Vol.
174,
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No.
3,
14,
1991
1991
BIOCHEMICAL
AND
BIOPHYSICAL
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COMMUNICATIONS Pages
Bioluminescent
Immunoassay Using a Monomeric Aequorin Conjugate
Teruya
Erikaku,
Shuhei
Zenno and Satoshi
1331-l
336
Fab’-Photoprotein Inouye*
Yokohama Research Center, Chisso Corporation, 2 Kamariya-cho, Kanazawa-ku, Yokohama 236, JAPAN Received
January
7, 1991
The photoprotein aequorinemitslight by an intermolecularreaction when mixed with Ca*+. To apply a bioluminescent immunoassaybasedon the light emissionproperty of aequorin, we preparedaequorin-antibody Fab’ conjugatesby a chemicalcross-linking technique. Recombinant apoaequorin was coupled with human tumor necrosisfactor-u (TNF-a) antibodies (polyclonal Fab’or monoclonal Fab’ fragments) usingN-succinimidyl4-(N-maleimidemethyl)cyclohexane-1carboxylate. The luminescentactivity of the Fab’aequorin conjugatewas about one-tenth that of aequorin. Using a monomericconjugate, humanTNF-a can be measuredat an attomole level by a sandwichimmunoassaytechnique. B 1991 Academic mess,Inc.
Aequorin is a photoprotein isolatedfrom the jellyfish, Aequorea Victoria (1) and hasbeen widely used as a biological calcium indicator (2). Aequorin is a complex of apoaequorin, coelenterazine(an imidazopyrazine compound)and molecularoxygen (3,4). Apoaequorinis made up of 189amino acid residuesarrangedin a singlepolypeptide chain, with three Ca2+-bindingsites
and three cystein residues(5). The luminescentreaction is triggered by Ca2+-bindingto aequorin and yields blue light (kmax=470 nm), CO;?,apoaequorinand coelenteramide(1,3). Aequorin can be reactivated by the incubation of apoaequorinwith coelenterazine,molecularoxygen, a reducing agent and EDTA (6). Previously, we cloned the cDNA for apoaequorin (5) expressed it in Escherichia coli (7,8) and purified the apoaequorin with a convenient procedure (8). The recombinant aequorin can be also usedas a calcium indicator (9). The apoaequorinis a stable protein (7,8) and this property is advantageousfor the cross-linking to other materials (such as enzymes, antibodiesand functional ligands) with a chemical cross-linker. Furthermore, the assay of aequorin is a highly sensitive (detection of photon), non-hazardous (no use of radioactive compound)and fairly rapid reaction (few secondreaction) (2,lO). Thus, the apoaequorinmay be a usefulprotein asa reporter enzyme for immunoassaysand hybridization assaysusing nucleic acid probes. Recently, we reported a bioluminescentimmunoassayusingthe protein A-aequorin fusion protein aseffectively asthe protein A-horseradishperoxidaseconjugate(11). For a more extensive *Presentaddress:The Whittier Institute, 9894 GeneseeAve., La Jolla, CA 92037. Abbreviations: DMF; N,N-dimethylformamide, DTNB; 5,5’-dithiobis(2-nitrobenzoic acid), IgG; Immunoglobulin G, PBM; N,N’-(1,2-Phenylene)bismaleimide, SMCC; N-succinimidyl 4-(Nmaleimidemethyl)cyclohexane-1-carboxylate,TNF; tumor necrosisfactor. 0006-291X/91
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application of aequorin in an immunoassay, we conjugated apoaequorin with the Fab’ fragment of an antibody using a chemical cross-linker and applied its conjugate in an immunoassay. As a model system, we describe here the preparation of the anti-human TNF-a Fab’-apoaequorin conjugates and their application in a bioluminescent immunoassay. MATERIALS
AND
METHODS
Materials Recombinant apoaequorin was produced in E.coli and purified as previously described (8). Coelenterazine, 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-3,7-dihy~oimidazol[ 1,2-alpyrazin-3one, was chemically synthesized in our laboratory according to the method of Inoue et al. (12). Nsuccinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC) was obtained from Zieben Chemical Co. (Tokyo, Japan). N,N’-(1,2-Phenylene)bismaleimide (PBM), 2mercaptoethlyamine-HCl, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) and N,Ndimethylformamide (DMF) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sephadex G-50 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Ultrogel AcA 44 was a product of LKB (Stockholm, Sweden). Polystyrene cuvettes were purchased from Labo Science Co., Ltd. (Tokyo,Japan). Centricon Microconcentrator was a product of Amicon (Beverly, MT). Immuno Pure F(ab’)z Preparation Kit was a product of Pierce (Rockford, IL). Biokine TNF Test Kit was from T Cell Sciences, Inc. (Cambridge, MA). Monoclonal and rabbit polyclonal anti-human TNF-a IgG were obtained from Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan). Bovine serum albumin (fraction V) was obtained from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). All other chemicals were of the highest grade commercially available. Buffers The buffers used in this experiment were as follows: Buffer A, 100 mM sodium phosphate buffer, pH 6.0; Buffer B, 100 mM sodium phosphate buffer, pH 7.0; Buffer C, 10 mM sodium phosphate buffer, pH 7.0, containing 0.1 % bovine serum albumin; Buffer D, 10 mM sodium phosphate buffer, pH 7.0, containing 100 mM NaCl; Buffer E, 300 mM Tris-HCl, pH 7.6, containing 100 mM EDTA. Introduction of maleimide mouns into aDoaeauorin The introduction of maleimidegroupsinto the amino or thiol residuesof apoaequorinwas perfomled by the method of Ishikawa et a1.(13). Apoaequorin (356 l.tg, 16.5 nmol) in 500 ~1 of buffer B was mixed with SMCC (165 ug, 494 nmol) dissolvedin 50 ul of DMF or with PBM (100 pg, 373 nmol) dissolved in 50 ~1 of DMF. The reaction mixture was incubated at 30” C for various lengths of time and was then subjected to gel filtration on a column (1.0 x 40 cm) of Sephadex G-50 using buffer A. Peak fractions of maleimide-apoaequorin were pooled and concentratedby a Centricon-10 Microconcentrator. Detemlinationof the averagenumberof maleimidegrouusintroducedinto auoaeauorin The protein concentration of the maleimide-apoaequorin was determined using the extinction coefficient at 280 nm of apoaequorin,which is 18.2 g-t.Lcm-l (2) and a molecular weight of 21,600 (8). The averagenumberof maleimidegroupsintroduced wasdeterminedby the method of Ishikawa et al. (13). The maleimidegroups of apoaequorinare reacted with a known amountof 2-mercaptoethylamine,and the remainingthiol groupsare measuredby Ellman’sreagent (14). Thus, 450 pl of the maleimide-apoaequorinsolution was mixed with 50 ~1 of 0.5 mM 2mercaptoethylamine/50mM EDTA, pH 6.0, and incubatedat 30” C for 20 min. Subsequently,10 pl of 10 mM DTNB in buffer B was addedto the above mixture and allowed to standfor 5 min at room temperature. By measuringthe absorbanceat 412 nm, the average number of maleimide groupsintroduced per apoae uorin moleculewas calculatedusing the molar extinction coefficient which is 13,600 mol-l-Lcm- P (14). Prenarationof anti-humanTNF-a Fab’ Monoclonal or polyclonal anti-human TNF-a IgG (Hayashibara Biochem. Lab.) was digested with immobilized pepsin according to the protocol of the Immuno Pure F(ab’)2 Preparation Kit, followed by dialysis against buffer A using dialysis tubing with a molecular weight cutoff range up to 50,000. The F(ab’)2 fragment obtained was concentrated by a Centricon- Microconcentrator and then converted to Fab’ fragment by the treatment with 21332
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mercaptoethylamine (13). The concentration of the Fab’ fragment was calculated by the absorbance at 280 nm, its extinction coefficient which is 1.48 g-l.Lcrn-l (15) and its molecular weight of 46,000 (16, 17). Conjueation of maleimide-apoaeauorin with anti-human TNF-a Fab’ The polyclonal or monoclonal Fab’ fragment (0.6 mg, 13 nmol) was incubated with an excess of maleimide-apoaequorin (1.26 mg, 58.3 nmol) in buffer A containing 5 mM EDTA at 4’ C for 24 hr. To separate the Fab’-apoaequorin conjugate from unconjugated apoaequorin and Fab’ fragment, the reaction mixture was subjected to gel filtration on a column (1 .O x 45 cm) of Ultrogel AcA 44 using the same buffer. Peak fractions of the Fab’-apoaequorin conjugate were pooled and concentrated by a CentriconMicroconcentrator. The protein concentration of the conjugate was determined by the dye-binding method of Bradford (18) which uses bovine y-globulin as the standard (Bio-Rad, Richmond, CA) Bioluminescent sandwich immunoassay techniaue Polystyrene cuvettes were coated with murine monoclonal anti-human TNF-a IgG (T Cell Sciences) according to the protocol of the Biokine TNF Test Kit. IgG-coated polystyrene cuvettes were incubated with human TNF-a standard (T Cell Sciences) in a total volume of 100 ~1 at 37” C for 5 hr with shaking and incubated an additional 16 hr at 4’ C. After incubation, the cuvettes were washed 3 times with buffer D and then incubated with the Fab’-apoaequorin conjugate (810 ng) in a total volume of 150 l.rl of buffer C, containing 100 mM NaCl, at 20” C for 4 hr with shaking. After aspirating the solution, the cuvettes were incubated twice with buffer D at 30’ C for 10 min with shaking and washed twice with buffer D. The luminescent activity of the Fab’-aequorin conjugate bound to human TNF-a was determined as described below. Assay of the luminescent activitv of aeuuorin The luminescent activity of aequorin was measured using a lumiphotometer model TD4000 (Labo Science, Tokyo, Japan). Various amounts of apoaequorin or sample solutions, 10 ~1 of buffer E, 1 ul of coelenterazine (200 ug/ml in methanol) and 1 ul of 2-mercaptoethanol were mixed and brought up to a volume of 100 ul with deionized water. The reaction mixture was incubated at 4” C for 14 hr. Subsequently, 10 ul of the above mixture was placed in a lumiphotometer and injected with 100 ul of 30 mM CaC12/30 mM Tris-HCl (pH 7.6). The luminescence was recorded and the values were presented in relative light units (r.1.u.). One r.1.u. is equivalent to the luminescent activity from 2.5 pg of native aequorin (8).
RESULTS
AND
DISCUSSION
In this paper, we described the introduction of maleimide groups into the thiol and amino groups of apoaequorin using a heterobifunctional
cross-linker
and the maleimide-apoaequorin
obtained was reacted with the Fab’ fragment of polyclonal and monoclonal anti-TNF-a. The crosslinked Fab’-apoaequorin conjugate was used for an immunoassay (Fig. 1). After the incubation with cross-linkers
for various lengths of time, the luminescent activity
of apoaequorin and the average number of maleimide groups per apoaequorin molecule were determined. As shown in Fig. 2, the luminescent activity of apoaequorin was effectively decreased by increasing the number of maleimide groups in apoaequorin. In the case of one maleimide group introduced per apoaequorin molecule, the luminescent activity was decreased to one-fourth for the thiol modification and one-half for the amino group. The modification of the thiol group in apoaequorin more greatly affected the luminescent activity than that of the amino group. Thus, the maleimide-apoaequorin
introduced into the amino group was used to conjugate with the Fab’
fragment of anti-TNF-a. To retain the luminescent activity and antigen-binding ability as highly as possible, it is necessary to prepare a monomeric conjugate of Fab’ fragment and apoaequorin (13). By controlling the reaction time to 5 min, the average number of maleimide groups introduced per apoaequorin molecule was approximately one. 1333
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Fieure 1, Schematic structures of modified apoaequorin conjugate. (A) Maleimide-apoaequorin modified with PBM. modified with SMCC. (C) Fab’-apoaequorin conjugate.
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and Fab’-apoaequorin (B) Maleimide-apoaequorin
The elution profile from the Ultrogel AcA 44 column of Fab’-apoaequorinconjugate was shown in Fig.3. The conjugate of Fab’-apoaequorinwas almost separatedfrom free apoaequorin and Fab’ fragment by gel filtration. The molecularweight of the conjugatewasestimatedto be 6070 kiloDaltons using a standardprotein of molecularweight (Pharmacia). This result indicatesthat one molecule each of Fab’ and apoaequorin were cross-linked to form a monomeric conjugate (Fig. 1-C). The specific activities of polyclonal and monoclonal Fab’-apoaequorinconjugates were 11.5 and 10.2 r.1.u. per ng of protein, respectively. Thesevalues are about one-tenth that of unmodified aequorin when computed on a molar basis. Theseconjugateswere stablefor at least one month at 4’ C. As shown in Fig. 4, the monomeric polyclonal and monoclonal Fab’-apoaequorin conjugates were used for the sandwich immunoassay of human TNF-a. In this assay, one attomole of human TNF-a can be measuredusing both conjugates. Further, the sensitivity of the 120-
60-
40
20 -
0-l 0
No. of Maleimide
1
2
Groups
per
3
Apoaequorin
4
Molecule
Fbure 2, Effects of the chemical modification on the luminescent activity of aequorin. Amino groups of apoaequorin were modified with SMCC (closed circles) and thiol groups of apoaequorin were modified with PBM (open circles). The incubation times with SMCC and PBM were 0, 10, 20, 30 min and 0, 3, 5, 10, 30 min, respectively. The luminescent activity of modified apoaequorin was expressed as the percentage of the activity of unmodified apoaequorin. The dashed vertical line represents the point of one maleimide molecule introduced per apoaequorin.
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600
A
1... A .z c 2al
0.08
s
0 06
2 s
0.05
.
0.07
.E
E
0.04
3 003
n
03 Fraction
Number
Human
TNF-a
(amolhube)
Figure 3, Elution profile from a Ultrogel AcA44 column of Fab’-apoaequorin conjugate. (A) Polyclonal Fab’-apoaequorin conjugate. Peak fractions (No.20,21) were used for the immunoassay. (B) Monoclonal Fab’-apoaequorin conjugate. Peak fractions (No l&19) were used for the immunoassay. Closed and opened squares represent the total activity of the luminescence and the absorbance at 280 nm, respectively. The flow rate was 0.41 ml/min and the fraction volume was 1.5 ml. Twenty microliters of each fraction was assayed as described in materials and methods. Figure 4, Bioluminescent sandwich immunoassay of human TNF-a using antihuman TNF-a Fab’ conjugated with apoaequorin. (A) the polyclonal anti-human TNF-* Fab’-apoaequorin conjugate. (B) the monoclonal anti-human TNF-a Fab’-apoaequorin conjugate. The assay mehtod was described in materials andmethods. Each point represents the mean of five determinations.
detection of human TNF-a using the Fab’-apoaequorinconjugate were approximately IOO-fold higher than that using either the rabbit polyclonal IgG-apoaequorin conjugate or the murine monoclonal IgG-horseradish peroxidase conjugate (data not shown). The decrease in the sensitivity using their IgG-conjugateswascausedby the nonspecificbinding of the Fc fragment as reported by Kato et al. (19). As in Fig. 4B, the luminescentintensity using monoclonal antibody waslower than polyclonal antibody. The low intensity may be due to the difference of antibodies. Previously, Inoue et al. (20) reported the sandwichimmunoassayof IgG usinga monomeric Fab’p-galactosidaseconjugate and the detection limit of humanIgG wasapproximately 100 attomolc. The difference of the detection sensitivity might be due to the difference in the ability of antibodies or in the nonspecific
adsorption
of apoaequorin
and p-galactosidase.
Recently,
Casadei et ul.
showedthat a fusion protein of aequorinand the IgG fragment of the specific antibody against the 4-hydroxy-3-nitrophenacetyl group could be used in an immunoassay(21). However, in this method, it is indispensableto isolate the specific IgG gene for the specific antigen. Thus, the 1335
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chemical cross-linked method of aequorin and the antibody is more suitable for the various species of antigen in an immunoassay. The sensitivity of the detection using the Fab’-apoaequorin conjugate
was high and suggested that aequorin is a beneficial
protein for the sensitive
immunoassay.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Shimomura, O., Johnson, F. H. and Saiga, Y. (1962) J. Cell. Comp. Physiol.59, 223. 239. Blinks, J. R., Prendergast,F. G. and Allen, D. V. (1976) Pharmacol. Rev. 28, l-93. Shimomura, 0. and Johnson,F. H. (1978) Proc. Nutl. Acad. Sci. USA. 75, 261 l-2615. Musicki, B., Kishi, Y. and Shimomura, 0. (1986) J. Chem. Sot. Chem. Commun. 21, 1566-1568. Inouye, S., Noguchi, M., Sakaki, Y., Takagi, Y., Miyata, T., Iwanaga, S., Miyata, T. and Tsuji, F. I. (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158. Shimomura, 0. and Johnson,F.H. (1975) Nature 256, 236-239. Inouye, S., Sakaki, Y., Goto, T. and Tsuji, F. I. (1986) Biocemistry 25, 8425-8429. Inouye, S., Aoyama, S., Miyata, T., Tsuji, F. I. and Sakaki, Y. (1989) J. Biocem. 105, 474-477. Shimomura, O., Inouye, S., Musicki, B. and Kishi, Y. (1990) Biochem. J. 270, 309312. Johnson,F. H. and Shimomura,0. (1978) Methods Enzymol. 57, 271-291. Zenno, S. and Inouye, S. (1990) Biochem. Biophys. Res. Commun.171, 169-174. moue, S., Sugiyama, S., Kakoi, H., Hasizume, K., Goto, T. and Iio, H.(1975) Chem. Lett. 141-144. Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y. and Ueno, T. (1983) J. Immunoassay4, 209-327. Ellman, G. E. (1959) Arch. Biochem. Biophys. 82, 70-77. Mandy, W. J. and Nisonoff, A. (1963) J. Biol. Chem.238,206-213. Jaquet, H. and Cebra, J. J. (1965) Biochemistry 4, 954-963. Utsumi, S. and Karush, F. (1965) Biochemistry 4,1766-1779. Bradford, M. M. (1976) Anal, Biochem. 72, 248-254. Kato, K., Fukui, H., Hamaguchi, Y. and Ishikawa, E. (1976) J. Zmmunology 116, 1554-1560. Inoue, S., Hashida, S., Tanaka, K., Imagawa, M. and Ishikawa, E. (1985) Anal. Lett. 18, 1331-1344. Casadei, J., Powell, M. J. and Kenten, J. H. (1990) Proc. Nutl. Acad. Sci. USA. 87, 2047-2051.
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1991
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Bioluminescent
Immunoassay Using a Monomeric Aequorin Conjugate
Teruya
Erikaku,
Shuhei
Zenno and Satoshi
1331-l
336
Fab’-Photoprotein Inouye*
Yokohama Research Center, Chisso Corporation, 2 Kamariya-cho, Kanazawa-ku, Yokohama 236, JAPAN Received
January
7, 1991
The photoprotein aequorinemitslight by an intermolecularreaction when mixed with Ca*+. To apply a bioluminescent immunoassaybasedon the light emissionproperty of aequorin, we preparedaequorin-antibody Fab’ conjugatesby a chemicalcross-linking technique. Recombinant apoaequorin was coupled with human tumor necrosisfactor-u (TNF-a) antibodies (polyclonal Fab’or monoclonal Fab’ fragments) usingN-succinimidyl4-(N-maleimidemethyl)cyclohexane-1carboxylate. The luminescentactivity of the Fab’aequorin conjugatewas about one-tenth that of aequorin. Using a monomericconjugate, humanTNF-a can be measuredat an attomole level by a sandwichimmunoassaytechnique. B 1991 Academic mess,Inc.
Aequorin is a photoprotein isolatedfrom the jellyfish, Aequorea Victoria (1) and hasbeen widely used as a biological calcium indicator (2). Aequorin is a complex of apoaequorin, coelenterazine(an imidazopyrazine compound)and molecularoxygen (3,4). Apoaequorinis made up of 189amino acid residuesarrangedin a singlepolypeptide chain, with three Ca2+-bindingsites
and three cystein residues(5). The luminescentreaction is triggered by Ca2+-bindingto aequorin and yields blue light (kmax=470 nm), CO;?,apoaequorinand coelenteramide(1,3). Aequorin can be reactivated by the incubation of apoaequorinwith coelenterazine,molecularoxygen, a reducing agent and EDTA (6). Previously, we cloned the cDNA for apoaequorin (5) expressed it in Escherichia coli (7,8) and purified the apoaequorin with a convenient procedure (8). The recombinant aequorin can be also usedas a calcium indicator (9). The apoaequorinis a stable protein (7,8) and this property is advantageousfor the cross-linking to other materials (such as enzymes, antibodiesand functional ligands) with a chemical cross-linker. Furthermore, the assay of aequorin is a highly sensitive (detection of photon), non-hazardous (no use of radioactive compound)and fairly rapid reaction (few secondreaction) (2,lO). Thus, the apoaequorinmay be a usefulprotein asa reporter enzyme for immunoassaysand hybridization assaysusing nucleic acid probes. Recently, we reported a bioluminescentimmunoassayusingthe protein A-aequorin fusion protein aseffectively asthe protein A-horseradishperoxidaseconjugate(11). For a more extensive *Presentaddress:The Whittier Institute, 9894 GeneseeAve., La Jolla, CA 92037. Abbreviations: DMF; N,N-dimethylformamide, DTNB; 5,5’-dithiobis(2-nitrobenzoic acid), IgG; Immunoglobulin G, PBM; N,N’-(1,2-Phenylene)bismaleimide, SMCC; N-succinimidyl 4-(Nmaleimidemethyl)cyclohexane-1-carboxylate,TNF; tumor necrosisfactor. 0006-291X/91
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application of aequorin in an immunoassay, we conjugated apoaequorin with the Fab’ fragment of an antibody using a chemical cross-linker and applied its conjugate in an immunoassay. As a model system, we describe here the preparation of the anti-human TNF-a Fab’-apoaequorin conjugates and their application in a bioluminescent immunoassay. MATERIALS
AND
METHODS
Materials Recombinant apoaequorin was produced in E.coli and purified as previously described (8). Coelenterazine, 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-3,7-dihy~oimidazol[ 1,2-alpyrazin-3one, was chemically synthesized in our laboratory according to the method of Inoue et al. (12). Nsuccinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC) was obtained from Zieben Chemical Co. (Tokyo, Japan). N,N’-(1,2-Phenylene)bismaleimide (PBM), 2mercaptoethlyamine-HCl, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) and N,Ndimethylformamide (DMF) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sephadex G-50 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Ultrogel AcA 44 was a product of LKB (Stockholm, Sweden). Polystyrene cuvettes were purchased from Labo Science Co., Ltd. (Tokyo,Japan). Centricon Microconcentrator was a product of Amicon (Beverly, MT). Immuno Pure F(ab’)z Preparation Kit was a product of Pierce (Rockford, IL). Biokine TNF Test Kit was from T Cell Sciences, Inc. (Cambridge, MA). Monoclonal and rabbit polyclonal anti-human TNF-a IgG were obtained from Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan). Bovine serum albumin (fraction V) was obtained from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). All other chemicals were of the highest grade commercially available. Buffers The buffers used in this experiment were as follows: Buffer A, 100 mM sodium phosphate buffer, pH 6.0; Buffer B, 100 mM sodium phosphate buffer, pH 7.0; Buffer C, 10 mM sodium phosphate buffer, pH 7.0, containing 0.1 % bovine serum albumin; Buffer D, 10 mM sodium phosphate buffer, pH 7.0, containing 100 mM NaCl; Buffer E, 300 mM Tris-HCl, pH 7.6, containing 100 mM EDTA. Introduction of maleimide mouns into aDoaeauorin The introduction of maleimidegroupsinto the amino or thiol residuesof apoaequorinwas perfomled by the method of Ishikawa et a1.(13). Apoaequorin (356 l.tg, 16.5 nmol) in 500 ~1 of buffer B was mixed with SMCC (165 ug, 494 nmol) dissolvedin 50 ul of DMF or with PBM (100 pg, 373 nmol) dissolved in 50 ~1 of DMF. The reaction mixture was incubated at 30” C for various lengths of time and was then subjected to gel filtration on a column (1.0 x 40 cm) of Sephadex G-50 using buffer A. Peak fractions of maleimide-apoaequorin were pooled and concentratedby a Centricon-10 Microconcentrator. Detemlinationof the averagenumberof maleimidegrouusintroducedinto auoaeauorin The protein concentration of the maleimide-apoaequorin was determined using the extinction coefficient at 280 nm of apoaequorin,which is 18.2 g-t.Lcm-l (2) and a molecular weight of 21,600 (8). The averagenumberof maleimidegroupsintroduced wasdeterminedby the method of Ishikawa et al. (13). The maleimidegroups of apoaequorinare reacted with a known amountof 2-mercaptoethylamine,and the remainingthiol groupsare measuredby Ellman’sreagent (14). Thus, 450 pl of the maleimide-apoaequorinsolution was mixed with 50 ~1 of 0.5 mM 2mercaptoethylamine/50mM EDTA, pH 6.0, and incubatedat 30” C for 20 min. Subsequently,10 pl of 10 mM DTNB in buffer B was addedto the above mixture and allowed to standfor 5 min at room temperature. By measuringthe absorbanceat 412 nm, the average number of maleimide groupsintroduced per apoae uorin moleculewas calculatedusing the molar extinction coefficient which is 13,600 mol-l-Lcm- P (14). Prenarationof anti-humanTNF-a Fab’ Monoclonal or polyclonal anti-human TNF-a IgG (Hayashibara Biochem. Lab.) was digested with immobilized pepsin according to the protocol of the Immuno Pure F(ab’)2 Preparation Kit, followed by dialysis against buffer A using dialysis tubing with a molecular weight cutoff range up to 50,000. The F(ab’)2 fragment obtained was concentrated by a Centricon- Microconcentrator and then converted to Fab’ fragment by the treatment with 21332
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mercaptoethylamine (13). The concentration of the Fab’ fragment was calculated by the absorbance at 280 nm, its extinction coefficient which is 1.48 g-l.Lcrn-l (15) and its molecular weight of 46,000 (16, 17). Conjueation of maleimide-apoaeauorin with anti-human TNF-a Fab’ The polyclonal or monoclonal Fab’ fragment (0.6 mg, 13 nmol) was incubated with an excess of maleimide-apoaequorin (1.26 mg, 58.3 nmol) in buffer A containing 5 mM EDTA at 4’ C for 24 hr. To separate the Fab’-apoaequorin conjugate from unconjugated apoaequorin and Fab’ fragment, the reaction mixture was subjected to gel filtration on a column (1 .O x 45 cm) of Ultrogel AcA 44 using the same buffer. Peak fractions of the Fab’-apoaequorin conjugate were pooled and concentrated by a CentriconMicroconcentrator. The protein concentration of the conjugate was determined by the dye-binding method of Bradford (18) which uses bovine y-globulin as the standard (Bio-Rad, Richmond, CA) Bioluminescent sandwich immunoassay techniaue Polystyrene cuvettes were coated with murine monoclonal anti-human TNF-a IgG (T Cell Sciences) according to the protocol of the Biokine TNF Test Kit. IgG-coated polystyrene cuvettes were incubated with human TNF-a standard (T Cell Sciences) in a total volume of 100 ~1 at 37” C for 5 hr with shaking and incubated an additional 16 hr at 4’ C. After incubation, the cuvettes were washed 3 times with buffer D and then incubated with the Fab’-apoaequorin conjugate (810 ng) in a total volume of 150 l.rl of buffer C, containing 100 mM NaCl, at 20” C for 4 hr with shaking. After aspirating the solution, the cuvettes were incubated twice with buffer D at 30’ C for 10 min with shaking and washed twice with buffer D. The luminescent activity of the Fab’-aequorin conjugate bound to human TNF-a was determined as described below. Assay of the luminescent activitv of aeuuorin The luminescent activity of aequorin was measured using a lumiphotometer model TD4000 (Labo Science, Tokyo, Japan). Various amounts of apoaequorin or sample solutions, 10 ~1 of buffer E, 1 ul of coelenterazine (200 ug/ml in methanol) and 1 ul of 2-mercaptoethanol were mixed and brought up to a volume of 100 ul with deionized water. The reaction mixture was incubated at 4” C for 14 hr. Subsequently, 10 ul of the above mixture was placed in a lumiphotometer and injected with 100 ul of 30 mM CaC12/30 mM Tris-HCl (pH 7.6). The luminescence was recorded and the values were presented in relative light units (r.1.u.). One r.1.u. is equivalent to the luminescent activity from 2.5 pg of native aequorin (8).
RESULTS
AND
DISCUSSION
In this paper, we described the introduction of maleimide groups into the thiol and amino groups of apoaequorin using a heterobifunctional
cross-linker
and the maleimide-apoaequorin
obtained was reacted with the Fab’ fragment of polyclonal and monoclonal anti-TNF-a. The crosslinked Fab’-apoaequorin conjugate was used for an immunoassay (Fig. 1). After the incubation with cross-linkers
for various lengths of time, the luminescent activity
of apoaequorin and the average number of maleimide groups per apoaequorin molecule were determined. As shown in Fig. 2, the luminescent activity of apoaequorin was effectively decreased by increasing the number of maleimide groups in apoaequorin. In the case of one maleimide group introduced per apoaequorin molecule, the luminescent activity was decreased to one-fourth for the thiol modification and one-half for the amino group. The modification of the thiol group in apoaequorin more greatly affected the luminescent activity than that of the amino group. Thus, the maleimide-apoaequorin
introduced into the amino group was used to conjugate with the Fab’
fragment of anti-TNF-a. To retain the luminescent activity and antigen-binding ability as highly as possible, it is necessary to prepare a monomeric conjugate of Fab’ fragment and apoaequorin (13). By controlling the reaction time to 5 min, the average number of maleimide groups introduced per apoaequorin molecule was approximately one. 1333
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Fieure 1, Schematic structures of modified apoaequorin conjugate. (A) Maleimide-apoaequorin modified with PBM. modified with SMCC. (C) Fab’-apoaequorin conjugate.
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and Fab’-apoaequorin (B) Maleimide-apoaequorin
The elution profile from the Ultrogel AcA 44 column of Fab’-apoaequorinconjugate was shown in Fig.3. The conjugate of Fab’-apoaequorinwas almost separatedfrom free apoaequorin and Fab’ fragment by gel filtration. The molecularweight of the conjugatewasestimatedto be 6070 kiloDaltons using a standardprotein of molecularweight (Pharmacia). This result indicatesthat one molecule each of Fab’ and apoaequorin were cross-linked to form a monomeric conjugate (Fig. 1-C). The specific activities of polyclonal and monoclonal Fab’-apoaequorinconjugates were 11.5 and 10.2 r.1.u. per ng of protein, respectively. Thesevalues are about one-tenth that of unmodified aequorin when computed on a molar basis. Theseconjugateswere stablefor at least one month at 4’ C. As shown in Fig. 4, the monomeric polyclonal and monoclonal Fab’-apoaequorin conjugates were used for the sandwich immunoassay of human TNF-a. In this assay, one attomole of human TNF-a can be measuredusing both conjugates. Further, the sensitivity of the 120-
60-
40
20 -
0-l 0
No. of Maleimide
1
2
Groups
per
3
Apoaequorin
4
Molecule
Fbure 2, Effects of the chemical modification on the luminescent activity of aequorin. Amino groups of apoaequorin were modified with SMCC (closed circles) and thiol groups of apoaequorin were modified with PBM (open circles). The incubation times with SMCC and PBM were 0, 10, 20, 30 min and 0, 3, 5, 10, 30 min, respectively. The luminescent activity of modified apoaequorin was expressed as the percentage of the activity of unmodified apoaequorin. The dashed vertical line represents the point of one maleimide molecule introduced per apoaequorin.
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600
A
1... A .z c 2al
0.08
s
0 06
2 s
0.05
.
0.07
.E
E
0.04
3 003
n
03 Fraction
Number
Human
TNF-a
(amolhube)
Figure 3, Elution profile from a Ultrogel AcA44 column of Fab’-apoaequorin conjugate. (A) Polyclonal Fab’-apoaequorin conjugate. Peak fractions (No.20,21) were used for the immunoassay. (B) Monoclonal Fab’-apoaequorin conjugate. Peak fractions (No l&19) were used for the immunoassay. Closed and opened squares represent the total activity of the luminescence and the absorbance at 280 nm, respectively. The flow rate was 0.41 ml/min and the fraction volume was 1.5 ml. Twenty microliters of each fraction was assayed as described in materials and methods. Figure 4, Bioluminescent sandwich immunoassay of human TNF-a using antihuman TNF-a Fab’ conjugated with apoaequorin. (A) the polyclonal anti-human TNF-* Fab’-apoaequorin conjugate. (B) the monoclonal anti-human TNF-a Fab’-apoaequorin conjugate. The assay mehtod was described in materials andmethods. Each point represents the mean of five determinations.
detection of human TNF-a using the Fab’-apoaequorinconjugate were approximately IOO-fold higher than that using either the rabbit polyclonal IgG-apoaequorin conjugate or the murine monoclonal IgG-horseradish peroxidase conjugate (data not shown). The decrease in the sensitivity using their IgG-conjugateswascausedby the nonspecificbinding of the Fc fragment as reported by Kato et al. (19). As in Fig. 4B, the luminescentintensity using monoclonal antibody waslower than polyclonal antibody. The low intensity may be due to the difference of antibodies. Previously, Inoue et al. (20) reported the sandwichimmunoassayof IgG usinga monomeric Fab’p-galactosidaseconjugate and the detection limit of humanIgG wasapproximately 100 attomolc. The difference of the detection sensitivity might be due to the difference in the ability of antibodies or in the nonspecific
adsorption
of apoaequorin
and p-galactosidase.
Recently,
Casadei et ul.
showedthat a fusion protein of aequorinand the IgG fragment of the specific antibody against the 4-hydroxy-3-nitrophenacetyl group could be used in an immunoassay(21). However, in this method, it is indispensableto isolate the specific IgG gene for the specific antigen. Thus, the 1335
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chemical cross-linked method of aequorin and the antibody is more suitable for the various species of antigen in an immunoassay. The sensitivity of the detection using the Fab’-apoaequorin conjugate
was high and suggested that aequorin is a beneficial
protein for the sensitive
immunoassay.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Shimomura, O., Johnson, F. H. and Saiga, Y. (1962) J. Cell. Comp. Physiol.59, 223. 239. Blinks, J. R., Prendergast,F. G. and Allen, D. V. (1976) Pharmacol. Rev. 28, l-93. Shimomura, 0. and Johnson,F. H. (1978) Proc. Nutl. Acad. Sci. USA. 75, 261 l-2615. Musicki, B., Kishi, Y. and Shimomura, 0. (1986) J. Chem. Sot. Chem. Commun. 21, 1566-1568. Inouye, S., Noguchi, M., Sakaki, Y., Takagi, Y., Miyata, T., Iwanaga, S., Miyata, T. and Tsuji, F. I. (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158. Shimomura, 0. and Johnson,F.H. (1975) Nature 256, 236-239. Inouye, S., Sakaki, Y., Goto, T. and Tsuji, F. I. (1986) Biocemistry 25, 8425-8429. Inouye, S., Aoyama, S., Miyata, T., Tsuji, F. I. and Sakaki, Y. (1989) J. Biocem. 105, 474-477. Shimomura, O., Inouye, S., Musicki, B. and Kishi, Y. (1990) Biochem. J. 270, 309312. Johnson,F. H. and Shimomura,0. (1978) Methods Enzymol. 57, 271-291. Zenno, S. and Inouye, S. (1990) Biochem. Biophys. Res. Commun.171, 169-174. moue, S., Sugiyama, S., Kakoi, H., Hasizume, K., Goto, T. and Iio, H.(1975) Chem. Lett. 141-144. Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y. and Ueno, T. (1983) J. Immunoassay4, 209-327. Ellman, G. E. (1959) Arch. Biochem. Biophys. 82, 70-77. Mandy, W. J. and Nisonoff, A. (1963) J. Biol. Chem.238,206-213. Jaquet, H. and Cebra, J. J. (1965) Biochemistry 4, 954-963. Utsumi, S. and Karush, F. (1965) Biochemistry 4,1766-1779. Bradford, M. M. (1976) Anal, Biochem. 72, 248-254. Kato, K., Fukui, H., Hamaguchi, Y. and Ishikawa, E. (1976) J. Zmmunology 116, 1554-1560. Inoue, S., Hashida, S., Tanaka, K., Imagawa, M. and Ishikawa, E. (1985) Anal. Lett. 18, 1331-1344. Casadei, J., Powell, M. J. and Kenten, J. H. (1990) Proc. Nutl. Acad. Sci. USA. 87, 2047-2051.
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