HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Norup, L. R. Articles by Juul-Madsen, H. R. Search for Related Content PubMed PubMed Citation Articles by Norup, L. R. Articles by Juul-Madsen, H. R.

An Assay for Measuring the Mannan-Binding Lectin Pathway of Complement Activation in Chickens

Department of Animal Health, Welfare and Nutrition, Faculty of Agricultural Sciences, University of Aarhus, P.O. Box 50, DK-8830 Tjele, Denmark

¹ Corresponding author: liselotter.norup{at}agrsci.dk

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
To investigate the ability of chicken mannan-binding lectin (cMBL) to work as a complement activator, a heterogeneous ELISA test was developed, in which the deposition of human complement factor 4 (C4) was used as a measure of complement activation ability. Serum from different experimental chicken lines was tested. The correlation between serum cMBL concentrations and human C4 deposition was high (correlation = 0.8549, P < 0.0001). There was no difference in C4 deposition among sera from the chicken lines when calculated as C4 deposition relative to the cMBL concentration.

Key Words: chicken • mannan-binding lectin • mannan-binding lectin-associated serine protease 2 • complement activation • complement factor 4 deposition

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The acute-phase protein mannan-binding lectin (MBL) is a glycoprotein belonging to the C-type lectin superfamily. Mannan-binding lectin is a carbohydrate-binding protein that selectively recognizes a number of structural oligosaccharide components on the surface of microorganisms in the presence of calcium (Turner, 1996). When MBL has bound to a microorganism, the molecule acts directly as an opsonin, or it forms a complex with the MBL-associated serine protease (MASP2). This is an initiating complex of the MBL pathway of complement activation. Thereby, MBL plays a major role in the first-line innate immune defense against bacteria, viruses, and parasites. Similar to mammals, chickens produce MBL in the hepatocytes, and the MBL production is up-regulated during the acute stages of infections (Nielsen et al., 1999; Laursen and Nielsen, 2000; Juul-Madsen et al., 2003). In humans it is well known that a number of mutations in the MBL gene generate various degrees of malfunction or deficiency of the MBL molecule (Jack et al., 1997; Madsen et al., 1998). In chickens large differences in basic serum MBL concentrations have been observed, and mean serum MBL among chickens lines varies from 6.6 to 18.8 µg/mL (L. R. Norup, N. C. Friggens, Department of Health, Welfare and Nutrition, Faculty of Agricultural Sciences, University of Aarhus, Tjele, Denmark, and P. Sørensen, Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aarhus, Tjele, Denmark, unpublished data). Such large variations in serum concentrations of MBL may be associated with varying functionality in the chicken MBL. In this study, the objective was to develop an assay for the investigation of chicken MBL as an activator of the complement cascade in chicken lines of experimental origin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Blood samples from chickens of a variety of experimental lines were analyzed for chicken mannan-binding lectin (cMBL) concentration and complement activation. Blood was drawn and allowed to coagulate at room temperature for approximately 2 h before centrifugation and removal of serum. The serum was stored at –20°C until use. The cMBL levels of serum samples were determined, and the samples were tested for their complement factor 4 (C4)-depositing capacity.

Antibodies and Proteins

Mouse monoclonal anti-cMBL antibody (HYB 182-01), biotinylated mouse monoclonal anti-cMBL antibody (HYB 182-01), biotinylated mouse antihuman C4b (HYB 162-02), and biotinylated mouse antihuman C4 (HYB 162-04) were all purchased from Statens Serum Institut (Co-penhagen, Denmark). Human C4 was purchased from Kem-En-Tec A/S (Copenhagen, Denmark).

Chicken MBL Assay

Microtiter wells (Maxisorp, Nunc, Roskilde, Denmark) were coated with 0.5 µg of anti-cMBL antibody (HYB 182-01) in 100 µL of 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂PO₄, pH 7.4. After incubation overnight at 4°C and washing in 10 mM Tris, 100 mM NaCl, 0.05% (vol/vol) Tween 20, pH 7.6 (TBST wash), residual protein-binding sites were blocked by 200 µL of 0.5% (vol/vol) Tween 20 in 10 mM Tris, 140 mM NaCl, pH 7.6 (TBS), for 45 min at room temperature. After washing in the TBST wash, 100 µL of diluted serum in TBS with 10 mM CaCl₂ and 0.05% Tween 20 was added to the wells. Wells receiving only buffer were used as negative controls, and a serially diluted serum (7.1 µg of cMBL/mL) was used as the standard. All dilutions were added in duplicate. After incubation for 1.5 h at room temperature and washing with the TBST wash, the wells received 0.1 µg of biotinylated mouse anti-cMBL (HYB 182-01) in 100 µL of TBS with 0.05% Tween 20. After incubation for 1.5 h at room temperature and washing with the TBST wash, horseradish peroxidase (HRP)-conjugated streptavidin (P0397, Dako, Glostrup, Denmark) diluted 20,000-fold in TBS with 0.05% Tween 20 was added. After 1 h of incubation and washing with TBST wash, the presence of HRP was detected by adding 100 µL of substrate solution [< 0.05% (wt/wt) 3,3',5,5'' tetra-methylbenzidin]. Color development was stopped with a 1 M solution of H₂SO₄ and determined by reading the absorbance at 450 nm with absorbance at 650 nm as reference. Intra- and inter-assay variations were 7.3 and 7.6%, respectively, for the high serum control, and 4.7 and 10.6%, respectively, for the low serum control. A 2-fold dilution series of a normal chicken serum (stored at –20°C in aliquots) was used as the standard.

Assay for Complement Deposition by the MBL Pathway

Microtiter wells (Maxisorp) were coated with 0.2 µg of mannan (Statens Serum Institut) in 100 µL of 15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6 (coating buffer). After incubation overnight at 4°C, residual protein-binding sites were blocked by adding 250 µL of 0.1% (wt/vol) BSA in 10 mM Tris, 140 mM NaCl, pH 7.6, for at least 2 h. After washing in 10 mM Tris, 140 mM NaCl, 5 mM CaCl₂, 0.05% Tween 20, pH 7.4 (TBST-Ca²⁺), dilutions of sera in 20 mM Tris, 1 M NaCl, 10 mM CaCl₂, 0.1% BSA, pH 7.4, 0.05% Triton X-100 (dilution buffer) were added. Wells receiving only buffer were used as negative controls, and all dilutions were added in duplicate. After incubation overnight at 4°C and washing with TBST-Ca²⁺, the wells received 0.25 µg of human C4 in 4 mM barbital, 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.1% NaN₃, pH 7.4 (barbital buffer). Wells were then incubated for 1.5 h at 37°C for decomposition of C4 in C4a and C4b and deposition of C4 and C4b on the surface of the wells. After washing with TBST-Ca²⁺, deposited C4 was detected by adding 0.06 µg of biotinylated monoclonal antibody (0.03 µg of HYB 162-02 and 0.03 µg of HYB 162-04) in 100 µL of TBST-Ca²⁺. The wells were incubated for 1.5 h at room temperature and then washed with TBST-Ca²⁺. Horseradish peroxidase-conjugated streptavidin (P0397, Dako) was then added in a 1:10,000-fold dilution in TBST-Ca²⁺ and incubated for 1 h at room temperature. After incubation, wells were washed in TBST-Ca²⁺, and HRP was detected by adding the substrate solution [< 0.05% (wt/wt) 3,3',5,5'' tetramethylbenzidin] at 100 µL per well. Color development was stopped with a 1-M solution of H₂SO₄ and determined by reading the absorbance at 450 nm, with absorbance at 650 nm as reference. Serial dilutions of a serum arbitrarily set at 1,000 mU/mL were used as a standard. Four sera with different mean values of complement deposition activity were used to assess intra- and interassay variations. Mean complement deposition activity, inter- and intraassay variations for those 4 sera were 1) 36.8 mU/mL, 2.8 and 3.3%; 2) 139.3 mU/mL, 1.3 and 7.6%; 3) 1,078.7 mU/mL, 7.9 and 8.2%; and 4) 1,557.4 mU/mL, 9.9 and 5.2%.

To evaluate a possible contribution from the classical pathway, some microtiter plates were coated with 1 µg of purified chicken IgG (I 4881, Sigma-Aldrich, Brøndby, Denmark) in 100 µL of coating buffer per well, and sera were diluted in either dilution buffer (1 M NaCl) or barbital buffer (0.145 M NaCl).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
In this study we established an assay to test the C4-and C4b-depositing capacity of the chicken MBL complex as a means of evaluating the complement-activating capacity of the chicken MBL-MASP complex. The assay was established on the basis of an existing human assay (Petersen et al., 2001) and adjusted to the chicken serum components. Laursen (1998) showed that it was possible to deposit C4 and C4b from human serum on the chicken MBL-MASP complex, but quantification of the complement activation capacity by the lectin pathway in chicken was not conducted. Our assay quantitively determines the ability of the chicken MBL-MASP complex to deposit human C4 and C4b.

For the preliminary tests, we chose 2 sera, 1 with a very high (35.7 µg/mL) and 1 with a very low (2.7 µg/mL) concentration of cMBL. These were tested in wells coated with either mannan or purified chicken IgG and diluted in either a hypertonic (1 M NaCl) or an isotonic (0.145 M NaCl) buffer. On the surface of the mannan-coated wells, the deposition of C4 and C4b was independent of the ionic strength of the dilution buffer (Figure 1). Under the same conditions, C4 and C4b deposition on the surface of the IgG-coated wells was small when the isotonic dilution buffer was used, and was almost totally inhibited by using the hypertonic buffer for serum dilution (Figure 2). Figures 1 and 2 show that nearly the entire contribution to C4 and C4b deposition from the classical pathway was inhibited when the buffer with high ionic strength was used for serum dilution (Figure 2), whereas the contribution from the lectin pathway was independent of the ionic strength in the serum dilution buffer (Figure 1). The present findings are in agreement with those found by Petersen et al. (2001) when they established the human assay for complement activation by MBL.

View larger version (14K): [in this window] [in a new window]   Figure 1. Deposition of complement factors C4 and C4b, determined in an ELISA in which mannan-coated microtiter wells were used for capturing chicken mannan-binding lectin (cMBL). Results are shown as the optical density (OD) at 450 nm, with 650 nm as the reference. Results were obtained from serial dilutions of serum, diluted either in buffer with isotonic (0.145 M NaCl; cMBL 2.7 µg/mL ——, cMBL 35.7 µg/mL ——) or hypertonic (1 M NaCl; cMBL 2.7 µg/mL ——, cMBL 35.7 µg/mL ——) ionic strength. Points represent means, and error bars represent standard deviations of duplicates (SD was less than 0.125 at all points).

View larger version (13K): [in this window] [in a new window]   Figure 2. Deposition of complement factors C4 and C4b in an ELISA with IgG-coated microtiter wells. Results are shown as the optical density (OD) at 450 nm, with 650 nm as the reference, and were obtained from serial dilutions of serum diluted in buffer with either isotonic (0.145 M NaCl; cMBL 2.7 µg/mL ——, cMBL 35.7 µg/mL ——) or hypertonic (1 M NaCl; cMBL 2.7 µg/mL ——), cMBL 35.7 µg/mL ——) ionic strength. Points represent means, and error bars represent the standard deviations of duplicates (SD was less than 0.110 at all points).

View larger version (14K): [in this window] [in a new window]   Figure 3. Deposition of complement factors C4 and C4b in an ELISA in which mannan-coated microtiter wells were used for capturing chicken mannan-binding lectin (cMBL). The results are shown as the optical density (OD) at 450 nm, with 650 nm as a reference, and were obtained from 3-fold serial dilutions of 14 sera from chickens with different cMBL concentrations. Sera equally represent cMBL concentrations ranging from 1.6 to 35.7 µg/mL, and dilution curves are displaced according to the cMBL level in the serum.

View larger version (10K): [in this window] [in a new window]   Figure 4. Correlation between the chicken mannan-binding lectin (cMBL) concentration and deposition of complement factors C4 and C4b (mU/mL) in sera from 170 chickens of experimental lines (correlation = 0.8549, P < 0.0001). Results were obtained from an ELISA in which mannan-coated microtiter wells were used for capturing cMBL. Serial dilutions of a serum arbitrarily set at 1,000 mU/mL were used as a standard. Sera used for determining the C4- and C4b-depositing capacity represent cMBL concentrations ranging from 1.6 to 32.3 µg/mL.

	ACKNOWLEDGMENTS

 
We wish to thank Jens Christian Jensenius and Lisbeth Jensen for introducing the human assay to us, and Claus Koch for supplying the anti-C4 antibodies.

Received for publication June 12, 2007. Accepted for publication July 5, 2007.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Jack, D., J. Bidwell, M. Turner, and N. Wood. 1997. Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene. Hum. Mutat. 9:41–46.[CrossRef][Web of Science][Medline]

Juul-Madsen, H. R., M. Munch, K. J. Handberg, P. Sørensen, A. A. Johnson, L. R. Norup, and P. H. Jørgensen. 2003. Serum levels of mannan-binding lectin (MBL) in chickens prior to and during experimental infection with avian infectious bronchitis virus (IBV). Poult. Sci. 82:235–241.[Abstract/Free Full Text]

Laursen, S. B. 1998. Mannan-binding lectin (MBL) in chickens. Identification, characterization, cloning and functional aspects. PhD Thesis. Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark.

Laursen, S. B., and O. L. Nielsen. 2000. Mannan-binding lectin (MBL) in chickens: Molecular and functional aspects. Dev. Comp. Immunol. 24:85–101.[CrossRef][Web of Science][Medline]

Madsen, H. O., M. L. Satz, B. Hogh, A. Svejgaard, and P. Garred. 1998. Different molecular events result in low protein levels of mannan-binding lectin in populations from Southeast Africa and South America J. Immunol. 161:3169–3175.

Nielsen, O. L., J. C. Jensenius, P. H. Jørgensen, and S. B. Laursen. 1999. Serum levels of chicken mannan-binding lectin (MBL) during virus infections; indication that chicken MBL is an acute phase reactant. Vet. Immunol. Immunopathol. 70:309–316.[CrossRef][Web of Science][Medline]

Petersen, S. V., S. Thiel, L. Jensen, R. Steffensen, and J. C. Jensenius. 2001. An assay for the mannan-binding lectin pathway of complement activation. J. Immunol. Methods 257:107–116.[CrossRef][Web of Science][Medline]

Turner, M. W. 1996. Mannose-binding lectin: The pluripotent molecule of the innate immune system. Immunol. Today 17:532–540.[CrossRef][Web of Science][Medline]

This article has been cited by other articles:

				L. Star, H. R. Juul-Madsen, E. Decuypere, M. G. B. Nieuwland, G. de Vries Reilingh, H. van den Brand, B. Kemp, and H. K. Parmentier Effect of early life thermal conditioning and immune challenge on thermotolerance and humoral immune competence in adult laying hens Poult. Sci., November 1, 2009; 88(11): 2253 - 2261. [Abstract] [Full Text] [PDF]

				L. R. Norup, T. S. Dalgaard, N. C. Friggens, P. Sorensen, and H. R. Juul-Madsen Influence of chicken serum mannose-binding lectin levels on the immune response towards Escherichia coli Poult. Sci., March 1, 2009; 88(3): 543 - 553. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Norup, L. R. Articles by Juul-Madsen, H. R. Search for Related Content PubMed PubMed Citation Articles by Norup, L. R. Articles by Juul-Madsen, H. R.