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Genes to Cells (2008) 13, 653-665. doi:10.1111/j.1365-2443.2008.01195.x
© 2008 Blackwell Publishing or its licensors
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Efficient production of monoclonal antibodies recognizing specific structures in Caenorhabditis elegans embryos using an antigen subtraction method

 Kazumasa Takeda, Chie Watanabe, Hiroshi Qadotaa, Momoyo Hanazawa and Asako Sugimoto*

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Monoclonal antibodies (mAbs) have been widely used to probe molecular components of specific cell types or cellular structures. We have developed a method to enrich antigens of low abundance in heterogeneous molecule mixtures by subtracting abundant antigens. The subtracted immunogen mixture is then used for immunization, which significantly increases the production of mAbs that exhibit specific staining patterns. By applying this "antigen subtraction" method to the embryonic extract of Caenorhabditis elegans, we have successfully isolated 35 mAbs that recognize specific structures, including P granules, muscles, the pharynx, and subsets of hypodermal cells; some of the mAbs revealed previously unreported cellular structures. This antigen subtraction approach can be used in various applications to produce mAbs against relatively scarce antigens in complex molecular mixtures. The mAbs will be useful tools for developmental and cell biological studies.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Monoclonal antibodies (mAbs) have been widely used as experimental tools in various fields of biological and medical sciences. One of the applications of mAbs is to visualize specific cellular structures or cell types in biological specimens. Such mAbs are not only useful as histological markers but also as biochemical probes to identify molecules that play important roles in particular cell types. In addition to their high specificity, another advantage of mAbs is that they can be produced in unlimited amounts and thus readily shared in the research community.

To produce mAbs that recognize specific structures (hereafter, specific mAbs), a so-called "shotgun" approach often has been used (Zipser & McKay 1981). In this approach, mAbs are indiscriminately produced against a complex mixture of molecules—for example, a tissue extract. Then the mAbs are screened by immunostaining according to researchers’ interest. This approach has been used to obtain mAbs that recognize specific organelles, cell types, or tissues of various experimental systems (Zipser & McKay 1981; Fujita et al. 1982). The shotgun approach has, however, a drawback in that it is difficult to isolate mAbs against proteins with low abundance in the immunogen mixtures. To isolate mAbs recognizing tissue- or cell-type-specific proteins, which tend to be less abundant than non-cell-type-specific proteins, generally a large number of hybridoma lines needs to be screened without any guarantee that the desired specific mAbs will be obtained. Therefore, to efficiently produce a wide variety of specific mAbs by the shotgun approach, a method to enrich molecules with low abundance is required.

Structure- and cell–type-specific mAbs have contributed to the studies of the nematode Caenorhabditis elegans by revealing its anatomical structures and in dissecting developmental processes. For example, mAbs against germ-line-specific granules, or P granules, have been extensively used as a germ-line marker (Strome & Wood 1982) and have contributed to the study of cell fate determination and asymmetric cell division (Strome & Wood 1983) and led to the identification of the P granule component PGL-1 (Kawasaki et al. 1998). A series of mAbs recognizing components of muscles and the hypodermis were isolated using partially purified muscle components (Francis & Waterston 1985, 1991). These mAbs revealed fine structures of each tissue and facilitated the understanding of the processes of tissue differentiation. Thus, mAbs recognizing specific structures have played crucial roles in advancing the molecular understanding of the biology of this organism. The number of such useful mAbs, however, remains limited.

We have now developed a method to enrich less-abundant antigens in heterogeneous immunogens and applied it to produce mAbs recognizing specific structures in the C. elegans embryo. This "antigen subtraction" method consists of two rounds of hybridoma production and screening. In the first round, crude embryonic extracts were used as immunogen, and hybridomas were screened by immunostaining. The mAbs that recognized abundant, non-cell-type-specific proteins were pooled and used to subtract these antigens from the embryonic extracts. The resultant protein mixtures, which should be enriched with less-abundant proteins, were then used for immunization in the second round. This procedure successfully produced 35 mAbs that recognize specific organelles, cell types, or tissues in C. elegans embryos. Staining patterns of these mAbs in embryos and adults were characterized, and some novel cellular structures were identified. We also identified an antigen of the mAb recognizing germ-line-specific granules called P granules. These mAbs will be highly useful as histological markers in the future studies of C. elegans. Furthermore, the antigen subtraction method will be applicable to other systems that require mAbs against low-abundance antigens.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Enrichment of low-abundance antigens by an antigen subtraction method

In a mAb shotgun approach in which a heterogeneous immunogen mixture is used, the abundance of each molecule in the immunogen generally correlates with the probability of antibody production in the host. Therefore, it is often difficult to obtain mAbs against molecules of low abundance, including molecules specific for subcellular structures or cell types. To circumvent this problem, we have developed an antigen subtraction method using two rounds of hybridoma production to enrich proteins of low abundance. We applied this method to produce mAbs that recognize organelle-, cell-type-, or tissue-specific molecules in C. elegans embryos (Fig. 1, Table 1).


Figure 1
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  		Figure 1  Strategy for antigen subtraction to efficiently produce mAbs recognizing molecules of low abundance in a complex molecular mixture. First round: When complex molecular mixtures (e.g. tissue extracts) are used as immunogen, mAbs recognizing abundant molecules are preferentially produced. The mAbs against abundant molecules can be pooled and used to subtract the corresponding antigens from the original immunogens to enrich for less-abundant molecules. Second round: The "subtracted" immunogens are used in the second round of immunization to improve the proportion of mAbs against low-abundance antigens in the original mixture.

 

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  		Table 1  Summary of screening of monoclonal antibodies
 
In the first round of hybridoma production, whole C. elegans embryonic extract was used as immunogen, and mAbs were screened by immunostaining of worm embryos. Of the hybridoma wells tested, approximately 60% (526 of 874) stained mixed-stage C. elegans embryos. However, only 3% of them (16 of 526) yielded a specific staining pattern; the rest generally exhibited uniform cytoplasmic staining in whole embryos, suggesting that those mAbs recognized abundant, non-cell-type-specific molecules. For the second round of hybridoma production, these non-cell-type-specific mAbs obtained in the first round were used as "eliminators" to subtract the corresponding abundant antigens from the embryonic extracts by immunoprecipitation.

Indeed, in the second round of hybridoma production using the subtracted extract as immunogen, approximately 50% (81 of 165) of the immunostaining-positive mAbs exhibited specific staining signals limited to particular structures or cell types. For further characterization, 35 hybridoma lines were selected and cloned (five from the first round, 28 from the second round and two from a previous hybridoma production using unsubtracted embryonic extract), and the resultant collection of mAbs was named the "KT mAb series" (Table 2).


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  		Table 2  Summary of KT mAbs
 
Specific structures and cell types recognized by KT mAbs

The 35 KT mAbs were analyzed with regard to how they stained various stages of C. elegans embryos (Table 2, Fig. 2). The largest mAb class comprised those recognizing P granules (13 mAbs). P granules are present specifically in germ cells and their precursors throughout C. elegans development (Strome & Wood 1982). Certain P granule–recognizing KT mAbs also stained other structures such as muscles and pharynx (Table 2 and see below). Other KT mAbs stained a variety of cell types (e.g. muscle, pharynx, intestine, sensory cilia and excretory pore), subcellular structures (e.g. centrosomes and nuclear membrane), or extracellular components (e.g. basement membrane and eggshell).


Figure 2
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  		Figure 2  Examples of staining patterns of KT mAbs in C. elegans embryos. Embryos were stained with KT mAbs and counterstained with DAPI (to stain nuclei) or mAb MH27 (recognizes AJM-1, which localizes to cell–cell junctions of epithelia). For each set, an image showing staining with the KT mAb (top) and a merged image with counterstain (bottom) is shown. Magenta, KT mAbs; green, DAPI (A–D) or MH27 (E–J). Scale bar: 10 µm. (A) KT23 staining of the nuclear membrane of approximately 16-cell stage embryo. (B) KT26 staining of the centrosomes of a 2-cell stage embryo. (C) KT2 staining of the P granules of a 2-cell stage embryo. (D) KT30 staining of the eggshell of approximately 300-cell stage embryo. (E) KT9 staining of the body wall muscle of a twofold stage embryo. (F) KT13 staining of the seam cells of a pretzel stage embryo. (G) KT27 staining of the C-lineage-derived hypodermis of a bean stage embryo. (H) KT16 staining of the pharynx of a pretzel stage embryo. (I) KT14 staining of the basement membrane of a threefold stage embryo. (J) KT31 staining of the sensory cilia and excretory pore of a 1.5-fold stage embryo.

 
Although these KT mAbs gave generally strong signals by immunostaining, only four of them (KT2, KT3, KT6 and KT35) gave specific bands by Western blotting of C. elegans extracts. The rest might recognize three-dimensionally folded structures that are destroyed by the denaturing conditions used for Western blotting. Twenty-six KT mAbs were of the IgM subclass, five were IgG and four were IgA.

KT mAbs that recognize the body wall muscle

Six KT mAbs (KT3, KT6, KT9, KT10, KT11 and KT12) recognized the body wall muscle or basement membrane between the body wall muscle and the hypodermis (Figs 3 and 4).


Figure 3
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  		Figure 3  Muscle-specific antibody staining of embryos at 1.5-fold and pretzel stage. (A) KT11, (B) KT12, (C) KT6, (D) KT9, (E) KT10, (F) KT3. Magnified view of the muscle region (boxed area) is also shown. KT11 and KT12 stained the cytoplasm of muscle cells. Two quadrants can be seen. KT9 and KT10 stained the basal face of muscle cells. Three quadrants can be seen. KT6 did not stain muscle cells in embryos, but they recognized P granules (arrowheads). KT3 stain P granules throughout embryogenesis, and dotted structures in muscle cells in late embryogenesis. The dashed lines indicate the shape of embryos. Scale bar: 10 µm (whole embryo), 2.5 µm (magnified view).

 

Figure 4
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  		Figure 4  Muscle-specific antibody staining of adults. (A, B) Schematic diagram of a body wall muscle cell of C. elegans (adapted from WormAtlas <http://www.wormatlas.org>). (A) A single muscle cell. (B) Magnified view of the section indicated in (A). The myofilament lattice is located close to the hypodermis and attached to the basement membrane at the dense body and M-line. The inner side of the muscle cells (muscle belly) contains nuclei and mitochondria. Myosin heavy chain A (MHC A) forms the central region of thick filament (Green). (C-F) KT mAb staining of adult muscles. (C) KT mAb staining at low-magnification (scale bar: 10 µm). (D) KT mAb staining at high magnification (scale bar: 5 µm). (E) Merged image of KT mAb (magenta) and anti–MHC A (green) staining. To visualize MHC A, the DM5.6 mAb or anti–green fluorescent protein (GFP) and myo-3::gfp animals were used for KT12 and KT6 or KT12 and KT6 staining, respectively. Overlap of the two antibody stains appears white. (F) Cross-section view of the images of (E). The arrow in (B) corresponds to the orientation of this view. (G) Schematic diagram of the recognized structure; magenta coloring indicates the structure stained by the KT mAb.

 
KT11 and KT12 showed cytoplasmic staining of muscle cells starting from the comma stage of embryogenesis (Fig. 3A,B). The signal gradually became filamentous by the pretzel stage (Fig. 3A,B). KT11 exhibited punctate staining of the myofilaments, whereas KT12 appeared to stain the entire myofilament. Both antibodies stained muscle fiber in adults as well (Fig. 4C). KT6 recognized only P granules in embryos (Fig. 3C), but in adults it additionally stained the body wall muscle (Fig. 4C).

Higher magnification of staining of adults revealed that KT11, KT12 and KT6 recognize distinct components in the myofilaments (Fig. 4D). KT11 labeling showed wider, diffuse bands, with a thin line of higher intensity in the center; KT12 staining appeared as double bands with narrow spacing, and KT6 staining produced thin bands containing evenly spaced unlabeled spots. To obtain information on the myofilament components recognized by these mAbs, we examined their staining patterns relative to myosin heavy chain A (MHC A), which localizes to the center of the A bands of the myofilament lattice (Miller et al. 1983) (Fig. 4B,E,F). MHC A, encoded by the myo-3 gene (Miller et al. 1986), was visualized with DM5.6 mAb or the myo-3::gfp transgene (Campagnola et al. 2002). MHC A bands coincided with the centerlines of the KT11 bands, indicating that KT11 antigen is localized at A bands (Fig. 4E–G). In contrast, KT12 and KT6 staining did not overlap with MHC A lines, and KT6 bands appeared to correspond to the narrow space between the double bands of KT12 (Fig. 4E–G). In addition, double staining of adults muscles with KT6 and MH35 mAb, which recognizes {alpha}-actinin localized to dense bodies (Francis & Waterston 1985), showed that the KT6 antigen is on I bands and that the blank spots in the staining correspond to dense bodies (data not shown). Thus, the KT6 antigen appeared to localize to the center of I bands, and the KT12 antigen to the outer edge of I bands or the border between A and I bands.

KT9 and KT10 staining first appeared at the comma stage and formed four lines that apparently corresponded to contact regions of muscle cells and the hypodermis (Fig. 3D,E). In adults, both mAbs stained basement membranes between body wall muscle cells and the hypodermis (Fig. 4C). With higher magnification, dots and lines with regular spacing were visible, and the staining patterns produced by KT9 and KT10 were indistinguishable (Fig. 4D). In double staining of adult muscles, the line stained by KT9 or KT10 co-localized with MHC A, suggesting that the line corresponded to the M line and the dots to dense bodies (Fig. 4E,G). The cross-section view of merged images indicated that KT9 and KT10 antigens were localized at the surface of muscle cells facing the hypodermis (Fig. 4F). These staining patterns were similar to those of muscle basement membrane attachment components, such as perlecan (UNC-52) (Rogalski et al. 1993).

KT3 stained P granules from the one-cell stage throughout embryogenesis. Its staining was limited to P granules until the comma stage, and in the 1.5-fold stage an undefined punctate signal was detected in the head region (Fig. 3F). Punctate KT3 signals in the muscle cells became apparent by the pretzel stage (Fig. 3F). In adults, the punctate KT3 signals were detected in body wall muscle cells (Fig. 4C). To identify the structures recognized by KT3, co-localization of KT3 signal with MHC A was examined. A cross-section view of the merged image revealed that KT3 antigens appeared to localize to the muscle belly and not at the myofilament lattice (Fig. 4F). We speculate that KT3 recognizes mitochondria or other organelles in the muscle belly.

KT mAbs that recognize subsets of hypodermal cells

The hypodermal (epidermal) cells are generated as six rows of cells on the dorsal surface of embryos (Simske & Hardin 2001). Cells of the two dorsal rows interdigitate to form a single layer of dorsal hypodermis. The cells of the lateral and ventral rows migrate over the surface of the embryo and meet along the ventral midline to enclose the embryo. After enclosure, cell shape change of hypodermal cells causes elongation of the entire body shape. During the elongation process, the majority of the dorsal cells and some of the ventral cells fuse to form syncytial cells. Two of the mAbs, KT27 and KT13, stained distinct subsets of hypodermal cells at specific periods during embryogenesis (Figs 2 and 5).


Figure 5
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  		Figure 5  Hypodermis-specific antibody staining of embryos. KT27 and KT13 show stage-specific staining patterns. (A) KT27 staining (left) and merged images (right) of double staining with KT27 (magenta) and MH27 (green) before interdigitation of dorsal hypodermal cells (top), at the bean stage (middle) and at the twofold stage (bottom). KT27 stains dorsal posterior hypodermal cells. The signal became undetectable after cell fusion of the dorsal hypodermis. Scale bar: 10 µm. (B) Schematic diagram of the hypodermis at about 390 min after the first cleavage (top, adapted from WormAtlas <http://www.wormatlas.org>) and the cell phylogeny starting from the "C" cell (bottom). KT27-stained cells are shown in magenta. (C) KT13 staining (left) and merged images (right) of double staining with KT13 (magenta) and MH27 (green) at the twofold stage (top) and pretzel stage (middle); enlarged images of the indicated boxed area in the pretzel stage images are shown at the bottom. Scale bar: 10 µm.

 
The KT27 signal was first detected before interdigitation of the dorsal rows, and the cytoplasm of posterior dorsal hypodermal cells was specifically stained (Fig. 5A). This staining lasted until the 1.5-fold stage, at the time of fusion of dorsal hypodermal cells. The 12 dorsal hypodermal cells #6–17, which ultimately fuse with each other and with other dorsal hypodermal cells to create the hyp7 syncytium, were KT27 positive. Notably, these KT27-positive cells corresponded to the entire set of hyp7 precursor cells derived from the C founder cell (Sulston et al. 1983) (Fig. 5B). The C cell lineage produces another hypodermal cell called hyp11, but because of its position at the most posterior side of the embryo, it was difficult to judge whether hyp11 expressed KT27 antigen. The remaining 65 hypodermal cells are derived from the AB lineage (Sulston et al. 1983) and were not stained with KT27. Timing of the loss of KT27 signal appeared to coincide with cell fusion of the dorsal hypodermis to generate the hyp7 syncytium. Thus, it is possible that the KT27 antigen diffused within the syncytium, making the signal undetectable.

KT13 recognized the lateral hypodermis (called seam cells) at the pretzel stage, but not at earlier stages (Figs 2 and 5C). Seam cells at the pretzel stage have a long rectangular shape, and the KT13 signals were stronger along the edge of the long sides of the rectangle (Fig. 5C). With higher magnification, The KT13 signal appeared as parallel fibrous structures along the short axis of each seam cell. To our knowledge, no such structures have been reported.

KT mAbs that recognize the apical side of the pharynx

The pharynx is a linear tube with two lobes contained within a single basal lamina (Albertson & Thomson 1976) (Fig. 6A). It is organized radially and shows threefold symmetry (Fig. 6B). The apical side of the pharyngeal epithelium faces toward the lumen, and is covered by cuticle. Adherens junctions are present near the junction between the apical and the lateral surfaces of pharyngeal epithelial cells, which are recognized by MH27 mAb (anti-AJM-1).


Figure 6
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  		Figure 6  Pharynx-specific antibody staining of embryos at the pretzel stage. KT16, KT17, KT19, KT20, KT22 and KT36 recognize the apical side of pharynx. (A) Schematic diagram of pharynx. (B) Schematic diagram of cross-section at the arrow indicated in (A). The pharynx shows threefold symmetry. Adapted from (Leung et al. 1999). (C) Immunostaining of pretzel stage embryos. Left: KT mAbs (magenta) and MH27 (green). Enlarged images of the indicated boxed areas (metacorpus and terminal bulb) are shown in (D) and (E). Right: KT mAbs. Arrow heads indicated the position of the pharyngeal–intestinal valve. Scale bar: 10 µm. (D) Metacorpus. (E) Terminal bulb.

 
Six KT mAbs (KT16, KT17, KT19, KT20, KT22 and KT36) recognized the apical side of the pharynx (Fig. 6C–E). In the pretzel stage embryos, KT16, KT17 and KT19 stained the apical (lumenal) surface of the pharynx, between the buccal cavity and the pharyngeal–intestinal valve (Fig. 6C–E). The signals of KT16 and KT17 became detectable after the threefold stage, while the KT19 signal was detected in the twofold embryos (data not shown). The KT20 signal was first detected in the comma stage as punctate staining in the apical region of the pharynx, and by the 1.75-fold stage the signal became a thick line (data not shown). In the pretzel stage embryo, KT20 staining was seen as a single dotted line between the buccal cavity and the pharyngeal–intestinal valve. The KT22 signal was detectable in the twofold embryo as a single line from the buccal cavity to the end of isthmus (data not shown); at the pretzel stage, the KT22 stained three lines from the buccal cavity to the anterior isthmus, which appeared to merge into a single line in the posterior isthmus region (Fig. 6C–E). No KT22 signal was detected in the terminal bulb and pharyngeal–intestinal valve. KT 36 showed punctate staining in the whole embryos and a dotted thin line from the procorpus through the isthmus region starting from the twofold stage (Fig. 6C–E).

Since the apical surface of the pharynx is lined with the specialized cuticle, we speculate that these antibodies might recognize some of its components.

KT3 recognizes PGL-3, a P granule component

As described above, KT3 recognized both muscle organelles and P granules (Figs 3F and 7A). To understand the nature of the molecule recognized by KT3, we determined the antigen. In Western blots against wild-type adult extracts, KT3 recognized a 75-kDa band (Fig. 7B) that was absent in Western blot of extracts from glp-4(bn2) mutant animals (Beanan & Strome 1992), which essentially do not have a germ-line (data not shown). These results indicated that this 75-kDa band corresponded to the P granule component recognized by KT3. We used mass spectrometric analysis of KT3 immunoprecipitate from wild-type worm extracts and identified the PGL-3 protein as a candidate for the 75-kDa protein recognized by KT3 (Fig. 7C). PGL-3 has been reported as a P granule component (Kawasaki et al. 2004).


Figure 7
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  		Figure 7  KT3 recognizes PGL-3, a component of P granules. (A) KT3 staining in wild-type and pgl-3 mutant embryos. (B) Western blotting against adult worm extracts. KT3 recognized a 75-kDa band in the wild-type (WT) extract but not in the pgl-3 mutant extract. A band of the same size was detected by polyclonal anti-PGL-3. Anti-tubulin was used as a loading control. (C) Polypeptides detected by mass spectrometry. The entire amino acid sequence of PGL-3 is shown. Detected fragments are boxed.

 
To confirm that the KT3 antigen was indeed PGL-3, Western blotting against pgl-3 mutants animals (Kawasaki et al. 2004) was conducted. In pgl-3 extracts, KT3 failed to recognize specific bands; furthermore, anti-PGL-3 polyclonal antibodies (Kawasaki et al. 2004) recognized a band of the same mobility as KT3 in the wild-type extract but not in a pgl-3 extract (Fig. 7B). We thus concluded that KT3 recognizes PGL-3.

In immunostaining of pgl-3 mutant embryos with KT3, the P granule signal was absent throughout embryogenesis, but the punctate muscle staining remained (Fig. 7A). Therefore, KT3 appeared to recognize a molecule other than PGL-3 that was not detected by Western blotting.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have developed an efficient antigen subtraction method of generating a panel of mAbs that recognize specific structures by modifying the shotgun mAb production approach (Zipser & McKay 1981). Use of a subtracted C. elegans embryo extract significantly increased the fraction of hybridomas producing organelle-, or cell-type-specific antibodies from 3% to 49% (Table 1). This approach can be used in various applications to produce mAbs against relatively scarce antigens in complex molecular mixtures. As a variation of the subtraction process, polyclonal antibodies against embryonic extracts might be used to remove abundant or highly antigenic components, although some portion of organelle- or cell-type-specific components inevitably will be removed by this procedure.

Immunostaining of C. elegans embryos and adults with the KT mAbs revealed some previously unidentified components of particular structures or cell types. For example, KT27 recognized C-lineage-derived hypodermal cells, and KT13 recognized previously unreported fibrous structures in seam cells that are detectable only beginning from the pretzel stage. KT mAbs specific for muscles distinguished substructures of muscle cells. Determining the identity of the antigens could lead to further understanding of the anatomy and cell differentiation of this animal.

Because a complex mixture of molecules was used as immunogen to produce these mAbs, their antigens are unknown. Generally protein antigens of mAbs can be determined by two methods: biochemical purification/mass spectrometry or expression cloning. We used the former to identify PGL-3 as the antigen of KT3. With the recent improvement of the sensitivity and throughput of experimental techniques and availability of genome-wide resources, we anticipate that the molecular targets of the remaining KT mAbs can be experimentally determined.

The complete genome sequence of C. elegans encodes about 20 000 proteins ( C. elegans Sequencing Consortium 1998). However, only a small subset of proteins has been analyzed for their precise expression and localization patterns. Large-scale analyses of spatiotemporal gene expression patterns using green fluorescent protein (GFP)-fused transgenes have been initiated (Hope et al. 2004; Dupuy et al. 2007), but current studies are mainly focused on promoter activities rather than protein localization patterns. This is mainly due to the relatively low throughput of construction of transgenic worms expressing translational GFP fusion proteins and of production of monoclonal/polyclonal antibodies targeting each protein. Therefore, the modified mAb shotgun approach presented here and systematic identification of the antigens of obtained mAbs might provide an alternative and complementary approach to reveal spatiotemporal protein expression patterns on a large scale.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Worm strains and culture conditions

Wild-type (N2 Bristol), RW1596 (myo-3(st386) V; stEx30), MT3188 (egl-17(n1377) X), SS104 (glp-4(bn2) I), and SS608 (pgl-3(bn104) V) strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN). Animals were maintained at 20 °C, except for SS104, which was raised at 25 °C.

Preparation of immunogen and antigen subtraction

Two rounds of mAb production were performed. To prepare C. elegans embryonic lysates as immunogen for the first round, embryos collected from adult worms by bleaching were thoroughly washed with cold M9 buffer and sonicated on ice. Preparations from approximately 1–3 x 105 embryos were used per mouse per injection. Hybridoma supernatants were screened by immunofluorescence microscopy and Western blotting. Hybridomas showing uniform staining of all cells in embryos were selected and categorized according to the patterns observed in Western blotting. Several pools of about 6–10 kinds of hybridoma supernatants were made so as to contain antibodies with a variety of Western blotting patterns.

For the second round of hybridoma production, "subtracted" embryonic lysates were prepared as follows. Approximately 1 x 106 worm embryos collected from adults by bleaching were suspended in 1 mL of cold M9 buffer containing 1 mM EGTA, 1 mM pAPMSF, and 200 µg/mL BSA, sonicated on ice and lysed with Triton X-100 (final 2% (wt/v)) at room temperature for 30 min. The lysates were then mixed with a suspension of Protein A-Sepharose CL-4B (200 mg) coupled with anti-mouse IgG (10 mg) and the pooled hybridoma supernatants described above (nearly 0.2–0.3 mL from each line), incubated for 1 h and centrifuged to remove the precipitate. The acetone-insoluble materials in the supernatants were collected with three volumes of cold acetone. These materials were suspended and used as the subtracted embryonic lysate immunogens for three mice per each injection.

Immunization and hybridoma production

Immunogens were injected intraperitoneally into Balb/c mice 4 times in the first round and 6 times in the second round at about 2–3-week intervals. Complete Freund's adjuvant was used in the initial immunization, and incomplete Freund's adjuvant was used in each boosting immunization except the last one. Three days after the last immunization, the spleen cells were removed from immunized mice and fused with mouse myeloma X63-Ag8, 6.5.3 cells by use of polyethylene glycol (the ratio of myeloma cells : spleen cells was approximately 1 : 5). Immediately after fusion the cells were diluted in hypoxanthine/aminopterin/thymidine (HAT) selection medium and distributed to 96-well plates (about 1 x 105 spleen cell equiv. per well per 0.1 mL) and incubated at 37 °C. Hybridoma supernatants were screened for antibody production by immunostaining against C. elegans embryos and Western blotting against C. elegans embryonic lysates. Hybridomas producing antibody with the desired specificity were cloned by limiting dilution at least 2 times, amplified and stored in liquid nitrogen.

Immunostaining

Embryos were collected by cutting gravid hermaphrodite adults of N2 or an egl-17 mutant. egl-17 animals were used to collect large numbers of embryos of diverse developmental stages because the egg laying defective phenotype of egl-17 adults causes accumulation of embryos in their uterus (Stern & Horvitz 1991). For immunostaining of embryos, conventional freeze-cracking and methanol-acetone fixation (Albertson 1984) was used (–20 °C methanol for 5 min, –20 °C acetone for 5 min). The samples were rehydrated before antibody staining by passing the slide through an acetone series (90%, 70%, 50% and 30%) at room temperature followed by transfer into PBS + 0.5% (wt/v) Tween 20 (PBST).

Adult worms were fixed with picric acid (Nonet et al. 1997) (for KT3, KT6, KT9, KT10 and KT12) or 2% paraformaldehyde (Finney & Ruvkun 1990) (for KT11). Fixation time with picric acid was 1 h at room temperature and was 1 h at 4 °C for formaldehyde.

For immunostaining, the slides were incubated overnight at 4 °C in a humid chamber with hybridoma supernatant at 1 : 2–1 : 10 dilution in PBST containing 0.5% BSA + 0.5% skim milk. For immunostaining of myo-3::gfp worms, rabbit polyclonal anti-GFP (Molecular Probes, Eugene, OR, A-6455) was used at 1 : 500 dilution. Secondary antibodies with fluorescent labels were used at 1 : 100–1 : 200 dilution and incubated for 2 h at room temperature. The secondary antibodies were immunoadsorbed with worm acetone powder to remove potential antibody fraction that recognize worm antigens, and the absence of nonspecific staining was confirmed by immunostaining of worm embryos. Secondary antibodies (from Molecular Probes) were as follows: goat anti-mouse IgG(H + L) Alexa Fluor 488, goat anti-mouse IgM Alexa Fluor 594, goat anti-rabbit IgG(H + L) Alexa Fluor 594. After immunostaining, DAPI was added to a final concentration of 1 µg/mL, and the samples were mounted with DABCO solution for microscopy.

Microscopy

Immunofluorescence images of embryos were acquired with an Olympus DSU disk scanning confocal microscope system with a 100x objective lens (U-PlanApo 1.35/Oil Iris). For each embryo, Z-series images (0.3–0.5 µM steps, 60–70 slices) were acquired and projected using a maximum intensity algorithm to produce a single integrated image using MetaMorph software (Molecular Devices).

Immunofluorescence images of adult worms were acquired with a laser-scanning confocal microscope LSM 510 (Zeiss) with a 63x (C-APOCHROMAT 1.2/W Korr) or 40x objective lens (C-APOCHROMAT 0.95/Korr). Z-series images (30–50) with 0.4 µM steps were captured. Images of the Z-stack projection and cross-sections were produced with the LSM software (Zeiss, Jena, Germany).

Identification of KT3 antigen

Wild-type (N2) and glp-4(bn2) mutant adult lysates were subjected to immunoprecipitation with KT3 mAb or control IgA (Beckman Coulter). The eluates were applied to SDS-PAGE, and a 75-kDa band detected only in the KT3-precipitated N2 lysates was subjected to liquid chromatography-tandem mass spectrometry (LC–MS/MS). Mass spectrometry data were analyzed with MASCOT software (Matrix Science, Boston, MA), and peptide fragments covering 37.7% (261 of 693) of PGL-3 were detected.

For Western blotting analysis, wild-type and pgl-3(bn104) adult animal extracts were prepared. Polyclonal anti-PGL-3 and DM1A anti-tubulin mAb (Sigma, , St Louis, MO) were used as control antibodies.


   Acknowledgements
 
The nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). The MH27 antibody developed by R. H. Waterston and DM5.6 antibody developed by H. Epstein were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by The University of Iowa, Department of Biological Sciences. We thank K. Shinmyozu (Mass Spectrometry Analysis subunit, RIKEN CDB) for mass spectrometry and Dr I. Kawasaki (University of Tokyo) for providing anti-PGL-3. This work was supported by Kakenhi (Grant-in-Aid for Scientific Research) on Priority Areas "Systems Genomics" from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yuji Kohara

aPresent address: Department of Pathology, Emory University, 615 Michael Street, Atlanta, GA 30322, USA. Back

* Correspondence: Email: sugimoto{at}cdb.riken.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 17 February 2008
Accepted: 26 March 2008





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