Bioconjugate Chem. 1004, 5,273-277
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TECHNICAL NOTES Peroxidase Labeling of IgMs Fragment of AB0 Blood Group Specific Mouse Monoclonal IgM Nobuhiro Yukawa, Hirokazu Matsuda, Yasuhisa Seo, Katsutoshi Suetomi, and Keiichi Takahama' Department of Legal Medicine, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan. Received January 14, 1994"
A method for peroxidase labeling of the monomeric subunit (IgMs) of AB0 blood group specific mouse monoclonal IgM is described. IgM was purified from a commercial monoclonal anti-B blood grouping reagent by a combination of salt precipitation, euglobulin precipitation, and gel filtration. IgM was mildly reduced with L-cysteine to yield SH-bearing IgMs. Finally, IgMs was conjugated to horseradish peroxidase, into which SH-reacting maleimide groups had been introduced using N-succinimidyl 6-maleimidohexanoate, through the selective reaction between SH of IgMs and maleimide groups of peroxidase.
INTRODUCTION Being produced under the requirement for efficient hemagglutination activity, most commercial monoclonal AB0 blood grouping reagents are of the IgM class. By combining ABO-specific mouse monoclonal IgM with an enzyme-labeled (anti-mouse IgM) IgG, forensic and medicolegal investigators developed many indirect enzyme immunoassays for ABH blood group substances in body fluids (1-3), and these have been proven to work quite accurately and reliably (4, 5). The encouraging results achieved by the indirect enzyme immunoassays evoked expectations for a simpler and more convenient direct enzyme immunoassay employing enzyme-labeled ABOspecific monoclonal IgM. This prompted us to develop a peroxidase-labeling method suitable for ABO-specific monoclonal IgM. EXPERIMENTAL PROCEDURES Scheme 1illustrates the principle of the method. Mouse IgM consists of five monomeric subunits (IgMs, H2Lz)l and one J chain joined by disulfide bonds at the penultimate cysteine of the H chain (6). The J chain, which accounts for approximately 2% of the total molecular weight of IgM (7), is not depicted in the scheme. Throughout the experiments, column chromatography (gel filtration) was performed at room temperature. Buffers used for equilibrating and running columns were degassed by an aspirator vacuum under sonication for 1520 min. The other buffers were not degassed. Purification of Monoclonal IgM. Mouse monoclonal IgM was purified by a combination of neutral salt @
Abstract published in Advance ACS Abstracts, April 15,
1994.
'Abbreviations used: IgMs, monomeric subunit of IgM pentamer; H and L, heavy and light (chains);Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetate; PBS, phosphate-bufferedsaline (10 mmol/L sodium phosphate buffer, pH 7.3, containing 0.145 mol/L NaC1); NC membrane, nitrocellulose membrane; Tween 20, polyoxyethylene sorbitan monolaurate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
precipitation, euglobulin precipitation upon dialysis under very low ionic strength conditions, and gel filtration described by Hayzer and Jaton (8). The amount of IgM was calculated from the absorbance at 278 nm by taking the extinction coefficient to be 1.35 g1.L.cm-l (9). One hundred mL of Bioclone anti-B (Lot No. BBB512D1, Ortho Diagnostics, Raritan, NJ) was precipitated with 20 g of NaZS04. The precipitate was dissolved in 4.0 mL of 0.155 mol/L NaCl and then thoroughly dialyzed against distilled water at 4 "C. The precipitate which formed was dissolved in 3.0 mL of 50 mmol/L tris(hydroxymethy1)aminomethane Tris-HC1 buffer, pH 8.0, containing 0.5 mol/L NaCl and subjected to gel filtration on a column (1.6 X 70 cm) of Ultrogel AcA 22 (IBF biotechnics, Villeneuve-la-Garenne, France) using the same buffer (Figure 1). The amount of IgM obtained was 6.1 mg. Preparation of Maleimide-Peroxidase. Maleimide groups were introduced into peroxidase using N-succinimidyl6-maleimidohexanoateaccording to Hashida et al. (10). The amount of maleimide-peroxidase was calculated from the absorbance a t 403 nm by taking the extinction coefficient and the molecular weight to be 2.275 g-1.L.cm-l and 40 000, respectively (11). Horseradish peroxidase (6.7 mg, Grade I, BoehringerMannheim GmbH, Mannheim, FRG) in 1.0 mL of 0.1 mol/L sodium phosphate buffer, pH 7.0, was mixed with 0.10 mL of 27.5 mmol/L N-succinimidyl6-maleimidohexanoate (Dojindo, Kumamoto, Japan) in N,N-dimethylformamide. After incubation at 30 "C for 45 min, the reaction mixture was subjected to gel filtration on a column (1.0 X 30 cm) of Sephadex G-25 medium (Pharmacia Fine Chemicals AB, Uppsala, Sweden) using 20 mmol/L sodium phosphate buffer, pH 6.8, containing 0.14 mol/L NaCl and 2 mmol/L ethylenediaminetetraacetate(EDTA). The average number of maleimide groups introduced per one peroxidase molecule was 1.5 (11). Mild Reduction of IgM To Yield Monomeric Subunit (IgMs). IgM was mildly reduced with L-cysteine to yield IgMs according to Hashimoto et al. (12). The amount of IgMs was calculated from the absorbance at 280 nm by taking the extinction coefficient and the molecular weight to be 1.29 gl-L-cm-l (13) and 180 000 (121,respectively.
1043-18Q2l94l29Q5-Q273$Q4.SQlQ 0 1994 American Chemical Society
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IgM (5.0 mg) in 1.2 mL of 0.2 mol/L Tris-HC1 buffer, pH 8.6, was mixed with 0.12 mL of 550 mmol/L L-cysteine (Nacalai Tesque Ltd., Kyoto, Japan) in the same buffer.2 After incubation at 27 "C for 10 min, the reaction mixture was cooled on ice and immediately subjected to gel filtration on a column (1.5 X 45 cm) of Ultrogel AcA 34 (IBF biotechnics) using 20 mmol/L sodium phosphate buffer, pH 6.8, containing 0.14 mol/L NaCl and 2 mmol/L EDTA at a flow rate of 21 mL/h (Figure 2). Fractions containing IgMs were pooled, concentrated with an ultrafiltration membrane (Diaflo PM-30, 25 mm in diameter, Amicon Corp., Danvers, MA), and immediately subjected to conjugation to maleimide-peroxidase. The amount of IgMs obtained was 1.7 mg. Conjugation of IgMs to Maleimide-Peroxidase. IgMs was reacted with maleimide-peroxidase to produce IgMs-peroxidase conjugate. The amount of IgMs-peroxidase conjugate was calculated from the absorbance at 280 nm and 403 nm (1I) by taking the extinction coefficient of IgMs (1.29 g-l-L-cm-l at 280 nm) (13)together with the molecular weight of IgMs (180 000) (12)and the extinction coefficient of peroxidase (0.73 g-l-L-cm-l at 280 nm and 2.275 gl.L.cm-' at 403 nm) together with the molecular weight of peroxidase (40 000) (11). IgMs (1.7 mg) in 1.5mL of 20 mmol/L sodium phosphate buffer, pH 6.8, containing 0.14 mol/L NaCl and 2 mmol/L EDTA was mixed with maleimide-peroxidase (1.1mg) in 0.43 mL of the same buffer and then concentrated at 4 OC with a microconcentrator (Centricon 30, Amicon Corp.) to a final volume of 0.26 mL. The concentration of IgMs and maleimide-peroxidase in the reaction mixture were 30 pmol/L and 90 pmol/L, respectively. After incubation at 4 "C for 16 h, the reaction mixture was subjected to gel filtration on the Ultrogel AcA 34 column (1.5 X 45 cm) using 100 mmol/L sodium phosphate buffer, pH 6.5. Fractions containing immunoreactive IgMs-peroxidase (Figure 3, fractions 35-48), which were determined as described below, were pooled. The amount of IgMsperoxidase obtained was 1.5 mg (1.01 mg as IgMs and 0.51 mg as peroxidase). The molar ratio of peroxidase to IgMs in the conjugate was hence calculated to be 2.3. Dot Immunoblotting. Fractions containing immunoreactive anti-B IgMs-peroxidase conjugate from gel filtration on an Ultrogel AcA 34 column were determined using the dot immunoblotting method (31, with modifications. Saliva from a blood group B ABH-secretor was diluted 100- to 3000-fold with phosphate-buffered saline (PBS), and 2 pL of the diluent was applied on a nitrocellulose (NC) membrane (0.45 pm, Bio-Rad laboratories, Richmond, CA). After blocking with a commercial buffer solution for an enzyme immunoassay containing casein (Block Ace, Snow Brand Co. Ltd., Sapporo, Japan), the NC membrane was incubated with anti-B IgMsperoxidase conjugate from each fraction (3.0 pg/mL as peroxidase) in Block Ace diluted 10-fold with distilled water at room temperature for 1 h. After the NC membrane was washed three times with 50 mmol/L TrisHC1 buffer, pH 7.5, containing 0.5 mol/L NaCl and 0.1 % Tween 20 (polyoxyethylene sorbitant monolaurate), peroxidase activity bound on the NC membrane was visualized %Cysteine solution was prepared just prior to use by dissolving L-cysteine (not L-cysteine monohydrochloride monohydrate) in 0.2 mol/L Tris-HC1 buffer, pH 8.6. The pH of the solution was not measured.
Yukawa et al.
with a commercialimmunostain kit for peroxidase (Konika HRP, Konika Corp., Tokyo, Japan).3 After the fractions containing immunoreactive a n t i B IgMs-peroxidase conjugate (Figure 3, fractions 35-48) were pooled, the antigen-binding activity of the a n t i B IgMsperoxidase conjugate was compared with that of the initial anti-B IgM by the dot blotting method. Blood group 0, A, B, and AB ABH-secretor saliva samples diluted with 10- to 30 000-fold with PBS were used as antigens. As for the anti-B IgMs-peroxidase conjugate, the procedure was the same as described above, and the concentration of IgMs-peroxidase conjugate was 5.9 pg/mL as IgMs or 3.0 pg/mL as peroxidase. As for anti-B IgM, an indirect method, in which IgM was used as primary antibody and affinity-purified goat (anti-mouse IgM) IgGperoxidase conjugate (Tago, Inc., Burlingame, CA) was used as secondary antibody, was adopted. The concentration of IgM was 5.9 pg/mL and the dilution of IgG-peroxidase conjugate was 1/500. The incubation times of the first incubation (IgM) and the second incubation (IgGperoxidase) were both 1 h. Purity and Stability of IgMs. IgMs prepared from another lot of Bioclone anti-B (100 mL, Lot No. BBB533A21, Ortho Diagnostics) was used for characterizing IgMs. In this lot, 2.3 mg of IgMs was obtained from 4.8 mg of IgM. The purity of IgMs was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (14). The separation on an 8-25% gradient gel followed by staining with Coomassie Brilliant Blue was performed using Phast System (Pharmacia Fine Chemicals AB). The gel was scanned at 660 nm using a Beckman spectrophotometer (DU-65) with Soft-Pac modules for determination of area and molecular weight (Beckman Instruments, Inc., Fullerton, CA). SH content and elution profile of gel filtration were examined for assessing the stability of IgMs in 20 mmol/L sodium phosphate buffer, pH 6.8, containing 0.14 mol/L NaCl and 2 mmol/L EDTA. IgMs eluted from an Ultrogel AcA 34 column (1.5 X 45 cm) at a flow rate of 21 mL/h (Figure 5a, fractions 40-5014 were pooled (0.24 mg/mL), concentrated to a volume of 1.5 mL (1.5 mg/mL), and then left standing on ice for 6 h, during which time the SH content in the IgMs was determined using 4,4'-dithiodipyridine (11, 15) as described below. Finally, the concentrated IgMs was mixed with N-ethylmaleimide (5 mmol/L in the mixture, NacalaiTesque Ltd.) for blocking the remaining SH groups and subjected to gel filtration on the same column at the same flow rate. Twenty pL of 5 mmol/L 4,4'-dithiodipyridine (Nacalai Tesque Ltd.) was added to 0.6 mL of the pooled IgMs before (0.24 mg/mL) and after concentration (rediluted to a concentration of 0.19 mg/mL from 1.5 mg/mL) in 20 mmoVL sodium phosphate buffer, pH 6.8, containing 0.14 mol/L NaCl and 2 mmol/L EDTA or to the same buffer as a reference. After 10 min incubation at 30 OC, the absorbance at 324 nm was measured and the number of dark blue color was developed by the heterocouplingreaction between a naphthol derivative and an aromatic amine derivative under the presence of H202.The exact chemical formulas of the derivatives were not available to the users. *IgMswas eluted more rapidly from a column of Ultrogel AcA 34 purchased recently (57% of the column volume, Figure 5) than from that purchased a few years before (65 75,Figure 2). Although this difference might be inevitable variation in gel fitration, we felt that some physicochemical properties of Ultrogel AcA 34 had been modified as we had similar experiences for Ultrogel AcA 44.
Technical Notes
Scheme 1
Bloconjugate Chem., Vol. 5, No. 3, 1994
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w-x lgMs lgMs
Mouse monoclonal IgM
@-NH2
-
Horseradish Horseradish peroxidase peroxidase
IgMs-peroxidase conjugate
Maleimide-peroxidase
N-Succinimidyl-6-maleimidohexanoate
0.4
Fraction No. ( 1.5 mL / tube )
Figure 1. Elution profile of crude IgM on an Ultrogel AcA 22 column (1.6 X 70 cm). IgM was eluted in the second peak (fractions
lgMs
Fraction No. ( 1.O mL /tube 1 Figure 2. Elution profile of mildly reduced IgM on an Ultrogel AcA 34 column (1.5 X 45 cm).IgMs was eluted in the second peak (fractions 48-57).
40-47). v
SH groups per one IgMs molecule was calculated by taking the molar extinction coefficient of 4-mercaptopyridine to be 19 800 (15). The initial IgM was also examined and found to have no free SH groups. RESULTS AND DISCUSSION Development oft he Method. Ishikawa and co-workers (16, 17) established an excellent method, the so-called “hinge method”, for the labeling of IgG with enzymes through the selective reaction between SH in the hinge of the Fab’ fragment of IgG and maleimide groups introduced into enzymes. A similar strategy was used for the coating of liposome with IgG by Martin and Papahajopoulos (18). They introduced maleimide groups into the liposome membrane and coupled the liposome to the Fab’ fragment of IgG. By the replacement of Fab’ with the monomeric subunit (IgMs) of IgM, Hashimoto et al. (12,19) were able to coat liposome with monoclonal IgM. The present method has been developed from a modification of these methods (Scheme 1). Monoclonal IgM. After enrichment by physicochemical means, the final purification of IgM was performed by gel filtration on an Ultrogel AcA 22 column. IgM was eluted in the second peak (Figure 1). IgM was subjected to mild reduction without further purification. Preparation of Monomeric Subunit (IgMs) of IgM. IgMs was prepared from IgM by mild reduction with L-cysteine, followed by gel filtration on an Ultrogel AcA 34 column. IgMs was eluted in the second peak (Figure 2). Hashimoto et al. (12) stated that it was necessary to seek the optimal concentration of IgM or L-cysteine in order to achieve a good yield of IgMs for each monoclonal IgM. In this experiment, the yield of the IgMs was 34772,
9
Fraction No. ( 1.O mL I tube )
Figure 3. Elution profile of the reaction mixture of IgMs and maleimide-peroxidase on an Ultrogel AcA 34 column (1.5 X 45 cm). Fractions35-48 contained immunoreactive IgMs-peroxidase conjugate. which was lower than that previously obtained (507% ) (12) but was sufficient for performing conjugation. Thus, we did not optimize the reducing conditions with trial and error. Purity and Stability of IgMs. SDS-PAGE of IgMs under reducing conditions showed H and L chain migrating at molecular masses of 79 and 27 kDa, respectively, which were similar to those (80and 25 kDa) reported by Knutson et al. (20), and one contaminant migrating at a molecular mass of 58 kDa (Figure 4). The nature of the contaminant was unknown. By densitography, the purity of the IgMs (H and L chains) was estimated to be 91-92 9%. The stability of IgMs before conjugation was assessed as follows. (1)Fractions containing IgMs eluted from an Ultrogel AcA 34 column (Figure 5a) were pooled and
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1
HC-
L-
2
3
Yukawa et ai.
Direct OSe
4 -94
ASe
kDa
-67
BSe
-43
ABSe
-30
I n d i rect
a a & %
OSe
-20
ASe
-14
Figure 4. SDS-PAGE of IgMs under reducing conditions using an 8-2576 gradient gel. Lanes 1, 2, and 3: IgMs (1,2, and 3 pg per lane, respectively). Lane 4: markers. For 1pg of IgMs, the areas of H chain (H), L chain (L), and a contaminant (C) on the densitogram comprised 61.7 % , 31.0%, and 7.3 % , respectively, and the purity of IgMs was hence calculated to be 92 % .Almost the same value (91%) was obtained for 2 pg, but a somewhat lower value (87%) was obtained for 3 pg probably due to a nonlinear increase in the areas.
ce I
0.10
0.05 0.00 20
70 Fraction No. ( 1.O mL / tube )
Figure 5. Changes in elution profile of IgMs after 6 h storage (b). IgM was mildly reduced and subjected to an Ultrogel AcA 34 column (1.5 X 45 cm), and fractions containing IgMs (40-50) (a) were pooled and concentrated. After being left on ice for 6 h, the concentrated IgMs was resubjected to the same column (b).
concentrated to a volume suitable for conjugation. The number of SH groups per one IgMs molecule was 7.2 for the pooled IgMs and was decreased to 6.6 after concentration. The concentrated IgMs was then left standing on ice for 6 h, during which time the values were found to remain essentially unchanged (6.5-6.7). (2) After 6 h of standing on ice, the IgMs was resubjected to gel filtration on the Ultrogel AcA 34 column (Figure 5b). The chromatogram showed a major peak corresponding to IgMs and a minor peak with larger molecular mass. The areas of the major and the minor peaks comprised 79% and 21 % ,respectively. These results (1and 2) indicated that IgMs was relatively stable, though some IgMs seemed to reassemble by oxidation. The number of SH per IgMs (H2L2) were larger (6.67.2) than that (2) expected from Scheme 1. Part of the difference (two SH per IgMs) seemed to be well explained by Clem and co-workers’ observations (21,22) that IgMs
0.01 0.03 0.1 0.3 Dilution
1
3
3
10
( x 10 - f o l d
30 )
Figure 6. Direct dot immunoblotting for 0,A, B, and AB ABHsecretor saliva samples (OSe, ASe, BSe, and ABSe) using anti-B IgMs-peroxidase conjugate (top) and indirect dot immunoblotting for the same samples using the initial anti-B IgM with (antimouse IgM) IgGperoxidase conjugate (bottom). Dilutions of saliva samples were from 10- to 30 000-fold. The detection limits both by the direct method and those by the indirect method were approximately 3000-fold dilution for BSe and 1000-fold dilution for ABSe.
prepared by mild reduction of IgM consisted of two covalent H-L chain halfmers which were noncovalently bound together. Cleavage of disulfide bonds within and between the H and L chains with resultant formation of smaller fragments (e.g., H2L) might also contribute to the difference. IgMs-Peroxidase Conjugate. IgMs was conjugated to maleimide-peroxidase through the selective reaction between SH of IgMs and maleimide groups of peroxidase and then subjected to gel filtration on an Ultrogel AcA 34 column. IgMs-peroxidase conjugate (the first broad peak) was separated from unconjugated peroxidase (the second peak) but not separated from unconjugated IgMs (Figure 3). The antigen-binding activity of IgMs-peroxidase conjugate tended to be less in the lower half section from the top (fraction 49) of the first broad peak. This decrease might be due to less or nonimmunoreactive complexes (e.g., H2L-peroxidases) with smaller sizes than IgMs (H2La)-peroxidase conjugate. The fractions from the upper half section (fractions 35-48) were pooled and used in the following experiment in order to confirm the conservation of antigen-binding activity of anti-B IgMsperoxidase conjugate. Antigen-Binding Activity of IgMs-Peroxidase Conjugate. Blood group 0,A, B, and AB ABH-secretor saliva samples were diluted 10-to 30 000-fold with PBS and were then subjected to both a direct dot immunoblotting using anti-B IgMs-peroxidase conjugate and an indirect dot immunob1ot:ing using the initial anti-B IgM with (antimouse IgM) IgG-peroxidase conjugate. There was no significant difference between the detection limits by the direct method and those by the indirect method (Figure 6). This indicated that no significant decrease in antigenbinding activity occurred for most part of the monoclonal anti-B IgM during conjugation. Fujiwara et al. (21)succeededin @-galactosidase-labeling of monoclonalanti-LewisxIgM, in which maleimide groups were introduced into IgM by the use of amino groups of IgM and then reacted with SH of @-galactosidase. The random use of amino groups, however, may possibly cause
Technical Notes
a decrease in antigen-bindingactivities of some monoclonal IgM due to the modification of amino groups which are located within or in close proximity to antigen binding sites (22). In the present method, although precise locations of SH groups used for conjugation were not determined, the SH groups released from disulfide bonds between monomeric subunits (IgMs) seemed much more accessible to maleimide-peroxidase than those released from other disulfide bonds (e.g., SH groups released from disulfide bonds between two H-L chain halfmers seemed hidden by noncovalent association of the two halfmers). Antigen-binding activities of most monoclonal IgM would thereby be conserved, and we hope the present method is applicable to other AB0 blood group specific monoclonal IgM. Anti-A IgMs-Peroxidase Conjugate. Mouse monoclonal anti-A IgM purified from Bioclone anti-A (Lot No. BAA 116G-1, Ortho Diagnostics) also could be labeled with peroxidase, and the results were briefly described. Anti-A IgM (7.9 mg) was reduced with L-cysteine at 27 “C for 10 min, in which the concentrations of IgM and L-cysteine were 3.8 mg/mL and 50 mmol/L, respectively. All IgMs obtained (3.6 mg) were reacted three times (mol/mol) with maleimide-peroxidase and resulted in 2.2 mg of immunoreactive anti-A IgMs-peroxidase conjugate (1.41 mg as IgMs and 0.76 mg as peroxidase). The molar ratio of IgMs to peroxidase in the conjugate was hence calculated to be 2.4. ACKNOWLEDGMENT
We are profoundly grateful to Professor Dr. Eiji Ishikawa, Dr. Takeyuki Kohno, Dr. Seiichi Hashida, and Dr. Koichiro Tanaka for their kind advice. We also thank to Professor Dr. Minoru Hamada and Dr. Hitoshi Takenaka for the use of a Beckman spectrophotometer. LITERATURE CITED (1) Bolton, S.,and Thorpe, J. W. (1986)Enzyme-linked immunosorbent assay for A and B water soluble blood group substances. J. Forensic Sci. 31, 27-35. (2) Takizawa, N., Ohba, Y., Mukoyama, R., Komuro, T., Mukoyama, H., and Takei, T. (1989)Determination of AB0 blood groups from saliva and saliva stains by an indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Nippon Hoigaku Zasshi 43, 294-302. (3) Pflug, W., Bassler, G., and Eberspacher, B. (1989)AB0 and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique. Forensic Sci. Int. 43, 171-182. (4) Bolton, S., and Thorpe, J. (1988) An examination of a contaminated seminal stain using absorption-elution and enzyme-linked immunosorbent assay (ELISA). J . Forensic. Sci. 33,797-800. (5) De Soyza, K. (1991) Evaluation of an enzyme linked immunosorbent assay (ELISA) method for AB0 and Lewis typing of body fluids in forensic samples. Forensic Sci. Int. 52, 65-76. (6) Davis, A. C., Roux, K. H., and Shulman, M. J. (1988)On the structure of polymeric IgM. Eur. J . Immunol. 18, 1001-1008. (7) Koshland, M. E. (1985)The coming of age of the immmunoglobulin J chain. Ann. Rev. Immunol. 3, 425-453.
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(8) Hayzer, D. J., and Jaton, J.-C. (1985)Immunoglobulin M (IgM). Methods Enzymol. 116,26-36. (9) Dombrink-Kurtzman, M. A., and Voss, E. W. (1988)Cryoprecipitation properties of a high-affinity monoclonal IgM antifluorescyl antibody. Mol. Immunol. 25, 1309-1320. (10) Hashida, S.,Imagawa, M., Inoue, S., Ruan, K.-H., and Ishikawa, E. (1984)More useful maleimide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge. J. Appl. Biochem. 6,56-63. (11) Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., and Ueno, T. (1983) Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J. Immunoassay 4, 209-327. (12) Hashimoto, Y., Sugawara, M., Kamiya, T., and Suzuki, S. (1986)Coating of liposomes with subunits of monoclonal IgM antibody and targeting of the Liposomes. Methods Enzymol. 121, 817-828. (13) Sunamoto, J.,Sato, T., Hirota, M., Fukushima, K., Hiratani, K., and Hara, K. (1987) A newly developed immunoliposome-An egg phosphatidylcholine liposome coated with pullulan bearing both a cholesterol moiety and an IgMs fragment. Biochim. Biophys. Acta 898, 323-330. (14) Laemmli, U. K. (1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (15) Grassetti, D. R., and Murray, J. F., Jr. (1967)Determination of sulfhydryl groups with 2,2’-or 4,4’-dithiodipyridine. Arch. Biochem. Biophys. 119, 41-49. (16) Yoshitake, S.,Yamada, Y., Ishikawa, E., and Masseyeff, R. (1979)Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide. Eur. J.Biochem. 101, 395-399. (17) Ishikawa, E. (1987)Development and clinical application of sensitive enzyme immunoassay for macromolecular antigens-A review. Clin. Biochem. 20, 375-385. (18) Martin, F.J., and Papahadjopoulos, D. (1982)Irreversible coupling of immunoglobulin fragments to preformed vesicles-An improved method for liposome targeting. J. Biol. Chem. 257, 286-288. (19) Hashimoto, Y., Sugawara, M., and Endoh, H. (1983)Coating of liposomes with subunits of monoclonal IgM antibody and targeting of the liposomes. J. Immunol. Methods 62,155-162. (20) Knutson, V. P., Buck, R. A., and Moreno, R. M. (1991) Purification of a murine monoclonal antibody of the IgM class. J . Immunol. Methods 136, 151-157. (21) Fujiwara, K., Matsumoto, N., Yagisawa, S., Tanimori, H., Kitagawa, T., Hirota, M., Hiratani, K., Fukushima, K., Tomonaga, A., Hara, K., and Yamamoto, K. (1988)Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewis’ using @+-galactosidase coupled to a monoclonal antibody of IgM isotype. J . Immunol. Methods 112,77-83. (22) O’Shannenssy, D. J., and Quarles, R. H. (1987) Review article-Labeling of the oligosaccharaide moieties of immunoglobulins. J. Immunol. Methods 99, 153-161. (23) Giles, R. C., Klapper, D. G., and Clem, L. W. (1983) Intramolecular heterogeneity of ligand binding by two IgM antibodies derived from murine hybridomas. Mol. Immunol. 20, 737-744. (24) Pascual, D., and Clem, L. W. (1988)Ligand binding by murine IgM antibodies: Intramolecular heterogeneity exists in certain, but not all, cases. Mol. Immunol. 25, 87-94.
