Targeting antigen-specific receptors on B lymphocytes to generate high yields of specific monoclonal antibodies directed against biologically active lower antigenic peptides within

Journal of Immunological Methods 251 (2001) 31–43www.elsevier.nl / locate / jim

Targeting antigen-specific receptors on B lymphocytes togenerate high yields of specific monoclonal antibodies directed

against biologically active lower antigenic peptides withinpresenilin 1

a , a b c*Masahiro Tomita , Hiroyuki Sugi , Kazuharu Ozawa , Tian Yow Tsong ,aTetsuro Yoshimura

aDepartment of Chemistry for Materials, Faculty of Engineering, Mie University, 1515 Kamihama-cho, Tsu, Mie 514-8507, JapanbChugai Pharmaceutical, Gotemba, Shizuoka 412-8513, Japan

cDepartment of Biochemistry, University of Minnesota, St. Paul, MN 55108-1022, USA

Received 21 July 2000; received in revised form 14 September 2000; accepted 4 December 2000

Abstract

We describe a targeting technique that selects antigen-specific receptors on B lymphocytes using antigen driven selectiveproduction of monoclonal antibodies which are directed against functional peptide sequences within the presenilin 1molecule that is believed to be related to the early-onset of familial Alzheimer’s disease. Three different peptide sequencesof presenilin 1 were constructed, one including the region around the amino acid position 300, where the putative cleavagesite exists and the other two present in the N- and C-terminal regions of that site. The efficiency in production of the desiredmonoclonal antibodies was at least 5–40-fold that obtained with the poly(ethylene glycol) (PEG)-mediated method. Inaddition, monoclonal antibodies directed against each of the peptide sequence displayed a high specificity for thecorresponding peptide, in contrast to the lack of success using the PEG method. Also, the selection of surfaceimmunoglobulin receptors on B lymphocytes by the peptides of interest was confirmed by immunofluorescent analysis. Herewe demonstrate that targeting B lymphocytes results in the successful and efficient production of highly specific monoclonalantibodies against the lower antigenic peptide sequences. 2001 Elsevier Science B.V. All rights reserved.

Keywords: B Lymphocytes; Biotin /avidin; Monoclonal antibodies; Targeting antigen-specific receptors; Peptide antigens; Presenilin 1

Abbreviations: BSA, bovine serum albumin; DMF, N,N-dimethylformamide; ELISA, enzyme-linked immunosorbent assay; FCS, fetalcalf serum; KLH, keyhole limpet hemocyanin; MBS, m-maleimidobenzoyl N-hydroxysuccinimide; NHS–biotin, N-hydroxysuccinimide–biotin; PBS, phosphate-buffered saline; PEF, pulsed electric field; PEG, poly(ethylene glycol); PVDF, polyvinylidene difluoride; RKmedium, RPMI 1640 containing kanamycin sulfate; Tricine /SDS–PAGE, tricine / sodium dodecyl sulfate–polyacrylamide gel electro-phoresis

*Corresponding author. Tel.: 181-59-231-9429; fax: 181-59-231-9430.E-mail address: [email protected] (M. Tomita).

0022-1759/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0022-1759( 01 )00299-X

32 M. Tomita et al. / Journal of Immunological Methods 251 (2001) 31 –43

1. Introduction Tomita and Tsong, 1990; Conrad and Lo, 1990;Tsong and Tomita, 1993; Tomita et al., 1996a,b).

¨Kohler and Milstein (1975) first described the The advantage of these chemical modifiers over theproduction of monoclonal antibodies against a given NHS–biotin is that they can selectively modify theantigen. It has since become clear that monoclonal Cys residues in the antigen. The modification ofantibodies are highly specific for the antigens of these residues is less detrimental to the antigenicityinterest, and are now being used not only for of proteins. Also, the antigen–MBS (or SPDP)–elucidating the biological functions of proteins, sugar avidin conjugates trigger no intermolecular B cellchains and lipids, but also for the diagnosis of many complexes. This approach led to the selective anddiseases and for therapeutic purposes. Their high highly efficient production of monoclonal antibodiesspecificity is of critical importance for these different against the protein molecules.applications. In the present study, we have focused on further

However, monoclonal antibodies specific for many developing the method to provide a procedure ap-important antigens remain unavailable, due to the plicable to any antigen. For this purpose, we em-difficulties of purifying the small amounts present in ployed peptide antigens. There are two points ofcells, or the instability of some antigens under note. The first is that the peptides can be constructedphysiological conditions. In addition, usage of the on the basis of the deduced amino acid sequences ofSendai virus- or poly(ethylene glycol) (PEG)-me- the proteins. The second point is that they can be

¨diated fusion methods (Kohler and Milstein, 1975; synthesized in large quantities and with exceptionalDe St. Groth and Scheidegger, 1980) causes non- purity. Here, we have selected functional peptidespecific fusion not only between spleen–myeloma sequences of presenilin 1, which has recently beenpairs of cells, but also spleen–spleen and myeloma– reported to be relevant to the early-onset of familialmyeloma pairs, resulting in lower production of the Alzheimer’s disease (Sherrington et al., 1995; Hardy,targeted hybridoma cells. Large numbers of growing 1997; Murayama et al., 1999; Guo et al., 1999; Chuihybridoma colonies need to be screened in order to et al., 1999; Wolfe et al., 1999).find those secreting the desired monoclonal anti- We first succeeded in targeting the antigen-specificbodies. receptors on B lymphocytes using the peptides. This

Some studies (Lo et al., 1984; Wojchowski and step determines the selective production of hybrid-Sytkowski, 1986; Hewish and Werkmeister, 1989; oma cells secreting monoclonal antibodies with highWerkmeister et al., 1991) have shown that using an efficiency and specificity.electrical pulse to induce cell fusion improves thePEG method. However, those protocols have notbeen generally accepted, either because they some- 2. Materials and Methodstimes lack reproducibility, due to the poor recoveryof antigen conjugates (Lo et al., 1984) or because of 2.1. Chemicalsthe direct modification of Lys residues in an antigenby N-hydroxysuccinimide (NHS)–biotin (Wojchow- N-Hydroxysuccinimide (NHS)–biotin and m-ski and Sytkowski, 1986; Hewish and Werkmeister, maleimidobenzoyl N-hydroxysuccinimide (MBS)1989; Werkmeister et al., 1991). The biotinylation of were purchased from Sigma. Roswell Park Memorialthe antigen would bring about a deterioration in Institute (RPMI) 1640 was from Nissui Pharma-immunogenicity, and also decrease the solubility of ceutical (Japan). Fetal calf serum (FCS) was ob-the antigen. Moreover, the biotinylated antigen may tained from Nippon Pharmaceutical (Japan). Allinduce intermolecular non-specific binding of the B other chemicals were of analytical reagent gradecells on the addition of streptavidin. unless otherwise stated.

To address this problem, we employed bifunction-al cross-linkers such as m-maleimidobenzoyl N-hy- 2.2. Cell linedroxysuccinimide (MBS) and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Tsong et al., 1988; PAI murine myeloma cells (Stocker et al., 1982)

M. Tomita et al. / Journal of Immunological Methods 251 (2001) 31 –43 33

were grown in complete RPMI 1640 medium sup- Ala–Gln–Glu–Arg–Asn–Glu–Cys, Ser–Lys–Asn–plemented with 10% FCS, 2 mM glutamine, 50 mM Ser–Lys–Tyr–Asn–Ala–Glu–Ser–Cys and Cys–b-mercaptoethanol and kanamycin sulfate (100 mg/ Val – Trp – Leu–Val–Asn–Met–Ala–Glu–Gly–Asp –ml) at 378C and humidified with 5%:95% CO /air Pro–Glu, termed peptide 1, peptide 2 and peptide 3,2

gas mixture. respectively. A cysteine residue was linked to the N-or C-terminus of each peptide for conjugation with

2.3. Antigen avidin.

A peptide antigen of presenilin 1 (positions 271– 2.5. Preparation of spleen cells319) was employed for the mouse immunization.Keyhole limpet hemocyanin (KLH) was attached to Spleen cells from an immunized mouse werethe peptide antigen as a carrier protein to enhance aseptically prepared according to the method re-antigenicity. This KLH-linked peptide antigen of ported previously (Tomita and Tsong, 1990), exceptpresenilin 1 is here termed presenilin 1 (271–319)– that they were washed with, and suspended in RPMIKLH. 1640 containing 100 mg/ml of kanamycin sulfate

(RK medium).2.4. Peptides for the selection of B lymphocytes

2.6. Preparation of peptide–avidin conjugateThree different peptide sequences of presenilin 1

at positions 271–280, 310–319 and 293–304 were As shown in Fig. 1, avidin (10 mg in 0.5 ml PBS)constructed for the selection of B lymphocytes was reacted with 5 ml of 0.15 M heterobifunctionalpreimmunized with presenilin 1 (271–319)–KLH. cross-linker, MBS (Kitagawa et al., 1983), for 0.5–1The amino acid sequences were Leu–Val–Glu–Thr– h at room temperature with gentle agitation. Five

Fig. 1. Production of peptide–avidin conjugates. Three different peptide sequences of presenilin 1 were conjugated with avidin using abifunctional cross-linker, MBS. Lysine residues in avidin and cysteine residues in each peptide were chemically linked, respectively, to NHSand maleimide functional groups in MBS in solution, as described in the text.


microliters of 1 M glycine were added to the reaction high-voltage pulse machine (BTX T820) for 10 ms,mixture to block excess unreacted NHS groups in four times, generating an electrical pulse with aMBS. After incubation for 30 min at room tempera- square wave.ture, 0.5 ml PBS was added and the reaction mixturewas dialyzed against PBS. The dialyzed 3.3 mg of 2.9. Purification of monoclonal antibodiesMBS–avidin conjugate in 0.33 ml PBS was thenreacted with 1 mg of each peptide dissolved in 0.67 Hybridoma cells secreting monoclonal antibodiesml in PBS. In this step the maleimido group in MBS against the desired peptides were grown intraperito-reacted with the free sulfhydryl group of the Cys neally in mice. Finally, the ascitic fluid containingresidue on the peptide. After incubation at room enriched monoclonal antibodies was purified bytemperature for more than 2 h with gentle agitation, protein G column chromatography.the reaction mixture was further kept at 48C over-night. Thereafter, peptide–MBS–avidin conjugate (1 2.10. Poly(ethylene glycol)(PEG) fusionmg/ml peptide) was dialyzed against PBS and keptat 2208C after sterilization. PEG fusion was performed according to De St.

Groth and Scheidegger (1980).2.7. Biotinylation of myeloma cells

2.11. Tricine /SDS–PAGE7 7PAI mouse myeloma cells (10 –5310 ) were

collected and washed with PBS containing kana- Tricine /SDS–PAGE was performed in 16.5% slabmycin sulfate (100 mg/ml). Twenty microliters of gels under reduced conditions (Schaegger and Von0.1 M NHS–biotin (1 mg NHS–biotin in 30 ml Jagow, 1987), and the separated proteins stainedDMF) were carefully added to 10 ml of myeloma with Brilliant blue G-250.cell suspension. In this step, myeloma cells werebiotinylated using NHS–biotin by modification of 2.12. Immunoblot analysisLys residues on their membrane surface proteins.After the mixture was incubated at room temperature The proteins separated by Tricine /SDS–PAGEfor 15–30 min with gentle agitation, biotinylated were transferred onto a polyvinylidene difluoridemyeloma cells were washed with RK medium and (PVDF) membrane at 100–200 mA for 2–3 h at 48Cused for electrofusion. (Towbin et al., 1979), soaked overnight in 1%

gelatin in PBS, rinsed 3 times with 0.05% Triton2.8. Pulsed electric field (PEF) method X-100 in PBS, and incubated for 1 h with an anti-

presenilin (271–319) antiserum, an anti-avidin an-The peptide–avidin conjugates (20 mg) prepared tiserum or anti-peptide 1, anti-peptide 2 and anti-

as described above were carefully added to the peptide 3 monoclonal antibodies. After three wash-spleen cell suspension in 5 ml RK medium and ings, the PVDF membranes were incubated for 1 h ingently mixed by stirring for 2 h at 48C. The cell 0.02% peroxidase conjugated with goat antibodysuspension containing B lymphocyte–peptide–avidin against mouse IgG (TAGO) and, after at least fivecomplex was washed with and suspended in RK washings, analyzed with the use of an Immunostainmedium. The B lymphocyte–peptide–avidin com- HRP-1000 set (Konica).plex was then combined with biotinylated myelomacells, as previously described (Tomita and Tsong, 2.13. Immunofluorescent analysis1990). Finally, B lymphocyte–peptide–avidin–biotin–myeloma cell complex was gently suspended Antigen-immunized B lymphocytes were selectedin 1–2 ml of isotonic sucrose containing 2 mM by peptide–avidin conjugates, and then B lympho-sodium phosphate buffer (pH 7.2), 0.1 mM CaCl cyte–peptide–avidin complexes were specifically2

and 0.1 mM MgCl . These cell mixtures were fused labelled with biotinylated phycoerythrin (PE). The221by imposing an electric field of a few kV cm by a resulting B lymphocyte–peptide–avidin–biotin–PE


complexes were analyzed by confocal laser micro- peptide–avidin conjugates in the same positions asscopy (Zeiss LSM410). In control experiments, B the bands observed in Fig. 2b. The results obtainedlymphocytes from a non-immunized mouse were here confirm that peptide–avidin conjugates wereemployed instead of those of the immunized mouse. successfully produced using a heterofunctional cross-

linker, MBS, and retained their antigenicities even2.14. Enzyme-linked immunosorbent assay (ELISA) after peptide conjugation to avidin.

Hybridoma cells secreting specific monoclonal 3.2. Selection of B lymphocytes by the peptide–antibodies against the given peptides were screened avidin conjugateby an ELISA method, as previously described(Tomita and Tsong, 1990). For the analyses of An immunofluorescent analysis was performed tospecificities of monoclonal antibodies against three clarify the selection of B lymphocytes targeted bydifferent peptide sequences, peptide 1–, peptide 2– the peptide. This is one of the critical steps in thisand peptide 3–avidin conjugates were used as an- method. As shown in Fig. 3a, the peptide conjugatedtigens. to avidin was able to target the surface antibodies on

B lymphocytes. In contrast, the same peptide conju-2.15. Determination of protein gate failed to recognize B lymphocytes from a non-

immunized mouse (Fig. 3b). This is strong evidenceProtein concentrations were determined according that the immunoglobulin receptors on B lymphocytes

to the method of Lowry et al. (1951) with BSA as a are stringently targeted by the peptide.standard.

3.3. Selective production of monoclonal antibodiesagainst the peptides3. Results

Fig. 4 schematically summarizes each step for the3.1. Immunoanalysis of peptide–avidin conjugatesselective production of monoclonal antibodiesagainst the peptides of presenilin 1 by the targetingIn an attempt to ensure high volume and selectivemethod, which is also known as the pulsed electricproduction of hybridoma cells secreting monoclonalfield (PEF) method. Firstly, B lymphocytes areantibodies against the biologically active peptides ofpreselected by the peptide of interest based onpresenilin 1, the B lymphocytes of interest weresurface immunoglobulin receptors on B lympho-stringently isolated in advance using three differentcytes. In this step, B lymphocytes immunized withpeptides. For this purpose, peptide–avidin conjugatesthe presenilin (271–319) were preferentially selectedwere prepared in order to target antigen-specificby the peptide–avidin conjugates in cell suspension.receptors on B lymphocytes. Fig. 2 shows the resultsSecondly, the power of the biotin–avidin technologyof immunoblot analyses of the three different pep-was harnessed. The peptide-selected B lymphocytestides of presenilin 1 chemically conjugated to avidin.were combined with myeloma cells by exploiting theEach peptide of interest conjugated to avidin cross-specificity and strength of the interaction betweenreacted with the antiserum directed against thebiotin and avidin. Finally, B lymphocyte–peptide–presenilin 1 (271–319). Three major immunoreactiveavidin–biotin–myeloma cell complexes were selec-bands were detected at around M 20 000, 36 000r tively fused using an electrical pulse.and more than 68 000, indicating that each peptide–

avidin was successfully recognized by the anti-pre-senilin 1 (271–319) antibody (Fig. 2b). Each band 3.4. Comparison of efficiency between the twocorresponds to subunits of the avidin molecule linked methodsto each peptide, showing one, two and four of thesubunits, respectively. Fig. 2c shows that the an- Table 1 shows a comparison of the efficiency oftiserum directed against avidin also recognized the production of monoclonal antibodies by the PEF and


Fig. 2. Immunoblot analyses of peptide–avidin conjugates. Three different peptides of presenilin 1 chemically conjugated with avidin wereseparated by Tricine /SDS–PAGE (a) and immunostained by anti-presenilin (271–319) antibody (b) and anti-avidin antibody (c). Afterseparating the peptide–avidin conjugates by Tricine /SDS–PAGE under reducing conditions, they were transferred to a PVDF membraneand specifically detected using anti-presenilin (271–319) and anti-avidin antibodies as described in the text. Presenilin (271–319) and avidinwere used as positive controls for the immunoblot analyses in (b) and (c), respectively. Lanes 1, m.w. markers; lanes 2, peptide 1–avidin;lanes 3, peptide 2–avidin; lanes 4, peptide 3–avidin; lanes 5, presenilin (271–319); lanes 6, avidin. Molecular weights are indicated on theleft (31000).

PEG-mediated methods. Clearly, the former has The maximum efficiency reached approximatelyadvantages in terms of efficiency, this being 5–40 80%. The PEF method could also produce thetimes greater than with the PEG-mediated method. desired hybridoma cells secreting monoclonal anti-


Fig. 3. Immunofluorescent analysis for the selection of B lymphocytes. In (a), B lymphocytes immunized by the peptide antigen wereselected by the peptide–avidin conjugate, and then labeled with biotinylated phycoerythrin (PE) as described in the text. In (b), a controlexperiment was carried out following the same method, except that B lymphocytes from a non-immunized mouse were used. Trans. andbio-PE indicate transillumination and biotin–phycoerythrin, respectively. In this experiment, human insulin was used as a peptide antigen.

bodies against three different peptide sequences of ed Bombyx mori cytoplasmic polyhedrosis viruspresenilin 1, despite the fact that the PEG method (BmCPV) as a control, because it is known to befailed to produce them. Moreover, we were able to strongly antigenic. The efficiency of generatingconfirm the feasibility of this protocol using an in antibodies against this antigen, however, was similarvitro immunization approach (Tomita et al., un- to or relatively lower than that obtained using thepublished results). In the PEG experiment, we select- peptide antigen, presenilin 1 (271–319).


Fig. 4. Schematic representation of the targeting method.

3.5. Specificities of monoclonal antibodies directed duced by the PEF method displayed a high spe-against three different peptide sequences cificity for single peptides. The monoclonal antibody

to peptide 1 exhibited high affinity for peptide 1,As shown in Fig. 5, monoclonal antibodies pro- without any cross-reactivity for peptides 2 and 3.

Table 1A comparison of the fusion efficiency of the PEF and PEG methods

Method Monoclonal antibody (A) Number of (B) Number of (B) /(A)3100colony-positive ELISA-positive (%)wells wells

aPEF Anti-peptide 1 15 7 46.7b68 33 48.5aAnti-peptide 2 7 3 42.9b14 11 78.6aAnti-peptide 3 28 10 35.7b12 6 50.0

PEG Anti-presenilin 1 147 12 8.2(271–319)

cAnti-BmCPV 417 15 3.6447 8 1.8395 27 6.8

a The pulse conditions were 2.0 kV/cm310 ms, 4 times.b The pulse conditions were 3.0 kV/cm310 ms, 4 times.c Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was used as antigen.


Fig. 5. ELISA procedure for the detection of the specificities of the monoclonal antibodies against the three different peptides of presenilin1. Peptide 1–, peptide 2– and peptide 3–avidin conjugates were used as antigens for detecting the specificities of monoclonal antibodiesdirected against each peptide as described in the text. An anti-mouse IgG (H1L) antibody conjugated with horseradish peroxidase was usedas a secondary antibody and visualized using o-phenylenediamine. Presenilin (271–319) was used as a positive control.

The same restricted specificities were found for the cytes can stringently isolate specific cells in advance,monoclonal antibodies directed against peptides 2 thereby leading to the selective production of mono-and 3. In contrast, the PEG-mediated method yielded clonal antibodies against the lower antigenic peptideno monoclonal antibodies against peptides 1, 2 or 3. sequences of the protein molecules of interest.It did, however, yield monoclonal antibodies directedagainst presenilin (271–319).

To further confirm the specificities of the mono- 4. Discussionclonal antibodies against the three different peptidesobtained by the PEF method, immunoblot analyses We have further developed targeting techniqueswere carried out. As shown in Figs. 6b–d, each for the selective production of monoclonal antibodiesmonoclonal antibody specifically cross-reacted with against peptide sequences. Our method resulted in athe corresponding peptide. On the other hand, mono- 5–40-fold higher efficiency in the production ofclonal antibodies produced by the PEG-mediated hybridoma cells secreting monoclonal antibodiesmethod showed cross-reactivity with presenilin against the given peptides, compared to results with(271–319) only and not with peptides 1, 2 and 3 (see the PEG-mediated method. The most critical point ofFig. 6a). These results confirm the data presented in this protocol is targeting antigen-specific receptorsFig. 5. on B lymphocytes by peptides conjugated with

Consequently, the PEF method results in efficient avidin. This ensures the involvement of the Bselective production of specific monoclonal antibo- lymphocytes of interest, even though the populationdies against the peptides of interest. One particular of immunized B lymphocytes is low. Another keyadvantage of this method is that targeting B lympho- point is that myeloma cells and peptide-selected B


Fig. 6. Immunoblot analyses for confirmation of the specificities of monoclonal antibodies against the three different peptides of presenilin1. Peptide 1–, peptide 2– and peptide 3–avidin conjugates were separated by Tricine /SDS–PAGE and immunostained by anti-presenilin(271–319) monoclonal antibody (a), anti-peptide 1 monoclonal antibody (b), anti-peptide 2 monoclonal antibody (c) and anti-peptide 3monoclonal antibody (d) as described in the text. Presenilin (271–319) was used as a positive control. Lanes 1, m.w. markers; lanes 2,presenilin (271–319); lanes 3, peptide 1–avidin; lanes 4, peptide 2–avidin; lanes 5, peptide 3–avidin. Molecular weights are indicated onthe left (31000).


lymphocytes are brought into contact by the strong strong binding (Green, 1962, 1963, 1975). In addi-and specific binding of avidin to the tethered biotin. tion, the thio ether chemical linkage between MBSFurthermore, it is worth noting that the peptide- and the peptide is extremely stable. Thus, thepreselected B lymphocyte and myeloma cell com- peptide–avidin conjugate could successfully select Bplexes were selectively fused by electrofusion with a lymphocytes immunized by the peptide antigen,square wave. In this final step, only B lymphocyte– producing B lymphocyte–peptide–avidin complexes.myeloma cell complexes linked by tight biotin– Avidin is composed of homotetrameric subunits,avidin bonds were preferentially fused. each of which can specifically bind biotin. The

–15In this study, we have focused on biologically association is rapid and extremely strong (K 510d

active peptide sequences from the presenilin 1 M), and effectively irreversible. The affinity of6molecule, especially a hydrophilic loop region avidin for biotin is approximately 10 -fold that

around amino acid position 300. It has been reported reported between antigen and antibody. Therefore, itthat this site is enzymatically cleaved (Mercken et was used here for specific binding with biotinylatedal., 1996; Thinakaran et al., 1996), and might play an myeloma cells in order to bring peptide-preselectedimportant role in the early-onset of familial Alzheim- B lymphocytes and myeloma cells into contact priorer’s disease (Hardy, 1997; Murayama et al., 1999; to electrofusion. In addition, the avidin appeared toGuo et al., 1999; Chui et al., 1999; Wolfe et al., enhance the display of the peptides to the surface1999). The three different peptide sequences of immunoglobulin receptors on the B lymphocytes.presenilin 1 were constructed on the basis of the The specific strong affinity between biotin and avidindeduced amino acid sequence of the protein mole- has also been employed for other selective interac-cule (Sherrington et al., 1995). The amino acid tions such as liposome–liposome (Ringsdorf andsequences of peptides 1 and 2 contain the N- and Simon, 1994; Chiruvolu et al., 1994; Schoen et al.,C-terminal regions of the putative cleavage site while 1996; Walker et al., 1997), liposome–protein (Loug-peptide 3 includes position 300 itself. There was no hrey et al., 1987, 1990; Rivnay et al., 1987) andoverlap. Each monoclonal antibody produced by this protein–protein (Kongtawelert and Ghosh, 1990;method exhibited stringent specificity for its own Muramatsu et al., 1996). Furthermore, the interactionpeptide. In contrast, the PEG fusion method failed to has been exploited for the selective purification ofproduce the desired monoclonal antibodies. Thus important proteins and biomolecules (Cronan, 1990;preselection of B lymphocytes by the peptides of Germino et al., 1993; Olejnik et al., 1995; Lesley andinterest is critical for the routine production of the Groskreutz, 1997) and in a new drug delivery systemdesired monoclonal antibodies. (Shin et al., 1997).

The use of the chemical modifier, MBS, has In conclusion we have shown that targeting tech-several advantages in the production of peptide– niques can result in the effective production ofavidin conjugates. One is that it retains heterofunc- monoclonal antibodies against lower antigenic pep-tional groups which can make covalent bonds with tide sequences of presenilin 1. It is probable thatboth Lys and Cys residues. Moreover, the chemical targeting B lymphocytes will lead to the efficientreaction can be carried out in solution, bringing production of monoclonal antibodies not only againstabout high recovery of the peptide–MBS–avidin peptides, but also against low molecular weightconjugate. Another advantage is that the modification biofunctional moieties such as sugar chains, lipidsof Lys residues in the avidin can shift the isoelectric and other biomolecules. Preliminary work suggestspoint from alkaline (pH 10) (Woolley and that this method should be applicable to someLongsworth, 1942) towards the neutral region, which environmental estrogens.inhibits the non-specific binding of the avidin tonegatively charged B lymphocytes and myelomacells. Furthermore, the chemical modification of Acknowledgementslysine residues in avidin does not affect its strongaffinity for biotin, since it has been reported that it is This work was supported in part by a Grant-in-Aidthe tryptophan residues which participate in the for Scientific Research on Priority Areas (A) to M.T.


Lesley, S.A., Groskreutz, D.J., 1997. Simple affinity purification(10145225, 11132231, 12019232) from the Ministryof antibodies using in vivo biotinylation of a fusion protein. J.of Education, Science, Sports and Culture of Japan.Immunol. Methods 207, 147.

PAI murine myeloma cells were kindly provided by Lo, M.M.S., Tsong, T.Y., Conrad, M.K., Strittmatter, S.M., Hester,Dr. Mamoru Nakanishi, Faculty of Pharmaceutical L.D., Snyder, S.H., 1984. Monoclonal antibody production by

receptor- mediated electrically induced cell fusion. Nature 310,Sciences, Nagoya City University. We appreciate the792.critical reading of the manuscript by Dr. Malcolm

Loughrey, H., Bally, M.B., Cullis, P.R., 1987. A non-covalentMoore. method of attaching antibodies to liposomes. Biochim. Bio-

phys. Acta 901, 157.Loughrey, H.C., Choi, L.S., Cullis, P.R., Bally, M.B., 1990.

Optimized procedures for the coupling of proteins to lipo-Referencessomes. J. Immunol. Methods 132, 25.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.Chiruvolu, S., Walker, S., Israelachvili, J., Schmitt, F.-J., Lec- Protein measurement with the Folin phenol reagent. J. Biol.

kband, D., Zasadzinski, J.A., 1994. Higher order self-assembly Chem. 193, 265.of vesicles by site-specific binding. Science 264, 1753. Mercken, M., Takahashi, H., Honda, T., Sato, K., Murayama, M.,

Chui, D.-H., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, F., Nakazato, Y., Noguchi, K., Imahori, K., Takashima, A., 1996.Ueda, O., Suzuki, H., Araki, W., Inoue, H., Shirotani, K., Characterization of human presenilin 1 using N-terminalTakahashi, K., Gallyas, F., Tabira, T., 1999. Transgenic mice specific monoclonal antibodies: evidence that Alzheimer muta-with Alzheimer presenilin 1 mutations show accelerated tions affect proteolytic processing. FEBS Lett. 389, 297.neurodegeneration without amyloid plaque formation. Nat. Muramatsu, H., Song, X.-J., Koide, N., Hada, H., Tsuji, T.,Med. 5, 560. Kadomatsu, K., Inui, T., Kimura, T., Sakakibara, S.,

Conrad, M.K., Lo, M.M.S., 1990. Facilitated cell fusion for Muramatsu, T., 1996. Enzyme-linked immunoassay for mid-hybridoma production. Methods Enzymol. 184, 641. kine, and its application to evaluation of midkine levels in

Cronan, Jr. J.E., 1990. Biotination of proteins in vivo. J. Biol. developing mouse brain and sera from patients with hepato-Chem. 265, 10327. cellular carcinomas. J. Biochem. 119, 1171.

De St. Groth, S.F., Scheidegger, D., 1980. Production of mono- Murayama, O., Tomita, T., Nihonmatsu, N., Murayama, M., Sun,clonal antibodies: strategy and tactics. J. Immunol. Methods X., Honda, T., Iwatsubo, T., Takashima, A., 1999. Enhance-35, 1. ment of amyloid b42 secretion by 28 different presenilin 1

Germino, F.J., Wang, Z.X., Weissman, S.M., 1993. Screening for mutations of familial Alzheimer’s disease. Neurosci. Lett. 265,in vivo protein-protein interactions. Proc. Natl. Acad. Sci. 61.

˜USA 90, 933. Olejnik, J., Sonar, S., Krzymanska-Olejnik, E., Rothschild, K.J.,Green, N.M., 1962. Spectroscopic evidence for the participation of 1995. Photocleavable biotin derivatives: a versatile approach

tryptophan residues in the binding of biotin by avidin. for the isolation of biomolecules. Proc. Natl. Acad. Sci. USABiochim. Biophys. Acta 59, 244. 92, 7590.

Green, N.M., 1963. Avidin, 3. The nature of the biotin-binding Ringsdorf, H., Simon, J., 1994. Snap-together vesicles. Naturesite. Biochem. J. 89, 599. 371, 284.

Green, N.M., 1975. Avidin. Adv. Protein Chem. 29, 85. Rivnay, B., Bayer, E.A., Wilchek, M., 1987. Use of avidin-biotinGuo, Q., Fu, W., Sopher, B.L., Miller, M.W., Ware, C.B., Martin, technology for liposome targeting. Methods Enzymol. 149,

G.M., Mattson, M.P., 1999. Increased vulnerability of hip- 119.pocampal neurons to excitotoxic necrosis in presenilin-1 Schaegger, H., Von Jagow, G., 1987. Tricine-sodium dodecylmutant knock-in mice. Nat. Med. 5, 101. sulfate-polyacrylamide gel electrophoresis for the separation of

Hardy, J., 1997. Amyloid, the presenilins and Alzheimer’s disease. proteins in the range from 1 to 100 kDa. Anal. Biochem. 166,Trends Neurosci. 20, 154. 368.

Hewish, D.R., Werkmeister, J.A., 1989. The use of an electropora- Schoen, P., Leserman, L., Wilschut, J., 1996. Fusion of reconsti-tion apparatus for the production of murine hybridomas. J. tuted influenza virus envelopes with liposomes mediated byImmunol. Methods 120, 285. streptavidin /biotin interactions. FEBS Lett. 390, 315.

Kitagawa, T., Fujiwara, K., Tomonoh, S., Takahashi, K., Koida, Sherrington, R., Rogaev, E.I., Liang, Y., Rogaeva, E.A., Levesque,M., 1983. Enzyme immunoassays of kanamycin group anti- G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T.,biotics with high sensitivities using anti-kanamycin as a Mar, L., Foncin, J.-F., Bruni, A.C., Montesi, M.P., Sorbi, S.,common antiserum: reasoning and selection of a heterologous Rainero, I., Pinessi, L., Nee, L., Chumakov, I., Pollen, D.,enzyme label. J. Biochem. 94, 1165. Brookes, A., Sanseau, P., Polinsky, R.J., Wasco, W., Da Silva,

¨Kohler, G., Milstein, C., 1975. Continuous cultures of fused cells H.A.R., Haines, J.L., Pericak-Vance, M.A., Tanzi, R.E., Roses,secreting antibody of predefined specificity. Nature 256, 495. A.D., Fraser, P.E., Rommens, J.M., St. George-Hyslop, P.H.,

Kongtawelert, P., Ghosh, P., 1990. A method for the quantitation 1995. Cloning of a gene bearing missense mutations in early-of hyaluronan (hyaluronic acid) in biological fluids using a onset familial Alzheimer’s disease. Nature 375, 754.labeled avidin-biotin technique. Anal. Biochem. 185, 313. Shin, S.-U., Wu, D., Ramanathan, R., Pardridge, W.M., Morrison,


S.L., 1997. Functional and pharmacokinetic properties of Tsong, T.Y., Tomita, M., 1993. Selective B lymphocyte-myelomaantibody-avidin fusion proteins. J. Immunol. 158, 4797. cell fusion. Methods Enzymol. 220, 238.

Stocker, J.W., Forster, H.K., Miggiano, V., Staehli, C., Staiger, G., Tsong, T.Y., Tomita, M., Lo, M.M.S., 1988. Pre-selection ofTakacs, B., Staehelin, T., 1982. Generation of two new mouse B-lymphocytes by antigen for fusion to myeloma cells bymyeloma cell lines ‘PAI’ and ‘PAI-O’ for hybridoma pro- pulsed electric field (PEF) method. In: Ohki, S., Doyle, D.,duction. Res. Disclosure 217, 155. Flanagan, T.D., Hui, S.W., Mayhew, E. (Eds.), Molecular

Thinakaran, G., Borchelt, D.R., Lee, M.K., Slunt, H.H., Spitzer, Mechanisms of Membrane Fusion. Plenum, New York, p. 223.L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Walker, S.A., Kennedy, M.T., Zasadzinski, J.A., 1997. Encapsula-Seeger, M., Hardy, J., Levey, A.I., Gandy, S.E., Jenkins, N.A., tion of bilayer vesicles by self-assembly. Nature 387, 61.Copeland, N.G., Price, D.L., Sisodia, S.S., 1996. Endo- Werkmeister, J.A., Tebb, T.A., Kirkpatrick, A., Shukla, D.D.,proteolysis of presenilin 1 and accumulation of processed 1991. The use of peptide-mediated electrofusion to selectderivatives in vivo. Neuron 17, 181. monoclonal antibodies directed against specific and homolo-

Tomita, M., Tsong, T.Y., 1990. Selective production of hybridoma gous regions of the potyvirus coat protein. J. Immunol.cells: antigenic-based pre-selection of B lymphocytes for Methods 143, 151.electrofusion with myeloma cells. Biochim. Biophys. Acta Wojchowski, D.M., Sytkowski, A.J., 1986. Hybridoma production1055, 199. by simplified avidin-mediated electrofusion. J. Immunol.

Tomita, M., Miyajima, S., Tsong, T.Y., 1996a. New method for Methods 90, 173.selective production of hybridoma cells. New Technol. Japan Wolfe, M.S., Xia, W., Ostaszewski, B.L., Diehl, T.S., Kimberly,24, 15. W.T., Selkoe, D.J., 1999. Two transmembrane aspartates in

Tomita, M., Miwa, M., Le, B., Miyajima, S., Tsong, T.Y., 1996b. presenilin-1 required for presenilin endoproteolysis and g-Selective production of monoclonal antibodies mediated by B secretase activity. Nature 398, 513.lymphocyte receptors. Protein Eng. 9, 819. Woolley, D.W., Longsworth, L.G., 1942. Isolation of an antibiotin

Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic factor from egg white. J. Biol. Chem. 142, 285.transfer of proteins from polyacrylamide gels to nitrocellulosesheets: Procedure and some applications. Proc. Natl. Acad.Sci. USA 76, 4350.

Documents

Targeting antigen-specific receptors on B lymphocytes to generate high yields of specific monoclonal antibodies directed against biologically active lower antigenic peptides within