956
Anal. Chem. 1966, 58,956-961
(19) Coon, R. L.; Lai, N. C. J.; Kamplne, J. P. J . Appl. Physiol. 1978, 4 0 , 625-629. (20) Neumark, J.; Bardeen, A.; Sulzer, E.; Kamplne, J. P. J. Neurosurg. 1975. 4 3 , 172-176. (21) Ollevler, C. N.; Berkenbosch, A.; QuanJer, P. H. H. Pflugers Arch. 1978, 373, 269-272. (22) Opdycke, W. N.; Parks, S. J.; Meyerhoff, M. E. Anal. Chim. Acta 1983, 155, 11-20. (23) Schulthess, P.; Shljo, Y.; Pham, H. V.; Pretsch, E.; Ammann, D.; SImon, W. Anal. Chim. Acta 1981, 131, 111-116. (24) Jensen, M. A.; Rechnitz, G. A. Anal. Chem. 1979, 51, 1972. (25) Guilbault, G. G.; Czarnecke, J. P.; Rahni, M. A. N. Anal. Chem. 1985, 5 7 , 2110-2116. (26) Meyerhoff, M. E.: Fratlcelll, Y. M.;Greenberg, J. A.: Rosen, J.; Parks,
(27) (28) (29)
(30)
S. J.; Opdycke, W. N. Clin. Chem. (Winston-Salem, N . C . ) 1982, 28, 1973-1978. Funck, R. J. J.; Morf, W. E.; Schultess, P.; Ammann, D.; Simon, W. Anal. Chem. 1982, 5 4 , 423-429. Anker, P.; Ammann, D.; Simon, W. Mlkrochim. Acta 1983, I , 237-242. Vanura, P.; Kuca, L. Collect. Czech. Chem. Commun. 1978, 4 1 , 2857-2877. Ross, J. W.; Rlseman, J. H.; Krueger, J. A. Pure Appl. Chem. 1973, 36, 473-487.
RECEIVED for review September 13,1985. Accepted November 25, 1985.
Homogeneous Enzyme-Linked Competitive Binding Assay for the Rapid Determination of Folate in Vitamin Tablets Leonidas G. Bachas and Mark E. Meyerhoff* Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
A rapid and sensltlve homogeneous enzyme-llnked competltive blndlng assay for folate that exhlblts unique dosereponse characteristics Is descrlbed. The method utlllzes a hlghly substituted glucose 6-phosphate dehydrogenase-folate conjugate In conjunctlon wlth folate blndlng proteln. I n the absence of folate, the enzyme conjugate Is inhiblted up to 70 %, I n presence of folate, actlvlty Is regalned In an amount proportlonal to the folate concentration. Such reversal occurs over an extremely narrow concentratlon range and thls range can be flne tuned to deslred values by varylng the concentratlon of folate blndlng proteln and/or the enzyme-folate conjugate. Theoretical models suggest that thls unusual behavior can be attributed to the favorable equilibrium constants between folate bindlng proteln and conjugate. The new assay Is shown to be preclse, accurate, selectlve, and useful for the measurement of folate In vitamin preparatlons. Speculation as to the advantageous use of thls novel assay in a yes/no type quality control situation is also presented.
The use of enzyme labels rather than radiotracers in the development of competitive binding assays for important biological molecules is a rapidly emerging area of bioanalytical research (1-3). The technique exploits the inherent chemical amplification of an enzyme-catalyzed reaction along with the selective molecular binding characteristics of certain proteins to achieve assays with high specificity and low detection limits. When antibodies are utilized as the binding reagent, the resulting methods are termed enzyme immunoassays (EIAs). Such methods overcome many of the problems associated with the more classical radioimmunoassay procedures (RIA) and are now being employed routinely in clinical chemistry laboratories (4). Enzyme immunoassays may be either heterogeneous (separation required) or homogeneous (no separation step). The homogeneous type methods are preferred since they often result in much faster assays. Ngo and Lenhoff (5) have reviewed the host of homogeneous EIA arrangements that have been proposed over the past decade. The most widely used approach is still the EMIT type assay pioneered by Ruben-
stein et al. (6) for the detection of haptenic species (low molecular weight drugs, hormones, etc.). In this case, the binding of hapten-enzyme conjugates by antihapten antibodies causes a modulation of the enzyme activity in solution. Usually, the modulation involves inhibition of enzyme activity and this inhibition is reversed in an amount proportional to the concentration of analyte in the sample (7, 8). Glucose 6-phosphate dehydrogenase (GGPDH) has been the enzyme label most often employed and a wide assortment of simple drug assays based on this system are commercially available from Syva Corp. (Palo Alto, CA). To date, antibodies have been the binders exclusively used in the EMIT type of assays. However, it is known that antibodies can exhibit considerable binding recognition toward the bridging group, which couples the hapten molecules to the enzyme label. This is particularly true when the same bridge is used to prepare the protein-hapten conjugate employed to elicit antibody production (9-11). Thus, the antibodies that form have often greater affinity for the enzyme labeled hapten than the unlabeled hapten and this can seriously diminish the detection capabilities (detection limits, steepness of dose-response curve, ED50 value, etc.) of the EMIT assays. While the effects of such bridging group recognition on the analytical properties of heterogeneous EIAs have been studied experimentally (9-1 I ) and, more recently, theoretically (12),such effects on EMIT type of assays have received only limited attention (13-15). We recently reported on the use of endogenous binding proteins rather than antibodies in the development of heterogeneous enzyme-linked competitive binding methods for folate, cyanocobalamin, and thyroxime (16,17). Since binding proteins do not recognize the bridging group, it was our belief that assays with improved analytical characteristics would result. Indeed, the folate assay (16) exhibited excellent detection limits, high sensitivity (Le., steepness of the descending portion of the dose-response curve), and a rather unique biphasic (hooked) dose-response curve. These characteristics were attributed to the binding properties of the immobilized folate binding protein. The purpose of this report is to extend this concept by describing our findings when folate binding protein (FBP) is utilized in solution (nonimmobilized) along with highly substituted GGPDH-folate conjugates in an EMIT
0003-2700/88/0358-0956$01,50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
Table I. Composition of Analyzed Multivitamin Tablets (Contents of Each Tablet)
vitamin vitamin B1 vitamin B2 vitamin B, vitamin B12 niacin niacinamide calcium &pantothenate biotin folic acid vitamin C p-aminobenzoic acid choline bitartrate inositol
folic acid B complex stress B with vitamin with C" complexb B12' 5 mg 5 mg
5 mg 37.5 pg 5 mg 45 mg 25 mg 15pg 0.2 mg 150 mg 15 mgd 50 mgd 50 mgd
5 mg
5 mg 5 mg 12.5 pg 50 mg
5 pg
50 mg
12.5pg 0.2 mg 75 mg 15 mg 50mg 50mg
0.8 mg
Wm. T. Thompson Co., Carson, CA. bBio-Genics,Woodland Hills, CA. Bio-Genics, Canoga Park, CA. No special claims are made for this content. type assay arrangement for folate. Folic acid is a vitamin required by living organisms for the normal red blood cell formation and it functions mainly as a cofactor in reactions involving one carbon transfer for purine and pyrimidine synthesis (18,19). Besides laborious microbiological assays (20),folate has been determined in multivitamin, preparations by a variety of methods, including colorimetric (21),HPLC (22),and differential pulse voltammetry (23). These techniques suffer varying degrees of interference by other vitamins or compounds present in the preparations, Consequently, prolonged extraction procedures are often required to partially or totally isolate folate before the analysis. The homogeneous method reported here can directly quantitate folate at levels less than lo-' mol L-l, which is suitable for the measurement of folate in multivitamin preparations. The new assay method is fast (a single test can be run from start to finish in 1 2 min) and requires minimal pretreatment of the sample. More importantly, the assay displays an unusually steep dose-response curve over a very narrow concentration range. The reasons for this unique dose-response behavior are examined in terms of the relative association constants for the reagents involved. Further, the implications of this effect are discussed in terms of the potential use of this assay in quality control situations. EXPERIMENTAL SECTION Apparatus. Enzyme activities were determined with a Gilford Stasar I11 spectrophotometer equipped with a vacuum-operated sampling system and a temperature-controlled cuvette (thermostated at 30 OC throughout the experiments). This spectrophotometer was interfaced with a Syva CP-5000 EMIT clinical processor (set on delay and measurement times of 30 s). A Syva pipettediluter (Model 1500)was used for pipetting the conjugate, binder, standards, and samples for the assays. It was set so that in all cases 50 p L of solution was pipetted and diluted with 100 pL of assay buffer (0.050 mol L-' Tris-HC1,O.lO mol L-l NaC1, 0.01% NaN,, pH 7.8). A Cary 219 spectrophotometer with auto-base-line correction was employed to record the spectra of the enzyme, haptens, and enzyme-folate conjugates. Reagents. Glucose 6-phosphate dehydrogenase from Leuconostoc rnesenteroides, FBP from bovine milk, and all other chemicals 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 compositions (given by the manufacturers) are shown in Table I. The dilutions of conjugate, binder, standards, and samples were made using assay buffer containing 0.10% gelatin (Tris-gelatin buffer). The dilutions of DL-5-methyltetrahydrofolicacid (MTHFA) were prepared with assay buffer containing 0.10% gelatin and 0.20% sodium ascorbate (Tris-ascorbate buffer).
957
Table 11. Parameters of Prepared Conjugates folate/enzyme degree of conjugaconjugate initial tion conj-23 conj-24 conj-25
200 400 800
3.1
conjugate % % concn,O residual inhibimol L-1 activity tionb 1.9 X 10" 2.2 x 104 2.2 X lo4
9.9 19.6
83 55 21
21
36 70
=Refer to protein concentration of the conjugate preparation (corresponds to a starting G6PDH content of 100 units). bTo determine the percent inhibition an excess of FBP was used with 1:200, 1:200, and 1:20 dilutions of the preparations of conj-23, coni-24, and coni-25, respectively. Preparation of GGPDH-Folate Conjugates. The G6PDHfolate conjugates were synthesized starting with folic acid and using a modified N-hydroxysuccinimide ester method (15, 16). Each conjugate was characterized by the degree of conjugation (average number of ligands per enzyme molecule) and by the residual enzymatic activity. The degree of conjugation and the protein concentration of the conjugate preparation were estimated from calculations involving absorbance measurements at two wavelengths (230 nm and 279 nm) (24). The residual enzymatic activity was calculated by comparing the activities of a conjugated enzyume to that of a solution containing unconjugated enzyme of the same protein concentration. Highly substituted conjugates were specifically prepared and tested for this work. The various parameters associated with their preparation as well as the corresponding degree of conjugation and percent residual activity are given in Table 11. Enzymatic Activity Determination. The rate of appearance of NADH was used to determine the activity of the G6PDH enzyme conjugates. The assay involves addition of 600 pL of a mixed @-NAD+and glucose 6-phosphate (G6P) substrate solution (0.010 mol L-' @-NAD+and 0.013 mol L-l G6P in assay buffer) to a 2-mL disposable beaker cup containing 50 pL of the conjugate solution (diluted with 100 p L assay buffer) and 100 pL (two times 50 pL) Tris-gelatin buffer (diluted with 200 p L (two times 100 p L ) assay buffer). After the solution was mixed, the absorbance at 340 nm was measured with the Gilford spectrophotometer. Measurements were taken after 30 s and 60 s and the enzymatic activity was calculated as the difference of these two absorption values. When a 1:200 dilution of conjugate was used, the enzymatic activity was calculated as the net change in absorbance measured after 30 s and 4.5 min. Association Study. A volume of 50 p L of a solution containing FBP in Tris-gelatin buffer (diluted with 100 pL assay buffer) was incubated for varying periods of time with 50 p L of an appropriate dilution of the enzyme conjugated ligand (diluted with 100 pL assay buffer) and 50 pL of Tris-gelatin buffer (diluted with 100 pL assay buffer). After this incubation 600 p L of the substrate solution was added and the enzymatic activity of the reaction mixture was measured as above. Binding Protein Dilution Curve. In order to determine the optimum concentration of the binding protein to be used in the assays, 50-pL portions of solutions containing different concentrations of FBP in Tris-gelatin buffer (diluted with 100 p L assay buffer) were incubated with 50 fiL of an appropriate dilution of the enzyme conjugated ligand (diluted with 100 pL of assay buffer) and 50 pL of Tris-gelatin buffer (diluted with 100 pL of assay buffer). After 10 min of incubation at room temperature, 600 p L of the substrate solution was added and the enzymatic activity was measured as above. A binder dilution curve was prepared by plotting percent inhibition vs. the amount of binder used. The percent inhibition (I)was calculated from the measured activity in the assay (AA3 and the activity of the conjugate at zero binder concentration (AA,) as follows: I=
AAo - AAf AAO
x 100
Preparation of Standard Solutions. A stock solution of folate was prepared by dissolving a given amount of folic acid in
958
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
Tris-gelatin buffer. A stock solution of MTHFA was prepared by dissolving the contents of a 5-mg ampule of the sodium salt of MTHFA in 5 mL of Tris-ascorbate buffer. The concentration of the stock solution was estimated spectrophotometrically from a 1:lOO dilution of this solution in 0.10 mol L-l phosphate buffer pH 7.0 (ena = 31.7 X 103L mol-' cm-' at pH 7.0 (25,26)).Standard solutions of both folate and MTHFA were prepared from dilutions of the corresponding stock solutions with Tris-gelatin buffer and Tris-ascorbate buffer, respectively. Equilibrium (Single Incubation) Assays for Folate. Fifty-microliter portions of the standards (diluted with 100 KL of assay buffer) were incubated in a disposable beaker cup with 50 KLof a conjugate solution (diluted with 100 pL of assay buffer) and 50 p L of a FBP solution (diluted with 100 p L of assay buffer) for 10 rnin in a single incubation mode of analysis (27). The resulting enzymatic activity of the mixture was measured as described above. A doseresponse curve was prepared by plotting percent inhibition vs. concentration of folate in the standards. The dose-response curve for MTHFA was prepared in the same way. Sample Preparation. Five tablets of the multivitamin preparation were weighed and finely powdered. On the basis of the claimed folic acid content (on the label), a quantity of the powder corresponding to approximately 0.2 mg of folic acid was weighed and shaken vigorously with 5 mL of 1.0 mol L-l NaOH for 15 min. The mixture was centrifuged at 2400 rpm for 10 min and the supernatant was recentifuged under the same conditions. A quantity of 4 mL of the supernatant was transferred to a 20-mL vial and the pH was adjusted to between 7 and 8 with 1.0 mol L-' HC1. This preparation was then made to volume in a 10-mL volumetric flask with Tris-gelatin buffer. Several dilutions of the sample were prepared and analyzed by the procedure outlined above. Recovery Study. For the recovery study, a quantity of the powdered Stress B complex multivitamin preparation (Bio-Genics, Woodland Hills, CA) corresponding to 0.2 mg (according to the content claimed on the vial label) was weighed and 250 pL or 500 pL of a 4.07 X IO4 mol L-' folate standard solution (in Tris-gelatin buffer) was added. The mixture was, then, shaken vigorously with 4.75 mL or 4.5 mL of 1.0 mol L-' NaOH, respectively, for 15 min. The rest of the procedure used was the same as above.
RESULTS AND DISCUSSION In our original work describing a heterogeneous folate assay (16), we indicated that the enzyme conjugates prepared for that study could not be inhibited by excess FBP in solution. At that time we suggested that perhaps the FBP molecule was too small (molecular weight of approximately 30 000 (28))to modulate the activity of the G6PDH-folate conjugate. However, in that work, conjugates containing only one to three folates per enzyme molecule were employed. Upon subsequent investigation, we found that conjugates prepared with much higher folate/enzyme ratios could, in fact, be inhibited up to 70% by either pure FBP or @-lactoglobulin(contains FBP (29)). Indeed, for homogeneous assay systems a high degree of conjugation of the enzyme conjugate (molecules of hapten attached per enzyme molecule) is desirable (8). This will increase the number of binder molecules that can bind to the conjugate, which will, in turn, enhance the probability of observing an induced modulation of the enzymatic activity. Enzymes requiring more than one substrate, such as glucose 6-phosphate dehydrogenase, are ideally suited for these assays because protein binding will more likely alter the association of at least one substrate to its active site. For this work, several G6PDH-folate conjugates were synthesized and the parameters (e.g., initial folate/enzyme ratio) used for the preparation of those that exhibited inhibition in the presence of FBP in solution are summarized in Table 11. The degree of conjugation, the percent residual activity, and the percent inhibition of the conjugates are also shown in this table. It can be seen that increasing the initial molar ratio of folate/enzyme resulted in greater degrees of conjugation (up to 19.6). Even though highly substituted, all
40
I
I
/d
c
.-2
-
C
40
I I/
I
2oi/ b 0
0 0
20
40
60
80
100
Amount of FBP, pg
Figure 2. Effect of varying the levels of FBP (micrograms FBP in assay 1:40 dilution mixture) on the inhibition of two dilutions of conj-25 (0, conj-25; 0, 1:20 dllution conj-25). These are dilutuions for the concentration of conj-25 glven in Table 11. Values shown are means of duplicate measurements. (Note: to calculate the ratio of moles of FBP/moles conk25, use an approxlmate molecular weight of FBP equal to 30 000.)
conjugates possessed residual activities that were sufficiently high for use in EMIT type arrangements. However, the 21 % and 36% inhibitions observed for conj-23 and conj-24, respectively, were not enough for the development of a homogeneous assay with adequate precision. Thus, the conjugate that showed the maximum inhibition (conj-25) was used in all subsequent homogeneous folate assays. The effect of changing the incubation time of the FBP with the conjugate was studied for a fixed FBP and conj-25 concentrations (association reaction). As shown in Figure 1,after an incubation time of 40 min a nearly maximum inhibition of the enzymatic activity was observed. However, 60% inhibition occurred after only 10 min, and this was deemed adequate for the development of a useful folate assay. Obviously, as also shown in Figure 1, if faster assays are desired, an even lower incubation time is possible at the expense of less total inhibition (i.e., no incubation period still yields approximately 41 % inhibition). The binder dilution curve for this conjugate was constructed by use of two different conjugate dilutions (1:20 and 1:40) of the original conjugate preparation and an incubation time of the binder with the conjugate of 10 min. Figure 2 demonstrates that for the same concentration of FBP, the less the conjugate concentration in the assay, the more the conjugate is inhibited by the binder. As suggested by Kabakoff and Greenwood (30)the amount of binder that corresponds to 85% of the maximum inhibition will result in a doseresponse curve
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986 959
*O
t -0.0
-7.0
-6.0
-5.0
l o g [Folate]. M 7
~ x I O - ~
ixiO-' [Folate],
2xiO-'
5xiO-'
M
Figure 3. Typical dose-response curve for folate also showing the f one standard deviation curves. Data were obtained with a 1:40 dilution conj-25 and a 0.25 mg/mL FBP solution. Quintuple measurements were used for calculating the standard deviation. The x axis refers to concentrations of folate in the standards. with the best detection capabilities. Thus, a 0.25 mg/mL FBP solution and a 1:40 dilution of conj-25 were used in order to obtain dose-response curves. A typical dose-response curve obtained under these conditions is given in Figure 3. As shown in this figure the inhibition induced by FBP can be reversed by the addition of unlabeled folate to the reaction mixture in a very unique manner; the dose-response curve is incredibly steep over an extremely narrow concentration range. Never before has such a dramatic dose-response curve been reported for any type of competitive binding assay. Figure 3 also includes information on the confidence limits, which were placed at f l standard deviation. The calculation of the standard deviation of the inhibition (sI)at a certain folate concentration level was based on standard deviations of both quintuple estimations of the activity of the uninhibited conjugate (so) and the measured conjugate activity in the presence of folate (sf)
where I, AAo, and AAfare as defined in the Experimental Section. The results of this analysis show that there is an almost constant standard deviation (with respect to percent inhibition) over the whole range of the dose-response curve with an average value of 3.3 (range of 2.6-3.7). As suggested by Robdard (31),dose-response curves with confidence limits placed at f l standard deviation are useful in calculating an approximate percent error on a dose estimate. For example, for the data in Figure 3, an inhibition of 40% will result in a folate estimate of 1.31 X lo4 mol L-l with an associated error of 7.1%. Similarly, on the steep portion of the dose-response curve an inhibition of 25% corresponds to a folate level of 1.74 X lo4 mol L-' and a much lower error of 4.1%. When the activity of the conjugate and of the incubation mixture was measured over a 2.5-min period (i.e., five 30-9 measurements) instead of 30 s,the average standard deviation dropped to 2.5 (range of 2.2-2.9). This improvement in the standard deviation is attributed to the averaging of the spectrophotometric noise. This noise is known to affect enzyme kinetics data, which are based on a two-point estimation of the reaction rate (32). In order to study the effect of FBP and conjugate concentrations on the response characteristic of the assay, three different conj-25 dilutions (1:20, 1:40, and 1:200) were used
Figure 4. Effect of varlous conjugate and FBP concentrations on the dose-response curve. Means of duplicate measurements were plotted. The reagent concentrations used were a follows: (A) (V)0.050 mg/mL FBP, 1:200 dilution conj-25; (B) (0) 0.25 mg/mL FBP, 1:20 dilution conj-25; (C) (A)0.25 mg/mL FBP, 1:40 dilution conj-25; (D) (0)0.50 mg/mL FBP, 1:20 dilution conj-25; (E) ( 0 ) 0.50 mg/mL FBP, 1:40 dilution conj-25. The percent inhibition was plotted against the logarithm of the folate concentration in the standards. (Figure 4). To evaluate the effect of combined changes in reagent concentrations on the dose-response curve, all four reagent combinations of two FBP solutions (0.50 mg/mL and 0.25 mg/mL) with two conj-25 dilutions (1:20 and 1:40) were employed. It can be observed (Figure 4) that halving the binder concentration will shift the dose-response curve to approximately half the EDb0value (EDs0 is defined as the effective dose for 50% of maximum response). The EDmvalue can be lowered even more (i.e., by approximately 10-fold) by using a 0.050 mg/mL FBP solution (a 10-fold lower content than the 0.50 mg/mL FBP solution). However, this occurs at the expense of decreasing the extent of the steep range of the dose-response curve. Figure 4 also demonstrates that for the same amount of FBP in the assay, a higher conjugate concentration will result in a dose-response curve with a lower EDm value. Carter and Meyerhoff (15) arrived at these same conclusions in their EMIT assay for cyclic AMP based on an anticyclic AMP antibody. This effect is predicted by theory (Figure 2 in ref 12) when for ratios of conjugate/binder less than one, the greater the conjugate concentration, the lower the EDbovalue. It should be noted, however, that this theoretical treatment was based on a heterogeneous assay mass action model. Modeling the homogeneous case is a far more complex task, which requires considerable knowledge of the mechanism of the homogeneous inhibition. Nonetheless, the heterogeneous model can serve as a basis to explain our observations at least to a first approximation. Assuming a molecular weight of 30 000 for FBP (28), the concentration of FBP in the 450 p L of the assay incubation mixture (when 50 pL of a 0.50 mg/mL FBP solution was used) is 1.8 x lo4 mol L-l. According to Hansen et al. (28) this concentration corresponds to a tetramer in solution. The same investigators reported that a 10-fold dilution in FBP (i.e., a 1.8 X lo-' mol L-l FBP solution) will result in a solution where FBP is in equilibrium between a monomeric and a dimeric form. By demonstrating that even with this dilute FBP solution the conjugate is still inhibited homogeneously (curve A in Figure 4), we suggest that the low molecular weight monomeric form of the binding protein is still large enough to inhibit the conjugate. The effect on the dose-response curve of varying both the concentrations of the enzyme conjugate and the binding protein in a fixed ratio while altering the dilutions of both reagents proportionately can also be seen in Figure 4. Curves A, C, and D form one family of such dose-response curves. The greater the concentrations of both conjugate and binder,
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
960
K Y n = IO
.\
Y
20
-
K*/K=l
10-
KP
Flgure 6. Effect of changes in the Kq and Kp * parameters on the shape of equilibrium mode heterogeneous assays for K*/K = 10 (bridging group recognition).
-8.0
-7.0
-6.0 -5.0 log(Concentration), M
-4.0
Flgure 7. Dose-response curves for folate and MTHFA using a 1:40 dilution of conj-25 and a 0.25 mglmL FBP solution. Values shown are averages of duplicate measurements. The x axis refers to the logarithm of concentrations of folate and MTHFA in standards. here. On the other hand, when a binder (e.g., antibody) exhibiting bridging group recognition is used as the specific binder (K* > K see ref 12), increasing the binder concentration will not increase the steepness of the dose-response curve to as large an extent (Figure 6). This may explain why such steep dose-response curves have not been observed in traditional antibody-based homogeneous assays. In reality, for the folate-FBP system described here, K* may be somewhat less than K and according to theoretical predictions, even steeper dose-response curves than those shown in Figure 5 may result. It should also be noted that the dose-response curves reported in Figures 4 and 5 are plotted as bound activity vs. log (dose) compared to the EMIT type curves, which are plotted as percent inhibition vs. log (dose). However, bound activity in heterogeneous assays is equivalent to percent inhibition in homogeneous arrangements when considering the effects of chemical equilibria. The percent cross-reaction of the assay system with the folate analogue MTHFA was also evaluated (Figure 7). As expected (33,34)the curve for MTHFA has a slightly higher ED50 value. The same was observed in our heterogeneous arrangement (35)at the same pH. The percent cross-reactivity (calculated as the ratio of the ED, value of the dose-response curve when using folate as the standard over the ED50 value when employing MTHFA standards, multiplied by 100) was found to be 85%. The corresponding cross-reactivity for the heterogeneous assay was 46% (35). It should be noted that folic acid is a relatively stable compound, whereas MTHFA can be oxidized easily and requires presence of antioxidants.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
Table 111. Vitamin Tablets Analysis a m t o f folate p e r tablet, m g manufacturer Thompson Bio-Genics Bio-Genics
vitamin tablet
B complex w i t h C stress B complex folic acid w i t h v i t a m i n BIZ
found"
claimed
0.201 0.217 0.827
0.200 0.200
regard to new vitamin B12and riboflavin methods, is currently under way. Registry No. GGPDH, 9001-40-5; folic acid, 59-30-3.
LITERATURE CITED
0.800
"Average o f t w o determinations.
Table IV. Vitamin Tablets Recovery Study m g o f folate manufacturer
vitamin tablet
found"
added
% recovery
Bio-Genics Bio-Genics
stress B complex stress B complex
0.096 0.041
0.090 0.045
107 91.3
" Average o f t w o determinations. Thus, all enzyme conjugates were prepared by use of the more stable folate analogue (folic acid). To demonstrate the value of the proposed assay for real sample determinations, analysis of three different multivitamin preparations was undertaken. From the values of the percent inhibition and the corresponding dose-response curve the concentration of folate in the dilution of the samples that falls on the steep portion of the dose-response curve was estimated graphically. Taking into accoun the dilution of the sample, the folate content of the vitamin tablets was evaluated (Table 111). Selectivity over a wide range of other vitamins was excellent as demonstrated by the accuracy of the folate assays in these rather complex samples (see Table I for compositions). It can be seen that the new method obtain values within less than 8.5% of the reported value. A recovery study was also performed for one of these multivitamin tablets by spiking the sample with 0.045 mg or 0.090 mg of folate. It is evident from the recovery values given in Table IV that the amounts of folate used to spike the samples could be recovered with satisfactory accuracy. Besides using the reported homogeneous assay to determine the folate content of real samples, the unique steepness of the dose-response curve should be useful in quality control situations where a decision on concentration being higher or lower than a given cutoff value must be made (YES/NO decision). Indeed, it has been demonstrated (Figure 4) that by simply varying the concentration of the assay reagents, the steep portion of the dose-response curve can be fine adjusted to a desired value over a wide range of folate concentrations. This behavior suggests that such an assay would be ideally suited for the control of folate content in pharmaceutical products, infant formulas, cereals, etc. More importantly, if such assay approach could be extended to other analytes by using different binding proteins and enzyme-ligand conjugates, then a whole new range of rapid and simple quality control tests may be possible. Work in this direction, particularly with
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RECEIVED for review October 2,1985. Accepted December 16, 1985. We gratefully acknowledge the National Science Foundation for supporting this work (Grant No. CHE8506695).
