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Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay
Journal of Immunological Methods 274 (2003) 271 – 279 www.elsevier.com/locate/jim
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Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay Rose Njemini a, Christian Demanet b, Tony Mets a,* b
a Geriatric Unit, Academic Hospital, Free University of Brussels (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium HLA and Molecular Hematology Laboratory, Academic Hospital, Free University of Brussels (VUB), Brussels, Belgium
Received 10 November 2002; accepted 15 December 2002
Abstract Heat shock proteins (Hsp) have been associated to several clinical relevant conditions. Currently used methods to determine Hsp 70 possess certain drawbacks. Therefore, we developed a cell lysate immunometric assay (CLIA) for the quantification of intracellular Hsp 70. This CLIA uses a combination of two distinct monoclonal antibodies that recognize different epitopes on the Hsp 70 molecule. A recombinant human Hsp 70 was used as the standard material. The detection range of the CLIA was 4 – 4000 ng/ml. The intra- and interassay coefficients of variation were, on average, 5% and 12%, respectively. The recovery varied between 81% and 116%. The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution (between 97% and 120%). The reliability of the CLIA was assessed by comparison with the values determined by flow cytometric procedure; these two sets of values showed a highly significant correlation (r = 0.896, p < 0.0001), indicating that the two methods are comparable. We conclude that this assay represents a low-cost alternative of the flow cytometric technique. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Heat shock protein 70; Cell lysates; ELISA; Flow cytometry
1. Type of research The heat shock protein (Hsp) family is composed of highly conserved proteins present in all organisms. These proteins are typically regarded as intracellular proteins although studies have shown that they can be secreted in to the peripheral circulation (Rea et al.,
Abbreviation: Hsp, heat shock protein(s). * Corresponding author. Tel.: +32-2-477-63-66; fax: +32-2477-63-64. E-mail address: [email protected] (T. Mets).
2001; Pockley et al., 1998; Njemini et al., submitted for publication). Under normal physiological conditions, Hsp are expressed at low levels (Craig and Gross, 1991) and play diverse roles as molecular chaperones (Soti and Csermely, 2000; Gething and Sambrook, 1992; Baler et al., 1992; Beckmann et al., 1990), preventing protein denaturation (Feder and Hofmann, 1999) or inappropriate polypeptide aggregation (Young, 1990). A variety of stressful stimuli, including physiological and environmental stressors, induce substantial increase in intracellular Hsp synthesis (Njemini et al., 2002; Foster and Brown, 1997; Mangurten et al., 1997; Morimoto et al., 1994; Ferm et al., 1992; Welch,
0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-1759(03)00004-8
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1992). Elevated levels of Hsp have also been observed during clinically relevant situations (Morimoto, 1998; Xu and Wick, 1996; Heufelder et al., 1992; Berberian et al., 1990; Kiessling et al., 1991). Several studies have shown the expression of Hsp, especially Hsp 70, after ischemia/reperfusion in the liver, brain, heart and kidney (Gingalewski et al., 1996; Emami et al., 1991; Knowlton et al., 1991; Simon et al., 1991; Nowak et al., 1990; Schiaffonati et al., 1990). Investigations of antigens involved in immune response against bacteria and parasites have revealed a variety of stress proteins among the known targets (Kaufmann, 1990). In addition, the Hsp family has been reported to elicit priming of antigen-specific cytotoxic T lymphocytes when bound to antigenic viral or tumor peptides (Song-Dong et al., 2001). In view of its role in inflammatory and infectious diseases, Hsp has been studied in a variety of medically relevant models or conditions such as hyperthermia (Li et al., 1995), hypertension (Lovis et al., 1994), toxic exposure to chemical agents (Witzmann et al., 1996), hypoxia (Chen et al., 1999), ischemia (Gray et al., 1999; Perdrizet et al., 1999), inflammation (Jacquier-Sarlin et al., 1994), autoimmunity (Feige and van Eden, 1996), cancer (Jaattela, 1999) and bacterial (Delogu et al., 1997; Nishimura et al., 1997) and viral (Kilgore et al., 1998) infections. The most commonly used methods for assessing Hsp 70 are the flow cytometric and Western blotting techniques (Njemini et al., 2002; Udelsman et al., 1993). Although flow cytometry provides the possibility of further characterising cells by immunophenotyping, this method is associated with substantial problems. The fluorochromes used for detection are not readily available for most Hsp. In addition, the flow cytometer is an expensive apparatus and is unavailable in many laboratories. Furthermore, flow cytometers process samples sequentially which is a slow process for a large number of samples. Eventhough Western blotting has been widely used in the detection of Hsp 70, this technique is subjective, as it depends on visual judgement of the banding pattern or becomes expensive when densitometry is applied. To address these problems, we have developed an Hsp 70 cell lysate immunometric assay (CLIA) that allows for reproducible, accurate and precise determination of Hsp 70 in cell lysates. This assay can handle multiple samples, is cost effective and can easily be adapted in any laboratory with relative ease.
2. Time required: 21 h 1. Coating plates: 14 h. 2. Washing plates and addition of blocking buffer: 5 min per plate. 3. Blocking plates: 1 h. 4. Washing plates and preparation of standards or samples: 45 min per plate. 5. Duration of standards or samples on plates: 2 h. 6. Washing plates and addition of rabbit polyclonal antibody: 5 min per plate. 7. Duration of rabbit polyclonal antibody on plates: 1 h. 8. Washing plates and addition of anti-rabbit immunoglobulin (Ig) peroxidase conjugate: 5 min per plate. 9. Duration of anti-rabbit immunoglobulin peroxidase conjugate on plate: 1 h. 10. Washing plates and addition of substrate: 5 min per plate. 11. Duration of substrate on plate: 30 min. 12. Putting stop solution on plates: 1 min per plate. 13. Reading plates on ELISA plate reader: 1 min per plate.
3. Materials 3.1. Special equipment
ELISA plates (Nunc ELISA MaxiSorp microwell plates, Fisher Scientific, Santa Clara, CA, USA) were read using a microplate reader (CERES 900C, BIO-TEC Instruments, Belgium).
3.2. Blood, reagents and antibodies 3.2.1. Blood Ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood from nine healthy volunteers was used for flow cytometric studies (Beckman-Coulter Epics XL, USA) and CLIA.
3.2.2. Reagents Culture medium (RPMI 1640), trypsin – EDTA (0.05% trypsin, 0.02% EDTA), L-glutamine, N-2hydroxyethyl-piperazine-NV-2-ethanesulphonic acid (HEPES) buffer, penicillin and streptomycin
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were purchased from Life Technologies (Gibco, Paisley, Scotland). Fetal calf serum (FCS) was from Biochrom (International Medical, Belgium). Bovine serum albumin (BSA) was from Roche (Boehringer, Mannheim, Germany). Ficoll was from Nycomed (Oslo, Norway). RIPA buffer (pH 7.5) consisted of NaCl 150 mM, 1% nonidet P40, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, phosphate inhibitors (10 mM Na3VO4, 50 mM NaF, 1 mM Na2P2O710H2O, 10 mM h-glycerophosphate, 10 mM p-nitrophenylphosphate) and a cocktail of protease inhibitors (1:1000). Fix and Perm cell permeabilization kit (reagents A and B) was from IMTEC (Diagnostics, Antwerpen, Belgium). Carbonate buffer consisted of 15 mM Na2CO3, 35 mM NaHCO3 and 3 mM NaN3. Phosphate-buffered saline (PBS) was made up of 135 mM NaCl, 1 mM KH2PO4, 40 mM Na2HPO42H2O and 3 mM KCl. Blocking solution consisted of 1% bovine serum albumin and 0.05% polyoxyethylene-sorbitan monolaurate (Tween 20, Sigma, USA) in PBS. Wash solution consisted of 0.05% polyoxyethylene-sorbitan monolaurate in PBS.
3.2.3. Antibodies Anti-CD14 phycoerythrin (PE) conjugated was from Becton Dickinson (Erembodegem, Belgium). The monoclonal antibody directed against the inducible form of Hsp 70, (clone C92F3A-5, SPA-810), recombinant human Hsp 70 (SPP-755) and the rabbit polyclonal anti-Hsp 70 antibody (SPA-812) were from StressGen Biotechnologies (Victoria, Canada). Anti-rabbit immunoglobulin peroxidase conjugate and o-phenylenediamine dihydrochloride (OPD) substrate were purchased from Sigma. Pure mouse IgG (isotype-matched control) was from Becton Dickinson. The F(abV)2 fragment of goat anti-mouse immunoglobulin (Ig) G (conjugated to fluorescein isothiocyanate (FITC)) used as secondary antibody was purchased from IMTEC.
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4. Detailed procedures 4.1. Cell preparation Recover peripheral blood mononuclear cells by density gradient centrifugation of EDTA blood on ficoll as described previously (Njemini et al., 2002). 1. Dilute EDTA blood two times with PBS and carefully layer over ficoll (1/3 ficoll to 2/3 blood) in a 10-ml test tube. 2. Centrifuge for 20 min at 500 g; the erythrocytes and granulocytes will sediment in the bottom while the lymphocytes and monocytes will remain in the interface between the ficoll and plasma layers. 3. Remove the cells and centrifuge again for 10 min at 100 g. 4. Wash the cells twice in PBS containing 1% BSA (PBS/BSA) at 900 g for 3 min. 5. Resuspend in RPMI 1640 supplemented with 10% FCS, 2 mM HEPES buffer, 2 mM L-glutamine, penicillin and streptomycin. 6. Stain cells immediately for intracellular Hsp 70 determination by flow cytometry or use cells to prepare cell lysates for Hsp 70 determination by CLIA. If heat shock is necessary. 7. Plate aliquots of cell suspension in 35-mm diameter Petri dishes (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) and incubate overnight in a 5% CO2 incubator at 37 jC. 8. The following morning, heat shock the cells for 1 h in a water bath, at the appropriate temperatures and allow them to recover at 37 jC in a 5% CO2 incubator. 9. After 4 h, harvest the cells and resuspend in PBS/ BSA. The suspension can be used directly for Hsp 70 determination by flow cytometry or to prepare cell lysates for Hsp 70 determination by CLIA. 4.2. Analysis of Hsp 70 by flow cytometry The procedure used for determining Hsp 70 was similar to that described previously (Njemini et al., 2002). 1. Incubate 100 Al of the cell suspension (about 1 106 cells) with 10 Al anti-CD14 PE-conjugated antibody for 15 min at 4 jC.
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2. Wash cells with PBS/BSA. 3. Fix them at room temperature in 75 Al of a solution containing formaldehyde (reagent A) for 15 min, according to the manufacturer’s prescription. Wash cells with PBS/BSA. 4. Permeabilize the cells with 75 Al of reagent B and at the same time add 10 Al of the primary Hspspecific antibody diluted 1:20 in PBS/BSA. 5. Incubate the cells for 15 min at room temperature. 6. Wash cells with PBS/BSA. 7. Resuspend the labelled cells in 75 Al of reagent B and 10 Al of the secondary antibody diluted 1:10 in PBS/BSA, and further incubate for 15 min in the dark at room temperature. 8. Wash cells with PBS/BSA. 9. Add 500 Al of Facsflow solution (Becton Dickinson, immunocytometry system, San Jose, CA). The samples can be analysed immediately or within a few hours (stored at 4 jC) using a flow cytometer. 4.3. Preparation of cell lysates 1. Centrifuge cells to pellet, aspirate media and wash cells four times with PBS/BSA at 900 g for 3 min. 2. Aspirate the supernatant from the final wash. 3. Resuspend the cell pellet in an appropriate volume of RIPA (100 Al RIPA to 200,000 cells). 4. Pipet up and down to break up the cell pellet until the cell suspension is homogenous and no clumps are visible. 5. Incubate for 30 min on ice with occasional mixing. 6. Centrifuge the extracts at 1600 g for 10 min in a 4 jC refrigerated centrifuge.
3. Block nonspecific binding sites by incubation with 150 Al of PBS/T containing 0.1% BSA (PBS/ T/BSA) for 1 h at 37 jC on a shaker. 4. Wash plates six times with PBS/T. 5. Add 100 Al of the reference preparation (diluted across columns 1 and 2 of plates, fivefold serial dilution) or samples (diluted 10 times) and incubate the plates for 2 h at 37 jC on a shaker. 6. Wash plates six times with PBS/T. 7. Add 100 Al of rabbit polyclonal anti-Hsp 70 (1:400) diluted in PBS/T/BSA for 1 h on shaker at 37 jC. 8. Wash plates six times with PBS/T. 9. Incubate plates with 100 Al of an anti-rabbit immunoglobulin peroxidase conjugate in PBS/T/ BSA (1:10,000) for 1 h on shaker at 37 jC. 10. Wash plates six times with PBS/T. 11. Add 200 Al of OPD substrate. 12. Stop reaction after 30 min with 50 Al of 1N H2SO4. 13. Determine the absorbance at 492 nm with background subtraction at 620 nm using a microplate
The supernatant collected is the cell lysate, which is now ready for analysis using the Hsp 70 CLIA. The resulting pellets can be discarded. 4.4. Cell lysate immunometric assay for detection of intracellular Hsp 70 1. In 96-well microwell plates, aliquot 100 Al per well of purified human recombinant Hsp 70 antibody (5 Ag/ml) diluted in 0.1 M carbonate buffer (pH 9.6). Cover plates with plate cover and place them at 4 jC overnight. 2. Wash plates six times with PBS containing 0.05% Tween 20 (PBS/T).
Fig. 1. Titration curve of Hsp 70 detection antibody against dilutions of Hsp 70 antigen. Each curve represents the titration of a different concentration (dilution factor: 200 – 25,600) of Hsp 70 detection antibody using the same dilution range of the antigen. The conjugate and capture conditions were kept constant throughout the experiment.
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5. Results
Fig. 2. Titration of capture Hsp 70 antibody against optimized antigen and detection antibody. A concentration of 5 Ag/ml was considered optimal for the capture system.
reader. The quantity of Hsp 70 in cells is estimated from the calibration curve. 4.5. Statistical analysis The Hsp 70 levels obtained using the two methods were compared by the Spearman Rank Test.
We have developed a CLIA for the detection of intracellular Hsp 70 concentration using the onestep-at-a-time procedure. All factors were kept constant except for one and the response was taken at different levels of the factor. Keeping the appropriate response level of the factor constant, a new factor was chosen and the procedure was repeated in the same manner. A titration curve of Hsp 70 detection antibody against dilutions of Hsp 70 antigen was generated to determine the optimal level of detection antibody (Fig. 1) and a titre of 1:400 was adopted. The titration of the capture antibody (Fig. 2) and the enzyme conjugate (Fig. 3) were optimized by preliminary checkerboard titration. An optimum concentration of 5 Ag/ml was considered for the capture antibody and a titre of 1:10,000 was adopted for the enzyme conjugate. Thereafter, the effect of other variables, such as temperature and incubation time, on the assay performance was systematically studied. To evaluate the assay, human recombinant Hsp 70 was used to generate a standard curve (Fig. 4). A fivefold dilution series was performed to set up the standard values. Each standard value was measured 12 times and the first two measurements (0 – 4000 ng/
Fig. 3. Assessment of optimized capture, antigen and detection system with different dilutions of conjugate. The initial concentration of the conjugate was 0.4 mg/ml and a conjugate dilution of 1:10,000 was adopted.
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measured value with that expected. The recovered value of Hsp 70 in spiked samples was between 81% and 116% of the expected value. In addition, dilution of cell lysates yielded parallel results with dilution between 97% and 120% of the expected value (data not shown). In order to relate the CLIA to the flow cytometric assay, PBMC were prepared from samples of nine volunteers. These cells were either treated immedi-
Fig. 4. Representative standard dose – response curve and precision profile for the Hsp 70 immunoassay. A fivefold dilution series was performed to set up the standard values (0 – 4000 ng/ml). Each standard value was measured 12 times and the first two measurements acted as the standard dose – response curve. The concentrations of the remaining 10 replicates of each standard value were determined from the standard curve and were used to generate the coefficient of variation profile.
ml) acted as the standard dose –response curve. The concentration of the remaining 10 replicates of each standard value was determined from the standard curve and was used to generate the coefficient of variation profile (Fig. 4). The sensitivity of the assay was 4 ng/ml and was defined as the concentration that gave an absorbance of the zero standard plus two standard deviations. To test the precision of the assay, experiments were conducted using the optimum conditions. The precision of the present CLIA was determined by assaying three samples 20 times on one plate (intraassay), and two samples 20 times in four individual assays using different batches of reagents (interassay). The average intraassay variation was 5%, and the average interassay variation was 12% (data not shown). Possible interference in the determination of intracellular Hsp 70 concentrations by other compounds was investigated by spiking cell lysates with known concentrations of purified Hsp 70 and comparing the
Fig. 5. Relationship between Hsp 70 analysis of PBMC by CLIA and of PBMC: monocytes and lymphocytes (A), pure monocyte (B) and pure lymphocyte (C) subpopulations, measured as MFI by flow cytometry.
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ately for Hsp 70 determination or were exposed to 39 or 42 jC for 1 h before Hsp 70 measurement by flow cytometry and CLIA. Fig. 5 shows a comparative evaluation of the two techniques. There was a highly significant correlation between the CLIA and the flow cytometric assay when the results of the total PBMC from flow cytometry were considered (r = 0.896, p < 0.0001) as well as when only the monocyte (r = 0.885, p < 0.0001) or the lymphocyte (r = 0.823, p = 0.0001) results from flow cytometry were taken in to account.
6. Discussion Hsp are highly abundant proteins that play fundamental roles in promoting cellular survival and maintenance of normal cellular function (Mayer and Bukau, 1998). The interaction of Hsp with cellular components has been shown to have therapeutic potentials. For example, Hsp 70 has been demonstrated to take part in pathogen clearance (Forsdyke, 1985) and to interfere with inflammatory responses (Munoz et al., 1996). Accordingly, some investigators have attributed a protective value to Hsp, as the administration of Hsp 70 can modulate the induction of arthritis (Kingston et al., 1996). Furthermore, in the clinical situation, the presence of circulating Hsp seems to be beneficial in juvenile chronic arthritis (de Graeff-Meeder et al., 1995) and severe ischemia/reperfusion injury (Kume et al., 1996). The need for a simple assay for determining Hsp 70 in cells has emerged with the increasing interest in therapeutic manipulation of Hsp for clinical trials. To date, although several methods for assaying Hsp 70 have been used for investigatory purposes, these techniques have certain drawbacks as pointed out in the introduction. In order to overcome these problems, we developed an Hsp 70 CLIA for the detection and quantification of intracellular Hsp 70. Our approach offers advantages over the conventional methods. Firstly, the assay constitutes a more cost-effective method, which can easily be amenable in many laboratories. Secondly, the assay allows the handling of multiple samples since a single plate can be used to assay 80 samples in single or 40 samples in duplicate.
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6.1. Troubleshooting The substrate, OPD, is very sensitive to light and temperature and must be dissolved in the dark immediately prior to use. It is advisable to put the substrate diluent (Milli-Q water) at 37 jC for 15 min before dissolving the OPD tablets. Given that ELISA is highly sensitive to temperature, differences in results due to drastic changes in environmental temperature were eliminated by carrying out the experiments in an incubator at a constant temperature of 37 jC. We realized that for samples with very high levels of the protein, a twofold diluted sample demonstrated a higher level of Hsp 70 compared to the same undiluted sample. Carpenter (1997) reported that it may be caused by antigen excess, in which a majority of the antigen binding sites are occupied, preventing completion of the sandwich. With regard to this problem and based on our linearity results, we decided to use a tenfold dilution of the samples. If samples generate lower values than the lowest standard, the samples should be reassayed at a lower sample dilution. Similarly, if samples generate higher values than the highest standard, the samples should be reassayed at a higher sample dilution. 6.2. Alternative and support protocols For the present CLIA for Hsp 70 measurement, we obtained average inter- and intraassay coefficients of variation of 5% and 12%, respectively. Tijssen (1985) suggested that the between test coefficient of variation of results obtained for reference samples under ideal conditions should be < 10%. However, a between assay coefficient of variation of 10– 15% was considered more usual by McLaren et al. (1981). The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution, suggesting the absence of any cellular interference. The standard recovery in different samples revealed that it is a precise assay with a high reproducibility. In order to assess the reliability of the present CLIA for quantification of intracellular Hsp 70, the levels of Hsp 70 obtained with this system were compared with those determined by the flow cytometric method. These two sets of values showed a
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highly significant correlation, indicating that the two methods are comparable. In conclusion, the described CLIA technique can be considered as a reliable and cost-effective diagnostic test that is applicable in low-technology settings. It may be relevant in the understanding of disease conditions that are accompanied by Hsp 70 production.
7. Quick procedure 1. Coat plates with capture antibody (overnight at 4 jC). 2. Wash plates. 3. Block plates (37 jC, shaker, 1 h). 4. Wash plates. 5. Put standards or samples on plates (37 jC, shaker, 2 h). 6. Wash plates. 7. Put rabbit polyclonal antibody on plates (37 jC, shaker, 1 h). 8. Wash plates. 9. Add anti-rabbit immunoglobulin peroxidase conjugate to plates (37 jC, shaker, 1 h). 10. Wash plates. 11. Add substrate to plates (37 jC, shaker, 30 min). 12. Stop reaction with H2SO4. 13. Read plates.
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R. Njemini et al. / Journal of Immunological Methods 274 (2003) 271–279 Kume, M., Yamamoto, Y., Saad, S., Gomi, T., Kimoto, S., Shimabukuro, T., Yagi, T., Nakagami, M., Takada, Y., Morimoto, T., Yamaoka, Y., 1996. Ischemic preconditioning of the liver in rats: implications of heat shock protein induction to increase tolerance of ischemia – reperfusion injury. J. Lab. Clin. Med. 128, 251. Li, G.C., Mivechi, N.F., Weitzel, G., 1995. Heat shock proteins, thermotolerance, and their relevance to clinical hyperthermia. Int. J. Hypertherm. 11, 459. Lovis, C., Mach, F., Donati, Y.R., Bonventre, J.V., Polla, B.S., 1994. Heat shock proteins and the kidney. Ren. Fail. 16, 179. Mangurten, A.B., Brader, K.R., Loos, B.M., Lee, E., Quiroga, A.I., Bathori, J., Lurain, J.R., Laszlo, A., Phillips, B., 1997. Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix. Cell Stress Chaperones 2, 168. Mayer, M.P., Bukau, B., 1998. Hsp70 chaperone systems: diversity of cellular functions and mechanism of action. Biol. Chem. 379, 261. McLaren, M.L., Lillywhite, J.E., Au, A.C., 1981. Indirect enzyme linked immunosorbent assay (ELISA): practical aspects of standardization and quality control. Med. Lab. Sci. 38, 245. Morimoto, R.I., 1998. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 12, 3788. Morimoto, R.I., Tissieres, A., Georgopoulos, C., 1994. Progress and perspectives on the biology of heat shock proteins and molecular chaperones. In: Morimoto, R.I., Tieeieres, A., Georgopoulos, C. (Eds.), The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1. Munoz, M.G., Jeremias, J., Witkin, S.S., 1996. The 60 kDa heat shock protein in human semen: relationship with antibodies to spermatozoa and Chlamydia trachomatis. Hum. Reprod. 11, 2600. Nishimura, H., Emoto, M., Kimura, K., Yoshikai, Y., 1997. Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-alpha-induced cell death. Cell Stress Chaperones 2, 50. Njemini, R., Abeele, M.V., Demanet, C., Lambert, M., Vandebosch, S., Mets, T., 2002. Age-related decrease in the inducibility of heat-shock protein 70 in human peripheral blood mononuclear cells. J. Clin. Immunol. 22, 195. Njemini, R., Demanet, C., Mets, T. Investigation of heat shock protein 70 in the peripheral circulation of patients using an
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optimized enzyme-linked immunosorbent assay. Submitted for publication. Nowak Jr., T.S., Bond, U., Schlesinger, M.J., 1990. Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia. J. Neurochem. 54, 451. Perdrizet, G.A., Shapiro, D.S., Rewinski, M.J., 1999. Surgical stress and the heat shock response: in vivo models of stress conditioning. Ann. N.Y. Acad. Sci. 874, 320. Pockley, A.G., Shepherd, J., Corton, J.M., 1998. Detection of heat shock protein 70 (Hsp70) and anti-Hsp70 antibodies in the serum of normal individuals. Immunol. Invest. 27, 367. Rea, I.M., McNerlan, S., Pockley, A.G., 2001. Serum heat shock protein and anti-heat shock protein antibody levels in aging. Exp. Gerontol. 36, 341. Schiaffonati, L., Rappocciolo, E., Tacchini, L., Cairo, G., BernelliZazzera, A., 1990. Reprogramming of gene expression in postischemic rat liver: induction of proto-oncogenes and hsp 70 gene family. J. Cell. Physiol. 143, 79. Simon, R.P., Cho, H., Gwinn, R., Lowenstein, D.H., 1991. The temporal profile of 72-kDa heat-shock protein expression following global ischemia. J. Neurosci. 11, 881. Song-Dong, M., Tong, G., Gao, G.F., Po, T., 2001. HBV-specific peptide associated with heat-shock protein gp96. Lancet 357, 528. Soti, C., Csermely, P., 2000. Molecular chaperones and the aging process. Biogerontology 1, 225. Tijssen, P., 1985. Processing of data and reporting of results of enzyme immunoassays. In Practice and Theory of Immunoassays. Elsevier, Amsterdam, p. 385. Udelsman, R., Blake, M.J., Stagg, C.A., Li, D.G., Putney, D.J., Holbrook, N.J., 1993. Vascular heat shock protein expression in response to stress. Endocrine and autonomic regulation of this age-dependent response. J. Clin. Invest. 91, 465. Welch, W.J., 1992. Related mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72, 1063. Witzmann, F.A., Fultz, C.D., Lipscomb, J.C., 1996. Toxicant-induced alterations in two-dimensional electrophoretic patterns of hepatic and renal stress proteins. Electrophoresis 17, 198. Xu, Q., Wick, G., 1996. The role of heat shock proteins in protection and pathophysiology of the arterial wall. Mol. Med. Today 2, 372. Young, R.A., 1990. Stress proteins and immunology. Annu. Rev. Immunol. 8, 401.
Protocol
Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay Rose Njemini a, Christian Demanet b, Tony Mets a,* b
a Geriatric Unit, Academic Hospital, Free University of Brussels (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium HLA and Molecular Hematology Laboratory, Academic Hospital, Free University of Brussels (VUB), Brussels, Belgium
Received 10 November 2002; accepted 15 December 2002
Abstract Heat shock proteins (Hsp) have been associated to several clinical relevant conditions. Currently used methods to determine Hsp 70 possess certain drawbacks. Therefore, we developed a cell lysate immunometric assay (CLIA) for the quantification of intracellular Hsp 70. This CLIA uses a combination of two distinct monoclonal antibodies that recognize different epitopes on the Hsp 70 molecule. A recombinant human Hsp 70 was used as the standard material. The detection range of the CLIA was 4 – 4000 ng/ml. The intra- and interassay coefficients of variation were, on average, 5% and 12%, respectively. The recovery varied between 81% and 116%. The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution (between 97% and 120%). The reliability of the CLIA was assessed by comparison with the values determined by flow cytometric procedure; these two sets of values showed a highly significant correlation (r = 0.896, p < 0.0001), indicating that the two methods are comparable. We conclude that this assay represents a low-cost alternative of the flow cytometric technique. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Heat shock protein 70; Cell lysates; ELISA; Flow cytometry
1. Type of research The heat shock protein (Hsp) family is composed of highly conserved proteins present in all organisms. These proteins are typically regarded as intracellular proteins although studies have shown that they can be secreted in to the peripheral circulation (Rea et al.,
Abbreviation: Hsp, heat shock protein(s). * Corresponding author. Tel.: +32-2-477-63-66; fax: +32-2477-63-64. E-mail address: [email protected] (T. Mets).
2001; Pockley et al., 1998; Njemini et al., submitted for publication). Under normal physiological conditions, Hsp are expressed at low levels (Craig and Gross, 1991) and play diverse roles as molecular chaperones (Soti and Csermely, 2000; Gething and Sambrook, 1992; Baler et al., 1992; Beckmann et al., 1990), preventing protein denaturation (Feder and Hofmann, 1999) or inappropriate polypeptide aggregation (Young, 1990). A variety of stressful stimuli, including physiological and environmental stressors, induce substantial increase in intracellular Hsp synthesis (Njemini et al., 2002; Foster and Brown, 1997; Mangurten et al., 1997; Morimoto et al., 1994; Ferm et al., 1992; Welch,
0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-1759(03)00004-8
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1992). Elevated levels of Hsp have also been observed during clinically relevant situations (Morimoto, 1998; Xu and Wick, 1996; Heufelder et al., 1992; Berberian et al., 1990; Kiessling et al., 1991). Several studies have shown the expression of Hsp, especially Hsp 70, after ischemia/reperfusion in the liver, brain, heart and kidney (Gingalewski et al., 1996; Emami et al., 1991; Knowlton et al., 1991; Simon et al., 1991; Nowak et al., 1990; Schiaffonati et al., 1990). Investigations of antigens involved in immune response against bacteria and parasites have revealed a variety of stress proteins among the known targets (Kaufmann, 1990). In addition, the Hsp family has been reported to elicit priming of antigen-specific cytotoxic T lymphocytes when bound to antigenic viral or tumor peptides (Song-Dong et al., 2001). In view of its role in inflammatory and infectious diseases, Hsp has been studied in a variety of medically relevant models or conditions such as hyperthermia (Li et al., 1995), hypertension (Lovis et al., 1994), toxic exposure to chemical agents (Witzmann et al., 1996), hypoxia (Chen et al., 1999), ischemia (Gray et al., 1999; Perdrizet et al., 1999), inflammation (Jacquier-Sarlin et al., 1994), autoimmunity (Feige and van Eden, 1996), cancer (Jaattela, 1999) and bacterial (Delogu et al., 1997; Nishimura et al., 1997) and viral (Kilgore et al., 1998) infections. The most commonly used methods for assessing Hsp 70 are the flow cytometric and Western blotting techniques (Njemini et al., 2002; Udelsman et al., 1993). Although flow cytometry provides the possibility of further characterising cells by immunophenotyping, this method is associated with substantial problems. The fluorochromes used for detection are not readily available for most Hsp. In addition, the flow cytometer is an expensive apparatus and is unavailable in many laboratories. Furthermore, flow cytometers process samples sequentially which is a slow process for a large number of samples. Eventhough Western blotting has been widely used in the detection of Hsp 70, this technique is subjective, as it depends on visual judgement of the banding pattern or becomes expensive when densitometry is applied. To address these problems, we have developed an Hsp 70 cell lysate immunometric assay (CLIA) that allows for reproducible, accurate and precise determination of Hsp 70 in cell lysates. This assay can handle multiple samples, is cost effective and can easily be adapted in any laboratory with relative ease.
2. Time required: 21 h 1. Coating plates: 14 h. 2. Washing plates and addition of blocking buffer: 5 min per plate. 3. Blocking plates: 1 h. 4. Washing plates and preparation of standards or samples: 45 min per plate. 5. Duration of standards or samples on plates: 2 h. 6. Washing plates and addition of rabbit polyclonal antibody: 5 min per plate. 7. Duration of rabbit polyclonal antibody on plates: 1 h. 8. Washing plates and addition of anti-rabbit immunoglobulin (Ig) peroxidase conjugate: 5 min per plate. 9. Duration of anti-rabbit immunoglobulin peroxidase conjugate on plate: 1 h. 10. Washing plates and addition of substrate: 5 min per plate. 11. Duration of substrate on plate: 30 min. 12. Putting stop solution on plates: 1 min per plate. 13. Reading plates on ELISA plate reader: 1 min per plate.
3. Materials 3.1. Special equipment
ELISA plates (Nunc ELISA MaxiSorp microwell plates, Fisher Scientific, Santa Clara, CA, USA) were read using a microplate reader (CERES 900C, BIO-TEC Instruments, Belgium).
3.2. Blood, reagents and antibodies 3.2.1. Blood Ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood from nine healthy volunteers was used for flow cytometric studies (Beckman-Coulter Epics XL, USA) and CLIA.
3.2.2. Reagents Culture medium (RPMI 1640), trypsin – EDTA (0.05% trypsin, 0.02% EDTA), L-glutamine, N-2hydroxyethyl-piperazine-NV-2-ethanesulphonic acid (HEPES) buffer, penicillin and streptomycin
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were purchased from Life Technologies (Gibco, Paisley, Scotland). Fetal calf serum (FCS) was from Biochrom (International Medical, Belgium). Bovine serum albumin (BSA) was from Roche (Boehringer, Mannheim, Germany). Ficoll was from Nycomed (Oslo, Norway). RIPA buffer (pH 7.5) consisted of NaCl 150 mM, 1% nonidet P40, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, phosphate inhibitors (10 mM Na3VO4, 50 mM NaF, 1 mM Na2P2O710H2O, 10 mM h-glycerophosphate, 10 mM p-nitrophenylphosphate) and a cocktail of protease inhibitors (1:1000). Fix and Perm cell permeabilization kit (reagents A and B) was from IMTEC (Diagnostics, Antwerpen, Belgium). Carbonate buffer consisted of 15 mM Na2CO3, 35 mM NaHCO3 and 3 mM NaN3. Phosphate-buffered saline (PBS) was made up of 135 mM NaCl, 1 mM KH2PO4, 40 mM Na2HPO42H2O and 3 mM KCl. Blocking solution consisted of 1% bovine serum albumin and 0.05% polyoxyethylene-sorbitan monolaurate (Tween 20, Sigma, USA) in PBS. Wash solution consisted of 0.05% polyoxyethylene-sorbitan monolaurate in PBS.
3.2.3. Antibodies Anti-CD14 phycoerythrin (PE) conjugated was from Becton Dickinson (Erembodegem, Belgium). The monoclonal antibody directed against the inducible form of Hsp 70, (clone C92F3A-5, SPA-810), recombinant human Hsp 70 (SPP-755) and the rabbit polyclonal anti-Hsp 70 antibody (SPA-812) were from StressGen Biotechnologies (Victoria, Canada). Anti-rabbit immunoglobulin peroxidase conjugate and o-phenylenediamine dihydrochloride (OPD) substrate were purchased from Sigma. Pure mouse IgG (isotype-matched control) was from Becton Dickinson. The F(abV)2 fragment of goat anti-mouse immunoglobulin (Ig) G (conjugated to fluorescein isothiocyanate (FITC)) used as secondary antibody was purchased from IMTEC.
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4. Detailed procedures 4.1. Cell preparation Recover peripheral blood mononuclear cells by density gradient centrifugation of EDTA blood on ficoll as described previously (Njemini et al., 2002). 1. Dilute EDTA blood two times with PBS and carefully layer over ficoll (1/3 ficoll to 2/3 blood) in a 10-ml test tube. 2. Centrifuge for 20 min at 500 g; the erythrocytes and granulocytes will sediment in the bottom while the lymphocytes and monocytes will remain in the interface between the ficoll and plasma layers. 3. Remove the cells and centrifuge again for 10 min at 100 g. 4. Wash the cells twice in PBS containing 1% BSA (PBS/BSA) at 900 g for 3 min. 5. Resuspend in RPMI 1640 supplemented with 10% FCS, 2 mM HEPES buffer, 2 mM L-glutamine, penicillin and streptomycin. 6. Stain cells immediately for intracellular Hsp 70 determination by flow cytometry or use cells to prepare cell lysates for Hsp 70 determination by CLIA. If heat shock is necessary. 7. Plate aliquots of cell suspension in 35-mm diameter Petri dishes (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) and incubate overnight in a 5% CO2 incubator at 37 jC. 8. The following morning, heat shock the cells for 1 h in a water bath, at the appropriate temperatures and allow them to recover at 37 jC in a 5% CO2 incubator. 9. After 4 h, harvest the cells and resuspend in PBS/ BSA. The suspension can be used directly for Hsp 70 determination by flow cytometry or to prepare cell lysates for Hsp 70 determination by CLIA. 4.2. Analysis of Hsp 70 by flow cytometry The procedure used for determining Hsp 70 was similar to that described previously (Njemini et al., 2002). 1. Incubate 100 Al of the cell suspension (about 1 106 cells) with 10 Al anti-CD14 PE-conjugated antibody for 15 min at 4 jC.
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2. Wash cells with PBS/BSA. 3. Fix them at room temperature in 75 Al of a solution containing formaldehyde (reagent A) for 15 min, according to the manufacturer’s prescription. Wash cells with PBS/BSA. 4. Permeabilize the cells with 75 Al of reagent B and at the same time add 10 Al of the primary Hspspecific antibody diluted 1:20 in PBS/BSA. 5. Incubate the cells for 15 min at room temperature. 6. Wash cells with PBS/BSA. 7. Resuspend the labelled cells in 75 Al of reagent B and 10 Al of the secondary antibody diluted 1:10 in PBS/BSA, and further incubate for 15 min in the dark at room temperature. 8. Wash cells with PBS/BSA. 9. Add 500 Al of Facsflow solution (Becton Dickinson, immunocytometry system, San Jose, CA). The samples can be analysed immediately or within a few hours (stored at 4 jC) using a flow cytometer. 4.3. Preparation of cell lysates 1. Centrifuge cells to pellet, aspirate media and wash cells four times with PBS/BSA at 900 g for 3 min. 2. Aspirate the supernatant from the final wash. 3. Resuspend the cell pellet in an appropriate volume of RIPA (100 Al RIPA to 200,000 cells). 4. Pipet up and down to break up the cell pellet until the cell suspension is homogenous and no clumps are visible. 5. Incubate for 30 min on ice with occasional mixing. 6. Centrifuge the extracts at 1600 g for 10 min in a 4 jC refrigerated centrifuge.
3. Block nonspecific binding sites by incubation with 150 Al of PBS/T containing 0.1% BSA (PBS/ T/BSA) for 1 h at 37 jC on a shaker. 4. Wash plates six times with PBS/T. 5. Add 100 Al of the reference preparation (diluted across columns 1 and 2 of plates, fivefold serial dilution) or samples (diluted 10 times) and incubate the plates for 2 h at 37 jC on a shaker. 6. Wash plates six times with PBS/T. 7. Add 100 Al of rabbit polyclonal anti-Hsp 70 (1:400) diluted in PBS/T/BSA for 1 h on shaker at 37 jC. 8. Wash plates six times with PBS/T. 9. Incubate plates with 100 Al of an anti-rabbit immunoglobulin peroxidase conjugate in PBS/T/ BSA (1:10,000) for 1 h on shaker at 37 jC. 10. Wash plates six times with PBS/T. 11. Add 200 Al of OPD substrate. 12. Stop reaction after 30 min with 50 Al of 1N H2SO4. 13. Determine the absorbance at 492 nm with background subtraction at 620 nm using a microplate
The supernatant collected is the cell lysate, which is now ready for analysis using the Hsp 70 CLIA. The resulting pellets can be discarded. 4.4. Cell lysate immunometric assay for detection of intracellular Hsp 70 1. In 96-well microwell plates, aliquot 100 Al per well of purified human recombinant Hsp 70 antibody (5 Ag/ml) diluted in 0.1 M carbonate buffer (pH 9.6). Cover plates with plate cover and place them at 4 jC overnight. 2. Wash plates six times with PBS containing 0.05% Tween 20 (PBS/T).
Fig. 1. Titration curve of Hsp 70 detection antibody against dilutions of Hsp 70 antigen. Each curve represents the titration of a different concentration (dilution factor: 200 – 25,600) of Hsp 70 detection antibody using the same dilution range of the antigen. The conjugate and capture conditions were kept constant throughout the experiment.
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5. Results
Fig. 2. Titration of capture Hsp 70 antibody against optimized antigen and detection antibody. A concentration of 5 Ag/ml was considered optimal for the capture system.
reader. The quantity of Hsp 70 in cells is estimated from the calibration curve. 4.5. Statistical analysis The Hsp 70 levels obtained using the two methods were compared by the Spearman Rank Test.
We have developed a CLIA for the detection of intracellular Hsp 70 concentration using the onestep-at-a-time procedure. All factors were kept constant except for one and the response was taken at different levels of the factor. Keeping the appropriate response level of the factor constant, a new factor was chosen and the procedure was repeated in the same manner. A titration curve of Hsp 70 detection antibody against dilutions of Hsp 70 antigen was generated to determine the optimal level of detection antibody (Fig. 1) and a titre of 1:400 was adopted. The titration of the capture antibody (Fig. 2) and the enzyme conjugate (Fig. 3) were optimized by preliminary checkerboard titration. An optimum concentration of 5 Ag/ml was considered for the capture antibody and a titre of 1:10,000 was adopted for the enzyme conjugate. Thereafter, the effect of other variables, such as temperature and incubation time, on the assay performance was systematically studied. To evaluate the assay, human recombinant Hsp 70 was used to generate a standard curve (Fig. 4). A fivefold dilution series was performed to set up the standard values. Each standard value was measured 12 times and the first two measurements (0 – 4000 ng/
Fig. 3. Assessment of optimized capture, antigen and detection system with different dilutions of conjugate. The initial concentration of the conjugate was 0.4 mg/ml and a conjugate dilution of 1:10,000 was adopted.
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measured value with that expected. The recovered value of Hsp 70 in spiked samples was between 81% and 116% of the expected value. In addition, dilution of cell lysates yielded parallel results with dilution between 97% and 120% of the expected value (data not shown). In order to relate the CLIA to the flow cytometric assay, PBMC were prepared from samples of nine volunteers. These cells were either treated immedi-
Fig. 4. Representative standard dose – response curve and precision profile for the Hsp 70 immunoassay. A fivefold dilution series was performed to set up the standard values (0 – 4000 ng/ml). Each standard value was measured 12 times and the first two measurements acted as the standard dose – response curve. The concentrations of the remaining 10 replicates of each standard value were determined from the standard curve and were used to generate the coefficient of variation profile.
ml) acted as the standard dose –response curve. The concentration of the remaining 10 replicates of each standard value was determined from the standard curve and was used to generate the coefficient of variation profile (Fig. 4). The sensitivity of the assay was 4 ng/ml and was defined as the concentration that gave an absorbance of the zero standard plus two standard deviations. To test the precision of the assay, experiments were conducted using the optimum conditions. The precision of the present CLIA was determined by assaying three samples 20 times on one plate (intraassay), and two samples 20 times in four individual assays using different batches of reagents (interassay). The average intraassay variation was 5%, and the average interassay variation was 12% (data not shown). Possible interference in the determination of intracellular Hsp 70 concentrations by other compounds was investigated by spiking cell lysates with known concentrations of purified Hsp 70 and comparing the
Fig. 5. Relationship between Hsp 70 analysis of PBMC by CLIA and of PBMC: monocytes and lymphocytes (A), pure monocyte (B) and pure lymphocyte (C) subpopulations, measured as MFI by flow cytometry.
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ately for Hsp 70 determination or were exposed to 39 or 42 jC for 1 h before Hsp 70 measurement by flow cytometry and CLIA. Fig. 5 shows a comparative evaluation of the two techniques. There was a highly significant correlation between the CLIA and the flow cytometric assay when the results of the total PBMC from flow cytometry were considered (r = 0.896, p < 0.0001) as well as when only the monocyte (r = 0.885, p < 0.0001) or the lymphocyte (r = 0.823, p = 0.0001) results from flow cytometry were taken in to account.
6. Discussion Hsp are highly abundant proteins that play fundamental roles in promoting cellular survival and maintenance of normal cellular function (Mayer and Bukau, 1998). The interaction of Hsp with cellular components has been shown to have therapeutic potentials. For example, Hsp 70 has been demonstrated to take part in pathogen clearance (Forsdyke, 1985) and to interfere with inflammatory responses (Munoz et al., 1996). Accordingly, some investigators have attributed a protective value to Hsp, as the administration of Hsp 70 can modulate the induction of arthritis (Kingston et al., 1996). Furthermore, in the clinical situation, the presence of circulating Hsp seems to be beneficial in juvenile chronic arthritis (de Graeff-Meeder et al., 1995) and severe ischemia/reperfusion injury (Kume et al., 1996). The need for a simple assay for determining Hsp 70 in cells has emerged with the increasing interest in therapeutic manipulation of Hsp for clinical trials. To date, although several methods for assaying Hsp 70 have been used for investigatory purposes, these techniques have certain drawbacks as pointed out in the introduction. In order to overcome these problems, we developed an Hsp 70 CLIA for the detection and quantification of intracellular Hsp 70. Our approach offers advantages over the conventional methods. Firstly, the assay constitutes a more cost-effective method, which can easily be amenable in many laboratories. Secondly, the assay allows the handling of multiple samples since a single plate can be used to assay 80 samples in single or 40 samples in duplicate.
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6.1. Troubleshooting The substrate, OPD, is very sensitive to light and temperature and must be dissolved in the dark immediately prior to use. It is advisable to put the substrate diluent (Milli-Q water) at 37 jC for 15 min before dissolving the OPD tablets. Given that ELISA is highly sensitive to temperature, differences in results due to drastic changes in environmental temperature were eliminated by carrying out the experiments in an incubator at a constant temperature of 37 jC. We realized that for samples with very high levels of the protein, a twofold diluted sample demonstrated a higher level of Hsp 70 compared to the same undiluted sample. Carpenter (1997) reported that it may be caused by antigen excess, in which a majority of the antigen binding sites are occupied, preventing completion of the sandwich. With regard to this problem and based on our linearity results, we decided to use a tenfold dilution of the samples. If samples generate lower values than the lowest standard, the samples should be reassayed at a lower sample dilution. Similarly, if samples generate higher values than the highest standard, the samples should be reassayed at a higher sample dilution. 6.2. Alternative and support protocols For the present CLIA for Hsp 70 measurement, we obtained average inter- and intraassay coefficients of variation of 5% and 12%, respectively. Tijssen (1985) suggested that the between test coefficient of variation of results obtained for reference samples under ideal conditions should be < 10%. However, a between assay coefficient of variation of 10– 15% was considered more usual by McLaren et al. (1981). The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution, suggesting the absence of any cellular interference. The standard recovery in different samples revealed that it is a precise assay with a high reproducibility. In order to assess the reliability of the present CLIA for quantification of intracellular Hsp 70, the levels of Hsp 70 obtained with this system were compared with those determined by the flow cytometric method. These two sets of values showed a
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highly significant correlation, indicating that the two methods are comparable. In conclusion, the described CLIA technique can be considered as a reliable and cost-effective diagnostic test that is applicable in low-technology settings. It may be relevant in the understanding of disease conditions that are accompanied by Hsp 70 production.
7. Quick procedure 1. Coat plates with capture antibody (overnight at 4 jC). 2. Wash plates. 3. Block plates (37 jC, shaker, 1 h). 4. Wash plates. 5. Put standards or samples on plates (37 jC, shaker, 2 h). 6. Wash plates. 7. Put rabbit polyclonal antibody on plates (37 jC, shaker, 1 h). 8. Wash plates. 9. Add anti-rabbit immunoglobulin peroxidase conjugate to plates (37 jC, shaker, 1 h). 10. Wash plates. 11. Add substrate to plates (37 jC, shaker, 30 min). 12. Stop reaction with H2SO4. 13. Read plates.
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