Enzyme-linked immunosorbent assay for an octapeptide based on a

Dual Detection of Peptides in a Fluorescence Binding Assay by Employing Genetically Fused GFP and BFP Mutants. Jennifer C. Lewis and Sylvia Daunert...
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Anal. Chem. 1993, 65, 1147-1151

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Enzyme-Linked Immunosorbent Assay for an Octapeptide Based on a Genetically Engineered Fusion Protein Allan Witkowski,' Sylvia Daunert,' Mark S. Kindy,t and Leonidas G. Bachas'J Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055,and Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084

Traditional chemical means of preparingenzymeligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs. Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase. The resulting enzymepeptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics. The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc. INTRODUCTION Enzyme immunoassays (EIAs) have been established as powerful analytical techniques because they combine the attractive selectivity properties of immunoassays with the use of cheap, highly purified, relatively stable, nonradioactive labels.',* The most common heterogeneous EIA technique is the enzyme-linked immunosorbent assay (ELISA). This procedure is based on a competition between an unlabeled ligand and an enzyme-ligand conjugate for a limited number of antibody binding sites. The higher the unlabeled ligand (analyte) concentrationin the sample, the less enzyme-labeled ligand associates with the binder and the lower the resulting enzymatic activity in the solid phase. The distribution of label between the antibody-bound and the free portion reflects, by comparison with standards, the concentration of ligand in the sample. The two main componenta of ELISAs are the enzymeligand conjugate and the antibody. A considerable amount of research has been focused toward the evaluation of antibodies? but surprisingly little emphasis is being placed on the controlled production of enzyme-ligand conjugates. The current methods of preparing enzyme-ligand conjugates usually involve chemical coupling of the ligand to free amino Department of Chemistry. Department of Biochemistry. (1) Gould, B. J. Anal. Proc. 1987,24, 136-137. (2) Howanitz, J. H. In Immunochemical Assays and Biosensor Technology for the 1990's; Nakamura, R. M., Kasahara, Y., Rechnitz, G. A., Eds.; ASM: Washington, DC, 1992; pp 23-35. (3) Nakamura, R. M. In Immunochemical Assays and Biosensor Technology for the 1990's; Nakamura, R. M., Kasahara, Y., Rechnitz, G. A., Eds.; ASM: Washington, DC, 1992; pp 205-227. +

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groups on the e n ~ y m e . However, ~ control of the number of ligands attached per enzyme and the exact site of attachment is very limited.5 Some control over the degree of conjugation can be achieved by varying the mole ratios of the reactants, but the conjugates produced are still heterogeneous. This problem is further complicated when the ligand has more than one reactive group through which it can be attached to the enzyme. Theoretical6 and experimental' studies have shown that monosubstituted conjugates provide the best assay performance. Therefore, ideally, conjugatesshould be prepared by attaching a single ligand to an enzyme at one specific location. One means of achieving this is through the production of fusion proteins. Fusion proteins are genetically engineered proteins in which altered DNA dictates the synthesis of a desired conjugate complex. The DNA strands for the components to be conjugated are enzymatically linked, including the appropriate signal sequencesthat allow for direct synthesis of the complex by bacteria after transformation. After incubation of the transformed culture, the fusion protein can be isolated and purified. The use of fused-gene technology in analytical chemistry has grown in recent years, with fusion proteins being used for immobilized metal ion affinity chromatography? hydrophobic interaction chromatography! and for immobilization to protein A in immunoassays.lOJ1 Initial studies by Mosbach and co-workers have shown that fusions of the relatively large human proinsulin with @-galactosidaseand alkaline phosphatase appear to have potential for use in ELISAs.lZJ3 However, it has yet to be shown that fusion proteins will be useful in ELISAs of much smaller ligands. Our approach has been to produce a fusion protein between a small peptide and an enzyme and then determine whether such a conjugate is viable for the development of ELISAs. Specifically, a synthetic octapeptide, Asp-Tyr-Lys-Asp-AspAsp-Asp-Lys, was combined with the bacterial alkaline phosphatase (BAP) enzyme (Figure 1). The octapeptide, which is the FLAG peptide utilized commercially in IBIKodak's FLAG Biosystem for fusion protein preparation, was chosen because it contains six carboxylic groups, three amino groups, and one hydroxyl group. These numerous functional groups would make the preparation of controlled conjugates very difficult by chemical means. In contrast, precise ~

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(4) Ullman, E. F.; Maggio, E. T. In Enzyme-Immunoassay; Maggio, E. T., Ed.; CRC Press: Boca Raton, FL, 1980; pp 71-104. (5) Ullman, E. F.; Maggio, E. T. In Enzyme-Immunoassay;Maggio, E. T., Ed.; CRC Press: Boca Raton, FL, 1980, pp 105-134. (6) Bachas, L. G.; Meyerhoff, M. E. Anal. Biochem. 1986, 156, 223238. (7) Daunert, S.;Payne, B. R.; Bachas, L. G. Anal. Chem. 1989, 62, 2160-2164. (8) Smith, M. C.; Furman, T. C.; Ingolia, T. D.; Pidgeon, C. J . Biol. Chem. 1988,236, 7211-7215. (9) Persson, M.; Bergstrand, M. G.; Bulow, L.; Mosbach, K. Anal. Biochem. 1988, 172, 330-337. (10) Kobatake, E.; Nishimori, Y.; Ikariyama, Y.; Aizawa, M.; Kato, S. Anal. Biochem. 1990, 186, 14-18. (11) Hunger, H. D.;Flachmeier,C.;Schmidt, G.; Behrendt, G.;Coutelle, C. Anal. Biochem. 1990, 187, 89-93.

0 1993 American Chemical Society

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was synthesized, purified, and sequenced by the University of Kentucky Macromolecular Center. All chemicals were of reagent grade or better and were used as received. All experiments were conducted at room temperature. Preparation and Isolation of the Peptide-BAP Protein. Bacteria (Escherichia coli strain JM109) were transformed with the peptide-BAP vector and then cultured to express the fusion protein according to the manufacturer's instructions. Specifically, the bacteria were grown in 500 mL of liquid broth supplemented Octapeptide A$p'-Tyr2-Lya3-Asp4-Asp'-Aape-AapT-Lyaewith 1.7 mM IPTG and 0.10 mg/mL ampicillin at 37 "C for 4 h. Linker - L e ~ ~ - ~ l ~ ' ~ - P h a ' ' - S e r ' ~ - A r q ' ~ - A s p ' ~ ~ I l a ~ ' - V a I ' ~ - A a p ' ~ - A r q ' ~ -Isolation S~r'~of the peptide-BAP protein was done by the osmotic shock procedure for periplasmic fractionation according to the Flgurr 1. N-Terminal amino acid sequence of the peptide-BAP fusion supplied directions. All molecular biology procedures were done protein. using standard protocols.14 Affinity Purification of the PeptideBAP Fusion Protein. The peptide-BAP protein was purified using 1mL of anti-FLAG M1 affinity gel packed into a rapid-flow column. the anti-FLAG MI affinity gel contains monoclonal antibodies against the chosen octapeptide immobilized onto agarose beads. Column preparation and affinity chromatography were conducted according to the manufacturer's instructions. Ten aliquots (1mL each) of a phosphate-buffered saline (PBS) solution (0.10 M NaH2P04, pH 7.4, with 0.15 M NaCl) also containing 2.0 mM EDTA (PBSI EDTA) were collected during the elution stage. All the fractions recovered were examined for the presence of protein based on their absorbance at 280 nm using a Perkin-Elmer (Norwalk, CT) Lac Lambda 6 spectrophotometer. Those fractions containing protein were pooled together and lyophilized. The lyophilized protein was resuspended in a small amount of 50 mM Tris-HC1, pH 8.0, containing 1.0 mM MgC12 and 0.10 mM Zn(Ac)2. This solution was then dialyzed against 1 L of the Tris-HC1 buffer using standard cellulose dialysis tubing (Spectrum Medical Industries; Los Angeles, CA)with a molecular weight cutoff of 12 000-14 000. The dialysis buffer was changed four times, and the final volume floori of peptide-BAP conjugate recovered was 2.66 mL. Flgurr 2. Plasmid map of the peptide-BAP vector. The vector is the Binder-Dilution Study. In order to determine how much 8.08-kb pFLAG1-BAP from IBI-Kodak. of the M1 affinity gel beads were necessary to use for each assay, a binder-dilution study was conducted. A stock solution of beads composition can be attained by recombinant DNA techniques. was prepared by pipeting 1 mL of beads (not including volume In this manner, defined monosubstituted conjugates were of shipping buffer) followed by rinsing and centrifuging three developed that are modified in a controlled manner at an consecutive times with 0.10 M glycine-HC1, pH 3.0, and then three additional times with PBS. After the final rinse, the beads exact site on the enzyme and that are part of a homogeneous were centrifuged, the supernatant was decanted, and the total population. volume was brought to 2 mL with PBS. A 1:lO and a 1:100 dilution were made from this stock bead solution. By using different EXPERIMENTAL SECTION volumes of these solutions, a range of bead quantities were tested. The beads were pipetted into conical polypropylene tubes Reagents. Tris(hydroxymethyl)aminomethane, free base (Evergreen Scientific; Los Angeles, CA), and the total volume (Tris), (ethylenedinitri1o)tetraacetic acid (EDTA), magnesium was brought to 600 pL with PBS/Ca (PBS with 1.16 mM CaC12). chloride hexahydrate, p-nitrophenyl phosphate (PNP or Sigma Next, 100 pL of a diluted peptide-BAP solution was added to 104 phosphatase substrate), anhydrous sodium monobasic phosthe tubes and incubated for 1 h with shaking. The tubes were (EDAC), phate, l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide then centrifuged, the supernatant was decanted, and they were and all proteins not specifically mentioned were obtained from rinsed three times with DEAiCa buffer (2.4 M DEA, pH 8.0,with Sigma (St. Louis, MO). Zinc acetate dihydrate, N-hydroxysuc0.057 mM MgCl2 and 1.0 mM CaC12). After the final rinse, most cinimide (NHS), dimethyl sulfoxide (DMSO), sodium bicarbonof the supernatant was removed with a disposable pipet tightly ate, and diethanolamine (DEA) were purchased from Aldrich fitted with a stopper set 6 cm from the tip. When placed in the (Milwaukee, WI). All other salts and glucose were from Fisher tubes, this pipet reproducibly removed the washing buffer, with (Cincinnati, OH). The liquid broth and isopropylthio-p-galac0.25 mL solution remaining in each tube. To determine the toside (IPTG) were from Gibco-BRL (Gaithersburg, MD), the amount of conjugate bound to the solid phase, 1.08 mL of DEA agar was from Difco (Detroit, MI),and the sodium dodecyl sulfate buffer (2.4 M DEA, pH 8.0, with 0.057 mM MgC12)and 670 pL (SDS) was from Calbiochem (San Diego, CA). Calcium chloride of AttoPhos substrate solution (36 mg of AttoPhos per 60 mL of dihydrate was purchased from MCB Reagents (Cincinnati, OH), DEA buffer) were added. This was incubated for 1h withshaking, the AttoPhos fluorescent substrate was from JBL Scientific (San centrifuged, and the supernatant was decanted into disposable Luis Obispo, CA),4-methylumbelliferyl phosphate (4-MUP) was fluorimeter cuvettes (Evergreen Scientific). The fluorescence from Kodak (Rochester,NY),and ammonia-freeglycine was from emission intensity was monitored at 560 nm with an excitation Eastman (Rochester, NY). A FLAG Biosystem containing the wavelength of 440 nm using a SPEX (Edison, NJ) Fluorolog pFLAG-1-BAP vector (Figure 2), anti-FLAG M l monoclonal F111A spectrometer. antibody, anti-FLAG M1 affinity gel, and a rapid-flow chromaDose-Response Curve. A dose-response curve was obtained tography column was purchased from IBI-Kodak (New Haven, by incubating 100 pL of various concentrations of free peptide CT). The BCA Protein Assay Reagent was obtained from Pierce with a fixed volume of stock beads for 1 h. Next, 100 pL of a (Rockford, IL). The unlabeled octapeptide used in these studies 1:lOOpeptide-BAP dilution was added and incubated for another 1

2

(12) Lindbladh, C.; Persson, M.; Bulow, L.; Stahl, S.; Mosbach, K. Biochem. Biophys. Res. Cornmun. 1987, 149, 607-614. (13) Peterhans, A.; Mecklenburg, M.; Meussdoerffer, F.; Mosbach, K. Anal. Biochem. 1987, 163, 470-475.

(14) Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory: ColdSpring Harbor, NY, 1982.

ANALYTICAL CHEMISTRY, VOL. 85, NO. 9, MAY 1, 1993

hour. This was followed by the rinsing and substrate reaction steps as described above. Chemical Conjugation of Peptide-BAP. The octapeptide was activated as an NHS ester for coupling to the BAP. The peptide and NHS were used in a 1.2-fold molar excess to EDAC. The peptide (9.6 pmol) was dissolved in 800 pL of dry DMSO with stirring for about 1.5 h. EDAC (80 pmol) and NHS (96 pmol) were dissolved in 2 mL of dry DMSO, and 200 pL of the EDAC/NHS solution was added to the solution containing the peptide. The reaction was run overnight at room temperature in the dark with stirring. The total reaction time was 17.5 h, and the final solution contained 8 pmol/mL NHS-peptide. To conjugate the activated ester to BAP, 100 units of BAP were dissolved in 2 mL of 0.10 M NaHCO.1, pH 8.0. From this stock solution, 900 pL (45 units) were taken for the reaction. The activated NHS-peptide was added to the BAP to give a 100:l peptide:BAP ratio, with 146 pL NHS-peptide added in small portions (50, 50, and 46 pL) at 10-min intervals. The reaction was run on ice with stirring, and the total reaction time was 6 h. The conjugate was then dialyzed against 1 L of DEA assay buffer, with three changes of buffer.

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Table I. Comparison of Different Substrates for the PeDtide-BAP Conjugate incubation conjugate substrate detection time (h) dilution S/Ba AttoPhos fluorescence 1 1:1oOoO 45 fluorescence 3 1:500 1.2 4-MUP absorbance 2 1:100 19 PNP S/B refers to the ratio between signal and background.

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RESULTS AND DISCUSSION Although the ELISA is a widely accepted analytical technique, several aspects of its development have yet to be addressed. One such issue is the lack of control in the preparation of enzyme-ligand conjugates. Taking advantage of the flexibility of genetic engineering, we have designed a method involving fusion protein techniques for preparing conjugates of precise composition for use in ELISAs. In this assay, the peptide-enzyme fusion protein and unlabeled peptide compete for a limited amount of monoclonal antibody that is immobilized on a solid support. The enzymatic activity on the solid phase can be correlated to the concentration of the unlabeled peptide in the sample through a dose-response (calibration) curve. We report here results that validate the feasibility of developing ELISAs for peptides using this approach. A peptide-BAP enzyme conjugate was prepared by attaching the C-terminus of the octapeptide to the N-terminus of BAP using standard molecular biology methods14 as described in the Experimental Section. Due to the vector cloning site used to insert the BAP, a linker peptide results between the octapeptide of interest and the enzyme (Figure 1). The desired peptide-enzyme fusion protein was isolated and, after affinity purification, was assayed for enzymatic activity. The activity of the peptide-BAP was determined by using P N P as the substrate and monitoring the absorbance at 405 nm. This experiment indicated that the stock conjugate solution contained 12.5units of enzyme/mL. The totalprotein content of the peptide-BAP solution was also analyzed using a standard bicinchoninic acid (BCA) analysis and bovine serum albumin as the protein standard. Based on the absorbance of the stock fusion protein, the recovered peptideBAP solution contains 0.24 mg/mL total protein. dThese data correspond to an activity of 53 units/mg of protein, which is comparable to the 48 unita/mg of protein reported by Sigma for type I11alkaline phosphatase from E. coli. Consequently, by comparison of the specific activities of peptide-BAP and unconjugated BAP, it may be concluded that the production of the peptide-BAP fusion does not affect the enzymatic activityof the conjugate. This is in agreement with extensive studies by Hoffman and Wright that indicate a minimum effect of N-terminal alteration on the activity of BAP.lS Studies were also conducted to determine the best substrate and the optimum conditions for the generation of the enzymatic signal. The substrates tested were P N P (300 pM

in the assay tube), 4-MUP (300 pM in the assay tube), and AttoPhos. A summary of the signal-to-background ratios obtained using the various substrates is shown in Table I. In this table, the time of incubation of the enzyme and substrate, the exact dilution of the labeled peptide used, and the relative increase in signal (absorbance or fluorescence intensity) over the background levels due to the presence of the enzymatic product are shown. It is evident from the data in Table I that AttoPhos is the best substrate for the peptide-BAP conjugate because it gives the largest increase in signal over background, while using the least amount of enzyme conjugate and the shortest time of incubation. Consequently, AttoPhos was chosen as the substrate for all subsequent assays. The manufacturers of AttoPhos recommend using a DEA buffer a t pH 10.0 for mammalian alkaline phosphatase, but based on earlier reports’6 a lower optimal pH was expected for BAP. Therefore, pH studies were conducted to examine the effect of pH on the enzymatic activity using AttoPhos as the substrate. As it can been seen in Figure 3, there is maximum enzymatic activity at pH 8.5, with a rapid decrease a t higher pH values. Since the activity at pH 8.0 was only slightly lower than at pH 8.5, and in order to avoid possible changes in activity induced by slight variations in the pH of the solution during the assay, the rest of the experiments were performed at pH 8.0. In order to determine the optimum amount of antibody beads to use in each assay, a binder-dilution study was conducted. These results are shown in Figure 4. The ideal quantity of binder (antibody coated beads) was determined to be 10 pL of stock beads solution when incubated with 100 pL of a 1:10 OOO dilution of peptide-BAP. This corresponds to the volume of beads that yields ca. 85 %I of the maximum signal. To avoid pipeting errors due to small volumes, the beads were delivered in 100-pLaliquote of a 1:lO bead dilution. It should be pointed out that the affinity beads were reused after regeneration according to the manufacturer’s instruction.

(15)Hoffman, C.S.;Wright,A. Proc. Natl. Acad. Sci. U.S.A. 1985,82, 5107-5111.

(16)Worthington Enzyme Manual; Worthington, C. C., Ed.;Worthington Biochemical Corporation: Freehold, NJ, 1988;pp 264-268.

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PH Flguro 3. Effect of the pH of the assay solution on the enzymatic actMty of the peptide-BAP conjugate. The sample solution contained 1.33 mL of MA, 670 pL of AttoPhos, and 100 pL of a 1: 10 000 dilution of peptMe-BAP and was incubated for 104 min. The fluorescence

signal is in counts per second (cps).

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993

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200 300 Volume of Beads (pL) Flgurr 4. Typical blnder-dllutlon curves obtained using different concentratlons of the peptide-BAP conjugate and different amounts of antlbody-coatedbeads: (0)fresh beads and 1:10 000 peptide-BAP dllutlon; (0)regenerated beads and 1: 100 peptide-BAP dilution.

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log [Peptide] Dose-response curves for the octapeptide generated by incubating a flxed concentration of the peptide-BAP conjugate and varylng concentratlons of unlabeled octapeptide in the presence of two separate dilutions of beads: ( 0 )100-pL stock beads; (0) 50-pL stock beads. AF refers to the change in fluorescence intensity. Flguro 5.

Although the manufacturer contends that the beads can be recycled repeatedly, significant retention of fusion protein was noticed even after regeneration. This was evident by an increase in the background signal when previously used beads were incubated with substrate in the absence of additional conjugate. Due to the retention of the peptide-BAP, along with the decreased binding ability, the amount of binder and conjugate used with beads that had been recycled had to be modified. The changes in bead binding appeared to level off after about seven usages, and a t that point a new binderdilution curve was prepared (Figure 4). The second curve, for which 100pL of a 1:100 dilution of peptide-BAP was used in the incubation step, shows that 100 pL of stock beads is necessary to obtain ca. 85% of the maximum signal. To evaluate the ability to use the peptide-BAP fusion protein in an ELISA-type competitive binding assay for the determination of the octapeptide, a dose-response curve for this peptide was constructed. Figure 5 shows the effect that varying amounts of unlabeled peptide in solution have on the fluorescence intensity observed when incubated in the presence of a fixed amount of enzyme-labeled peptide. A typical sigmoidal dose-response curve is observed. The smaller the amount of unlabeled analyte present in solution, the higher the amount of peptide-BAP conjugate bound on the beads. Therefore, at low peptide concentrations, more enzymatic activity is measured on the solid phase. The limit of detection for this assay is 1 X lo-; M using 100pL of beads. As expected, when the amount of beads is decreased, a shift

log [Peptide]

Dose-response curves for the octapeptide comparing peptide-BAP conjugates prepared by NHS-activatlon (0)and genetic ( X ) means. Equivalent amounts of BAP activity were used In both cases, with the amount of actlvity correspondlng to 100 pL of a 1:100 dilution of genetically Synthesized FLAGBAP. A volume of 100 pL of stock beads was used for both conjugates. AF refers to the change in fluorescence intensity. Flgurr 6.

toward lower detection limits is observed (see Figure 5). In this case, the concentration of antibody binding sites is lower because less beads are present. Since the assay is performed in a sequential binding manner, less peptide is now necessary to achieve the same percentage of the binding site saturation. This results in lower detection limits for the peptide. It is important to mention that the reduction in number of binding sites also decreases the maximum enzymatic signal achievable. The pooled standard deviation for the data in Figure 5 is 3.4 X lo6 cps for the curve using 100 pL of M1 beads and 3.7 X IO6 cps using 50 pL of M1 beads. This represents an error in the maximum fluorescence of 3% and 7 % for 100 and 50 p L of beads, respectively. In order to provide a comparison between genetically engineered conjugates and conjugates prepared with the conventional method of NHS-carbodiimide activation, the octapeptide was also chemically conjugated to BAP. The ratio of peptide:BAP chosen was 100:1, which is typical of ratios generally used in the chemical preparation of enzymeligand conjugates.17 These two differently prepared conjugates were used to construct dose-response (calibration) curves (Figure 6). An identical number of enzymatic units was used for both of these curves. As can be seen in Figure 6, the genetically prepared conjugate performs significantly better in the peptide assay. The curve for the genetically engineered peptide-BAP is much steeper, yielding much greater sensitivity, which is in agreement with the theoretically predicted behavior for monosubstituted vs multisubstituted conjugates.6 Another feature of these curves is that the total activity on the solid phase is much lower when using the conventionally prepared conjugate. This is attributed to improper orientation of the attached peptides and/or crowding of the peptides on the enzyme surface. In addition, the conjugate prepared by the NHS-carbodiimide-mediated reaction may also have a lower binding constant, allowing some attached conjugate to leach off the beads during the washing steps. Thus, genetically synthesized peptide-BAP conjugates show definite analytical advantages over ones prepared with the conventional NHS activation chemistry. In conclusion, we have described a novel approach to immunoassays for peptides based on genetically engineered fusion proteins. Because their production is dictated simply by the genetic sequence introduced into the bacteria, fusion proteins provide a simple, reproducible means of preparing (17) Bachas, L. G.; Lewis, 56, 1723-1726.

P. F.: Meyerhoff, M. E.Anal. Chem. 1984,

ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993

monosubstituted enzyme-ligand conjugates. Such an approach also leads to the production of homogeneous populations of conjugates and enables exact control of the site of modification on the enzyme. Since antibodies have lower apparent affinities for monosubstituted conjugates than for multisubstituted ones, an improvement in the analytical characteristics should be achieved without any loss in selectivity. An additional advantage of this approach is the preparation of conjugates with no batch-to-batch variability that may lead to increased long-term assay reproducibility. Assays developed in this manner can be used to detect numerous compounds of interest, such as biological peptides, peptide drugs, hormones, receptors, etc. Work is underway that extends this approach to other recombinant enzymes and peptides.

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ACKNOWLEDGMENT This work was supported by a grant from the National Institutes of Health (GM-40510). A.W. gratefully acknowledges the support of a National Science Foundation Graduate Fellowship; an American Chemical Society, Division of Analytical Chemistry Fellowship sponsored by the Society for Analytical Chemists of Pittsburgh; and a Presidential Fellowship from the University of Kentucky. S.D. acknowledges grant support from the Van Slyke Society of the American Association for Clinical Chemistry.

RECEIVED for review September 4, 1992. Accepted January 4, 1993.