Anal. Chem. 1987, 5 9 , 837-841
wavelengths) devices than in thick ones. For thick substrate devices, experimental evidence indicates that they will not prove useful as chemical sensors in liquids due to the enormous degree of Rayleigh wave attenuation. Finally, while it is clear that thin substrate SAW devices can propagate acoustic energy a t their design frequencies with liquids on their top surfaces and may be useful as chemical sensors, such devices likely do not operate by the same mechanism that has been put forth for gas-phase operation of thick substrate devices as chemical sensors.
ACKNOWLEDGMENT Helpful discussions with V. Ristic of the University of Toronto are gratefully acknowledged. We also thank H. van de Vaart and Janpu Hou of Allied Corporate Technology and R. Steinberg of Allied/Bendix for their comments and J. Kauffman of Allied/Bendix for her skillful fabrication of the devices.
LITERATURE CITED (1) (2) (3) (4) (5)
Konash, P. L.; Bastiaans, G. J. Anal. Chem. 1980, 35, 1929-1931. Nomura, T.; Minemura, A. Nippon Kagaku Kaishi 1980, 1261. Nomura, T.; Okuhara, M. Anal. Chim. Acta 1982, 742, 281-284. Bruckenstein, S.; Shay, M. €lechochim. Acta 1985, 30, 1295-1300. Hager, H. H.; Verge, P. D. Sens. Actuators 1985, 7 , 271-283.
837
(6) Kanazawa, K. K.; Gordon, J. G. Anal. Chem. 1985. 57, 1771-1772. (7) Kanazawa, K. K.; Gordon, J. G. Anal. Chim. Acta 1985, 775, 99-105. (8) Thompson, M.; Arthur, C. L.; Dhallwai, 0 . K. Anal. Chem. 1986, 58, 1206-1 209. (9) Thompson, M.; Dhaliwal, G. K.; Arthur, C. L.; Caiabrese, G. S. I€€€ Trans. UFFC, in press. (10) Hlavay, J. J.; Guilbauk, G. 0. Anal. Chem. 1977, 49, 1890-1898. (11) Giassford, A. P. M. J . Vac. Sci. Techno/. 1978, 75, 1836-1843. (12) Crane, R. A. and Fischer, 0. J . Phys. D . 1979, 72, 2019-2026. (13) Wohttjen, H. Sens. Actuators 1984, 5 , 307-325. (14) Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336. (15) White, R. M. R o c . I€€€ 1970, 58, 1238-1276. (16) Siobodnick. A. J. J . Appl. Phys. 1972, 4 3 , 2565-2568. (17) Campbell, J. J.; Jones, W. R. I€€€ Trans. Sonlcs Ulhason. 1970, SU-77,71-76. (18) Smith, W. R.; Pedler. W. F. I€€€ Trans. Microwave Theory Tech. 1975, MTT-23, 853-864. (19) Farneii. G. W. In Surface Wave Filters; Matthews, H., Ed.; Wiiey-Interscience: New York, 1977; Chapter 1, pp 17-27. (20) Milsom, R. F.; Redwood, M.; Reilly, N. H. C. I n Surface Wave Filters; Matthews. H.. Ed.; Wiley-Interscience: New York, 1977; ChaDter 2, pp 55-108. (21) Ristic, V. M. Principles of Acoustic Devices; Wiley-Interscience: New York, 1983; p 260. (22) Wagers, R. S. Proc. I€€€ 1978, 6 4 , 699-702. (23) Chuang, C. T.; White R. M.; Bernstein, J. J. I€€€ flecton. Device Lett. 19s:2 , EDL-3, 145-148.
RECEIVED for review June 24,1986. Accepted November 5, 1986. Instrumentation Laboratory provided financial support for this research.
Homogeneous Enzyme-Linked Assay for Vitamin B,, C. D. Tsalta and M. E. MeyerhofP Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
A rapld and sensnlve homogeneous enzyme-llnked competltlve blndlng assay for vitamin B,, Is descrlbed. The assay utlllzes a glucose6phosphatedehydrogenase-B,, conjugate In conjunctlon with R-protein, a naturally occurrlng blnder of B12. I n the absence of the vltamln, the catalytic activity of the enzyme-B,, conJugate Is lnhlblted to 360%. I n the presence of B,,, actMty Is regalned In an amount proportional to the concentration of vltamln B,, In the sample. Such reactlvatlon occurs over a very narrow B,, concentration range and this range can be controlled by varying the amount of blnder used In the assay tube. The extremely steep dose-response behavior provkles a yes/no type B,, detectlon system. The new assay Is shown to be preclse, accurate, selectlve, and useful for the dlrect measurement of B,, In vltamln tablets.
Enzyme-linked competitive binding assays involving selective antibody reagents are now routinely used to detect a wide variety of biomolecules (1-5). Homogeneous enzyme multiplied immunoassay techniques (EMIT) (6-8) rely on the ability of analyte-specific antibodies to inhibit the catalytic activity of enzyme-analyte conjugates in solution. In the presence of free analyte (from the sample), the activity of the enzyme is regained in an amount proportional to the concentration of the analyte present. Because no separation step is required, the resulting assays are simple and easily automated. Recently (9), we have examined the use of natural binding proteins in place of antibodies in such homogeneous assay arrangements. For example, when folate binding protein was
used in conjunction with glucose-6-phosphate dehydrogenase (G6PDH)-folate conjugates, a rapid and highly selective assay for folate was devised. The method was unique in that the response to folate occurred over an extremely narrow concentration range. Such steep dose-response behavior had not been observed previously for conventional antibody-based systems. Thus, we attributed such response characteristics to the fact that, unlike antibodies, natural binding proteins probably have somewhat greater affinity for the unconjugated analyte from the sample than the enzyme-labeled analyte molecules. Theoretical treatment of this case of unequal affinities under the conditions normally employed in homogeneous enzyme-immunoassays (high binder to conjugate ratios), predicts very steep dose-response curves (9). We now describe an enzyme-linked homogeneous assay for determination of vitamin B12(cyanocobalamin (CNCbl)) which also exhibits extremely steep dose-response behavior. The assay employs G6PDH-B12 conjugates with low degrees of ligand substitution (ca. seven vitamins per enzyme). These same conjugates are capable of being inhibited up to 77% in the presence of R-protein, a natural binder of B12. When standards or samples containing BIZare added to the assay mixture, the activity of the conjugates is regained over a very narrow concentration range of the vitamin and this range can be adjusted by merely changing the amount of reagents used in the assay tube. The final assay is shown to be rapid, precise, and selective enough to accurately detect vitamin B12 in multivitamin tablets.
EXPERIMENTAL SECTION Apparatus. Enzyme activities were measured with a Gil-
ford-Stasar I11 spectrophotometer equipped with a vacuum operated sampling system and a temperature-controlled cuvette
0003-2700/87/0359-0637$01.50/00 1987 American Chemical Society
838
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987 r-----1 CONH, b
Table I. Composition of Analyzed Multivitamin Tablets (Contents of Each Tablet) vitamin vitamin Bl vitamin B2 vitamin B6 vitamin B12 niacin niacinamide calcium d-pantothenate biotin folic acid vitamin C p-aminobenzoic acid choline bitartrate inositol
B complex with C" mg
5 mg 37.5 pg
0.2 mg
_ _ I _ _ -
I
\
J
- - _ - - - - - - --
co
CONH,
0.8 mg
150 mg 15 mg 50 mg 50 mg
"Wm. T. Thompson Co., Carson, CA.
Park, CA.
5 gg
,
\
m=
5 mg 45 mg 25 mg 15 wg
r--
folic acid with vitamin B12*
mg 3
-
II CH, '
.-
e NHCH2CHCH, I
?
"Bio-Genics, Canoga
I
__
(maintained a t 30 "C throughout the experiments). This spectrophctometer was connected to a Syva CP-5000 EMIT clinical processor for automatically setting the reading intervals and recording the absorbance values. A Cary 219 spectrophotometer with auto-base-line correction was used for recording the spectra of the GGPDH, vitamin B12and enzyme-Bls conjugates. Reagents. The solvents dimethyl sulfoxide (Me2SO) and N,N-dimethylformamide (DMF) were obtained from Aldrich (Milwaukee, WI). GGPDH from Leuconostoc nesenteroides, porcine R-protein (non-intrinsic factor), hog intrinsic factor (IF), vitamin B,, and all other biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and were of the highest purity available. The vitamin preparations analyzed were commercially available and their manufacturers and compositions are listed in Table I. Substrate solutions were prepared in 0.05 mol L-' Tris-HC1,O.lO mol L-' NaC1, 0.01% (w/v) NaN3, pH 7.8 buffer (assay buffer) whereas conjugates, standards, and binding protein solutions were prepared in the same buffer also containing 0.1% (w/v) gelatin (Tris-gel buffer). Preparation of G6PDH-Bl2 Conjugates. In order to attach vitamin B12to the enzyme, free carboxyl groups had to be created on an otherwise unreactive Blz molecule (Figure 1). Mild hydrolysis of B12was performed yielding a mixture of mono-, di-, and tricarboxylic acids (10,11). Only the three monocarboxylic acids (b, d, and e from Figure 1) resulting from the hydrolysis of the three propionamide chains of the molecule were isolated by ion-exchange chromatography according to the procedure of Anton et al. (12). This procedure is outlined in Figure 2. Further treatment of this mixture to obtain only the e-monocarboxylic acid of CNCbl (12) was not pursued since the heterogeneity of the derived conjugate preparation (containing a mixture of the three different monocarboxylic acid derivatives) should not be recognized by the binder (R-protein) used in this assay. Indeed, it is known that R-protein is not capable of differentiating among the three isomers (13-15). The G6PDH-B12 conjugates were prepared by a modified N-hydroxysuccinimide (NHS) ester method (see Figure 2) (16, 17). Reaction variables studied included initial ratios of hydrolyzed B12to enzyme, the pH of the conjugation buffer, and the reaction time. Each conjugate was characterized by the degree of conjugation (average number of haptens per enzyme molecule), by its residual enzymatic activity, and by the percent inhibition induced by excess R-protein. The degree of conjugation and the protein concentration of each conjugate were estimated from absorbance data at two wavelengths (230 nm for the enzyme and 360 nm for vitamin B12). Table I1 summarizes such data for 10 conjugates prepared under various conditions. Enzymatic Activity and Maximum Percent Inhibition Determinations. The rate of formation of NADH was used t o determine the activity of the enzyme-conjugates (GGPDH-BlJ. The assay consisted of adding 100 p L of @-NAD+solution (0.063 mol L-'), 100 pL of glucose-6-phosphate (G6P) solution (0.10 mol L-'i and 800 uL of assay buffer to a disposable 2-mL beaker cup
I
X =CN
Y=di met hyl benzi midazole Flgure 1.
Structure of cyanocobalamin (vitamin Bi2) molecule M
H
I
I
-
(
i
i l \
r \ rt I
Figure 2.
I i i k
(
3
1
1
I1
\
t l t r 5
?
I
I
\ I , )
P
T I
I,
~
Reaction sequence for preparation of GGPDH-B,, conju-
gates. containing 100 pL of the conjugate solution and 150 pL of Tris-gel buffer. The reaction mixture was quickly mixed and the absorbance values at 340 nm were recorded twice, after 20 and 80 s. The difference in absorbance over this 60-s inverval was used as a measure of the enzymatic activity. Maximum percent inhibition data (Table 11)for each conjugate were obtained as follows: 50 g L of an R-protein solution (5.3 X mol L-l) and 100 g L of Tris-gel buffer were substituted for the 150 pL of the %-gel buffer used above. In addition, a 40-min incubation interval preceded the addition of the substrate solution to the assay mixture. The percent inhibition (%I) was then calculated from the measured absorbance change (AA,)and from the absorbance data obtained at zero binder concentration (~I-4~1, using the following formula: %I =
A'% - Q
AAO
I
x 100
Association Time Study. Fifty microliters of R-protein solution (5.3 X lo-' mol L-') was incubated for varying time intervals with 100 pL of a solution of G6PDH-B12 conjugate B-4 (7.5 X mol L-l) and 100 pL of Tris-gel buffer. After this incubation step, the substrates were added and the enzymatic activity was determined as described above. Binding Protein Dilution Curve. Fifty-microliter portions of different concentration solutions of R-protein were incubated with 100 p L of conjugate B-4 and 100 pL of Tris-gel buffer for 20 min. Again, after the incubation, the substrates were added
ANALYTICAL CHEMISTRY, VOL. 59, NO.
6,MARCH 15, 1987
839
Table 11. Parameters of G6PDH-BI2 Conjugates initial B12Ienz molar ratio
degree of conjugation (Bdenz)
100/1
%
70
resid activity
inhibition by R-protein" 19 46 65 77
conj
conj rxn conditions
B-1 B-2 B-3
200/ 1 300/ 1 400/ 1
2.8 4.9 6.4 7.3
100
pH 9.4 7 h a t 4 "C
M-1 M-2 M-3
pH 7.3 7 h a t 4 "C
400/ 1 600/1 8OOjl
4.2 5.9 6.8
99 88 80
P-1
pH 1.3 18 h at 4 "C
400/1 600/1 800/1
4.3 5.8 6.6
87
B-4
P-2 P-3
"All conjugates were diluted so that their protein concentration was 7.5 X dilution of the coniueate solutions were used.
and the enzymatic activity was measured as outlined above. A binder dilution curve was constructed by plotting percent inhibition vs. amount of R-protein added. Preparation of Standard Solutions. Stock solutions of vitamin Blz as well as cobinamide dicyanide [(CN),Cbi] were prepared by dissolving a given amount of the substances in assay buffer. The concentrations of these stock solutions were determined spectrophotometricallyusing a molar extinction coefficient for (CN),Cbi at 367 nm, ES7 = 30680 L mol-' cm-', and for CNCbl at 361 nm, E'361= 28 060 L mol-' cm-' (23). Standard solutions were then prepared by diluting the stock solutions with Tris-gel buffer. Homogeneous Assays for Vitamin B12. The assays were performed by mixing 100 pL of the standard solutions of the vitamin with 100 pL of conjugate B-4 solution and 50 pL of R-protein solution for 20 min at room temperature. After this period, the enzyme activity was determined as described above. Dose-response curves were constructed by plotting percent inhibition vs. the logarithm of concentration of vitamin B12in the standards. Vitamin Tablets Analysis. Two tablets of the multivitamin preparation or five tablets of the folic acid with vitamin B12 preparation (see Table I) were weighed and mixed with 60 mL of cold distilled-deionizedwater for 5 min in a blender (18). After the total volume was brought up to 100 mL, the solution was shaken for 1h at 4 "C in order to fully extract the vitamin (18). The suspension was then centrifuged twice at 2400 rpm for 10-min intervals. Several dilutions of the final supernatant were made with Tris-gel buffer and these samples were analyzed according to the homogeneous assay protocol described above (in place of standards). The concentrations were determined graphically from prior calibration curves (taking into account dilutions) and are reported as the average of three determinations. Recovery Study. For this study, five folic acid plus vitamin B12tablets were ground with 420 pL or 840 KLof a 8.8 X lo4 mol L-' B12standard solution. The rest of the procedure was the same as for the analysis of the unspiked vitamin tablets. R E S U L T S AND DISCUSSION A host of competitive binding radioassays which employ several different B,, specific binders (IF, R-protein, and Transcobalamin-11) for determining vitamin Blz levels in biological samples have been described previously (19-24). While radioimmunoassay techniques have also been proposed (25,26), such methods have not gained wide use due to difficulties in preparing appropriate antibodies toward the B,, molecule. T o our knowledge, there have been no attempts to utilize enzyme labels in place of radiotracers (57C0)for devising either heterogeneous or homogeneous B12 assays. In the new homogeneous enzyme-linked competitive binding assay described here, we use R-protein as the B12binder. Although R-protein is not as selective as intrinsic factor in recognizing physiological forms of Blz, the former was chosen for this work because of its reduced cost and greater stability.
87 83 81
37
60 70 40 56 67
84 79
mol L-I. For the percent inhibition data, 100 pL of a 1:2
60
56
I/ , 0
10
20
30
40
Incubation t i m e , rnin
Figure 3. Time study of the association of the G6PDH-B,2 conjugate B-4 (0.6X lo-' mol L-' in assay tube) with R-protein (2.1 X mol L-' in assay tube). Incubation times varied from 0 to 40 min. Data points are means of duplicate measurements.
Further, for potential B12 determinations in multivitamin tablets, or foods, it will be shown that R-protein offers adequate selectivity. For any homogeneous enzyme-linked competitive binding method, the properties of the required enzyme-analyte conjugate reagent are quite critical. Therefore, in preliminary studies we prepared many G6PDH-Blz conjugates under a variety of reaction conditions. As shown in Table 11, higher initial B,,/enzyme ratios yield higher degrees of ligand substitution and greater conjugate inhibition by excess R-protein. It was also found that a greater degree of conjugation occurred by keeping the reaction mixture a t p H 9.4 rather than a t pH 7.3. All of the conjugates had very high residual activities (379% ; in comparison to an unconjugated enzyme-control of same protein-concentration). Conjugate B-4, with an estimated 7.3 BIzresidues per enzyme, and a maximum inhibition of 77% by R-protein, was used in all subsequent experiments. The association kinetics of the interaction of R-protein with conjugate B-4 were investigated. As shown in Figure 3, while 77% inhibition could be achieved after 40 min of incubation, >50% could be realized without any incubation period a t all (zero time = time necessary t o mix reagents and monitor enzyme activity). This suggests that the binding kinetics are fast enough to devise a very rapid B12 assay. However, for subsequent assays, we chose to use a preliminary incubation period of 20 min (which yields 2 3 7 0 % inhibition) so that a nearly maximum change in signal could be observed over the concentration range of B12 standards tested. In order to determine the optimum concentration of Rprotein to be used in competitive binding assays of B12,a binding protein dilution curve was constructed (Figure 4) by
840
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
D
U
c + 0
s
60 50
-
40
-
30 20
o
n 0
r
, 02
,
I
,
04
,
,
06
Amount of R-protein.
I
,
oa
I
I
I
I I2
u9
1
~
-
20 J
0
-1
i -a
-6
-7 Lop [CNCbl]
in stondordr.
l
o / -9
-8
-7
-6
-5
Log [CNCbll in 8tandardr. M
Flgure 4. Binding protein dilution curves obtained by incubating varying amounts (microgramsof R-protein in assay tube) of R-protein with two different dilutions of conjugate 8-4 (+, 1:400 dilution, corresponding to 0.3 X lo-’ mol L-’ in assay tube; 0,1:200 dilution, corresponding to 0.6 X lo-’ mol L-’ of conjugate in assay tube). The points on the curves are means of duplicate measurements.
40
-
H
Flgure 5. Typical dose-response curve for E,, plotted along with the f 1 standard deviation curves, calculated using quintuple measurements for each standard. The final reagent concentrations used in the assay tube were 0.3 X lo-’ mol L-l 8-4 conjugate and 4.2 X lo-’ mol L-’ R-protein. using two different concentrations of conjugate B-4. It can be seen that, a t low binder levels, for a given R-protein concentration, the more dilute conjugate is inhibited to a greater extent. However, as the concentration of R-protein increases, inhibition of both conjugate dilutions reaches the same saturation point. To achieve desirable sensitivity in the actual Blz assays, the 1:400 dilution of conjugate B-4 and 0.485 pg/tube of R-protein (4.2 X lo4 mol L-l based on a molecular weight of 92 500 for R-protein (27))were used in the initial analytical applications of the assay system (corresponds to 260% inhibition a t zero dose of B12). By use of the above concentration of R-protein and the 1:400 dilution of conjugate B-4, doseresponse to Blzstandards was determined. Figure 5 shows the typical calibration curve obtained under these conditions. As with most homogeneous enzyme immunoassays, inhibition of the G6PDH-Blz conjugate is lost as the concentration of analyte (BIZ)increases in the reaction mixture. However, the regeneration in activity occurs over a very narrow concentration range of Blz standards. This extremely narrow “ON-OFF” range makes it difficult to accurately prepare standards which fall on the steepest portion of the curve. Nonetheless, as also shown in Figure 5, this “ON-OFF” behavior is very reproducible. The standard deviation over the entire Blz concentration range tested varied from f1.2 to 3.0% inhibition, with an average value of 2.1 %. Calculations of these standard deviations of the percent inhibition values were carried out by use of the
Flgure 6. Effect of varying the R-protein concentration on the doseresponse curves for Elf. The concentration of 6-4 conjugate was fixed at 0.3 X lo-’ mol L- (1:200 dilution) in the assay tube. The final R-protein concentrations in the tube for each curve were as follows: (A) 1.1 X lo-’ mol L-’, (E) 4.2 X lo-’ mol L-’, (C) 1.1 X mol L-’, (D) 2.1 X lo-’ mol L-’. Data points are means of duplicate measurements. formula described by Bachas and Meyerhoff (9) and were based on quintuple measurements for each standard. From this curve, it is possible to estimate the degree of precision for concentration measurements of samples containing B12. For example, from Figure 5, an inhibition value of 53% would correspond to a concentration of B12in the sample of 1.25 X M with an associated relative precision value of ft4.9%. Obviously, the relative precision value in terms of concentration units would increase dramatically in regions where the dose-response curve flattens. The steep dcaeresponse curve shown in Figure 5 resembles those first obtained in our earlier folate work (9). Since conventional homogeneous enzyme immunoassay methods do not exhibit similar behavior, at that time, we attributed these unique dose-response characteristics to the use of natural binding proteins rather than antibodies in such assay arrangements. Theoretical models suggest that the steep assay curves should occur when the binder shows a higher association constant toward the free analyte than the enzyme-labeled analyte (9). This is likely to occur when the binder does not exhibit recognition of the bridge group which links the analyte to the enzyme label. Natural binding proteins are a class of binding reagents that should possess this property. The fact that this new BIZassay system also exhibits such “ON-OFF” type dose-response curves supports our original hypothesis. For the B,, assay, dose-response curves obtained with a fixed amount of conjugate can be moved to lower or higher detection limits, simply by varying the binder concentration. Figure 6 illustrates this point. As shown, a 4-fold reduction in binder concentration improves the detection limits by almost l decade. I t should be noted that reducing the binder concentrations also results in a lowering of the percent inhibition obtained and this appears to be the limiting factor in further improving the detection limits of the assay. This point can readily be seen by comparing curves A and D in Figure 6. Comparing these same curves also illustrates how decreasing the binder concentration, while lowering detection limits, reduces the steepness of the curves. With respect to selectivity, Figure 7 illustrates that when R-protein is used as the binder reagent, similar dose-response curves are obtained for B12 and (CN)&bi. The latter is a nonphysiologic analogue of the vitamin in which the 5,6-dimethylbenzimidazole ligand attached on position 6 on the cobalt atom (see Figure 1)has been replaced by a cyano group. In preliminary experiments, improved selectivity for CNCbl vs. dicyanocobinamidewas obtained when IF was used in place
ANALYTICAL CHEMISTRY, VOL. 59,
NO.6,
MARCH 15, 1987
841
60
In summary, we have reported on the development of a very 3 simple, precise, sensitive, and selective homogeneous assay -
50
-
40
-
30
-
20
-
10
-
O'
for vitamin Biz. While applied here only for the detection of Blz in vitamin tablets, we believe that these same reagents and detection principles could be adapted to automated flow technologies in a manner that would allow for the measurement of B,, on a continuous basis in cell culture media or other biological samples. Further, the very steep dose-response behavior exhibited by this assay could prove valuable in devising a simple yes/no type BIZdetection system for quality control of the vitamin BIZcontent in pharmaceutical and food products. Registry No. GGPDH, 9001-40-5;vitamin BI2, 68-19-9.
L
I
0,
LITERATURE CITED Figure 7. Dose-response curves for CNCbl(0) and (CN),Cbi (+) usin a 0.3 X lo-' mol L-' concentration of 5 4 conjugate and a 4.2 X 10-
8
mol L-' concentration of R-protein In the assay tube. Means of duplicate measurements are plotted.
Table 111. Vitamin Tablets Analysis pg o f v i t a m i n
BIZ per
tablet v i t a m i n tablet
B complex
with C folic acid w i t h BIZ
found"
claimed
37.9
37.5
5.1
5.0
Average of three determinations.
Table IV. Vitamin Tablets Recovery Study pg
of v i t a m i n B1,
v i t a m i n tablet
found"
added
% recovery
folic acid w i t h BIZ folic acid with B,,
10.7
10.0 5.0
107 109
5.4
Average o f three determinations.
of R-protein as the binder reagent. Indeed, intrinsic factor was found to homogeneously inhibit the GGPDH-B,, conjugates but to a lesser extent than R-protein (up to 50%). This may be due to the smaller size of the IF molecule (molecular weight of 50000-60000 vs. molecular weight of 92000-115000 for R-protein ( 2 7 , B ) )was well as to a partial loss of IF binding capacity attributable to instability of this protein in solution (20). Further, less total inhibition is likely due to the heterogeneity of the acid derivatives used for the preparation of the conjugates. (IF has lower binding affinities for certain of the derivatives (29-33).) The selectivity of the assay using R-protein as the binder was further evaluated by determining vitamin Blz content in multivitamin tablets. Two different vitamin tablet preparations were analyzed as described in the Experimental Section. Table I11 shows the results of such determinations. In view of the accuracy of these measurements and the complexity of the multivitamin preparation, it is apparent that even when R-protein is used as the binder the new assay exhibits high selectivity for Biz over most other vitamins. Recovery studies were also performed where the vitamin sample to be analyzed was spiked with two different volumes of vitamin B12standards. The amounts added (5 and 10 mg) could be recovered after analysis with sufficient accuracy (Table IV).
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RECEIVED for review September 29,1986. Accepted November 20, 1986. We gratefully acknowledge the National Science Foundation for supporting this work (Grant No. CHE8506695).
