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
Table 1. Composition of analyzed samples
Content B complex a
B complex b Folic acid c
(rag/5 fl oz)
Vitamin B 2
Vitamin B 6
Para-amino benzoic acid
a Bio-genics, Woodland Hills, CA
b The Kroger Co., Cincinnati, OH
~ Makers of KAL, Inc., Woodland Hills, CA
Similac, Ross Laboratories, Columbus, OH
* Contains numerous inorganic salts
Preparation of G6PDH-B12 Conjugate
A G6PDH-B12 conjugate containing approximately 6.4 B12 molecules per
enzyme was synthesized from monocarboxylic acid derivatives of B12 using
the procedure described in an earlier paper . This conjugate possessed
83% of its original enzymatic activity following the conjugation reaction.
Preparation of R-Protein Solid-Phase Reagent
R-protein solid-phase beads were prepared by covalently attaching
0.485 mg of R-protein to 900 mg of commercial CNBr activated Sepharose
4B particles according to the method suggested by the manufacturer .
Briefly, the activated gel was swollen and washed in 1 mM HC1 solution (in
order to remove dextran and lactose, which are added to the activated gel to
preserve its activity under freeze-drying). The protein to be coupled
was dissolved in coupling buffer (0.1 M NaHCO3, pH 8.3, 0.5 M NaC1)
and then added to the gel suspension and incubated for 2 h. Excess
protein was washed away and the remaining active groups in the
beads were blocked using glycine. After the coupling was completed
and the remaining active groups blocked, the excess blocking reagent
and adsorbed protein were washed away by alternatively washing the
beads with high and low pH buffer solutions. The solid-phase was
68 C.D. Tsalta et al.
finally resuspended in a 1 : 5 suspension (ratio of beads to total volume of
Tris-gel buffer) and stored at 4~ This suspension was further diluted
(1:66) with Tris-gel buffer for use as a reagent in the heterogeneous
competitive binding assay.
The solid phase binding beads could be stored for at least three months
without significant changes in their ability to bind G6PDH-B12 conjugates.
However, attempts to regenerate (by washing with an alkaline glycine
buffer, pH 12.9 ) the solid-phase after use in the B12 assays resulted in
beads that retained only 30% of their initial conjugate binding capacity.
Thus, fresh solid-phase reagent was always used for each B12 assay and to
obtain calibration data.
Heterogeneous Assays for Vitamin B12
The assays were carried out in a single incubation (equilibrium) mode. This
was accomplished by mixing 100#1 of the standard or sample solution with
100 yl of a conjugate solution and 200 #1 of an R-protein suspension with
400 r Tris-gel buffer. After an incubation period of 2.5 h, the beads were
centrifuged and washed three times with assay buffer. One hundred r of
each enzyme substrate, G6P (0.10 M) and fl-NAD + (0.063 M), and 800 j.tl of
assay buffer were then added and the tubes were incubated again for 2 h.
After this time, the beads were centrifuged and the absorbance of the super-
natant was measured at 340 nm.
Sample Preparations and Vitamin B12 Determinations
Infant formula. Samples of the liquid infant formula were used as is or
diluted 1 : 2 with Tris-gel buffer.
Vitamin tablets. For the folic acid with B12 vitamin preparation, 12 tablets
were ground and the amount equivalent to 3 tablets was weighed and mixed
with deionized H20 in a 50 ml centrifuge tube. The solution was shaken,
end to end, for 1 h at 4 ~ C. The suspension was then centrifuged four times
at 2400 rpm for 10 rain intervals. The supernatant was transferred, after
each centrifugation step, to a 100 ml volumetric flask. The volume was then
completed up to 100 ml with deionized H20. Several dilutions of the super-
natant were made with Tris-gel buffer. For the B-complex preparations
(with and without Vitamin C), 10 tablets were ground together and the
amount equivalent to one tablet was weighed and added to a 1000 ml volu-
metric flask. The volume was completed to mark with deionized H20 and
this solution was stirred for 24 h at 4 ~ C. From this sample solution, further
dilutions in Tris-gel buffer were prepared.
The resulting samples were stored at 4~
These samples were analyzed according to the heterogeneous assay
protocol described above, along with the standards. The unknown concen-
trations were estimated graphically from the calibration curve. Only the
steep portion of the dose response curve was used for analytical purposes.
and protected from light.
A Solid-Phase Enzyme-Linked Assay for Vitamin B12 69
For the recovery study, a concentrated infant formula solution was
spiked with 100 ~1, 500 r or 1500/.tl of a 3.5 • 10-7MB12 standard solution.
These spiked solutions were then diluted 1 : 5 with Tris-gel buffer. The rest
of the procedure was the same as for the analysis of the unspiked infant
Results and Discussion
Since the binding interaction of G6PDH-B12 and R-protein in solution
previously had been found to induce inhibition of enzymatic activity ,
we initially wanted to determine whether the immobilized form of R-protein
would yield an analogous effect. To investigate this, varying amounts of the
solid-phase R-protein reagent were incubated for 30 min with a given
amount of G6PDH-B12 conjugate (1.25 x 10 -9 M), in the presence of the
substrates G6P and fl-NAD +. After centrifugation, the absorbance of the
supernatant was measured and compared to that obtained for the same
experiment in the absence of solid-phase R-protein (full activity). While
nearly 30% inhibition was observed in the presence of excess (i. e., 30 r
absolute volume) solid-phase R-protein (compared to 65% for the same
conjugate when soluble R-protein is employed ) insignificant effects
(2--4% inhibition) were found when the amount of solid-phase R-protein
equalled that normally employed in the solid-phase enzyme-linked compet-
itive assay protocol (2--4/.tl; limiting reagent). Thus, we concluded that the
inhibition induced by the immobilized R-protein would not cause problems
when attempting to utilize G6PDH-Bn conjugates in devising a heteroge-
neous competitive binding assay for Ba2.
I I I
' ; ' s ; ,, ,;',;' b'
abs. voL of beods (pL)
Fig. 1. G6PDH-B12 conjugate (9.4x 10-1~ M) binding as a function of increasing immo-
bilized R-protein solid-phase (volume refers to settled volume of R-protein beads). Data
points are average of two determinations