Mikrochim. Acta [Wien] 1989, I, 65--73
y by Springer-Verlag 1989
A Solid-Phase Enzyme-Linked Assay for Vitamin B12
Catherine D. Tsalta*, Sara A. Rosario, Geun Sig Cha,
Leonidas G. Bachas**, and Mark E. Meyerhoff***
Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA
Abstract. A new solid-phase enzyme-linked competitive binding assay
for vitamin B12 (cyanocobalamin) is described. The assay is based on the
competition between analyte B12 molecules and a glucose-6-phosphate
dehydrogenase-vitamin B12 conjugate for a limited number of R-protein
binding sites immobilized on sepharose particles. After appropriate
incubation and washing steps, the enzyme activity bound to the solid-
phase is inversely related to the concentration of B12 in the sample.
Under optimized conditions, the method can detect B12 in the range of
3 x 10-1~ x 10 -8 M (using 100 #1 sample) with high selectivity over
other biological molecules.
Key words: vitamin B12 (cyanocobalamin), competitive binding assays,
enzyme-vitamin conjugates, vitamin tablet analyses.
Enzyme-immunoassays (EIA) are attractive alternatives to classical radio-
immunoassay procedures for the detection of biomolecules at trace levels.
Modern heterogeneous EIA methods are often based on the competition
between the analyte and enzyme-labeled analyte molecules for antibody
binding sites immobilized on a solid phase [1, 2]. After appropriate sepa-
ration and washing steps, the measured enzyme catalytic activity bound to
the solid support is inversely proportional to the concentration of analyte in
the sample. While selective antibodies have been the reagents most often
used to devise such methods, we recently demonstrated the advantages of
using immobilized natural binders [3--5] in place of antibodies, particularly
in cases where the preparation of antibodies toward the analyte is difficult.
We now report an extension of this new concept by describing a very simple
solid-phase enzyme-linked method suitable for the direct determination of
vitamin B12 (cyanocobalamin, CNCbl).
* Current address: Kallestad Lab., Inc., 200 Lake Hazeline, Chaska, MN 55318, USA
** Current address: Department of Chemistry, University of Kentucky, Lexington, KY
*** To whom all correspondence should be addressed
66 C.D. Tsalta et al.
The two bioanalytical methods most often used to detect B12 include
microbiological assays [6, 7] and radioassay competitive binding methods
[7--10]. The microbiological method is very slow (1--2 days) and yields
only semiquantitative values. The competitive binding technique involves
the use of a 57Co-cobalamin in conjunction with various vitamin B12
selective binders (R-protein, Intrinsic Factor, and TranscobalaminII)
insolubilized on solid supports [7--13]. While extremely sensitive, the
radioassay method is plagued by the need to use and dispose of radio-
Recently , we introduced a new homogeneous enzyme-linked assay
for vitamin B~2 based on the inhibition of glucose-6-phosphate dehy-
drogenase-vitamin B12 (G6PDH-Ba2) conjugates by soluble R-protein. This
homogeneous method was quite rapid and selective; however, even under
optimized conditions, the proposed assay could only detect B12 at levels
>10 nM. In the assay described here, we report results obtained when
using an immobilized form of R-protein in conjunction with similar
G6PDH-Bu conjugates. By employing a heterogeneous assay protocol, the
detection capabilities of the enzyme-linked competitive binding method are
improved significantly (down to 0.3 nM). The final assay is selective for B~2
and useful for the direct determination of B~2 in infant formula and vitamin
Enzyme activities were measured with a Gilford (Oberlin, OH) Stasar III Spectrophotometer
equipped with a vacuum-operated sampling system and temperature-controlled cuvette. The
cuvette chamber was maintained at 30 ~ C. The spectrophotometer was interfaced with a Syva
CP-5000 EMIT Clinical Processor.
Porcine R-protein (non-intrinsic factor), vitamin B12 , glucose-6-phosphate dehydrogenase
(G6PDH) (from Leuconostoc mesenteroides), as well as all other biochemicals were obtained
from Sigma Chemical Co. (St. Louis, MO).
Substrate solutions (glucose-6-phosphate, fl-NAD +) were prepared in 0.050M
Tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer, pH 7.8, containing
0.10 M NaC1 and 0.01% (w/v) NaN3 (assay buffer). Conjugates, standards, and binding
protein solutions were prepared in the same buffer also containing 0.1% (w/v) gelatin
(Tris-gel buffer). Gelatin was added to reduce non-specific adsorption of the enzyme-B12
conjugate or analyte B12 molecules onto the walls of the test tubes and the solid-phase R-
protein beads. The vitamin preparations and the infant formula analyzed were commercially
available and their manufacturers and compositions are listed in Table 1.
A standard stock solution of vitamin B12 was prepared by dissolving a given amount of
cyanocobalamin in assay buffer. The concentration of this stock solution was determined
spectrophotometrically using a molar extinction coefficient for CNCbl at 361nm,
E361 = 28060 1 mo1-1 cm -1. Standards solutions of B12 in the range of 10-11--10 6 Mwere
prepared by diluting this stock solution with Tris-gel buffer.
A Solid-Phase Enzyme-Linked Assay for Vitamin B12
carboxylic acid isomers of B12 [19, 20]), synthesis of new conjugates
prepared with only the "e"-monocarboxylic acid derivative of B12 , rather
than a mixture of monocarboxylic acids, will be required.
Acknowledgements. The authors gratefully acknowledge support of this work by the National
Science Foundation (Grant # 8506695).
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Received September I0, 1987. Revision December 7, 1987.