Anal. Chem. 1984, 56,1723-1726
1723
CORRESPONDENCE Cooperative Interaction of Immobilized Folate Binding Protein with Enzyme-Folate Conjugates: An Enzyme-Linked Assay for Folate Sir: The use of enzyme labels as alternatives to radioisotopes for quantitating antigens, antibodies, and haptens has resulted in the development of enzyme immunoassays which overcome some serious drawbacks inherent to traditional radioimmunoassays ( I , 2). The sensitivity of enzyme-immunoassay systems for ligands (haptens) is limited partly by the immunological bridging group recognition by antibodies toward the enzyme-labeled ligands. Antibodies, prepared by immunizing animals, recognize not only the hapten and the carrier protein that is used for immunization but also the bridging group that covalently couples the hapten to the protein. This bridging group is also often present in the enzymehapten conjugates used in the assays and thus, affiiity constants between the antibody and the respective enzymelabeled ligand are sometimes greater than those of the antibody and the free ligand (3-5). Naturally occurring binding proteins, however, have similar or higher affinity constants for free ligands than enzyme-labeled ligands. Thus, it should be possible to use such binding proteins rather than antibodies in enzyme linked methods and this approach may result in assays with improved analytical Characteristicswhen compared to conventional immunoglobulin-based assays. In this correspondence, preliminary data on such an enzyme linked assay for folate are reported. The assay uses folate binding protein (FBP) as the specific binder and the enzyme glucose 6phosphate dehydrogenase (G6P-DH) as the label. Folates occur naturally within cells of mammals, bacteria, and plants (6). In mammalian organisms they mainly function as vital cofactors in reactions involving one carbon transfer for purine and pyrimidine synthesis (7-9). The levels of folate in the serum and red blood cells (RBC) can be used in the diagnosis of megaloblastic anemia and to evaluate nutrition (10). The normal levels of serum and RBC folate for humans are 5-19.8 ng/mL and 184-492 ng/mL, respectively (11). The assay described here appears to have adequate detection limits to perform assays of folate in these samples without the need of radiotracer labels which have been used traditionally for folate determinations (12). Further, it will be shown that even lower detection capabilities of the enzyme-labeled assay may be possible by taking advantage of the unique cooperative binding characteristics of immobilized FBP. EXPERIMENTAL SECTION Apparatus. Enzyme assays were performed at 340 nm with a Gilford Stasar I11 spectrophotometer equipped with a vacuum-operated mpling system and temperature-controlledcuvette. The cuvette chamber was maintained at 30 OC. A Cary 219 spectrophotometer with autobaseline correction was employed to record the spectra of the enzyme, folate, and conjugates. Polypropylene test tubes were used throughout the study. Reagents. The solvents dimethyl sulfoxide (MezSO) and NJV-dimethylformamide (DMF) were obtained from Aldrich. G6P-DH from Leuconostoc mesenteroides and FBP from bovine milk, as well as all other chemicals, were obtained from Sigma Chemical Co. and were of the highest purity available. Deionized, distilled water was used throughout the study. Preparation of GGP-DH-Folate Conjugates. The G6PDH-folate conjugates were synthesized by the N-hydroxysuccinimideester method (13). Equimolar amounts of folic acid 0003-2700/84/0356-1723$01.50/0
and N-hydroxysuccinimide (NHS) were reacted overnight at 4 "C under stirring with l-ethyl-3-(3'-(dimethylamino)propyl)carbodiimide hydrochloride (EDAC) in dry Me2SO/DMFsolvent to form the activated folate ester. To ensure complete reaction in the desired direction a 20% excess of both folic acid and NHS with respect to EDAC was used. This i s because free EDAC can cross-link protein and, thereby, affect the efficiency of the conjugation reaction. To prepare the G6P-DH-folate conjugates,the activated folate ester solution was added in aliquots at 5-min intervals into a solution of 100 units of enzyme which was previously dialyzed in HCO3-/C0:- buffer (0.10mol L-' NaHC03, pH 9.4). The protein solution was kept at 0 OC during the addition of the activated ester. The conjugation reaction was run for 6 h in the dark at 4 OC under stirring while the active site of the enzyme was protected during the conjugation reaction by adding an excess of glucose 6-phosphate (G6P) and @-NADHcompared to the enzyme present. Finally, the conjugate was dialyzed against the assay buffer (0.0500 mol L-' Tris-HC1,0.100 mol L-' NaC1, 0.01% NaN3, pH 7.8). Contrary to previous reports concerning the preparation of ligand-enzymeconjugates via the NHS ester reaction, it was found that a mixed solvent system of Me2S0and DMF yielded better conjugates. This is because folic acid has a higher solubility in room temperature MezSOthan in DMF which allows use of less solvent during the preparation of the activated ester. Moreover, presence of MezSOalters the enzymatic activity to a lesser degree than DMF. However, pure MePSOhas a freezing point of 18.5 OC, whereas the recommended temperature for the reaction of forming the activated ester is 0-4 OC. Therefore, a mixed solvent was used. A mixture of Me2SO/DMF of 2.3/1 does not freeze at 4 "c under the reaction conditions. Conjugates prepared in such a solvent mixture had better residual enzymatic activity compared to those prepared in DMF alone. Each conjugate was characterized by the degree of conjugation (average number of folates per enzyme) and by the residual enzymatic activity. The degree of conjugation and the concentration of the conjugate in the preparation were determined spectrophotometricallyby employing two simultaneous equations (14). A G6P-DH molecular weight of 103 700 was assumed (15). The residual enzymatic activity was calculated by comparing the activities of a conjugated enzyme to that of a solution containing unconjugated enzyme of the same protein concentration. Several G6P-DH-folate conjugates were prepared as described above. A conjugate (conj-15) prepared by using a 1101 initial mole ratio of folic acid activated ester to enzyme was found to have a degree of conjugation of 3.2 and a residual activity of 74% and was used in all subsequent studies. Enzymatic Activity Determination. The rate of appearance of NADH, measured by the change in absorbance at 340 nm per unit time, is used to determine the activity of the enzyme conjugate. The assay involves addition of 100 FL of @-NAD+substrate (0.060 mol L-' @-NAD+in assay buffer), 100 p L of G6P substrate (0.10 mol L-l G6P in assay buffer), and 100 p L of the enzyme solution to a disposable sample cup containing 800 pL of assay buffer. After agitation the reaction mixture is aspirated into the thermostated flow cell of the Gilford spectrophotometer. Absorption measurements were taken after a 30-8 delay to allow for temperature equilibration. FBP-Beads Preparation. Folate binding beads were prepared by covalently attaching 2 mg of FBP to 800 mg of CNBr-activated Sepharose 4B beads as suggested by the manufacturer (16). To determine their binding capacity for the conjugate, volumes of 0 1984 American Chemical Society
1724
ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST
1984 log (ng of folate In tube) -2
-1
I ’
0
I-.--
I
4
0.4
c
I
/ 5
10
Volume 01 FBP-beads,
15
20
I
pl
Flgure 1. Folate-enzyme conjugate binding as a function of increasing immobilized FBP concentration (volume refers to settled volume of FBP-beads). Binding of conjugate to beads in the absence of immobilized FBP was negligible.
FBP-beads (50 pL or more of various diluted suspensions) were incubated with 100 pL of 1.5 X lo-* mol L-l conjugate. The final volume of each tube was brought to 800 pL with assay buffer, corresponding to a concentration of 1.8 x IO4 mol L-’ conjugate in the tube. After incubation for 30 min at room temperature the tubes were centrifuged and the supernatants were discarded. The beads were washed three times with 2 mL of assay buffer. The pellet was finally resuspended in a total volume of 1.1mL containing 100 pL of each substrate solution and incubated for 30 min at room temperature. After centrifugation the absorbance in the supernatant was determined. The complex of folate with FBP can be readily dissociated by lowering the pH to
