 is released from endogenous binders therefor by use of a liquid releasing agent which includes an organic liquid, which does not destroy vitamin B.sub.12, a reducing agent which does not destroy vitamin B.sub.12, and cyanide ions.

In accordance with another aspect of the present invention, there is provided an improved assay for vitamin B.sub.12 in a serum wherein the vitamin B.sub.12 is released from endogenous binders by the use of a liquid releasing agent which includes an organic liquid which does not destroy vitamin B.sub.12, a reducing agent which does not destroy vitamin B.sub.12 and cyanide ions.

In accordance with a further aspect of the present invention there is provided a kit for the assay of vitamin B.sub.12 which includes vitamin B.sub.12 tracer, vitamin B.sub.12 binder and a liquid releasing agent for releasing vitamin B.sub.12 from endogenous binders which is comprised of an organic liquid which does not destroy vitamin B.sub.12, a reducing agent which does not destroy vitamin B.sub.12, and cyanide ions.

Applicant has found that by the use of a liquid releasing agent in accordance with the invention it is possible to release vitamin B.sub.12 from endogenous serum proteins without using the elevated temperatures heretofore required to effect heat release of the vitamin B.sub.12. Thus, vitamin B.sub.12 can be released from endogenous binder at temperatures lower than 100.degree. C.; e.g., it is possible to effect heat release at room temperature, although higher temperatures could be employed.

The organic liquids which are used in the B.sub.12 releasing agent of the present invention are preferably water miscible to facilitate the subsequent assay. In addition, the organic liquid should be one which when diluted in the subsequent assay does not materially affect the B.sub.12 binding capacity of the B.sub.12 binder employed in the assay; i.e., the binder is capable of providing sufficient B.sub.12 binding capacity in the presence of the assay diluted organic liquid to provide an effective B.sub.12 assay. The organic liquid is generally one of which is known in the art to be suitable for use as a water miscible organic solvent. As representative examples of suitable organic liquids, there may be mentioned: ketones, such as acetone; alcohols; in particular alkanols such as methanol, ethanol, etc., ethers, such as dioxane, tetrahydrofuran, etc.; and miscellaneous liquids such as dimethylformamide, dimethylsulfoxide, etc. It is to be understood that the releasing agent may include one or more of such organic liquids. The selection of a particular liquid is deemed to be within the scope of those skilled in the art from the teachings herein.

The reducing agent employed as one of the components of the vitamin B.sub.12 releasing agent may be either an organic or inorganic reducing agent which does not destroy vitamin B.sub.12. As representative examples of suitable reducing agents there may be mentioned: dithiothreitol, dithioerythritol, monothioglycol, thiodiglycol, thioglycollic acid, cysteine, homocysteine, glutathione, mercaptoethanol, sulfhydryl reducing agents or inorganic reducing agents such as sodium sulfite, sodium dithionate, sodium sulfide, sodium metabisulfite, with the organic reducing agents being preferred. The reducing agent component could be a reducing agent which is normally known to be suitable for the destruction of vitamin B.sub.12 in that, in some cases, in the amounts employed in the releasing agent of the present invention, such reducing agents do not destroy vitamin B.sub.12. As a result, such reducing agents are included within the scope of the present invention.

The cyanide ion component is provided by a suitable cyanide compound such as a cyanide salt; e.g., potassium cyanide.

The three components are employed in an amount effective to denature B.sub.12 endogenous serum proteins and to release vitamin B.sub.12 therefrom. In general, the ratio of reducing agent to organic liquid is from 0.00001:1 to 0.1:1, preferably from 0.00025:1 to 0.00075:1. The ratio of cyanide ion to organic liquid is generally from 0.00001:1 to 0.1:1, and preferably from 0.00025:1 to 0.00075:1 The selection of optimum amounts is deemed to be within the scope of those skilled in the art from the teachings herein.

The releasing agent may be employed as an aqueous solution of the organic liquid, reducing agent and cyanide ions. In such cases, the ratio of organic liquid to water is generally from 4:1 to 6:1.

The releasing agent is added to the serum in an amount effective to denature vitamin B.sub.12 -binding endogenous protein and to release vitamin B.sub.12 therefrom. In general, the releasing agent is added to provide a ratio of organic liquid to serum of at least 0.5:1, and preferably at least 1.28:1, with the ratio of organic liquid to serum generally being no greater than 10:1. The use of excess amounts of releasing agent may adversely affect the subsequent B.sub.12 assay, and as a result, the releasing agent is preferably not employed in quantities which are much greater than the effective minimum amount for release of vitamin B.sub.12 from the endogenous binder.

The serum is contacted with the releasing agent for a time sufficient to release vitamin B.sub.12 from endogenous binder. In general, the contacting time is about 15 minutes, although longer times could be employed. The vitamin B.sub.12 may be released from endogenous binder at room temperature, although higher temperatures could be employed; e.g., up to 37.degree. C.

The vitamin B.sub.12 releasing agent may be incorporated into a kit for the assay of vitamin B.sub.12 which includes, in addition to the releasing agent, a vitamin B.sub.12 binder and a vitamin B.sub.12 tracer. The kit may further include suitable standards, buffer(s) and an adsorbent for separating unbound B.sub.12 and B.sub.12 tracer, from B.sub.12 and B.sub.12 tracer bound to B.sub.12 binder.

The vitamin B.sub.12 binder may be either a naturally occurring binder or an antibody to vitamin B.sub.12, produced by a procedure known in the art for raising antibodies. As representative examples of natural binders, there may be mentioned: saliva, chicken serum, intrinsic factor, etc., with intrinsic factor being preferred. The selection of a suitable binder is deemed to be within the scope of those skilled in the art from the teachings herein.

The binder may be unsupported or supported on a suitable support for use in a so-called solid phase assay. Thus, for example, the binder may be included in the kit adsorbed on or covalently bound to a solid support; e.g., a tube, solid particles, sheet, etc.

The vitamin B.sub.12 tracer is vitamin B.sub.12 or appropriate analog thereof (one which is specifically bound by the B.sub.12 binder) labeled with a suitable "tag" or "label." The label or tag, as known in the art, may be a radioisotope, an enzyme or a fluorescent material. In general, the tracer is radiolabeled; in particular, .sup.57 Co. The tracer could also be a radioiodinated analog of vitamin B.sub.12 ; e.g., as disclosed in U.S. Pat. No. 3,981,863.

In accordance with the vitamin B.sub.12 assay, the serum is incubated with the releasing agent, preferably at room temperature for a time sufficient to denature the serum B.sub.12 endogenous binders and release vitamin B.sub.12 therefrom. Subsequently, the serum sample is diluted with a suitable buffer (the pH of the assay may vary over a wide range of pH), with such dilution minimizing the affect of the releasing agent components on the binder employed in the assay. The sample is contacted with vitamin B.sub.12 tracer and binder, and the unbound tracer and B.sub.12 is separated from the B.sub.12 and tracer bound to the B.sub.12 binder. In the case where the B.sub.12 binder is not supported on a solid support, the free B.sub.12 and free tracer may be separated by the use of a particulate adsorbent; e.g., dextran coated charcoal, ion exchange resin, etc.

The following example is illustrative of the invention; however, the scope of the invention is not to be limited thereby:

The following example of the assay is one in which the assay is performed at pH 9.3. It may also be done at any pH known in the art.

The following reagent kit is used in the assay.

1. Tracer

0.75 u Ci Vitamin B.sub.12 [.sup.57 Co], human serum albumin, sodium borate, dextran and preservatives.

2. Binder

Hog Intrinsic Factor formulated for a trace binding of 55.+-.15%, human serum albumin, dextran and preservatives.

3. Standards

Containing human serum albumin, sodium borate, sodium chloride and preservatives.

3A Standard A--Zero level

3B Standard B--100 pg/ml B.sub.12

3C Standard C--200 pg/ml B.sub.12

3D Standard D--400 pg/ml B.sub.12

3E Standard E--1000 pg/ml B.sub.12

3F Standard F--2000 pg/ml B.sub.12

4. Releasing Agent

82.5% Acetone: 17.5% water solution which contains 0.05% Potassium Cyanide and 0.05% dithiothreitol.

5. Buffer

pH 9.3 Sodium Borate with 6.25 .mu.g Potassium Cyanide/ml.

6. Dithiothreitol Solution 5%

6A. Assay Buffer

A mixture of 1 ml of Reagent 6 to 50 ml of Reagent 5.

7. Dextran Coated Charcoal Suspension

4.4.+-.0.1 G dextran charcoal dry mix (1:10), suspending agent and sodium chloride in 100 ml distilled water.

The Standard Curve covers the range of 100 to 2000 pg/ml of Vitamin B.sub.12. A "Blank" (tubes 3 and 4) is used to correct for background counts and radioactive tracer which is not adsorbed onto the charcoal.

This invention is particularly advantageous in that it permits release of vitamin B.sub.12 from endogenous binders without the necessity of heating to elevated heat releasing temperatures. The ability to effect such release at lower temperatures; e.g., room temperature, shortens the overall assay procedure, and it eliminates the previous heating and cooling step required during such assays.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.