Bioeonjgate Chem. 1002, 3,471-476
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ARTICLES Transition Metal Carbonyl Labeling of Proteins. A Novel Approach to a Solid-Phase Two-Site Immunoassay Using Fourier Transform Infrared Spectroscopy Anne Varenne,+Michele Salmain,+Chantal Brisson,: and G6rard Jaouen*v+ Ecole Nationale Supbrieure de Chimie de Paris, 11,rue Pierre et Marie Curie, 75231 Paris Cedex 05, France, and Clonatec, 60, rue de Wattignies, 75580 Paris Cedex 12, France. Received February 4,1992
Labeling of bovine serum albumin (BSA) and anti-human thyroid stimulating hormone (hTSH) monoclonal antibodies (mAbs) was performed using (N-succinimidyl4-pentynoate) hexacarbonyldicobalt (NSCoz(CO)& Conditions of couplingwere different depending on the protein to be labeled, denaturation of the mAbs occuring with high percentages of organic solvent in the reaction mixture. The influence of reaction time and initial concentration of NSCO2(CO)6 was examined. They were both shown to affect the final coupling rate of the metal carbonyl probe. Preservation of the immunoreactivity toward 1251-hTSHwas observed for five conjugates having different NSCoz(CO)6: mAb molar ratios when compared to unmodified and peroxidase-labeled mAbs. Finally, a preliminary study of the quantitative detection of the metal carbonyl mAbs on microtiter wells was achieved using Fourier transform infrared spectroscopy.
INTRODUCTION The conjugation of metal-containing species to proteins and especially to antibodies has been described for a large variety of applications. Progress in fundamental biochemistry can be expected from protein topography, enzyme mechanism (Kostic, 1988), and 3-D structural analysis by X-ray crystallography (Petsko, 1985). In the case of medical diagnostics, radiometal labeling of monoclonal antibodies1 offers a promising tool for in vitro (Goldenberg et al., 1980;Mirzadeh et al., 1990)and in vivo studies (Alvarez et al., 1988). In the case of quantitative assays, Miles and Hales (1965) had for the first time underlined the interest of using labeled antibodies in the assay of polypeptide antigens. Radiohalogens were the first probes used in immunometric assays. But rapidly, “cold” probes were suggested, giving birth for example to the popular ELISA tests where enzymes are conjugated to antibodies (Engvall, 1980). Quantitative assays based on metallic probes are still little developed, since Cais et al. proposed the term “metalloimmunoassay”in 1977. However, many detection techniques can be associated to this type of labeling. When the probe is a coordination complex, atomic absorption spectroscopy (Cais,1980,Chbret andBrossier, 1986;Mariet and Brossier, 19901, electrochemistry (Weber and Purdy, 1979;DiGleria et al., 1986),and fluorescence (Hemmila et al., 1984; Diamandis and Morton, 1988) have been described as detection techniques for antigen assays. A few years ago, we suggested the use of metal carbonyl moieties as markers for biological molecules (Jaouen et t
Ecole Nationale Supbrieure de Chimie de Paris. Clonatec.
Abbreviations used: BSA, bovine serum albumin; mAb, monoclonal antibody; hTSH, human thyroid stimulating hormone; NSCo2(CO)~:(N-succinimidyl4-pentynoate)hexacarbonyldicobalt; FT-IR,Fourier transform infrared; PBS, phosphatebuffered saline; NS, number of scans; POX, (horse-radish) peroxidase.
al., 1985). The development of sensitive and easy-to-use Fourier transform infrared spectrometers should encourage this concept. Recently, we proposed the term “carbonyl metalloimmunoassay” (CMIA) to designate assays using labeled antigens (Salmain et al., 1991a, 1992). In the case of low molecular weight biomolecules like hormones and drugs, we synthesized metal carbonyl derivatives which still kept a very good recognition for the target protein (receptor or antibody) (VessiBres et al., 1988; Gruselle et al., 1989). Additionally, a detection limit of 0.3 pmol of Co2(CO)6-labeledestradiol in solution has been obtained by optimizing the infrared measurements (Salmain et al., 1991b). In order to enhance the sensitivity, an interesting solution is to increase the number of Coz(CO)6 fragments bound to the biomolecule. This idea, which is hardly applicable to haptens, could be easily applied to proteins (antibodies), which possess many potential reactive sites. CMIA tests for haptens and also for antigens could then be designed. Unfortunately, direct methods of complexation are not compatible with biological media where proteins are strictly soluble. We recently developed an indirect method of introduction of Coz(CO)6 fragments on biomolecules based on a Bolton-Hunter-like reagent, (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt (NSCO2(CO)6). In this paper, we describe the first example of labeling of proteins by Co2(CO)6 fragments, an immunoreactivity study of the metal carbonyl conjugates, and a preliminary study of FT-IR detection of the conjugates on microtiter plates. EXPERIMENTAL PROCEDURES
Materials, Reagents, and Instrumentation. Inorganic salts were obtained from Merck (Darmstadt, Germany). Buffers were prepared from demineralized water as follows: phosphate-buffered saline (PBS),0.01 M, 0.14 0 1992 Amerlcan Chemical Society
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M NaCl, pH = 7.2; carbonate buffer, 0.1 M, pH = 9.6. Acetonitrile and ethanol were obtained from Prolabo (Paris, France). Brillant Blue G and bovine serum albumin (fraction V) were obtained from Sigma (St. Louis, MO). Anti-hTSH monoclonal antibodies JOSS 2-2 and microtiter wells (Maxisorb, Nunc, Denmark) were a gift from Dr. Brisson (CLONATEC, Paris, France). (N-Succinimidyl 4-pentynoate)hexacarbonyldicobalt NSCOZ(CO)~, was synthesized as described previously (Salmain et al., 1991a). UV-visible data were measured on a Uvikon 860 spectrophotometer (Kontron, Switzerland). FT-IR measurements were obtained with a Michelson 100 spectrometer (Bomem, Quebec, Canada) equipped with a liquid nitrogen cooled InSb detector. Data were treated with a PC-AT computer (NEC) connected to the interferometer and the Bomem Spectracalc software package for peak absorbance measurements. Labeling of BSA with NSCoz(CO)6. BSA (21 nmol) in 1mL of PBS was added to various quantities of NSCo2(CO)6 in a fresh acetonitrile solution. The volume was brought to 2 mL with acetonitrile (final percentage 50% and the pH was adjusted to 9.6 with carbonate buffer. The mixture was incubated at 4 "C for 18 h. Purification and Analysis of the Conjugates. The conjugates were purified on a G-25 Sephadex PDlO gel filtration column (Pharmacia, Upsala) following the procedure recommended (elution with PBS). The first 10 fractions of 1 mL were collected and assayed for the presence of protein and CO~(CO)~. The presence of protein in each tube was qualitatively and quantitatively determined by the Coomassie Blue method and the presence of C02(CO)6fragments by FT-IR spectroscopy as follows: 20 pL of each sample tube was dropped on 15 mg of KBr powder (IR grade, Prolabo, France) and lyophilized. The powder was recovered and pressed into a 3-mm KBr disk. Tubes containing both species were pooled and analyzed. The protein concentration was remeasured using the CoomassieBlue method. The concentration of covalently bound Co&O)6 fragments was determined either by UV spectroscopy at 348 nm, assuming an absorption coefficient of 3700 L mol-' cm-l, or by FT-IR spectroscopy as described above (v = 2052 cm-l). For the labeling of BSA, the measures were confirmed by atomic absorption spectroscopy. The final Coz(C0)e:protein molar ratio was compared to the initial Coz(C0)~:proteinmolar ratio in order to calculate the coupling yield. Labeling of JOSS 2-2 by NSCo2(CO)6. JOSS 2-2 (6.6 nmol) in 1mL of PBS was added to 165 nmol of NSCo2(CO)6 in 165 pL of ethanol. The volume was brought to 2 mL with PBS and the pH was adjusted to 9.6 with carbonate buffer. The mixture was incubated for 15 h and the immunoconjugate was purified and analyzed as described above. Effect of Reaction Time on the Labeling of JOSS 2-2 by NSCo~(CO)6. A number of 2-mL mixtures containing 6.6 nmol of JOSS 2-2 and 231 nmol (35 equivalents) of NSCoz(CO)6 with a find percentage of ethanol of 8% were incubated for variable periods at 4 OC. The immunoconjugates were purified and characterized as described above. Effect of Concentration of NSCOz(CO)6 on the Labeling of JOSS 2-2. A number of 2-mL mixtures containing 6.6 nmol of JOSS 2-2 and 10-45 equiv of NSCo2(CO)6(final ethanol percentage = 8%)were incubated for 3.5 or 15 h at 4 "C. The immunoconjugates were purified and characterized as described above. Study of the Immunoreactivity of the Conjugates. Variable dilutions of JOSS 2-2, peroxidase-labeled JOSS
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Figure 1. Protein elution profile (Coomassie Blue method), Coz(CO)6 marker elution profile (infrared spectroscopic detection; 3-mm KBr pellets), and Co2(CO)6 free marker elution profile (infrared spectroscopic detection; 3-mm KBr pellets).
2-2, Or [cO~(co)~]~-Joss 2-2 ( X = 4, 11, 22, 25, 29, 31) were assayed with lZ5I-hTSH. After precipitation of the bound fractions by addition of anti-mouse IgG antibody, the binding of the tracer was plotted versus the dilution of the unconjugated or conjugated mAbs. The nonspecific binding of 1251-prolactinto the mAbs (unconjugated or conjugated) was studied in the same manner. Quantitative Detection of [Coz(CO)&rJOSS 2-2 on Microtitration Wells by FT-IR Spectroscopy. A number of 26.8-pL samples of variable concentrations of [cO~(co)~]~rJoss 2-2 in PBS were deposited on microtiter wells, quantities ranging from 4 to 33 pmol of protein. The solvent was evaporated off in a vacuum dessicator for 1h and immediately analyzed by FT-IR spectroscopy (NS = 10). After baseline correction, the absorbance of the 2052-cm-l uC0 stretching band was plotted versus the quantity of protein deposited. RESULTS AND DISCUSSION
Preliminary studies of protein labeling with the metal carbonyl probe NSCo2(CO)6 were achieved with BSA as the model protein (Scheme I). NSCO2(CO)6 is not readily soluble in water like other N-succinimidyl esters, so it had to be dissolved in a miscible organic solvent. Acetonitrile was chosen at first and added to the incubation mixture in a 1:lproportion. Unreacted NSCo2(C0)6was removed by gel filtration chromatography on a Sephadex G-25 column (ready-to-use PDlO), and the efficiency of this purification step was checked by a test chromatography of free NSCoz(C0)s alone and by extensive dialysis of the conjugate after gel filtration chromatography. The purification of the reaction mixture was followed by infrared spectroscopy of the COZ(CO)~ marker and by colorimetry of the BSA which reacted with CoomassieBlue. Both elution profiles are superposed as shown in Figure 1,together with the elution profile of the free maker alone. The Coz(CO)6 marker gives two chromatographic peaks. By comparison of the elution profile of free NSCO2(CO)6 alone, the marker eluted in tubes 5-9 is attributed to the free (unconjugated) form, so the two peaks correspond to the bound and free fractions, respectively. The BSA provides one peak, the form of which can be exactly superposed to the bound marker fraction peak (tubes 1-4). A coupling rate was calculated for each of the fractions (1-41, from standard straight lines for BSA and free marker obtained under the same conditions. It was found to be constant except for tube 4 which contains both forms of marker (free and conjugated), indicating that the labeling procedure provides conjugates with homogeneouscoupling rates. Coupling rates measured by UV (348 nm) and IR
Transltlon Metal Carbonyl Labeling of Proteins
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Scheme I. Preparation of the Metal Carbonyl-Protein Conjugates
40
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HCFC-CH,CYCONH COZ(CO)~
k
Protein
Table I. Effect of Concentration of NSCoz(C0)6 on the Labeling of BSA. NSCo&O)6:BSA ratio initial final % coupling yieldb 10 8 80 30 26 87 40 37 92 60 40 67 M; temperature = 4 OC; reaction time = 15 0 [BSA] = 1.05 X h; pH = 9.6;volume of reaction = 2 mL (acetonitrile = 50%). * Calculated from (finalNSCo&O)6:BSA ratio)/(hitialNsco&O)~j: BSA ratio) X 100.
(2052 cm-l) were identical, indicating that the 2052-cm-' peak absorbance is proportional to the number of COZ(CO)6fragments bound to the protein. On the other hand, the coupling rate measured from the 2091-cm-l band is unexpectedly higher. In this case, the absorbance of the bound Coz(CO)6 fragments cannot be correlated to that of the free ones. We already observed such a difference of intensity in another series Of COz(CO)6-dkyne complexes (Salmain et al., 1991b), indicating a greater dependence of the intensity of this particular band with the substituent. After pooling tubes 1-3, the average rate of COZ(CO)S fragments bound to BSA was assayed by UV spectroscopy, assuming the same e value as for the free NSCOz(CO)6. This measure was confirmed by atomic absorption spectrophotometry (considered here as the reference method), justifying the above approximation. The coupling ratio remeasured after dialysis was identical. The other parameters, i.e. pH, reaction time, and protein concentration were not optimized. We examined the influence of the initial NSCOz(C0)s: BSA molar ratio on the final coupling yield (Table I). The yields reported are very high except for 60 equiv of NSCo2(CO)6 where a final molar ratio of 40 to 1 is measured. At pH = 9.6, NSCoz(C0)s is highly reactive toward free amino groups and is not subject to hydrolysis. Conjugates with a desired coupling rate can then be obtained by choosing the right initial equivalents of NSCoz(CO)6. Among the 59 lysine residues contained in the primary structure of BSA, only 40 seem to be reactive under the conditions described. We suspect that the 40 residues are those located at the surface of protein which are readily accessible to solvent and reagents. The same value of 40 has been recently pointed out by Diamandis and Morton (1988) when they labeled BSA with an excess of BCPDA chelate. In addition, Bauminger and Wilchek (1970)noted earlier that only 30-35 out of the 59 lysine residues of BSA are usually accessible for coupling with haptens. Coupling of NSco~(Co)6 to anti-hTSH mAbs JOSS 2-2 was tried first under the same conditions of reaction described for BSA. This experiment was unsuccessful and instead we observed a rapid denaturation (precipitation)
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Figure 2. Effect of reaction period on the coupling rate of NSCa(CO)6to JOSS 2-2. Experimental conditions: [JOSS2-23 = 3.3 X
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of the protein. We checked that this phenomenon was essentially due to the presence of a high percentage (50% ) of acetonitrile in the incubation mixture and not to the presence of the probe. Organic solvents are well-known to induce this kind of behavior, but we can still notice a great difference between BSA and JOSS 2-2. In a second experiment, we replaced acetonitrile by ethanol and reduced the final percentage of organic solvent to 8% in the reaction medium. The labeling of JOSS 2-2 was then successfully performed with 25 equiv of NSCOZ(CO)G leading to a final coupling of 22 COZ(CO)~ fragments per protein molecule after a 15-h incubation time. By reducing the percentage of organic solvent in the mixture, we were able to label, with a high yield, the monoclonal anti-hTSH antibody with the metal carbonyl probe, indicating that the protein contains at least 22 lysine residues accessible to the reagent. However, after several experiments, the results did not appear reproducible, so we went on studying the kinetics of the coupling reaction of NScOz(co)~jto Jess 2-2. In a second set of experiments for the labeling of JOSS 2-2, we studied the influence of the reaction time on the coupling rate for an initial NSCOZ(CO)~:JOSS 2-2 molar ratio of 351. The results are presented in Figure 2. In 1 h, 27 out of the 35 equiv of NSCOz(CO)6 added were coupled to the protein, indicating the reaction is complete in a very short period. The coupling rate remained approximately constant between 1 and 15 h of reaction. Protein recovery rates were about 5040% of the initial molar quantity reacted even with the low percentage of ethanol used. A reaction time of 3.5 h seems a good compromise to obtain an efficient labeling of JOSS 2-2. We finally studied the effect of concentration of NSCoz(CO)6 on the labeling of JOSS 2-2 for different reaction times. In Figure 3, we present the plots of find NSCO~(CO)G: JOSS 2-2 molar ratio versus initial NSCOZ(CO)~:JOSS 2-2 molar ratio for 3.5 and 15 h. The overall coupling yields are as high as for the other experiments except for the 15-h reaction time, for which they are low and unreproducible irrespective of the initial ratio. The coupling yield drops to 89% for 35 initial equivalents of NSCoz(CO)6 and a 3.5 h reaction time. But when the initial NSCoz(CO)6:JOSS 2-2 molar ratio reaches 45 the coupling rate is only 29, indicating a possible saturation value of the lysine sites accessible to the metal carbonyl reagent of approximately 30 residues. As for BSA, immunoconjugates of various coupling rates can be easily obtained by selecting the proper value of
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Table 11. Effect of the NSCoz(C0)s:BSA Ratio on the Immunoreactivity of the Protein ~~
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of two Iz51 atoms can be incorporated per protein without loss of immunoreactivity (Johnson et al., 1960). As for 0 10 20 30 40 50 fluorescent dyes, conjugates with coupling ratios in the initial C02(C0)6: J O S S 2-2 ratio range of 4-6 are the best choice, essentially because of Figure 3. Effect of concentrationof NSCoz(CO)6on the labeling fluorescencequenching associated with higher substitution of JOSS 2-2. Experimentalconditions: [JOSS 2-21 = 3.3 X lO+ degrees (Brinkley, 1992). A coupling rate of 31:l is a very M; temperature = 4 O C ; pH = 9.6; volume of reaction = 2 mL positive point for the improvement of FT-IR detection (ethanol 8%). sensitivity. For heterogeneous assays, the use of antibody/antigen loss 2-2 coated on solid phases can considerably simplify the 4 wx.ioss 2.2 procedures. Associated with two-site immunometric asCoUC0)bJOSS 2.2 says, polystyrene microtiter wells are widely used especially when the probe is an enzyme. We chose to study the feasability of detection of metal carbonyl labeled antibodies on this solid phase because it seemed well-adapted for the design of an easy (ready-to-use) assay. The first step was to examine the FT-IR transmission spectrum of a polystyrene well. In Figure 5 is presented its whole IR spectrum and an expansion of the 2200-1800-~m-~ region where vC0 bands are expected. Two major observations can be drawn from these two spectra. 0 . . . . . . . . , . ......., . . . . . . . . , . ...., . 10 100 1000 10000 100000 1000c0c First, polystyrene is an extremely absorbent material, but 1 / dilution a spectral window (inset of Figure 5) can be observed Figure 4. Immunoreactivity of JOSS 2-2, POX-JOSS 2-2, and between 2200 and 2000 cm-'. Second, the alkyne-Cozc o ~ ( c o ) ~ - J o2-2 s s (coupling ratio = 22). Dilution curves (CO)6 fragments are completely compatible with an IR (- -). obtained in the presenceof lZ5I-hTSH(-) and lZ5I-prolactin detection on the polystyrene plate because they usually show their three vC0 bands at wavenumbers above 2000 initial equivalents of NSCo&O)6. This is a very accute cm-l. point because the sensitivity of the FT-IR detection will be globaly proportional to the number of C O ~ ( C O ) ~ The introduction of alkyne-Coz(CO)6 fragments on the mAb JOSS 2-2 modifies its IR spectrum as illustrated on fragments covalently bound to the protein. The introFigure 6 inset. When compared to the IR spectrum of duction of such fragments on the monoclonal antibody is unmodified JOSS 2-2, that of [co2(co)~1-Joss 2-2 likely to interfere with the binding of the antigen, here presents three extra bands at 2093,2052, and 2023 cm-', hTSH. So the preliminary study concerning immunocorresponding to stretching vibrations of the CO ligands conjugate requires an evaluation of their biological activity bound to the metals and characteristic of the metallofor different coupling rates. carbonyl fragments. Polystyrene being a highly absorbent The biological study of the metal carbonyl conjugates material made it necessary to record the IR spectra on a was assessed by performing a dilution assay of the mAbs very sensitive apparatus. For this purpose, we chose an in the presence of 1251-hTSHas the tracer (specificbinding) InSb detector, previously shown as the most sensitive in and in the presence of 1251-prolactin(nonspecific binding). the 2000-cm-1 region (Salmain et al., 1991b). The dilution curves obtained from unconjugated JOSS In order to detect the metal carbonyl conjugates on the 2-2, POX-JOSS 2-2, and c o ~ ( c o ) ~ - J2-2 o ~(with ~ a microtiter wells, we deposited a constant volume of coupling ratio of 22) are reported Figure 4. different concentrations of protein in PBS and evaporated Moreover, the binding of Iz51-hTSHor 1251-prolactinto the solutions in a vacuum dessicator. Wells were immeequal dilutions of metal carbonyl conjugates with coupling diately analyzed. A blank well where a same volume of ratios of 4,11,25,29 and 31 was measured by comparison PBS had been evaporated was used for the reference to unconjugated mAb (Table 11). Using this assay, we spectrum. cannot observe any significant difference of reactivity between unmodified and conjugated mAbs with variable A typical spectrum of the conjugate deposited on a coupling ratios. microtiter well is shown in Figure 6. Again, each of the The lysine residues involved in the conjugation of the three bands can be clearly seen at 2093, 2054, and 2023 Coz(CO)6 fragments do not seem to interfere during the cm-'. Actually, only the 2054-cm-1 band provides a good binding of hTSH to JOSS 2-2 and the labeling of the metal linearity because the 2093 cm-' band is too low and the carbonyl probes does not alter the specificity of binding 2023 cm-' band too close to the polystyrene cutoff. The of the antibody. It is interesting to notice that a relatively plot of the 2052-cm-l band absorbance versus the quantity high number of metal carbonyl fragments coupled are still of conjugate deposited is reported in Figure 7. It presents compatible with a good immunoreactivity. This finding a fairly acceptable correlation coefficient over the range is remarkable since for radioisotopic labeling, a maximum of study. Without precisely optimizing the IR measure1
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No. 8, 1992 475
Bloconlugate Chem., Vol. 3,
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The coupling of biologically active proteins with an organometallicentity was performed in nondenaturating conditions with a specific labeling agent analogous to the Bolton-Hunter reagent. In the case of the monoclonal antibody, the percentage of organic solvent used to solubilize this reagent had to be carefully optimized in order to minimize the precipitation of the protein. We showed that the coupling ratio for both BSA and JOSS 2-2 reaches a plateau, indicating a saturation of the number of lysine residues involved in the coupling reaction with the metal carbonyl fragments. A good biological activity expressed as the immunoreactivity of the metal carbonyl conjugates toward 1261-hTSHwas retained even with high coupling rate conjugates, indicating that they may be included in an hTSH assay procedure. The IR detection of the modified mAb was performed on classical microtiter plates and this preliminary study showed that a fairly good linearity could be obtained between the quantity of conjugate deposited and the absorbance read a t 2052 cm-l.
I / 10
20
30
40
[Co2(CO)6]-JOSS 2-2 quantlty (pmol/well)
Figure 7. IR quantitative detection of c o ~ ( c o ) ~ - J o 2-2 s s on
microtiter wells ( u = 2054 cm-l; NS = 10; y = -2.0124e-3 + 5.2841e4x; r2 = 0.949).
menta, we detected 4 pmol of [COZ(CO)S]ZTJOSS 2-2 on the microtiter wells. The position of the microtiter well must be carefully checked between the reference and the sample spectra in order to have a good compensation. Without a special mounting plate that could have kept the wells in the right position, it was extremely difficult
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Solid-phase immunoassays are now feasible once a special IR mounting plate for the microtiter plates is available. ACKNOWLEDGMENT
This work was supported by Clonatec (Paris, France) who graciously supplied the monoclonal antibodies, the microtiter plates, and the gel filtration columns. We wish to thankF. Robillard from Clonatec for the immunological studies. This research was financially supported by an ANVAR grant. M.S. also wants to thank Dr. IS. Butler for discussion and correction of the manuscript. LITERATURE CITED Alvarez, V. L., Dwight Lopes, A., Rodwell, J. D., McKearn T. J., and Stuart, F. P. (1988) Radioimmunoscintigraphy and radioimmunotherapy in nude mouse models. Antibodymediated delivery systems (J.D. Rodwell, Ed.) pp 99-118, Marcel Dekker, New York. Bauminger, S. J., and Wilchek, M. (1980)The use of carbodiimides in the preparation of immunizing conjugates. Methods Enzymol. 70, 151-159. Brinkley, M. (1992)A brief survey of methods for preparing protein conjugates with dyes, haptens and cross-linking reagents. Bioconjugate Chem. 3,2-13. Cais, M. (1980)Specific binding assay method and reagent means. U S . Patent No. 4,205,952.Cais, M., Dani, S., Eden, Y., Gandolfi, O., Horn, M., Isaacs, E. E., Josephi, Y., Saar, Y., Slovin, E., and Snarsky, L. (1977) Metalloimmunoassav. Nature 270. 534-535. Cheret, P., and Broslier, P. (1986)'Metalloimmunoassay of antidepressant drugs: Production and characterization of antiserum. Res. Commun. Pathol. Pharmacol. 54,237-253. Diamandis, E. P., and Morton, R. C. (1988) Time-resolved fluorescence using an europium chelate of 4,7-bis-(chlorosulfophenyl)-l,l0-phenantroline-2,9-dicarboxylic acid (BCPDA). J. Immunol. methods 112,43-52. Di Gleria, K., Allen, H., Hill, O., McNeil, J., and Green, M. J. (1986)Homogeneous ferrocene-mediated amperometric immunoassay. Anal. Chem. 58, 1203-1205. Engvall, E. (1980) Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 70,419-439. Goldenberg, D. M.,Edmundkim, E., Deland, F. H., Bennett, S., and James Prime, F. (1980)Radioimmunodetection of cancer with radioactive antibodies t o carcinoembryonic antigen. Cancer Res. 40,2984-2992.
Varenne et el. Gruselle M., Deprez, P., Vessibres, A., Greenfield, S.,Jaouen, G., Larue, J. C.,andThouvenot,D. (1989)Cobaltandmolybdenum carbonyl clusters in immunology. Synthesis and binding properties of mycotoxin derivatives of zearalenone. J. Organomet. Chem. 359,C53-C56. Hemmila, I., Dabuku, S., Mukkala, V. M., Siitari, H., andLovgren, T. (1984)Europeium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 353-343. Jaouen, G., VessiBres, A,, Top, S., Ismail, A. A,, and Butler, I. S. (1985)Metal-carbonyl fragments as a new class of markers in molecular biology. J. Am. Chem. SOC.107,4778-4780. Johnson, A., Day, E. D., and Pressman, D. (1960)The effect of iodination on antibody activity. J. Immunol. 84,213-220. Kostic, N. M. (1988)Selective labeling, cross-linking and cleavage of proteins with transition-metal complexes. Comm. Inorg. Chem. 8,137-162. Mariet, F., and Brossier, P. (1990)A new power assiociation for immunoassay:organometallic label conjugated to solvent s e p aration method. Res. Commun. Chem. Pathol. Pharmucol. 68,251-262. Miles, L. E., and Hales, C. N. (1968)Labelled antibodies and immunological assay systems. Nature 219,186-189. Mirzadeh, S.,Brechbiel, M. W., Atcher, R. W., and Gansow, D. A. (1990)Radiometal labeling of immunoproteins: Covalent linkage of 2-(4-isothiocyanatobenzyl)diethylenetriaminepen taacetic acid ligands to immunoglobulin. Bioconjugate Chem. 1 , 59-65. Petsko, G. H. (1985) Preparation of heavy-metal derivatives. Methods Enzymol. 114, 147-156. Salmain, M., VessiBres, A,, Butler, I. S., and Jaouen, G. (1991a) (N-Succinimidyl 4-pentynoate)hexacarbonyl:a transitionmetal carbonyl complex having similar uses to the BoltonHunter reagent. Bioconjugate Chem. 2,13-15. Salmain, M., VessiBres, A., Jaouen, G., and Butler, I. S. (1991b) Fourier Transform infrared spectroscopic method for the quantitative trace analysis of transition metal carbonyl-labeled bioligands. Anal. Chem. 63,2323-2329. Salmain, M., VessiBres, A., Brossier, P., Butler, I. S., and Jaouen, G. (1992)Carbonylmetalloimmunoassay (CMIA) a new type of non-radioisotopic immunoassay. Principles and application to phenobarbital assay. J. Immunol. Methods 148,65-75. VessiBres, A., Jaouen, G., Gruselle, M., Rossignol, J. L., Savignac, M., Top, S., and Greenfield, S. (1988)Synthesis and receptor binding of polynuclear organometallic estradiol derivatives. J. Steroid Biochem. 30,301-305. Weber, S.G., andPurdy, W. C. (1979)Homogeneousvoltammetric immunoassay: A preliminary study. Anal. Lett. 12,1-9. Registry No. N S C O ~ ( C O )132178-38-2; ~, TSH, 9002-71-5.
