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. 2000 Jan;5(1):14–20. doi: 10.1043/1355-8145(2000)005<0014:PACOPA>2.0.CO;2

Preparation and characterization of polyclonal antibodies against human chaperonin 10

Maria J Somodevilla-Torres 1, Narelle C Hillyard 1, Halle Morton 1, Dianne Alewood 2, Judy A Halliday 2, Paul F Alewood 2, David A Vesey 1, Michael D Walsh 3, Alice C Cavanagh 1,4
PMCID: PMC312905  PMID: 10701835

Abstract

Abstract Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.

INTRODUCTION

Early pregnancy factor (EPF) was first described as a factor that appeared in maternal serum within 24 hours of fertilization in all mammalian species tested and that persisted for at least the first half of gestation (Morton et al 1987). Subsequent studies demonstrated that EPF is not confined to pregnancy; activity can be detected in serum during the processes of normal tissue renewal, such as liver regeneration (Quinn et al 1994), as well as in pathologic situations, such as the development of cancer (Quinn 1991; Quinn and Morton 1992). It can be demonstrated in vitro that the appearance of EPF in the extracellular compartment closely parallels cellular growth, and that the induction of growth arrest or differentiation causes the rapid disappearance of EPF (Quinn et al 1990).

Before our identification of EPF as a homologue of chaperonin 10 (Cpn10; Cavanagh and Morton 1994), we raised monoclonal antibodies to that protein partially purified from a medium conditioned by the human choriocarcinoma cell line BeWo (Athanasas et al 1989; Quinn et al 1990). These antibodies were invaluable neutralizing agents and enabled us to determine that EPF is not only closely associated with cellular growth but is also required for normal embryonic development (Athanasas et al 1989; Athanasas-Platsis et al 1991), establishment and maintenance of tumors (Quinn and Morton 1992), and normal liver regeneration (Quinn et al 1994). As low-affinity immunoglobulinM antibodies , however, their utility was limited. Furthermore, antibody production by the hybridomas was unstable (Quinn et al 1990). These characteristics were considered to result from the essential growth-regulatory properties of EPF. Hybridomas, like the parent myeloma, both produce EPF and require it for growth; hence, the only hybrid cells capable of surviving production of these EPF-neutralizing agents were those producing the least effective antibodies. Once the EPF amino acid sequence was established, we undertook production of polyclonal antibodies to synthetic peptides that corresponded with different parts of this sequence to produce a more robust array of reagents.

The need for such tools was made even more compelling by the actual identity of the EPF protein sequence. Seventy percent of the amino acid sequence of the molecule isolated from human platelets was determined, and except for a single residue now known to represent a species difference, this sequence was found to be identical to that of rat Cpn10 (Hartmann et al 1992). Cpn10 functions in eubacteria and within eukaryote mitochondria and plastids as an accessory molecule to chaperonin 60 (Ellis and van der Vies 1991). The Escherichia coli forms of these molecules (the products of the GroE operon) are known as GroES and GroEL, respectively (Zeilstra-Ryalls et al 1991). Further studies determined that human platelet–derived EPF and rat mitochondrial Cpn10 were functionally interchangeable in vitro, but GroES did not exhibit activity in the EPF bioassay (Cavanagh and Morton 1994). Using probes based on the human protein sequence, we cloned a cDNA from a human melanoma library (Summers et al 1996), thus confirming the amino acid sequence identity between platelet-derived EPF and Cpn10. This apparent identity between an extracellular molecule with growth-regulatory and immunomodulatory properties and a molecular chaperone targeted to intracellular organelles raises unprecedented regulatory and mechanistic questions. In this study, we describe the production, assessment, and application of antibodies to selected epitopes of Cpn10 that will be useful tools in the search for answers to such questions.

RESULTS AND DISCUSSION

Antibody response to Cpn10-derived synthetic peptides displays an unusual pattern

During the initial attempts at antibody production, short synthetic peptides corresponding with the N-terminal sequence of Cpn10 (residues 1–11) and an internal sequence (residues 33–44) were each conjugated to ovalbumin and administered according to a standard immunization schedule (Johnstone and Thorpe 1996). Rabbits did not respond to booster doses of antigen, and with time, specific antibody production declined (data not shown). With a C-terminal peptide corresponding to residues 87–101, the response was more in accord with the pattern expected, with specific antibody production gradually increasing in response to booster doses of antigen (data not shown). Unfortunately, this particular peptide did not appear to be very immunogenic, because 2 of the 3 rabbits failed to give any response. Nevertheless, it was possible to determine that the antiserum obtained from this single animal did not neutralize activity in the EPF bioassay, in contrast to the antisera obtained from the animals that were immunized with the N-terminal and the internal peptides (Table 1).

Table 1.

 Production and characterization of anti-Cpn10 antibodies

graphic file with name i1355-8145-005-01-0014-t01.jpg

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To investigate this phenomenon further, an alternative immunization schedule that produces the antilymphocyte sera used in the EPF bioassay (Morton et al 1973) was applied, and the response of the animals to ovalbumin alone or together with conjugates of the N-terminal and internal peptides and a longer C-terminal peptide (residues 77–101) was studied (Fig 1). A response to booster doses of N-terminal and internal peptide could be elicited under these conditions (Fig 1B,C), but hyperimmunization was not induced. In addition, the overall trend of antibody production was downward, and very limited quantities of antiserum of only moderate titer could be harvested (average titer against peptide, 0.33 × 104; average titer against whole molecule, 103). This failure of hyperimmunization could be seen with the antipeptide response and with the response to the conjugation partner ovalbumin. In contrast, response to the C-terminal peptide (Fig 1D) and to ovalbumin alone (Fig 1A) followed a more usual pattern. As observed previously, this unusual pattern of decline in antibody response despite booster doses of antigen correlated with the capacity to neutralize activity in the EPF bioassay (Table 1). This bioassay is based on the capacity of EPF to alter the response of T cells to antilymphocyte serum in vitro. EPF can affect other aspects of T-cell function, such as the delayed-type hypersensitivity reaction (Noonan et al 1979), but it remains to be determined what aspect of the complex interplay of T- and B-cell function involved in antibody production is affected by exposure to these particular epitopes of Cpn10 during the immunization process. That an effect could be observed on bystander antibodies as well as on those elicited against the particular epitopes suggests the involvement of some fundamental aspect of the immune response, such as maintenance of B-cell memory.

Fig 1.

Fig 1.

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 Antibody response of rabbits to ovalbumin conjugates of chaperonin 10 (Cpn10)–derived synthetic peptides. Ovalbumin alone (A) or conjugates of an N-terminal peptide 1–11cys (B), an internal peptide cys33–44 (C), and a C-terminal peptide cys77–101 (D) were administered at the times indicated by the arrows. Peptides corresponding with the indicated residues in human Cpn10, with an additional cys at the position indicated, were synthesized and conjugated to ovalbumin as in Table 1. Rabbits (n ≥ 3 for each peptide) were immunized with peptide conjugate in Freund's adjuvant (complete for the first injection, incomplete thereafter; CSL Ltd, Melbourne, Australia) that was administered by subcutaneous injection at multiple sites. Each total dose consisted of approximately 50 μg of synthetic peptide. Serum was obtained 7–14 days post-injection commencing with the third injection and tested using enzyme-linked immunsorbent assay in doubling dilutions with a starting dilution of 1 in 103. Plates were coated with peptide conjugated to bovine serum albumin (5 μg/mL), and bound antibody was detected with biotinylated donkey antirabbit immunoglobulin [F(ab′)2 fragment] followed by biotin/streptavidin/peroxidase complex (Amersham Life Science, Amersham, UK) according to the manufacturer's instructions. Antibody titer was recorded as the highest dilution of serum, giving an absorbance reading ≥0.1

When considered with our previous passive immunization studies using monoclonal EPF-neutralizing antibodies, these investigations confirm that circulating Cpn10, as its alter ego EPF, can have profound biologic effects. Nevertheless, considered from the practical starting point of this study (ie, production of an array of useful antibodies), further investigation was clearly necessary. A variety of longer peptides were thus synthesized and administered as ovalbumin conjugates using the modified immunization schedule. The properties of the elicited antibodies are summarized in Table 1. Higher titer antisera that were better able to recognize the whole molecule were obtained (average titer against both peptide and whole molecule, 0.6–2.5 × 105), but production of EPF-neutralizing antibodies still declined with time (data not shown). The rate of decline was not so dramatic as that seen with the shorter peptides, however, and reasonable quantities of antiserum could be harvested. All peptides spanning residues 1–76 of the molecule produced EPF-neutralizing antibodies, suggesting that this region of the molecule is involved in binding to lymphocytes. In contrast, antibodies against residues 77–101 were nonneutralizing, indicating that the C-terminal quarter of the molecule is directed away from the binding site.

Production of antibodies against the full-length recombinant Cpn10 molecule was also undertaken. Several forms of immunogen were tried, including the unmodified molecule, a fusion protein with glutathione-S-transferase added at the N-terminus (produced in pGEX-2T vector; Pharmacia Biotechnology, Uppsala, Sweden), and a molecule with a 6-histidine tag added to the C-terminus (produced in pET21d vector; Novagen, Madison, WI), which was immobilized on a metal, charged gel. None was successful in raising antibodies (data not shown). In contrast, the whole molecule conjugated to ovalbumin via an additional C-terminal cysteine produced high-titer antibodies in abundance (titer, 106; average yield of affinity-purified antibody, 1 mg/mL serum; average yield of antipeptide antibodies, 0.07–0.25 mg/mL serum). As was seen with α77–101 (Fig 1D), production of the nonneutralizing α1–101 reached a plateau after approximately 2 months (data not shown). After affinity purification, the utility of these antibodies as biologic probes was assessed, and these results are summarized in Table 1 and illustrated in Figures 2 and 3.

Fig 2.

Fig 2.

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 Detection of chaperonin 10 (Cpn10) in complex protein mixtures by immunoprecipitation and immunoblotting. Representative examples of at least 2 separate experiments are shown. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of lysate from ∼107 surface iodinated MOLT4 human T-leukemia cells precipitated with 1.5 μg of the anti-Cpn10 antibodies α1–101 (lane 1), α1–28 (lane 2), α77–101 (lane 3), the control antibody antiovalbumin (lane 4), and α1–101 after pretreatment of lysate with antiovalbumin (lane 5). In addition, 125I-Cpn10 (∼20 pg; lane 6) was analyzed in parallel. MOLT 4 cells were grown in RPMI (GibcoBRL, Rockville, MD) and 10% fetal bovine serum (FBS), harvested in the logarithmic growth phase, and washed twice with RPMI. Four samples of 3 × 107 cells were each surface iodinated, lysed, and immunoprecipitated as follows: Cells were resuspended in 0.1 mL of Hanks buffered salt solution (HBSS) and 0.1 mL of 0.1 M sodium phosphate buffer (pH, 7.4). Five Iodobeads (Pierce; rinsed thoroughly with HBSS) and 1.2 mCi carrierfree Na125I (Amersham Life Science) were then added, and the cells were shaken gently at r/t for 15 minutes. Cells were next transferred to a fresh tube, and 500 nmol mandelic acid was added as an iodine scavenger. After standing on ice for approximately 5 minutes, cells were washed twice with ice-cold RPMI and 0.01% sodium azide and then lysed (2 hours at 4°C) with 1 mL of ice-cold lysis buffer (0.01 M sodium phosphate buffer [pH, 7.4], 1% Triton X-100, 5 mM MgCl2, 0.01% sodium azide containing 10 μg/mL each of phenylmethylsulfonylflouride (PMSF), pepstatin, and leupeptin). After removal of cellular debris by centrifugation, lysates were pooled, precleared with 400 μL of ProteinA-Sepharose (Pharmacia Biotechnology, Piscataway, NY), and divided into 12 equal portions. Each portion was precipitated (overnight at 4°C) with 1.5 μg of either the antibodies described previously or (data not shown) 1 of the anti-Cpn10 antibodies (α1–11, α33–44, α29–56, or α57–76; see Table 1), followed by 20 μL of ProteinA-Sepharose. In a further experiment, lysate from 107 iodinated cells was precleared with 40μL of ProteinA-Sepharose and 5 μg of antiovalbumin, then precipitated with 1.5 μg of α1–101. Precipitated material was analyzed using SDS-PAGE under reducing conditions with 10%–20%, precast Tris-Tricine gels (Novex, San Diego, CA, USA). In addition, 125I-Cpn10 (∼20 pg, iodinated using the Iodogen technique) was analyzed in parallel. Protein was visualized by staining with Coomassie Blue, and radiolabeled material was visualized by autoradiography (BIOMAXTM MS; Eastman Kodak, Rochester, NY, USA; 8 day exposure to −80°C). (B) Purified recombinant Cpn10 in amounts of 0.5 ng (lanes 1, 4, and 7), 5 ng (lanes 2, 5, and 8), and 50 ng (lanes 3, 6, and 9) was diluted in normal human serum (dilution, 1:20) and probed with α77–101 (lanes 1–3), α1–28 (lanes 4–6), and α1–101 (lanes 7–9). Samples (10 μL) were separated by SDS-PAGE as in part A and electroblotted onto Immobilon P membranes (Millipore Corporation, Bedford, MA, USA) using a NaHCO3/Na2CO3 buffer system (Dunn et al 1986; 60 V for 1 hour at 4°C). Membranes were probed with anti-Cpn10 antibodies (1 μg/mL, 1 hour, r/t) as described previously and with other anti-Cpn10 antibodies as in part A (data not shown), followed by peroxidase-linked donkey antirabbit immunoglobulin (1/2000; Amersham Life Science, Amersham, UK) and ECL™ detection reagents (Amersham) according to the manufacturer's instructions. In a control experiment, primary antibody was omitted (data not shown). BIOMAX ML film (Eastman Kodak) was exposed to membranes for 30 seconds

Fig 3.

Fig 3.

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 Immunolocalization of early pregnancy factor (EPF)/chaperonin 10 (Cpn10) in normal and malignant human colonic tissue. Immunostaining of colorectal carcinoma (A–C) with arrows indicating intense apical membrane reactivity (B) and staining of material within a malignant gland space (C) are shown. Also shown is a junctional specimen (D) of colorectal carcinoma (Ca) and normal colon (N). Original magnifications: (A) 300×, (B–D) 120×. Paraffin sections (3–4 μm) were affixed to Superfrost Plus® adhesive slides (Menzel-Gläser, Braunschweig, Germany) and air dried overnight at 37°C. Sections were dewaxed in xylol and rehydrated through descending graded alcohols to Tris-buffered saline (TBS; 0.05 M Tris, 0.15 M NaCl) at a pH of 7.2 to 7.4. Sections were transferred to 0.01 M citric acid buffer at a pH of 6 and boiled twice for 5 minutes each time, allowed to cool, and then transferred to TBS (Shi et al 1991). Endogenous peroxidase activity was inhibited by incubating sections in 1.0% H2O2 and 0.1% NaN3 in TBS for 10 minutes. Nonspecific antibody binding was inhibited by incubating the sections in 4% skim milk powder in TBS for 15 minutes. The sections were then placed in a humidified chamber and incubated with 10% normal (nonimmune) goat serum (Zymed Corp., San Francisco, CA, USA) for 20 minutes. Excess normal serum was decanted from the sections, and the primary antibody (αAc1–28, 1 μg/mL) was applied overnight at room temperature. After this and subsequent incubations, the sections were thoroughly washed in several changes of TBS. Sections were then incubated with prediluted biotinylated goat–antirabbit immunoglobulins (Zymed) for 30 minutes, then with streptavidin–horseradish peroxidase conjugate (Zymed) for 15 minutes and washed in 3 changes of TBS for 5 minutes each time. Color was developed in 3,3′-diaminobenzidine (Sigma) with H2O2 as a substrate for 5 minutes, and then sections were washed in running tap water, lightly counterstained in Mayer's hematoxylin, dehydrated through ascending graded alcohols, cleared in xylene, and mounted using DePeX (BDH Gurr, Poole, UK)

Use of anti-Cpn10 antibodies in enzyme-linked immunosorbent assay

A sandwich enzyme-linked immunosorbent assay (ELISA) was used to assess the capacity of various combinations of antibodies to detect Cpn10 (Table 1). For all the combinations scored, capture and detection antibody species were interchangeable except for α29–56 and α57–76. These antibodies were effective only as the detection species, indicating that binding capacity is compromised by immobilization of the antibodies on plastic. The most sensitive combinations were antipeptide antibodies α77–101 with either α29–56 or α57–76, αAc1–28 with α29–56, and anti–whole molecule α1–101 with self. The ELISA using any of these combinations had a lower detection limit of approximately 500 pM, which is not sufficiently sensitive to detect EPF in serum. Some improvement in sensitivity might be achieved with greater signal amplification, but the real limitation is the affinity of the primary antibodies. An apparent Ka of approximately 107 M−1 indicates that these antibodies are of moderate to high affinity, but values that are several orders of magnitude higher can be achieved (Allauzen et al 1995). Presumably, these values will be required to push the detection limits of an immunoassay into the low pM range, as has been achieved for a variety of cytokines and growth factors. Peptides spanning the entire length of the Cpn10 molecule were used in the experiments described earlier, but none was successful in generating antibodies with this order of affinity. When considered with our previous experience regarding monoclonal antibody production (Athanasas et al 1989; Quinn et al 1990), this suggests that preparation of anti-Cpn10 antibodies with very high affinity will be extremely difficult and require novel strategies. Nevertheless, the basic sandwich ELISA is a valuable research tool and is extremely useful, for example, in assessing labeling procedures (eg, iodination) and in determining the serum concentrations of therapeutic doses of protein.

Use of anti-Cpn10 antibodies for protein localization

The versatility of the antibodies in the techniques that are useful in localizing proteins was next examined. In the first set of experiments, immunoprecipitation and immunoblotting techniques were used to detect EPF/Cpn10 in complex protein mixtures (Fig 2). Proteins on the surface of intact MOLT4 human T-leukemia cells were iodinated and the cells lysed, and the capacity of the anti-Cpn10 antibodies to precipitate labeled material was then examined (Fig 2A). Three antibodies, α1–101, α1–28, and α77–101, were able to detect a labeled band of the appropriate size, with α1–101 being considerably more efficient than the other antibodies. (With longer exposure times, some other antibodies, as indicated in Table 1, also gave positive results.) Comparison with the precipitation pattern of the control antibody antiovalbumin revealed that the molecular recognition pattern of these anti-Cpn10 antibodies is precise, with only a single species being specifically precipitated. This can be seen clearly when nonspecific background has been removed by the inclusion of irrelevant antibody in the preclearing step. The specifically precipitated species migrates in an identical position to pure Cpn10, providing further evidence for the specificity of the antibodies. To examine the utility of the antibodies in immunoblotting, normal human serum was spiked with varying amounts of purified recombinant Cpn10, and proteins were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene diflouride (PDVF) membrane, which was then probed with various antibodies (Fig 2B). The most sensitive antibody was α77–101, which readily detected 5 ng antigen (0.5 pmol, ie, the amount loaded on the gel). Clear evidence of Cpn10 dimer in lane 3 of Figure 2 (as well as higher order oligomers on heavily exposed gels; data not shown) and its absence in lanes 6 and 9, which contained the same protein mixture but developed with αAc1–28 and α1–101, respectively, suggests that α77–101 preferentially recognizes oligomeric forms of the molecule. Cpn10 functions as a noncovalent heptamer, and even under the highly denaturing conditions of SDS-PAGE, dimeric and, to a lesser extent, higher order oligomeric forms of the molecule persist and can be revealed with very sensitive detection methods (see lane 6 in Fig 2A for evidence of Cpn10 dimer). In contrast, the other useful antibodies, αAc1–28 and α1–101, appeared to more accurately reflect monomeric:oligomeric ratios in the samples.

Several antibodies were examined in immunohistochemical techniques, with αAc1–28 exhibiting outstanding properties (Table 1). This antibody was then used to probe the distribution of EPF/Cpn10 in normal and malignant human colonic tissue (Fig 3). Immunoreactivity with αAc1–28 was most pronounced in the carcinoma cells, in which punctate and diffuse cytoplasmic staining was observed (Fig 3A). The vesicular structures almost certainly correspond to mitochondria, being relatively evenly distributed throughout the cytoplasm rather than having a particular spatial distribution suggestive of secretory vesicles, Golgi apparatus, or endoplasmic reticulum. Infrequently, evidence of intense apical membrane staining (Fig 3B) and staining within secreted material was seen in malignant gland spaces (Fig 3C), suggesting either active secretion or release of EPF/Cpn10. The punctate cytoplasmic staining pattern was also present in normal colon epithelial and other cells, such as smooth muscle cells and leukocytes; however, diffuse cytoplasmic staining was almost invariably absent in nontumor cells. This can be seen clearly in Figure 3D, in which dense and diffusely stained areas of malignancy to the left of the field contrast strongly with the punctate pattern of staining in the background, normal tissue. The overall staining pattern seen in these studies is entirely in accord with the known biology of EPF and Cpn10. In normal tissue, immunoreactive material is localized to the mitochondrion, which is in accord with the constitutive activity of Cpn10. A prominent feature of malignant tissue is extramitochondrial staining, and this is evident throughout the cytoplasm as well as in secretory membranes and secretory glandular spaces. The presence of immunoreactive material in these locations is consistent with the extracellular appearance of EPF during neoplastic growth and provides the first direct demonstration of the coincidence of protein and EPF activity in the extracellular compartment.

These antibodies now form a valuable resource. In addition to assisting with further investigation of the biology of EPF, they can be used to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10.

Acknowledgments

Funding for these studies was provided by CSL Ltd, Melbourne, Australia, and the Australian Research Council.

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