Preparation and characterization of polyclonal antibodies against human chaperonin 10

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.

Fig 1.

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

Fig 2.

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 (BIOMAX^TM 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.

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)