Hybridoma antibodies as specific probes to Drosophila melanogaster yolk polypeptides

Insect Biochem. Vol. 16, No. 5, pp. 789-795, 1986 Printed in Great Britain

0020-1790/86$3.00+0.00 Pergamon Journals Ltd

HYBRIDOMA ANTIBODIES AS SPECIFIC PROBES TO DROSOPHILA MELANOGASTER YOLK POLYPEPTIDES SHUENN-JUE WU and MICHAELMA* Department of Entomology, University of Maryland, College Park, MD 20742, U.S.A. I

(Received 26 August 1985; revised and accepted 6 November 1985)

Al~ract--Hybridoma antibodies to Drosophila melanogaster soluble yolk proteins (YPs) were developed by both/n vivo and/n vitro immunizations followed by the fusion of SP2/0-Agl4 cells and splenocytes of BALB/c mice. Rabbit antiserum was made female specific by affinity column with male proteins as ligand. The binding sites of these hybridoma antibodies and rabbit antibodies towards different YP components were identified with a combination of gel electrophoresis, Western blotting and immunohistochemical staining. A double antibody sandwich enzyme-linked immunosorbent assay was developed with monoclonal antibodies from 2 cell lines and alkaline phosphatase labelled rabbit polyclonal antibodies as primary and secondary antibodies respectively. Yolk polypeptide levels in the haemolymph can be monitored in individual insect samples. Key Word Index: Hybridoma, monoclonai antibodies, Drosophila melanogaster, ELISA and vitellogenesis

INTRODUCTION Vitellogenesis is the physiological process of yolk synthesis and deposition in the developing ovary which is common to all egg laying animals. Besides being an important component of insect reproductive physiology, vitellogenesis provides an important model system for research on the hormonal control of gene expression, membrane transport and molecular evolution. Despite the extensive background information on the morphological, genetic and hormonal studies of vitellogenesis in Drosophila there are a lot of unresolved problems especially in the area of hormonal regulation (Posflethwait and Jowett, 1981; Bownes, 1982; Hagedorn, 1985). Many of these problems are due to the lack of a sensitive and specific method to quantify yolk polypeptides (YP) or vitellogenin. YP gene probes have been used successfully to monitor the levels of transcripts present in fat bodies (Bownes et al., 1983) and follicular epithelial cells (Brennan et al., 1982). However, the relationship between the levels of transcripts, YP synthesis and YP uptake into developing oocytes remains unclear. It is important to realize that after the formation of YP transcripts, there are co-translational and post-translational events such as presequence removal, glycosylation, disulphide bond formation, hydroxylation, carboxylation, phosphorylation, lipidation, methylation and ADPribosylation modifications (Ghelis and Yon, 1982). For these reasons, detection of YP transcripts does not necessarily represent concurrent appearance of the gene product. In the study of factors affecting vitellogenesis, we should be monitoring events involving the dynamics and localization of gene product or yolk polypeptides. Thus a specific and accurate YP quantification procedure is crucial to the success *To whom reprint requests should be addressed.

of studies such as hormonal control of vitellogenesis despite the advantages of being able to monitor specific YP transcripts. The objectives of this study were: (1) to develop specific monoclonal antibodies to Drosophila melanogaster YPs with both/n vitro (Ma et al., 1984b) and in rive immunizations as part of the cell fusion protocol established by Kohler and Milstein (1975); (2) to develop a rabbit polycional antibody that is highly specific to the three major yolk polypeptides; (3) to synthesize a specific enzTme-antibody conjugate to YPs; (4) to develop a double antibody sandwich ELISA procedure that would detect minute quantities of YPs in individual flies; and (5) to monitor YP levels of female flies at different times post-eclosion. MATERIALS AND METHODS

Development of monoclonal antibodies to Drosophila yolk protein Soluble yolk protein preparation. Laboratory stocks of Drosophila melanogaster (Oregon R) were maintained on Formula 4-24 Instant Drosophila medium (Carolina Biological Supply Co.). The soluble yolk protein was prepared by dissecting ovaries from female adults 72 hr after emergence, homogenizing in 0.15 M NaC1 and centrifugation for 20 rain at 45,000g and 4°C. This preparation is used for immunization and subsequent initial screening of monoclonal antibodies. Another yolk protein preparation was purified according to the procedures described by Mintzas and Kambyseltis (1982). The binding sites of the monoclonal antibodies were identified by a combination of gel electrophoretic separation of yolk polypeptides, Western blotting and immunohistochemical methods. Immunization. Ma et al. (1984b) reported an /n vitro immunization protocol that will enhance hybridoma production to picomoles of antigen. EL-4 supcmatant was added to complete RPMI 1640 medium for promoting antigen-activated B-ceU proliferation. Two BALB/c mice

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SHUENN-JUEWU and MICHAELMA

were immunized with this in vitro protocol. Another two mice were immunized by successive intraperitoneal injections according to the procedure described in Ma et aL (1984a). Splenocytes were harvested from these two groups of mice for cell fusion experiments. Cell fusion. Cell fusion was performed by modifying the protocol established by Kohier and Milstein (1975) (Ma et aL, 1984a). Wells with hybridomas producing antibodies to the Drosophila antigen were identified using the indirect ELISA (Voller et aL, 1979). Two cell fusion experiments were performed yielding 14 positive clones from the fusion of SP2/0-AgI4 cells and splenocytes that were immunized/n vitro. The same procedure was followed with the splenocytes collected from the mice immunized in vivo and two high affinity clones~were selected for further characterization. All the cell lines were propagated for ascites production and liquid nitrogen vapour phase cryogenic storage. Characterization of monoclonal antibodies to yolk polypeptides Gel electrophoresis. Different components of the D. melanogaster soluble yolk protein were analysed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) with the MINI-SLAB vertical gel electrophoresis system (Idea Scientific Co.). Protein samples in sample buffer (2% SDS, 10% sucrose, 2mMTris-HCl buffer at pH 8.3) were heated at 100°C water bath for 2-3 min. The slab gel (I00 x 150 x 0.5 mm) contained 8% acrylamide with a 3% stacking layer. Samples were then applied to the wells at 2-8/~l/well (equivalent to 1-4/~g/well). Gel analysis was carried out initially at 150 V for 30min and then increased to 300 V for 2 hr. Gel patterns, after staining with Coomassie Blue, were recorded and analysed by a Joyce Loebl Chromsean 3 densitometer. Western blotting and immunohistochemical staining. Transfer of proteins from the gel to nitrocellulose membrane (0.45/~m) was modified from the procedures developed by Towbin et al. (1979) and Burnette (1981). In our studies, we chose a different blocking agent for better reduction of nonspecific background staining. The nitrocellulose paper strips were incubated in a milk-based cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), which contained 5% (w/v) nonfat dry milk and 0.0001% methiolate in phosphate buffered saline (PBS; Johnson et al., 1984). Protein bound by monoclonal antibodies were detected by appearing pink over a white background (O'Conner and Asman, 1982). Enzyme linked immunosorbent assay Primary antibodies. Two cell lines, VT-15 A4 and VT74 El, were selected because of their affinity to the three major yolk polypeptides of Drosophila. Ascites fluids of these two cell lines were produced and the monoclonal antibodies were concentrated by ammonium sulphate precipitation followed by centrifugation and dialysis. The coating concentration of the primary antibodies was 100 #g/ml. Female-specific rabbit antiserum as secondary antibodies. Rabbit antiserum to the soluble yolk protein was obtained through four injections using the immunization protocol described by Kobat and Mayer (1961). The antibody titer was shown to have an ELISA end point of 1:400,000 with the indirect method (Voller et al., 1979). This antiserum was made female specific by an affinity column which coupled Drosophila male protein to CNBr-scpharose (Pharmacia). Five hundred males were homogenized in coupling buffer (0.1 M NaHCO3, 0.5 M NaCI) at pH 8.3 followed by centrifugation to collect the supernatant. The male extract was coupled to the activated CNBr-Sepharose with approx. 5-10 rag protein per ml of gel. The remaining active groups were blocked with 0.2 M glycine buffer at pH 8. The antiserum was checked for possible erossreactivity with male

protein using the Western blotting procedure described previously. The immunoglobulin subclass G of this female-specific antiserum was collected by a protein A column procedure (Goding, 1978). The antibodies were then conjugated with alkaline phosphatase using the two-step glutaraldehyde method modified from Voller et al. (1979). Double antibody sandwich ELISA. Polyvinyl 96-well microplate (Falcon) was coated overnight at 4°C with monoclonal antibodies (50 #l/well) from the two chosen cell lines as the primary antibody. The microplate was then blocked with BLOTTO for 1 hr at room temperature. A dilution series of Drosophila soluble yolk protein was used as reference standard. The samples were incubated in the coated plates for I hr followed by three washings of PBSTween (PBS + 0.005% Twecn 20) with Skatron Microwash (Skatron Co.). The rabbit antibody-enzyme conjugate was added as the secondary antibodies (5/ag/ml) for 1 hr. After six washings, p-nitrophenyl phosphate in 10% diethanolamine-HCl(1 mg/ml, pH 9.8) was added. The colorimetric readings were recorded 1 hr later with a Biotek ELISA reader. Haemolymph sample preparation. Haemolymph samples were prepared by carefully dissecting adult females in 50 #1 of PBS without breaking the ovaries. The drop of PBS was then transferred to an Eppendorf microfuge tube and centrifuged at 10,000g to remove debris. Samples were collected at different times for measurement of the vitellogenin levels with the double antibody sandwich ELISA method. At least five female haemolymph samples were analysed for each time period.

RESULTS Successful cell fusions were achieved with BALB/c mice spleens that were immunized with both the in vitro and in vivo protocols. Fourteen positive clones were selected from the cell fusion with in vitro technique and two from the in vivo method. Immunoglobulin subclass identification for the representative subclones of each original cell line revealed only three IgM producing clones (VT-29 G8, VT-65 GI and VT-7 A1) obtained with the in vitro method. The rest of the cell lines produced various subclasses of IgG (Table 1). With 8% SDS-PAGE gel analysis of the soluble ovarian protein, we were able to visualize the three major yolk polypeptides (ranging from 45,000 to 47,000 daltons) which were referred to as YP 1, YP 2 and Y P 3 (Fig. 1A). Computerized densitometry showed that these three yolk polypeptides made up 45.15% of the total soluble ovarian proteins. The YP 1, YP 2 and YP 3 were present in 15.49, 9.09 and 16.57% respectively (Fig. IC). Representative subclones were characterized for their binding to the ovarian proteins with a combination of gel electrophoresis, Western blotting and immunohistochemical staining. Ten cell lines were shown to bind specifically to the three major yolk polypeptides with blot patterns similar to the one shown in Fig. lB. Two cell lines produced monoclonal antibodies that have affinity to only YP 1 and Y P 2 , while four lines reacted to other ovarian proteins (Table 1). The rabbit polyclonal antibodies preparation was checked for its specificity to female protein by the same blotting and staining procedures used for

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D. melanogaster y o l k p o l y p e p t i d e s Table 1. Summary of the Western blotting experiment. Affinity of hybridoma antibodies from different cell lines to D. melanogaster yolk proteins Yolk polypcptides Hybridoma cell lines

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In vitro immunization: VT-I D9 VT-2 BI 1 VT-7 AI + VT-15 A4 + VT-17 F7 + VT-23 B2 VT-28 H i 0 + VT-29 G8 + VT-3I C3 + VT-33 G6 + V T 4 7 BI I + VT-62 El + VT-65 O l + VT-74 El + In vivo immunization: W-01F8-A3 + W - 0 1 F6

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monoclonal antibodies. A blot pattern was obtained with insignificant amount of crossreactivity to other ovarian proteins. Since this female-specific antibody is protein-A purified immunoglobulin G, therefore it would make an excellent secondary antibody when conjugated with alkaline phosphatase in an ELISA procedure. The sensitivity of the double antibody sandwich ELISA was found to be around 0.1 #g/ml with soluble ovarian protein as standard (Fig. 2). The variation of YP levels was monitored at different times post-eclosion (Fig. 3). A rapid increase of YP concentration in the haemolymph started at 2 hr after emergence from approx. 20 to 150 ng/fly within the first day. The peak was found to be 180 ng/fly at 48 hr and stayed at about the same level up to 72hr post-cclosion. Haemolymph samples, obtained from both pharate females and males processed in the same manner, were found to have no detectable concentration of YPs.

DISCUSSION

Measurement of vitellogenin levels with ELISA Immunochemical methods have been extremely important in Vitellogenesis research (Engelmann, 1970, 1979). In all cases, the development of a specific antiserum is needed for detecting low levels of vitellogenin. In the past 15 years, ELISA has emerged as a major laboratory technique for the quantification of protein antigens due to its sensitivity and reliability. Ma et al. (1984a, 1986) incorporated the use of monoclonal antibodies as primary antibodies in an indirect double antibody sandwich ELISA for m e a suring vitellogenin of Aedes aegypti and Aedes atropalpus. With this immunoassay, the dynamics of vitellogenin was monitored in individual mosquito preparations. Our double antibody sandwich ELISA procedure for YP measurement compared favorably with a detection limit of 0.1 ng//~l. Most rabbit antisera used in Drosophila vitellogenesis research were not absorbed with male haemolymph protein to ensure specificity to YPs (Gavin and Williamson, 1976; Kambysellis, 1977; Warren and Mahowald, 1979; Hames and Bownes, 1978). Bownes and Northiger (1981) demonstrated that their rabbit antiserum had no crossreactivity to male haemolymph protein with Ouchterlony immunodiffusion. However, we have found that detection limit of the Ouchterlony :immunodiffusion method is around microgram levels. This can result in significant interference in a sensitive ELISA procedure which detects nanogram levels of antigen. Thus, we chose to examine crossreactivity of our polyclonal rabbit antiserum with gel electrophoresis of female ovarian protein followed by Western blotting and immunohistochemical staining. We have also found the procedure of male protein adsorption, developed by Tanaka (1977), was insufficient in making the antiserum specific to YPs. Only with affinity purification, with male protein as ligand, a polyclonal preparation can be obtained that is highly specific to YP I, YP 2 and YP 3 as shown by the Western blot results (Fig. I B). The combination of monoclonal antibodies as the primary antibody and an enzymelabelled polyclonal antibody as secondary antibody

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Fig. 3. Yolk polypeptide concentration in the haemolymph of individual D. melanogasterfemales collected at different times after adult eclosion. Each point represents the mean of at least 5 insectsand bars represent standard error around the mean.

SHUENN-JuE WU and MICHAELMA

794

provides a two tier selection process which increases the specificity of the ELISA procedure. This was evident in the extremely low background in the pharate females and males. Temporal pattern o f vitellogenin production It is important to establish the normal pattern of vitellogenesis in studies involving endocrine control mechanisms. Any experimental manipulations that produce deviations from the norm can be detected and interpreted correctly. Our result is in good agreement with similar studies by Kambysellis (1977) who used a haemagglutination inhibition method to determine vitellogenin levels from individual insect samples. In that study, vitellogenin per fly was reported to be 4.6 ng at 0 - 1 5 m i n post-eclosion, increases to 41.8 ng/fly at 6 hr and finally 148.5 ng/fly at 24 hr. Jowett and Postlewait (1980) identified the active period of YP synthesis as the first 24 br posteclosion with an in vivo radioactivity incorporation method. The yolk polypeptide haemolymph profile generated with our ELISA method concurs with their observation of YP synthetic activity. We believe the ability to determine YPs in individual insects will permit more flexibility in future experimental designs for elucidating hormonal control mechanisms. In conclusion, we have established the double antibody sandwich ELISA, employing both monoclonal and YP-specific rabbit polyclonal antibodies, to be reliable in measuring individual female haemolymph vitellogenin content. Although gene probes are valuable for determining the YP-mRNA synthetic activity at the transcriptional level, the results cannot be extrapolated directly as YP produced. Thus, a sensitive measure for YP is not only important for complementing studies involving vitellogenin gene expression, but also in the elucidation of events that are under endocrine control. Acknowledgements--This research was supported by the College of Agricultural and Life Sciences, University of Maryland at College Park; and the Maryland Agricultural Experiment Station Scientific Article No. A4217, Contribution No. 7203.

REFERENCES

Bownes M. (1982) Hormonal and genetic regulation of vitellogenesis in Drosophila. Q. Rev. Biol. 57, 247-274. Bownes M. and Nothiger R. (1981) Sex determining genes and vitellogenin synthesis in Drosophila melanogaster. Molec. gen. Genet. 182, 222-228. Bownes M., Blair M., Kozma R. and Dempster M. (1983) 20-Hydroxyecdysone stimulates tissue-specific yolkprotein gene transcription in both male and female Drosophila. J. Embryol. exp. Morph. 78, 249-269. Brennan M. D., Weiner A. J., Goralski T. J. and Mahowald A. P. (1982) The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster. Devl Biol. 89, 225-236. Burnette W. (1981) Western Blotting: Electrophoretic transfer of proteins from SDS-PAGE to unmodified nitrocellulose and radiographic detection with antibodies and radio-iodinated protein A. Analyt. Biochem. 112, 195-203. Englemann F. (1970) The Physiology of lnsect Reproduction. Pergamon Press, Oxford.

Englemann F. (1979) Insect vitellogenin:identification, biosynthesis, and role in vitellogensis. In Advances in Insect Physiology (Edited by Treherne J. E., Berridge M. J. and WigglesworthV. B.), Vol. 14. Academic Press, New York. Gavin J. and Williamson J. H. (1976) Synthesis and deposition of yolk protein in adult Drosophila melanogaster. J. Insect Physiol. 22, 1457-1464. Ghelis C. and Yon J. (1982) Protein Folding. Academic Press, New York. Goding J. W. (1978) Use of staphylococcal protein A as an immunological reagent. J. immun. Meth. 20, 241-253. Hagedorn H. H. (1985) The role of ecdysteroids in reproduction. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), pp. 205-262. Pergamon Press, Oxford. Haines B. D. and Bownes M. (1978) Synthesis of yolk proteins in Drosophila melanogaster. Insect Biochem. 8, 319-328. Johnson D. A., Gautsch J. W., Sportsman J. R. and Elder J. H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Analyt. Techn. 1, 3-8. Jowett T. and Postlethwait J. H. (1980) The regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog. Devl Biol. 80, 225-234. Kabat E. A. and Mayer M. M. (Ed.) (1961) Preparation of antigens for immunization and injection schedules. In Experimental Immunochemistry, 2nd edn, pp. 871-872. Thomas, Springfield. Kambysellis M. P. (1977) Genetic and hormonal regulation of vitellogenesisin Drosophila. Am. Zool. 17, 535-549. Kennett R. H., Mckearn T. J. and Bechtel K. B. (1980) Monoclonal antibodies. In Hybridoma: A New Dimension in Biological Analyses. Plenum Press, New York. Kohler G. and Milstein A. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 496-497. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Ma M., Newton P. E., Gong H., Kelly T. J., Hsu H., Mailer E. P. and Borkovec A. B. (1984a) Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis. J. Insect Physiol. 30, 529-536. Ma M., Wu S-J., Howard M. and Borkovec A. B. (1984b) Enhanced production of mouse hybridomas to picomoles of antigen using EL-4 conditioned media in an in vitro immunization protocol. In vitro 20, 739-742. Ma M., Gong H., Newton P. E. and Borkovec A. B. (1986) Monitoring Aedes aegypti vitellogenin production and uptake with hybridoma antibodies. J. Insect Physiol. 32, 207-213. Mintzas A. C. and Kambysellis M. (1982) The proteins of Drosophila melanogaster: isolation and characterization. Insect Biochem. 12, 25-33. O'Connor C. G. and Ashman L. K. (1982) Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for detection of the specificity of monoclonal antibodies to protein mixtures. J. immun. Meth. 54, 267-271. Postlethwait J. and Jowett T. (1981) Regulation of vitellogenesis in Drosophila. In Regulation of Insect Development and Behaviour (Edited by Schnal F., Zabza A., Meen J. J. and Cymborowski B.), pp. 591-627. Wrociaw Technical University Press. Tanaka A. (1977) Immunohistochemical studies of vitellogenin during embryogenesis in the cockroach Blattela germanica. J. Embryol. exp. Morph. 38, 49-62. Towbin H., Staeheln R. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedures and some applications. Proc. natn. Acad. Sci. U.S.A. 76, 4350-4354.

D. melanogaster yolk polypeptides Voller A., Bidwell D. and Bartlett A. (1979) The enzyme linked immunosorbent assay (ELISA). In A Guide with Abstracts of Microplate Applications. Dynatech Laboratories Inc. Publication.

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Warren G. T. and Mahowald A. P. (1979) Isolation and partial chemical characterization of the three major yolk polypeptides from Drosophila melanogaster. Devl Biol. 68, 130-139.