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Quantitation of bovine macrophage colony-stimulating factor in bovine serum by ELISA
Veterinary Immunology and Immunopathology 95 (2003) 103–111
Quantitation of bovine macrophage colony-stimulating factor in bovine serum by ELISA K. Yoshiharaa,*, K. Oshimab, Y. Munetaa, R. Kikumaa, C. Yayotac, T. Hiraic, N. Satohd, S. Matsuurad, Y. Kikyod, M. Satoha, C. Kubotae, S. Inumarua, Y. Yokomizoa, Y. Moria a National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan National Agricultural Research Center for Western Region, 60 Yoshinaga, Kawai, Ota, Shimane 694-0013, Japan c Hokkaido Animal Research Center, 5-39 Shintokunishi, Shintoku, Kamikawa, Hokkaido 081-0038, Japan d Ibaraki Prefectural Livestock Hygiene Service Center, 5-17-26 Manabe, Tsuchiura, Ibaraki 305-0051, Japan e Kagoshima Prefectural Cattle Breeding and Genetic Institute, 2200 Tsukino, Osumi, Kagoshima 899-8212, Japan
b
Received 10 September 2002; received in revised form 18 March 2003; accepted 8 May 2003
Abstract We established an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of bovine macrophage colonystimulating factor (M-CSF) and used it to measure the serum M-CSF levels in bovine fetuses and calves. The average serum M-CSF level was 2:7 1:5 ng/ml in 39 calves under 100 days old, and 1:8 0:8 ng/ml in 15 cattle between 101 and 418 days old. Fetal sera samples (n ¼ 6) prepared from cattle between 150 and 280 days of gestational age had a higher average level of M-CSF (8:8 1:4 ng/ml). Alteration in serum M-CSF levels in each individual calf was also measured. The serum levels of M-CSF in calves at 0–1 day after birth ranged from 0.52 to 7.3 ng/ml. During the period 113–125 days after birth, serum levels were around 1:4 0:39 ng/ml. Although serum M-CSF levels generally decreased as the age of calves advanced, differences among individuals, especially among newborn calves, were observed. # 2003 Elsevier B.V. All rights reserved. Keywords: Bovine M-CSF; Bovine CSF-1; ELISA
1. Introduction
Abbreviations: M-CSF, macrophage colony-stimulating factor; CSF-1, colony-stimulating factor 1; GM-CSF, granulocyte-macrophage colony-stimulating factors; G-CSF, granulocyte colonystimulating factor; rBo, recombinant bovine; AP, alkaline phosphatase; CFA, complete Freund’s adjuvant; PNPP, p-nitrophenyl phosphate * Corresponding author. Tel.: þ81-29-838-7857; fax: þ81-29-838-7907. E-mail address: [email protected] (K. Yoshihara).
Macrophage colony-stimulating factor (M-CSF), which is also called colony-stimulating factor 1 (CSF-1), is a hematopoietic growth factor that regulates the survival, proliferation, and differentiation of the mononuclear phagocyte lineage (Stanley et al., 1983; Clark and Kamen, 1987). Serum M-CSF level is markedly higher than granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF), though they are all
0165-2427/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-2427(03)00103-X
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members of the same colony-stimulating factor family regulating the proliferation and differentiation of progenitor cells for granulocytes and macrophages. Although the serum GM-CSF and G-CSF levels were lower than 100 pg/ml, serum M-CSF level was in the range of 2.3–5.7 ng/ml in humans (Yong et al., 1992; Saito et al., 1992; Roth, 1991; Watari et al., 1989; Omori et al., 1992). Several reports found the serum M-CSF levels in newborn infants to be higher than those in adults (Saito et al., 1992; Roth, 1991; Roth and Stanley, 1995; Ishii et al., 1995). Moreover, serum M-CSF levels are significantly elevated during pregnancy in humans and mice, and are associated with increased numbers of monocytes and neutrophils (Saito et al., 1992; Bartocci et al., 1986; Yong et al., 1992). Not only the physiological changes in serum M-CSF levels but also the inflammatory response causes alteration in the M-CSF concentration (Klebl et al., 2001; Roth et al., 1997; Igarashi et al., 1999; Sweet et al., 2002). Measurements of serum M-CSF levels are essential to investigation of the roles of MCSF in inflammatory response and the development of the immune system. Therefore, we constructed an ELISA for bovine M-CSF and measured the serum M-CSF levels in calves.
2. Materials and methods 2.1. Preparation of standard bovine M-CSF The recombinant bovine M-CSF (rBoM-CSF) used in this study was described in our previous report (Yoshihara et al., 1998). Ammonium sulfate was dissolved to 50% saturation into the recombinant virusinfected insect cell supernatant and stirred overnight at 4 8C. The precipitate was discarded after centrifugation at 12,000 g for 10 min at 4 8C, then the supernatant was brought to 70% saturation with ammonium sulfate and stirred overnight at 4 8C. The resulting precipitate was collected by centrifugation at 12,000 g for 10 min at 4 8C, dissolved in PBS, and dialyzed overnight against PBS at 4 8C. The dialyzed 50–70% ammonium sulfate fraction was analyzed by SDSPAGE on 12.5% polyacrylamide gel, and the content of rBoM-CSF was found by image analysis using an Image Master 1D Prime (Amersham). The amount of total protein was decided by using BCA protein assay
reagent (Pierce, Rockford, IL) following the manufacturer’s instructions. This 50–70% ammonium sulfate fraction was used as a standard M-CSF for ELISA. 2.2. Generation of hybridoma and purification of mAb Recombinant BoM-CSF was accumulated in serum-free medium (Expression Five SFM) by insect cells (BTI TN5B1-4) infected with baculovirus carrying bovine M-CSF cDNA (Yoshihara et al., 1998). To adjust the protein concentration to 1 mg/ml, we concentrated the supernatant 12-fold by ultrafiltration using Microcon 3 (molecular weight cut-off is 3000 Da; Amicon, Inc., Beverly, MA). A 6-week-old BALB/c mouse was immunized by one i.p. injection of 100 ml of the concentrated supernatant (100 mg of total protein) emulsified in an equal volume of complete Freund’s adjuvant (CFA, Difco Laboratories Inc., Detroit, MI) followed by one i.p. injection of the concentrated supernatant emulsified in an equal volume of incomplete Freund’s adjuvant (Difco Laboratories Inc.) at 2 weeks interval. A 0.5 ml (100 mg) booster of the concentrated supernatant was injected i.v., and fusion was carried out 3 days later as described previously. Hybridoma supernatants were screened by ELISA using plates coated with the supernatant including rBoM-CSF. As a result, 40 hybridoma supernatants were judged positive. To evaluate whether hybridoma antibodies detected rBoM-CSF or other components in the supernatants of the insect cells, we used Western blot analysis for the second screening. Because rBoM-CSF was secreted in dimeric form, the insect cell culture supernatant including rBoM-CSF was separated on SDS-PAGE under reducing or non-reducing conditions and transferred to Immobilon PVDF membrane (Millipore, Bedford, MA). After the membrane had been blocked with Block Ace (Yukijirushi, Tokyo, Japan), hybridoma supernatants were applied on the membrane and incubated for 1 h at room temperature. After washing with TBS-T (25 mM Tris, 0.14 M NaCl, 5 mM KCl, 0.05% Tween 20, pH 7.4), horseradish peroxidaseconjugated anti-mouse IgM þ G antibodies (American Qualex Manufacturers, San Clemente, CA) were reacted for 1 h at room temperature. The results were visualized with ECL Western blotting detection
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reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK), following the manufacturer’s instructions. Instead of the hybridoma supernatants, we used anti-human M-CSF polyclonal antibody (Genzyme, Cambridge, MA) as the indicator for positions of rBoM-CSF. This anti-human M-CSF polyclonal antibody could detect rBoM-CSF though the signal was very weak. Finally, we obtained two kinds of hybridomas secreting anti-M-CSF mAb. The isotypes of the mAb were identified using a mouse monoclonal antibody isotyping kit (Amersham Pharmacia Biotech) and were identified as IgG1 and IgM. The hybridoma secreting the IgG1 class antibody (A4) was cloned by limiting dilution. Cloned hybridoma was cultured in serum-free medium (ASF 301, Ajinomoto, Tokyo, Japan) for 5 days, then mAb accumulated in medium was purified with protein A column (HiTrap Protein A HP, Amersham Pharmacia Biotech). We also performed Western blotting on the standard M-CSF, which was separated on SDS-PAGE gel under reducing and non-reducing condition then transferred onto Immobilon PVDF membrane (Millipore). The membrane was blocked in Block Ace overnight at 4 8C, washed with TBS-T, then incubated for 1 h at room temperature with 8 mg/ml of A4 in Block Ace diluted 10-fold with H2O. After washing, the membrane was incubated in a 1:1000 dilution of the anti-mouse IgG labeled alkaline phosphatase (AP) (Southern Biotechnology Associates, Inc., Birmingham, AL) for 1 h at room temperature. After being washed with TBS-T, blots were visualized with BCIP/NBT color development solution (Bio-Rad Laboratories, Hercules, CA) following the manufacturer’s instructions. 2.3. Preparation and purification of polyclonal antibody Antisera against rBoM-CSF were prepared in a rabbit. The rabbit was immunized with a subcutaneous injection of 0.325 mg of the concentrated supernatant emulsified in an equal volume of CFA. Three further immunizations were administered at 2-week intervals, and blood was collected 10 days after the last immunization. The purification of IgG fraction was carried out using protein A-Sepharose affinity chromatography following precipitation by ammonium sulfate at 33% saturation, and the IgG fraction was then dialyzed against PBS. In order to clarify what molecules were
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detected by these polyclonal antibodies, Western blotting was performed according to the method described in the previous section. Briefly, each 2.5 ml of the supernatants of insect cells infected with recombinant virus or wild-type virus was separated on SDS-PAGE gel under reducing conditions, then transferred onto a membrane. The membranes were incubated in the presence of 8 mg/ml of polyclonal antibodies or monoclonal antibody, A4. 2.4. ELISA ELISA plates (Nunc, Roskilde, Denmark) were coated with 100 ml of coating solution (5 mg/ml of A4 in 50 mM carbonate buffer, pH 9.6) and incubated overnight at 4 8C. After the plates had been washed twice with TBS-T, 100 ml of the serum samples were added in duplicate and left for 1 h at room temperature. After four washings, 100 ml of anti-BoM-CSF polyclonal antibody (0.65 mg/ml in TBS-T) was added, then the samples were incubated for 1 h at room temperature. The plates were washed four times, incubated for 1 h at room temperature with 100 ml of biotin-conjugated goat anti-rabbit IgG antibodies (Biosource, Camarillo, CA), diluted 1/20,000 in TBS-T, and again washed four times. One hundred microliters of AP conjugated streptavidin (Vector Laboratories, Inc., Burlingame, CA) diluted 1/1000 in TBS-T was added, and the samples were incubated for 1 h at room temperature. After four washings, the AP activity retained in the wells was assayed by the addition of 100 ml of PNPP (p-nitrophenyl phosphate, Sigma, St. Louis, MO) for 15 min at room temperature. The reaction was stopped by adding 50 ml of 150 mM EDTA, and the absorbance was measured at 409 nm in a microtiter plate reader (MTP-120 Microplate Reader, Corona Electric, Ibaraki, Japan). In order to confirm the specificity of ELISA, the supernatants of insect cells infected with recombinant virus carrying bovine IFNg (Murakami et al., 2001) or M-CSF cDNA and with wild-type virus that had been diluted 10,000, 100,000, and 1,000,000 times with PBS were measured. 2.5. Bovine serum The sera from six fetuses at 152–278 days of gestational age, their umbilical arteries and veins,
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and the sera from five of the corresponding dams (serum from the sixth dam could not be obtained) were obtained at a slaughterhouse. The sera from 29 Holstein calves were obtained at 1–82 days after birth from dairy farmers, and the sera from 21 Japanese black cattle at 31–418 days old were obtained from the National Agricultural Research Center for the Western Region. To investigate the alteration in serum M-CSF levels in each individual calf, we collected sera periodically from 13 calves bred in the Hokkaido Animal Research Center.
3. Results We previously reported that rBoM-CSF with a molecular weight of 34 kDa was secreted in dimeric form in the insect cell culture supernatant (Yoshihara et al., 1998). Here, a monoclonal antibody, A4, which we established in this study, bound to the 34 kDa band and the 68 kDa band in the SDS-PAGE performed under reducing and non-reducing conditions, respectively (Fig. 1). These results indicate that the monoclonal antibody A4 specifically bound to rBoM-CSF. Density analysis of the SDS-PAGE result revealed that the concentration of M-CSF in the dialyzed 50– 70% ammonium sulfate fraction (standard M-CSF) was 86.4% (Fig. 2). Because the total protein concentration of this fraction was 800 mg/ml, we estimated
Fig. 1. Western blotting of rBo-M-CSF. Standard M-CSF was electrophoresed on a SDS-polyacrylamide gel under a reducing condition (A) and under a non-reducing condition (B). Blots were probed with an anti-bovine M-CSF monoclonal antibody, A4. MW: molecular weight marker.
Fig. 2. SDS-PAGE analysis of standard M-CSF. Five microliters of standard M-CSF was electrophoresed on an SDS-polyacrylamide gel under the reducing condition. Density analysis of the SDSPAGE result revealed that the concentration of M-CSF in standard M-CSF was 86.4%. MW: molecular weight marker.
the M-CSF concentration to be 700 mg/ml, and used that as the standard M-CSF for the ELISA. Although the polyclonal antibodies detected the components in the supernatant of the insect cells infected with wild-type virus, the results of density analysis showed that more than 80% of the polyclonal antibodies reacted to rBoM-CSF (Fig. 3, lane 1). Moreover, A4 did not detect any components in the wild-type supernatant (Fig. 3, lane 4). The specificity of ELISA was confirmed by using the diluted supernatants of the insect cells infected with the wild-type virus or the recombinant virus carrying bovine IFNg. An increase in the absorbance of the IFNg and wildtype supernatants was not confirmed (Fig. 4). A typical standard curve is shown in Fig. 5. We consider the sensitivity range of this ELISA to be from 0.1 to 40 ng/ml. Changes in M-CSF levels in sera obtained from Holstein calves (n ¼ 29) and from Japanese black calves (n ¼ 21) are shown in Fig. 6A and B, respectively. The serum M-CSF levels decreased gradually in both groups. All of the sera samples were divided into two groups by the postnatal age, then the averages were calculated (Fig. 7). The average serum M-CSF level in 39 calves under 100 days old was 2:7 1:5 ng/ml, and that in 15
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from 0.52 to 7.3 ng/ml. During the period 113–125 days after birth, the serum levels concentrated around 1:4 0:39 ng/ml.
4. Discussion
Fig. 3. Western blotting of rBoM-CSF. The supernatants of the insect cells infected with recombinant virus (lanes 1 and 3) or wildtype virus (lanes 2 and 4) were electrophoresed on an SDSpolyacrylamide gel under a reducing condition. Blots were probed with polyclonal antibodies (lanes 1 and 2) or a monoclonal antibody (lanes 3 and 4) used for ELISA. MW: molecular weight marker.
cattle between 101 and 418 days old was 1:8 0:8 ng/ ml. Sera samples obtained from fetal calves at 150– 280 days of gestational age had a high average level of M-CSF (8:8 1:4 ng/ml). The alteration in serum M-CSF levels in each individual calf is shown in Fig. 8. The serum levels of M-CSF in calves at 0–1 day after birth scattered
We established hybridomas secreting anti-M-CSF mAb and developed an ELISA for bovine M-CSF. Since the concentrated supernatants including rBoM-CSF were used in order to produce polyclonal antibodies, and the 50–70% ammonium sulfate fraction of the supernatants including rBoM-CSF was used as a standard M-CSF, the components except for rBoM-CSF in the standard M-CSF have the possibility of increasing the optical density values. However, more than 80% of the polyclonal antibodies reacted with rBoM-CSF (Fig. 3), and the optical density values of the supernatant of the insect cells infected with the wild-type virus or recombinant virus carrying bovine IFNg were hardly detected (Fig. 4). Thus, the possibility of the enhancement of the optical value due to these components can be ignored. Three human M-CSF cDNA clones, each encoding a different length M-CSF polypeptide (a, 256 amino acids; b, 554 amino acids; and g, 438 amino acids), have been isolated from a single M-CSF gene (Kawasaki et al., 1985; Wong et al., 1987; Cerretti et al., 1988). The biologically active form of M-CSFa is shorter than that of M-CSFb (Pandit et al., 1992). In cattle, M-CSFa and M-CSFb cDNAs have been cloned previously (Yoshihara et al., 1998), and in
Fig. 4. The specificity of ELISA. ELISA specificity was tested by using the diluted supernatants of the insect cells infected with the wild-type virus or the recombinant virus carrying BoM-CSF or bovine IFNg. The data are the means S:D: from duplicate wells.
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Fig. 5. Standard curve for bovine M-CSF ELISA. Data are the means S:D: from duplicate wells.
Fig. 6. Relationship between serum M-CSF levels and postnatal age: (A) Holstein at 1–82 days after birth; (B) Japanese black cattle at 31–418 days after birth. Regression analysis was performed to determine the relationship between individual M-CSF levels and postnatal age.
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Fig. 7. M-CSF levels in fetuses and calves. Calves were divided into two groups by postnatal age. Samples were assayed in duplicate. Data are shown with the median, mean S:D: for each group.
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the same manner as human, M-CSFa is short part of amino acid sequence of M-CSFb. Since we used b form of M-CSF as an antigen to produce monoclonal and polyclonal antibodies, it is not clear whether the a form could be detected. However, based on the fact that several reports have found M-CSFb to be the dominant form in human serum (Suzu et al., 1991; Hanamura et al., 1988), we consider the alteration in serum M-CSF levels found in this study to indicate the actual amount of change. However, an examination to confirm whether A4 can detect bovine M-CSFa seems necessary. Several reports found that serum M-CSF levels in the fetus were higher than those in human adults (Roth and Stanley, 1995; Saito et al., 1992) and mice (Roth and Stanley, 1996). In the case of cattle, the serum levels in bovine fetuses were much higher than those in calves (Fig. 5). Several newborn calves maintained high serum levels, but the levels in five newborn calves were lower than 1.1 ng/ml (Fig. 6). It was not clear whether the M-CSF levels in these calves were also low when they were fetuses or whether levels decreased immediately after birth. Hara et al. described that the maximal concentrations of M-CSF in human milk were 10- to 100-fold higher than those in the serum (Hara et al., 1995). Although it is not clear what range of M-CSF concentrations are present in bovine milk,
Fig. 8. Change in serum M-CSF levels by postnatal age for calves in which M-CSF levels were measured on four separate days. The measured values for each calf are connected with an identifying line. Samples were assayed in duplicate. Data are shown with the mean S:D: for each group.
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Fig. 9. Serum M-CSF levels in six fetuses and the corresponding dams. The bars of the same pattern show the measured values for each fetus and its dam. Samples were assayed in duplicate. Data are shown with the median, mean S:D: for each group (the number of samples of dams’ serum is 5).
intake of M-CSF through the milk might influence M-CSF concentration in serum of calves. Because infectious diarrhea and pneumonia are major causes of death in neonatal calves and therefore a cause significant economic losses in the beef and dairy industries, investigations of the immunological activity of M-CSF and of the cellular immunity of calves showing low M-CSF level are warranted. Is M-CSF capable of crossing the placenta? M-CSF was found to cross the placenta in the mouse (Roth et al., 1998), but was found not to cross in the human (Saito et al., 1992). In the case of cattle, M-CSF levels in serum obtained from the umbilical artery and umbilical vein were almost the same (Fig. 9). This result may indicate that bovine M-CSF does not pass through the placenta, or that, even if bovine M-CSF can pass through the placenta, it does not actively move. Therefore, M-CSF in the serum is very probably produced in the bovine fetus. In this study, we described the establishment of an ELISA for bovine M-CSF and the evaluation of serum M-CSF levels in calves. Although the serum M-CSF levels decreased as the age of calves advanced, there were differences among individuals, and especially among newborn calves. This ELISA for bovine M-CSF should be useful for investigation of the role of M-CSF in the inflammatory response. Moreover, when bovine M-CSF levels in bovine sera and bovine standard M-CSF were assayed by
using a human M-CSF ELISA kit (R&D Systems Inc., Minneapolis, MN), the cross-reactivity was not actually recognized (data not shown). Because commercially available ELISA for human that are designed to have the low species cross-reactivity, ELISA established in this study should be a powerful tool to measure bovine M-CSF levels.
Acknowledgements This work was supported by a Grant-in-aid from the Recombinant Cytokine Project (PCR2002-2110), provided by the Ministry of Agriculture, Forestry and Fisheries, Japan.
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Quantitation of bovine macrophage colony-stimulating factor in bovine serum by ELISA K. Yoshiharaa,*, K. Oshimab, Y. Munetaa, R. Kikumaa, C. Yayotac, T. Hiraic, N. Satohd, S. Matsuurad, Y. Kikyod, M. Satoha, C. Kubotae, S. Inumarua, Y. Yokomizoa, Y. Moria a National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan National Agricultural Research Center for Western Region, 60 Yoshinaga, Kawai, Ota, Shimane 694-0013, Japan c Hokkaido Animal Research Center, 5-39 Shintokunishi, Shintoku, Kamikawa, Hokkaido 081-0038, Japan d Ibaraki Prefectural Livestock Hygiene Service Center, 5-17-26 Manabe, Tsuchiura, Ibaraki 305-0051, Japan e Kagoshima Prefectural Cattle Breeding and Genetic Institute, 2200 Tsukino, Osumi, Kagoshima 899-8212, Japan
b
Received 10 September 2002; received in revised form 18 March 2003; accepted 8 May 2003
Abstract We established an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of bovine macrophage colonystimulating factor (M-CSF) and used it to measure the serum M-CSF levels in bovine fetuses and calves. The average serum M-CSF level was 2:7 1:5 ng/ml in 39 calves under 100 days old, and 1:8 0:8 ng/ml in 15 cattle between 101 and 418 days old. Fetal sera samples (n ¼ 6) prepared from cattle between 150 and 280 days of gestational age had a higher average level of M-CSF (8:8 1:4 ng/ml). Alteration in serum M-CSF levels in each individual calf was also measured. The serum levels of M-CSF in calves at 0–1 day after birth ranged from 0.52 to 7.3 ng/ml. During the period 113–125 days after birth, serum levels were around 1:4 0:39 ng/ml. Although serum M-CSF levels generally decreased as the age of calves advanced, differences among individuals, especially among newborn calves, were observed. # 2003 Elsevier B.V. All rights reserved. Keywords: Bovine M-CSF; Bovine CSF-1; ELISA
1. Introduction
Abbreviations: M-CSF, macrophage colony-stimulating factor; CSF-1, colony-stimulating factor 1; GM-CSF, granulocyte-macrophage colony-stimulating factors; G-CSF, granulocyte colonystimulating factor; rBo, recombinant bovine; AP, alkaline phosphatase; CFA, complete Freund’s adjuvant; PNPP, p-nitrophenyl phosphate * Corresponding author. Tel.: þ81-29-838-7857; fax: þ81-29-838-7907. E-mail address: [email protected] (K. Yoshihara).
Macrophage colony-stimulating factor (M-CSF), which is also called colony-stimulating factor 1 (CSF-1), is a hematopoietic growth factor that regulates the survival, proliferation, and differentiation of the mononuclear phagocyte lineage (Stanley et al., 1983; Clark and Kamen, 1987). Serum M-CSF level is markedly higher than granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF), though they are all
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members of the same colony-stimulating factor family regulating the proliferation and differentiation of progenitor cells for granulocytes and macrophages. Although the serum GM-CSF and G-CSF levels were lower than 100 pg/ml, serum M-CSF level was in the range of 2.3–5.7 ng/ml in humans (Yong et al., 1992; Saito et al., 1992; Roth, 1991; Watari et al., 1989; Omori et al., 1992). Several reports found the serum M-CSF levels in newborn infants to be higher than those in adults (Saito et al., 1992; Roth, 1991; Roth and Stanley, 1995; Ishii et al., 1995). Moreover, serum M-CSF levels are significantly elevated during pregnancy in humans and mice, and are associated with increased numbers of monocytes and neutrophils (Saito et al., 1992; Bartocci et al., 1986; Yong et al., 1992). Not only the physiological changes in serum M-CSF levels but also the inflammatory response causes alteration in the M-CSF concentration (Klebl et al., 2001; Roth et al., 1997; Igarashi et al., 1999; Sweet et al., 2002). Measurements of serum M-CSF levels are essential to investigation of the roles of MCSF in inflammatory response and the development of the immune system. Therefore, we constructed an ELISA for bovine M-CSF and measured the serum M-CSF levels in calves.
2. Materials and methods 2.1. Preparation of standard bovine M-CSF The recombinant bovine M-CSF (rBoM-CSF) used in this study was described in our previous report (Yoshihara et al., 1998). Ammonium sulfate was dissolved to 50% saturation into the recombinant virusinfected insect cell supernatant and stirred overnight at 4 8C. The precipitate was discarded after centrifugation at 12,000 g for 10 min at 4 8C, then the supernatant was brought to 70% saturation with ammonium sulfate and stirred overnight at 4 8C. The resulting precipitate was collected by centrifugation at 12,000 g for 10 min at 4 8C, dissolved in PBS, and dialyzed overnight against PBS at 4 8C. The dialyzed 50–70% ammonium sulfate fraction was analyzed by SDSPAGE on 12.5% polyacrylamide gel, and the content of rBoM-CSF was found by image analysis using an Image Master 1D Prime (Amersham). The amount of total protein was decided by using BCA protein assay
reagent (Pierce, Rockford, IL) following the manufacturer’s instructions. This 50–70% ammonium sulfate fraction was used as a standard M-CSF for ELISA. 2.2. Generation of hybridoma and purification of mAb Recombinant BoM-CSF was accumulated in serum-free medium (Expression Five SFM) by insect cells (BTI TN5B1-4) infected with baculovirus carrying bovine M-CSF cDNA (Yoshihara et al., 1998). To adjust the protein concentration to 1 mg/ml, we concentrated the supernatant 12-fold by ultrafiltration using Microcon 3 (molecular weight cut-off is 3000 Da; Amicon, Inc., Beverly, MA). A 6-week-old BALB/c mouse was immunized by one i.p. injection of 100 ml of the concentrated supernatant (100 mg of total protein) emulsified in an equal volume of complete Freund’s adjuvant (CFA, Difco Laboratories Inc., Detroit, MI) followed by one i.p. injection of the concentrated supernatant emulsified in an equal volume of incomplete Freund’s adjuvant (Difco Laboratories Inc.) at 2 weeks interval. A 0.5 ml (100 mg) booster of the concentrated supernatant was injected i.v., and fusion was carried out 3 days later as described previously. Hybridoma supernatants were screened by ELISA using plates coated with the supernatant including rBoM-CSF. As a result, 40 hybridoma supernatants were judged positive. To evaluate whether hybridoma antibodies detected rBoM-CSF or other components in the supernatants of the insect cells, we used Western blot analysis for the second screening. Because rBoM-CSF was secreted in dimeric form, the insect cell culture supernatant including rBoM-CSF was separated on SDS-PAGE under reducing or non-reducing conditions and transferred to Immobilon PVDF membrane (Millipore, Bedford, MA). After the membrane had been blocked with Block Ace (Yukijirushi, Tokyo, Japan), hybridoma supernatants were applied on the membrane and incubated for 1 h at room temperature. After washing with TBS-T (25 mM Tris, 0.14 M NaCl, 5 mM KCl, 0.05% Tween 20, pH 7.4), horseradish peroxidaseconjugated anti-mouse IgM þ G antibodies (American Qualex Manufacturers, San Clemente, CA) were reacted for 1 h at room temperature. The results were visualized with ECL Western blotting detection
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reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK), following the manufacturer’s instructions. Instead of the hybridoma supernatants, we used anti-human M-CSF polyclonal antibody (Genzyme, Cambridge, MA) as the indicator for positions of rBoM-CSF. This anti-human M-CSF polyclonal antibody could detect rBoM-CSF though the signal was very weak. Finally, we obtained two kinds of hybridomas secreting anti-M-CSF mAb. The isotypes of the mAb were identified using a mouse monoclonal antibody isotyping kit (Amersham Pharmacia Biotech) and were identified as IgG1 and IgM. The hybridoma secreting the IgG1 class antibody (A4) was cloned by limiting dilution. Cloned hybridoma was cultured in serum-free medium (ASF 301, Ajinomoto, Tokyo, Japan) for 5 days, then mAb accumulated in medium was purified with protein A column (HiTrap Protein A HP, Amersham Pharmacia Biotech). We also performed Western blotting on the standard M-CSF, which was separated on SDS-PAGE gel under reducing and non-reducing condition then transferred onto Immobilon PVDF membrane (Millipore). The membrane was blocked in Block Ace overnight at 4 8C, washed with TBS-T, then incubated for 1 h at room temperature with 8 mg/ml of A4 in Block Ace diluted 10-fold with H2O. After washing, the membrane was incubated in a 1:1000 dilution of the anti-mouse IgG labeled alkaline phosphatase (AP) (Southern Biotechnology Associates, Inc., Birmingham, AL) for 1 h at room temperature. After being washed with TBS-T, blots were visualized with BCIP/NBT color development solution (Bio-Rad Laboratories, Hercules, CA) following the manufacturer’s instructions. 2.3. Preparation and purification of polyclonal antibody Antisera against rBoM-CSF were prepared in a rabbit. The rabbit was immunized with a subcutaneous injection of 0.325 mg of the concentrated supernatant emulsified in an equal volume of CFA. Three further immunizations were administered at 2-week intervals, and blood was collected 10 days after the last immunization. The purification of IgG fraction was carried out using protein A-Sepharose affinity chromatography following precipitation by ammonium sulfate at 33% saturation, and the IgG fraction was then dialyzed against PBS. In order to clarify what molecules were
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detected by these polyclonal antibodies, Western blotting was performed according to the method described in the previous section. Briefly, each 2.5 ml of the supernatants of insect cells infected with recombinant virus or wild-type virus was separated on SDS-PAGE gel under reducing conditions, then transferred onto a membrane. The membranes were incubated in the presence of 8 mg/ml of polyclonal antibodies or monoclonal antibody, A4. 2.4. ELISA ELISA plates (Nunc, Roskilde, Denmark) were coated with 100 ml of coating solution (5 mg/ml of A4 in 50 mM carbonate buffer, pH 9.6) and incubated overnight at 4 8C. After the plates had been washed twice with TBS-T, 100 ml of the serum samples were added in duplicate and left for 1 h at room temperature. After four washings, 100 ml of anti-BoM-CSF polyclonal antibody (0.65 mg/ml in TBS-T) was added, then the samples were incubated for 1 h at room temperature. The plates were washed four times, incubated for 1 h at room temperature with 100 ml of biotin-conjugated goat anti-rabbit IgG antibodies (Biosource, Camarillo, CA), diluted 1/20,000 in TBS-T, and again washed four times. One hundred microliters of AP conjugated streptavidin (Vector Laboratories, Inc., Burlingame, CA) diluted 1/1000 in TBS-T was added, and the samples were incubated for 1 h at room temperature. After four washings, the AP activity retained in the wells was assayed by the addition of 100 ml of PNPP (p-nitrophenyl phosphate, Sigma, St. Louis, MO) for 15 min at room temperature. The reaction was stopped by adding 50 ml of 150 mM EDTA, and the absorbance was measured at 409 nm in a microtiter plate reader (MTP-120 Microplate Reader, Corona Electric, Ibaraki, Japan). In order to confirm the specificity of ELISA, the supernatants of insect cells infected with recombinant virus carrying bovine IFNg (Murakami et al., 2001) or M-CSF cDNA and with wild-type virus that had been diluted 10,000, 100,000, and 1,000,000 times with PBS were measured. 2.5. Bovine serum The sera from six fetuses at 152–278 days of gestational age, their umbilical arteries and veins,
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and the sera from five of the corresponding dams (serum from the sixth dam could not be obtained) were obtained at a slaughterhouse. The sera from 29 Holstein calves were obtained at 1–82 days after birth from dairy farmers, and the sera from 21 Japanese black cattle at 31–418 days old were obtained from the National Agricultural Research Center for the Western Region. To investigate the alteration in serum M-CSF levels in each individual calf, we collected sera periodically from 13 calves bred in the Hokkaido Animal Research Center.
3. Results We previously reported that rBoM-CSF with a molecular weight of 34 kDa was secreted in dimeric form in the insect cell culture supernatant (Yoshihara et al., 1998). Here, a monoclonal antibody, A4, which we established in this study, bound to the 34 kDa band and the 68 kDa band in the SDS-PAGE performed under reducing and non-reducing conditions, respectively (Fig. 1). These results indicate that the monoclonal antibody A4 specifically bound to rBoM-CSF. Density analysis of the SDS-PAGE result revealed that the concentration of M-CSF in the dialyzed 50– 70% ammonium sulfate fraction (standard M-CSF) was 86.4% (Fig. 2). Because the total protein concentration of this fraction was 800 mg/ml, we estimated
Fig. 1. Western blotting of rBo-M-CSF. Standard M-CSF was electrophoresed on a SDS-polyacrylamide gel under a reducing condition (A) and under a non-reducing condition (B). Blots were probed with an anti-bovine M-CSF monoclonal antibody, A4. MW: molecular weight marker.
Fig. 2. SDS-PAGE analysis of standard M-CSF. Five microliters of standard M-CSF was electrophoresed on an SDS-polyacrylamide gel under the reducing condition. Density analysis of the SDSPAGE result revealed that the concentration of M-CSF in standard M-CSF was 86.4%. MW: molecular weight marker.
the M-CSF concentration to be 700 mg/ml, and used that as the standard M-CSF for the ELISA. Although the polyclonal antibodies detected the components in the supernatant of the insect cells infected with wild-type virus, the results of density analysis showed that more than 80% of the polyclonal antibodies reacted to rBoM-CSF (Fig. 3, lane 1). Moreover, A4 did not detect any components in the wild-type supernatant (Fig. 3, lane 4). The specificity of ELISA was confirmed by using the diluted supernatants of the insect cells infected with the wild-type virus or the recombinant virus carrying bovine IFNg. An increase in the absorbance of the IFNg and wildtype supernatants was not confirmed (Fig. 4). A typical standard curve is shown in Fig. 5. We consider the sensitivity range of this ELISA to be from 0.1 to 40 ng/ml. Changes in M-CSF levels in sera obtained from Holstein calves (n ¼ 29) and from Japanese black calves (n ¼ 21) are shown in Fig. 6A and B, respectively. The serum M-CSF levels decreased gradually in both groups. All of the sera samples were divided into two groups by the postnatal age, then the averages were calculated (Fig. 7). The average serum M-CSF level in 39 calves under 100 days old was 2:7 1:5 ng/ml, and that in 15
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from 0.52 to 7.3 ng/ml. During the period 113–125 days after birth, the serum levels concentrated around 1:4 0:39 ng/ml.
4. Discussion
Fig. 3. Western blotting of rBoM-CSF. The supernatants of the insect cells infected with recombinant virus (lanes 1 and 3) or wildtype virus (lanes 2 and 4) were electrophoresed on an SDSpolyacrylamide gel under a reducing condition. Blots were probed with polyclonal antibodies (lanes 1 and 2) or a monoclonal antibody (lanes 3 and 4) used for ELISA. MW: molecular weight marker.
cattle between 101 and 418 days old was 1:8 0:8 ng/ ml. Sera samples obtained from fetal calves at 150– 280 days of gestational age had a high average level of M-CSF (8:8 1:4 ng/ml). The alteration in serum M-CSF levels in each individual calf is shown in Fig. 8. The serum levels of M-CSF in calves at 0–1 day after birth scattered
We established hybridomas secreting anti-M-CSF mAb and developed an ELISA for bovine M-CSF. Since the concentrated supernatants including rBoM-CSF were used in order to produce polyclonal antibodies, and the 50–70% ammonium sulfate fraction of the supernatants including rBoM-CSF was used as a standard M-CSF, the components except for rBoM-CSF in the standard M-CSF have the possibility of increasing the optical density values. However, more than 80% of the polyclonal antibodies reacted with rBoM-CSF (Fig. 3), and the optical density values of the supernatant of the insect cells infected with the wild-type virus or recombinant virus carrying bovine IFNg were hardly detected (Fig. 4). Thus, the possibility of the enhancement of the optical value due to these components can be ignored. Three human M-CSF cDNA clones, each encoding a different length M-CSF polypeptide (a, 256 amino acids; b, 554 amino acids; and g, 438 amino acids), have been isolated from a single M-CSF gene (Kawasaki et al., 1985; Wong et al., 1987; Cerretti et al., 1988). The biologically active form of M-CSFa is shorter than that of M-CSFb (Pandit et al., 1992). In cattle, M-CSFa and M-CSFb cDNAs have been cloned previously (Yoshihara et al., 1998), and in
Fig. 4. The specificity of ELISA. ELISA specificity was tested by using the diluted supernatants of the insect cells infected with the wild-type virus or the recombinant virus carrying BoM-CSF or bovine IFNg. The data are the means S:D: from duplicate wells.
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Fig. 5. Standard curve for bovine M-CSF ELISA. Data are the means S:D: from duplicate wells.
Fig. 6. Relationship between serum M-CSF levels and postnatal age: (A) Holstein at 1–82 days after birth; (B) Japanese black cattle at 31–418 days after birth. Regression analysis was performed to determine the relationship between individual M-CSF levels and postnatal age.
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Fig. 7. M-CSF levels in fetuses and calves. Calves were divided into two groups by postnatal age. Samples were assayed in duplicate. Data are shown with the median, mean S:D: for each group.
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the same manner as human, M-CSFa is short part of amino acid sequence of M-CSFb. Since we used b form of M-CSF as an antigen to produce monoclonal and polyclonal antibodies, it is not clear whether the a form could be detected. However, based on the fact that several reports have found M-CSFb to be the dominant form in human serum (Suzu et al., 1991; Hanamura et al., 1988), we consider the alteration in serum M-CSF levels found in this study to indicate the actual amount of change. However, an examination to confirm whether A4 can detect bovine M-CSFa seems necessary. Several reports found that serum M-CSF levels in the fetus were higher than those in human adults (Roth and Stanley, 1995; Saito et al., 1992) and mice (Roth and Stanley, 1996). In the case of cattle, the serum levels in bovine fetuses were much higher than those in calves (Fig. 5). Several newborn calves maintained high serum levels, but the levels in five newborn calves were lower than 1.1 ng/ml (Fig. 6). It was not clear whether the M-CSF levels in these calves were also low when they were fetuses or whether levels decreased immediately after birth. Hara et al. described that the maximal concentrations of M-CSF in human milk were 10- to 100-fold higher than those in the serum (Hara et al., 1995). Although it is not clear what range of M-CSF concentrations are present in bovine milk,
Fig. 8. Change in serum M-CSF levels by postnatal age for calves in which M-CSF levels were measured on four separate days. The measured values for each calf are connected with an identifying line. Samples were assayed in duplicate. Data are shown with the mean S:D: for each group.
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Fig. 9. Serum M-CSF levels in six fetuses and the corresponding dams. The bars of the same pattern show the measured values for each fetus and its dam. Samples were assayed in duplicate. Data are shown with the median, mean S:D: for each group (the number of samples of dams’ serum is 5).
intake of M-CSF through the milk might influence M-CSF concentration in serum of calves. Because infectious diarrhea and pneumonia are major causes of death in neonatal calves and therefore a cause significant economic losses in the beef and dairy industries, investigations of the immunological activity of M-CSF and of the cellular immunity of calves showing low M-CSF level are warranted. Is M-CSF capable of crossing the placenta? M-CSF was found to cross the placenta in the mouse (Roth et al., 1998), but was found not to cross in the human (Saito et al., 1992). In the case of cattle, M-CSF levels in serum obtained from the umbilical artery and umbilical vein were almost the same (Fig. 9). This result may indicate that bovine M-CSF does not pass through the placenta, or that, even if bovine M-CSF can pass through the placenta, it does not actively move. Therefore, M-CSF in the serum is very probably produced in the bovine fetus. In this study, we described the establishment of an ELISA for bovine M-CSF and the evaluation of serum M-CSF levels in calves. Although the serum M-CSF levels decreased as the age of calves advanced, there were differences among individuals, and especially among newborn calves. This ELISA for bovine M-CSF should be useful for investigation of the role of M-CSF in the inflammatory response. Moreover, when bovine M-CSF levels in bovine sera and bovine standard M-CSF were assayed by
using a human M-CSF ELISA kit (R&D Systems Inc., Minneapolis, MN), the cross-reactivity was not actually recognized (data not shown). Because commercially available ELISA for human that are designed to have the low species cross-reactivity, ELISA established in this study should be a powerful tool to measure bovine M-CSF levels.
Acknowledgements This work was supported by a Grant-in-aid from the Recombinant Cytokine Project (PCR2002-2110), provided by the Ministry of Agriculture, Forestry and Fisheries, Japan.
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