Journal of lmmunological Methods, 156 (1992) 79-83
79
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06491
Chicken antibodies: a tool to avoid interference by complement activation in ELISA A n d e r s Larsson, P~ir-Erik Wej~ker, Per-Olof Forsberg and Tomas Lindahl Department of Clinical Chemistry, UniversityHospital, S-751 85 Uppsala, Sweden (Received 31 March 1992, revised received 2 June 1992, accepted 8 June 1992)
MicroELISA plates coated with mammalian IgG will activate the human complement system. It has been shown that this activation of the complement system may interfere in solid-phase immunometric assays, and that there is a difference between IgG from different species and between different IgG subclasses in their ability to activate the human complement system. We have studied the ability of mammalian IgG and avian IgG to activate the human complement system. We show that chicken IgG do not activate the human complement system, and chicken IgG can thus be used in solid-phase immunometric assays to reduce interference by complement activation. Key words: Complement component; C3; C4; C5; Chicken antibody; ELISA
Introduction
The use of two-site immunoassays in clinical chemistry has increased greatly over the past few years, due to the speed and sensitivity of the assays. However, there have been several reports of endogenous interference by components in the test samples (Boscato and Stuart, 1988; Johnson and Page Faulk, 1976; Weber et al., 1990). The problem of interference will increase as the sensitivity of the assays increases. Endogenous interferences are difficult to predict and to eliminate, since they vary both between patients and in the same patient from time to time. The most well known of these interferences is heterophilic IgG antibodies that have been demonstrated in up to 40% of patient samples (Boscato and Stuart, Correspondence to: A. Larsson, Department o f Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. Tel.: 46-18-663000; Fax: 46-18-552562.
1986). Rheumatoid factor (RF) and human antimouse IgG antibodies (HAMA) in the samples may react with mammalian polyclonal or monoclonal antibodies and cause false positive results in sandwich assays. We have previously shown that chicken antibodies can be used to avoid interference due to RF (Larsson et al., 1991) or HAMA (Larsson and Mellstedt, 1992). When normal human serum is added to microELISA plates coated with human IgG, the complement system in the samples is activated and the complement components are bound to the antibodies (Larsson and Sj6quist, 1989). It has been shown that immune precipitates can be re-solubilized by complement (Miller and Nussenzweig, 1975), and that complement present during the antigen-antibody reaction prevents the formation of insoluble complexes (Schifferli et al., 1982). Recent reports show that complement activation may interfere with antigen binding to the capture antibody in a immunometric TSH assay (K~ipyaho et al., 1989).
80
IgG from different species have different ability to activate the human complement system (Gigli and Austen, 1971). We have therefore studied the ability of IgG from different species to activate the human complement system. We show that microtiter plates coated with chicken antibodies do not activate the human complement system. Chicken antibodies can thus be used in immunological assays to avoid interference due to complement activation.
Materials and methods
Antisera Purified mouse IgG and rat IgG were obtained from Sigma Chemical Co. (St. Louis, MO) and human IgG was obtained from Kabi (Stockholm, Sweden). Rabbit IgG was purified from rabbit serum by affinity chromatography on Protein ASepharose. The chicken antibodies, rabbit antichicken IgG and biotinylated chicken anti-human IgG, anti-human C3, anti-human C4, anti-human C5 and anti-C-reactive protein (CRP) were provided by Immunsystem, Uppsala, Sweden. Test sera Sera or plasma from healthy blood donors were used. The sera were stored at - 70° C until assayed. Heat inactivated serum was prepared by incubating serum at 56°C for 30 min. Hydrazine treatment of serum was performed as previously described (Larsson and Sj6quist, 1988).
M NaCI, 1 mM MgC12, 0.15 mM CaCI2, pH 7.4) were added to the wells in duplicate. The plates were incubated for 2 h at 37°C, and then washed three times with NaC1-Tween. 200 /zl of the biotinylated antibodies, diluted 1/1000 in PBS, were added. The plates were incubated for 2 h at 37°C. After washing three times with NaC1Tween, 200 /xl of streptavidin-alkaline phosphatase (Pierce Europe, Netherlands), diluted 1/1000 in PBS, were added. After 2 h of incubation at 37°C, the plates were washed and 200/xl of 1 m g/ m l of p-nitrophenyl phosphate in 1 M diethanolamine, 0.5 mM MgC12, pH 9.8 were added. The plates were incubated for 25 min at room temperature in the dark. The plates were read in a Titertek Multiskan MC 340 (Flow Laboratories, Scotland).
Results
Binding of complement components to IgG coated plates When normal serum was added to plates coated with mammalian IgG the complement system was activated and C3 was bound to the plates (Fig. 1). Low binding was seen in heat inactivated serum. Low binding was also observed with any of 2.0
1.5
1.0
Binding of complement components to IgG MicroELISA plates (Dynatech M 129 A) were coated with 100 /xg human IgG, 10 ~g mouse IgG or 10 txg rat I g G / m l of 0.1 M NaHCO 3 pH 9.5, or 100 /~g chicken I g G / m l of PBS (0.15 M NaC1, 0.02 M NaH2PO 4, 0.02% NaN 3, pH 7.2) for 2 h at 37°C, or overnight at 4°C. The plates coated with mouse and rat IgG were blocked with 100 txg bovine serum albumin (BSA)/ml of PBS for 2 h at 37°C, or overnight at 4°C. The plates were washed three times with NaC1-Tween (0.9% NaC1, 0.05% Tween 20, 0.02% NaN 3) with a Wellwash 4 washing machine (Denley Instruments, Billingshurst, UK). 200/zl of the serum in various dilutions in VBS (0.045 M barbital, 0.15
i
0.5 0
1:390,625
1:15,625 1:625 Dilution of human serum
1:25
Fig. 1. Binding of h u m a n complement component C3 to microtiter plates coated with mammalian or avian IgG. Various dilutions of normal h u m a n serum or heat inactivated h u m a n serum were added to the plates. Biotinylated anti-hum a n C3 antibody and steptavidin-alkaline phosphatase conjugate were used for detection of bound C3. The absorbance values of the heat inactivated serum were subtracted from the values of the normal h u m a n serum. H u m a n IgG (filled circle), mouse IgG (filled square), rabbit IgG (grey square), rat IgG (white square), chicken IgG (white circle).
81
methods section with biotinylated chicken antiCRP in dilution 1/1000. No difference in the results was observed between samples diluted with or without EDTA. Thus, complement activation does not influence the CRP results.
1.2
~o
0.8
i
0.4
g
Discussion
0
1:390 625
1:15,625
1:625
1:25
Dilution of human serum
Fig. 2. Binding of human complement component C5 to microtiter plates coated with mammalian or avian IgG. Various dilutions of normal human serum or heat inactivated human serum were added to the plates. Biotinylated anti-human C5 antibody and streptavidin-alkaline phosphatase conjugate were used for detection of bound C5. The absorbance values of the heat inactivated serum were subtracted from the values of the normal human serum. Human IgG (filled circle), mouse IgG (filled square), rabbit IgG (grey square), rat IgG (white square), chicken IgG (white circle).
the serum when the plates were coated with chicken IgG or ovalbumin. No difference was observed between plates coated with chicken IgG or ovalbumin. Similar results were obtained with C4 and C5 (Fig. 2). Addition of EDTA to the buffer inhibited the binding of complement components to mammalian IgG. Human IgG could be detected bound to plates coated with chicken IgG or ovalbumin with the highest concentrations of serum (results not shown). Thus, the complement components bound to chicken IgG and ovalbumin with the highest concentration of serum are probably due to activation of the complement system by human IgG bound to the plate.
C-reactive protein (CRP) ELISA MicroELISA plates (Dynatech M 129 A) were coated overnight at 4°C with 2.5/zg affinity-purified chicken anti-CRP/ml of 0.1 M NaHCO 3 pH 9.5. The plates were then blocked with 100 /xg chicken I g G / m l of PBS (0.15 M NaCI, 0.02 M NaH2PO4, 0.02% NAN3, pH 7.2) for 2 h at 37°C. The sera were added to the wells in five-fold dilutions in VBS or VBS with 10 mM EDTA. All sera were run in duplicate. The CRP ELISA was performed as described in the materials and
Many immunological assays utilize an IgG antibody bound to a solid phase to capture the antigen. The antigen and the antibody thus form an immune complex. The human complement system can be activated via the classical pathway by antigen-antibody complexes. The initial step in this activation is the binding of Clq to the complex. A later step in the complement cascade is the activation of C4 and the formation of C4b. It has been shown that C4b and the antibody form a very stable complex and that approximately 80% of the C4b bound to IgG is located in the Fab region (Campbell et al., 1980). C3 is bound to the antigen-antibody complex via a similar binding as for C4. It is likely that a protein bound to the Fab region may influence the antigen binding region. It has been proposed that the result of the incorporation of C4 and C3 into the antigen-antibody lattice leads to a reduction in the effective valency of antigen and antibody (Lachmann and Walport, 1987). Complement activation may thus inhibit the binding of the antigen to the antibody. It has also been shown that complement binds to human, murine or rabbit antibodies bound to a solid surface (Larsson and Sj6quist, 1989; K~ipyaho et al., 1989), and that complement activation interferes with antigen-binding in a sandwich radioimmunoassay and may depress the TSH values with up to 40%. There are great differences between the ability of antibodies from different species to activate complement from other species. It has previously been shown that chicken antibodies are unable to activate the complement system in sera from rabbit, guinea pig, ox, rat, goat, cat or dog. A slight complement activation was observed with pig serum (Gigli and Austen, 1971). In this work we show that microtiter plates coated with human, mouse, rabbit or rat IgG were strong activators of the complement system
82
in h u m a n serum and that C4b and C3b are bound to the plates coated with mammalian IgG. Little or no activation was observed with microtiter plates coated with chicken I g G or ovalbumin. This activation is due to activation of the complement system by human I g G unspecifically bound to the plate. H e a t inactivation, addition of E D T A or hydrazine treatment of serum inhibited the binding of C4b, C3b and C5 to all I g G tested. We used a C R P as a model for an antigen specific assay utilizing chicken IgG as capture antibody. There were no significant differences in the C R P results when analyzing normal human serum or heat inactivated serum, with or without addition of E D T A . Thus, assays with chicken antibodies as capture antibodies are not influenced by an active complement system in the samples. E D T A can also be used to avoid complement interference, but it has a couple of disadvantages. E D T A can not be used together with europium or samarium labelled antibodies, as this will block the reaction. It is also practical that assays in clinical laboratories can be performed with more than one kind of serum or plasma sample. Immunization of chicken is similar to the immunization of rabbits and chicken antibodies can then be easily purified from egg yolk (Jensenius et al., 1981; L6sch et al., 1986). The amount of antibody produced in egg yolk from a chicken is higher than the amount produced in a rabbit during a similar time. Thus, there is no problem in producing chicken antibodies for the laboratory, and there are also many chicken antibodies commercially available. Chicken antibodies seem to be very stable. The antibody loss in I g G fractions stored at 4°C in 0.9% NaC1, 0.1% N a N 3 after 6 - 7 years storage was less than 5% determined by precipitation in tubes. The affinity of chicken antibodies are similar to the affinity of guinea pig and mouse antibodies in E L I S A (Lee et al., 1991). We have previously reported that chicken antibodies can be used in immunological methods to reduce false positive results due to heterophilic antibodies such as rheumatoid factors (Larsson et al., 1991) or h u m a n anti-mouse I g G antibodies after in vivo treatment with mouse monoclonal antibodies (Larsson and Mellstedt., 1992). In this work we show that chicken antibodies also can be
used to avoid false negative results due to complement activation.
Acknowledgements The authors wish to thank Ms. Camilla Blackh a m m a r for excellent technical assistance. This work was supported by the Swedish Medical Research Council (project no. 09875), the Swedish Society of Medicine and Tore Nilssons Foundation.
References Boscato, L.M. and Stuart, M.C. (1986) Incidence and specificity of interference in two-site immunoassays. Clin. Chem. 32, 1491. Boscato, L.M. and Stuart, M.C. (1988) Heterophilic antibodies, a problem for all immunoassays. Clin. Chem. 34, 27. Campbell, R.D., Dodds, A.W. and Porter, R.R. (1980) The binding of human complement component C4 to antibody-antigen aggregates. Biochem. J. 189, 67. Gigli, I. and Austen, K.F. (1971) Phylogeny and function of the human complement system. Ann. Rev. Microbiology 25, 309. Jensenius, J.C., Andersen, I., Hau, J., Crone, M. and Koch, C. (1981) Eggs, conveniently packaged antibodies. Methods for purification of yolk IgG. J. Immunol. Methods 46, 63. Johnson, P.M. and Page Faulk, W. (1976) Rheumatoid factor, its nature, specificity and production in rheumatoid arthritis. Clin. Immunol. Immunopathol. 6, 416. K~ipyaho, K., Tanner, P. and Weber, T. (1989) Effect of complement binding on a solid-phase immunometric TSH assay. Scand. J. Clin. Lab. Invest. 49, 211. Lachmann, P.J. and Walport, M.J. (1987) Deficiency of the effector mechanisms of the immune response and autoimmunity. Ciba Found. Symp. 129, 149. Larsson, A. and Sj6quist, J. (1988) Quantification of circulating immune complexes by chicken anti-C4 microELISA.J. Clin. Lab. Immunol. 27, 39. Larsson, A. and Sj6quist, J. (1989) Binding of complement component Clq, C3, C4 and C5 to a model immune complex in ELISA.J. Immunol. Methods 119, 103. Larsson, A., Karlsson-Parra, A. and Sj6quist, J. (1991) Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors. Clin. Chem. 37, 411. Larsson, A. and Mellstedt, H. (1992) Chicken antibodies, a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodies. Hybridoma 1, 33. Lee, K., Ametani, A., Shimizu, M., Hana, H., Yamamoto, T. and Kaminogawa, S. (1991) Production and characterization of anti-human insulin antibodies in the hen's egg. Agric. Biol. Chem. 55, 2141.
83 L6sch, U., Schranner, I., Wanke, R. and Jurgens, L. (1986) The chicken egg, an antibody source. J. Vet. Med. 33, 609. Miller, G.W. and Nussenzweig, V. (1975) A new complement function, solubilization of antigen-antibody aggregates. Proc. Natl. Acad. Sci. USA 72, 418. Schifferli, J.A., Woo, P. and Peters, D.K. (1982) Complement-
mediated inhibition of immune precipitation. 1. Role of the classical and alternative pathways. Clin. Exp. Immunol. 47, 555. Weber, T.H., K~ipyaho, K.I. and Tanner, P. (1990) Endogenous interference in immunoassays in clinical chemistry. A review. Scand. J. Clin. Lab. Invest. 50, Suppl 201, 77.
79
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06491
Chicken antibodies: a tool to avoid interference by complement activation in ELISA A n d e r s Larsson, P~ir-Erik Wej~ker, Per-Olof Forsberg and Tomas Lindahl Department of Clinical Chemistry, UniversityHospital, S-751 85 Uppsala, Sweden (Received 31 March 1992, revised received 2 June 1992, accepted 8 June 1992)
MicroELISA plates coated with mammalian IgG will activate the human complement system. It has been shown that this activation of the complement system may interfere in solid-phase immunometric assays, and that there is a difference between IgG from different species and between different IgG subclasses in their ability to activate the human complement system. We have studied the ability of mammalian IgG and avian IgG to activate the human complement system. We show that chicken IgG do not activate the human complement system, and chicken IgG can thus be used in solid-phase immunometric assays to reduce interference by complement activation. Key words: Complement component; C3; C4; C5; Chicken antibody; ELISA
Introduction
The use of two-site immunoassays in clinical chemistry has increased greatly over the past few years, due to the speed and sensitivity of the assays. However, there have been several reports of endogenous interference by components in the test samples (Boscato and Stuart, 1988; Johnson and Page Faulk, 1976; Weber et al., 1990). The problem of interference will increase as the sensitivity of the assays increases. Endogenous interferences are difficult to predict and to eliminate, since they vary both between patients and in the same patient from time to time. The most well known of these interferences is heterophilic IgG antibodies that have been demonstrated in up to 40% of patient samples (Boscato and Stuart, Correspondence to: A. Larsson, Department o f Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. Tel.: 46-18-663000; Fax: 46-18-552562.
1986). Rheumatoid factor (RF) and human antimouse IgG antibodies (HAMA) in the samples may react with mammalian polyclonal or monoclonal antibodies and cause false positive results in sandwich assays. We have previously shown that chicken antibodies can be used to avoid interference due to RF (Larsson et al., 1991) or HAMA (Larsson and Mellstedt, 1992). When normal human serum is added to microELISA plates coated with human IgG, the complement system in the samples is activated and the complement components are bound to the antibodies (Larsson and Sj6quist, 1989). It has been shown that immune precipitates can be re-solubilized by complement (Miller and Nussenzweig, 1975), and that complement present during the antigen-antibody reaction prevents the formation of insoluble complexes (Schifferli et al., 1982). Recent reports show that complement activation may interfere with antigen binding to the capture antibody in a immunometric TSH assay (K~ipyaho et al., 1989).
80
IgG from different species have different ability to activate the human complement system (Gigli and Austen, 1971). We have therefore studied the ability of IgG from different species to activate the human complement system. We show that microtiter plates coated with chicken antibodies do not activate the human complement system. Chicken antibodies can thus be used in immunological assays to avoid interference due to complement activation.
Materials and methods
Antisera Purified mouse IgG and rat IgG were obtained from Sigma Chemical Co. (St. Louis, MO) and human IgG was obtained from Kabi (Stockholm, Sweden). Rabbit IgG was purified from rabbit serum by affinity chromatography on Protein ASepharose. The chicken antibodies, rabbit antichicken IgG and biotinylated chicken anti-human IgG, anti-human C3, anti-human C4, anti-human C5 and anti-C-reactive protein (CRP) were provided by Immunsystem, Uppsala, Sweden. Test sera Sera or plasma from healthy blood donors were used. The sera were stored at - 70° C until assayed. Heat inactivated serum was prepared by incubating serum at 56°C for 30 min. Hydrazine treatment of serum was performed as previously described (Larsson and Sj6quist, 1988).
M NaCI, 1 mM MgC12, 0.15 mM CaCI2, pH 7.4) were added to the wells in duplicate. The plates were incubated for 2 h at 37°C, and then washed three times with NaC1-Tween. 200 /zl of the biotinylated antibodies, diluted 1/1000 in PBS, were added. The plates were incubated for 2 h at 37°C. After washing three times with NaC1Tween, 200 /xl of streptavidin-alkaline phosphatase (Pierce Europe, Netherlands), diluted 1/1000 in PBS, were added. After 2 h of incubation at 37°C, the plates were washed and 200/xl of 1 m g/ m l of p-nitrophenyl phosphate in 1 M diethanolamine, 0.5 mM MgC12, pH 9.8 were added. The plates were incubated for 25 min at room temperature in the dark. The plates were read in a Titertek Multiskan MC 340 (Flow Laboratories, Scotland).
Results
Binding of complement components to IgG coated plates When normal serum was added to plates coated with mammalian IgG the complement system was activated and C3 was bound to the plates (Fig. 1). Low binding was seen in heat inactivated serum. Low binding was also observed with any of 2.0
1.5
1.0
Binding of complement components to IgG MicroELISA plates (Dynatech M 129 A) were coated with 100 /xg human IgG, 10 ~g mouse IgG or 10 txg rat I g G / m l of 0.1 M NaHCO 3 pH 9.5, or 100 /~g chicken I g G / m l of PBS (0.15 M NaC1, 0.02 M NaH2PO 4, 0.02% NaN 3, pH 7.2) for 2 h at 37°C, or overnight at 4°C. The plates coated with mouse and rat IgG were blocked with 100 txg bovine serum albumin (BSA)/ml of PBS for 2 h at 37°C, or overnight at 4°C. The plates were washed three times with NaC1-Tween (0.9% NaC1, 0.05% Tween 20, 0.02% NaN 3) with a Wellwash 4 washing machine (Denley Instruments, Billingshurst, UK). 200/zl of the serum in various dilutions in VBS (0.045 M barbital, 0.15
i
0.5 0
1:390,625
1:15,625 1:625 Dilution of human serum
1:25
Fig. 1. Binding of h u m a n complement component C3 to microtiter plates coated with mammalian or avian IgG. Various dilutions of normal h u m a n serum or heat inactivated h u m a n serum were added to the plates. Biotinylated anti-hum a n C3 antibody and steptavidin-alkaline phosphatase conjugate were used for detection of bound C3. The absorbance values of the heat inactivated serum were subtracted from the values of the normal h u m a n serum. H u m a n IgG (filled circle), mouse IgG (filled square), rabbit IgG (grey square), rat IgG (white square), chicken IgG (white circle).
81
methods section with biotinylated chicken antiCRP in dilution 1/1000. No difference in the results was observed between samples diluted with or without EDTA. Thus, complement activation does not influence the CRP results.
1.2
~o
0.8
i
0.4
g
Discussion
0
1:390 625
1:15,625
1:625
1:25
Dilution of human serum
Fig. 2. Binding of human complement component C5 to microtiter plates coated with mammalian or avian IgG. Various dilutions of normal human serum or heat inactivated human serum were added to the plates. Biotinylated anti-human C5 antibody and streptavidin-alkaline phosphatase conjugate were used for detection of bound C5. The absorbance values of the heat inactivated serum were subtracted from the values of the normal human serum. Human IgG (filled circle), mouse IgG (filled square), rabbit IgG (grey square), rat IgG (white square), chicken IgG (white circle).
the serum when the plates were coated with chicken IgG or ovalbumin. No difference was observed between plates coated with chicken IgG or ovalbumin. Similar results were obtained with C4 and C5 (Fig. 2). Addition of EDTA to the buffer inhibited the binding of complement components to mammalian IgG. Human IgG could be detected bound to plates coated with chicken IgG or ovalbumin with the highest concentrations of serum (results not shown). Thus, the complement components bound to chicken IgG and ovalbumin with the highest concentration of serum are probably due to activation of the complement system by human IgG bound to the plate.
C-reactive protein (CRP) ELISA MicroELISA plates (Dynatech M 129 A) were coated overnight at 4°C with 2.5/zg affinity-purified chicken anti-CRP/ml of 0.1 M NaHCO 3 pH 9.5. The plates were then blocked with 100 /xg chicken I g G / m l of PBS (0.15 M NaCI, 0.02 M NaH2PO4, 0.02% NAN3, pH 7.2) for 2 h at 37°C. The sera were added to the wells in five-fold dilutions in VBS or VBS with 10 mM EDTA. All sera were run in duplicate. The CRP ELISA was performed as described in the materials and
Many immunological assays utilize an IgG antibody bound to a solid phase to capture the antigen. The antigen and the antibody thus form an immune complex. The human complement system can be activated via the classical pathway by antigen-antibody complexes. The initial step in this activation is the binding of Clq to the complex. A later step in the complement cascade is the activation of C4 and the formation of C4b. It has been shown that C4b and the antibody form a very stable complex and that approximately 80% of the C4b bound to IgG is located in the Fab region (Campbell et al., 1980). C3 is bound to the antigen-antibody complex via a similar binding as for C4. It is likely that a protein bound to the Fab region may influence the antigen binding region. It has been proposed that the result of the incorporation of C4 and C3 into the antigen-antibody lattice leads to a reduction in the effective valency of antigen and antibody (Lachmann and Walport, 1987). Complement activation may thus inhibit the binding of the antigen to the antibody. It has also been shown that complement binds to human, murine or rabbit antibodies bound to a solid surface (Larsson and Sj6quist, 1989; K~ipyaho et al., 1989), and that complement activation interferes with antigen-binding in a sandwich radioimmunoassay and may depress the TSH values with up to 40%. There are great differences between the ability of antibodies from different species to activate complement from other species. It has previously been shown that chicken antibodies are unable to activate the complement system in sera from rabbit, guinea pig, ox, rat, goat, cat or dog. A slight complement activation was observed with pig serum (Gigli and Austen, 1971). In this work we show that microtiter plates coated with human, mouse, rabbit or rat IgG were strong activators of the complement system
82
in h u m a n serum and that C4b and C3b are bound to the plates coated with mammalian IgG. Little or no activation was observed with microtiter plates coated with chicken I g G or ovalbumin. This activation is due to activation of the complement system by human I g G unspecifically bound to the plate. H e a t inactivation, addition of E D T A or hydrazine treatment of serum inhibited the binding of C4b, C3b and C5 to all I g G tested. We used a C R P as a model for an antigen specific assay utilizing chicken IgG as capture antibody. There were no significant differences in the C R P results when analyzing normal human serum or heat inactivated serum, with or without addition of E D T A . Thus, assays with chicken antibodies as capture antibodies are not influenced by an active complement system in the samples. E D T A can also be used to avoid complement interference, but it has a couple of disadvantages. E D T A can not be used together with europium or samarium labelled antibodies, as this will block the reaction. It is also practical that assays in clinical laboratories can be performed with more than one kind of serum or plasma sample. Immunization of chicken is similar to the immunization of rabbits and chicken antibodies can then be easily purified from egg yolk (Jensenius et al., 1981; L6sch et al., 1986). The amount of antibody produced in egg yolk from a chicken is higher than the amount produced in a rabbit during a similar time. Thus, there is no problem in producing chicken antibodies for the laboratory, and there are also many chicken antibodies commercially available. Chicken antibodies seem to be very stable. The antibody loss in I g G fractions stored at 4°C in 0.9% NaC1, 0.1% N a N 3 after 6 - 7 years storage was less than 5% determined by precipitation in tubes. The affinity of chicken antibodies are similar to the affinity of guinea pig and mouse antibodies in E L I S A (Lee et al., 1991). We have previously reported that chicken antibodies can be used in immunological methods to reduce false positive results due to heterophilic antibodies such as rheumatoid factors (Larsson et al., 1991) or h u m a n anti-mouse I g G antibodies after in vivo treatment with mouse monoclonal antibodies (Larsson and Mellstedt., 1992). In this work we show that chicken antibodies also can be
used to avoid false negative results due to complement activation.
Acknowledgements The authors wish to thank Ms. Camilla Blackh a m m a r for excellent technical assistance. This work was supported by the Swedish Medical Research Council (project no. 09875), the Swedish Society of Medicine and Tore Nilssons Foundation.
References Boscato, L.M. and Stuart, M.C. (1986) Incidence and specificity of interference in two-site immunoassays. Clin. Chem. 32, 1491. Boscato, L.M. and Stuart, M.C. (1988) Heterophilic antibodies, a problem for all immunoassays. Clin. Chem. 34, 27. Campbell, R.D., Dodds, A.W. and Porter, R.R. (1980) The binding of human complement component C4 to antibody-antigen aggregates. Biochem. J. 189, 67. Gigli, I. and Austen, K.F. (1971) Phylogeny and function of the human complement system. Ann. Rev. Microbiology 25, 309. Jensenius, J.C., Andersen, I., Hau, J., Crone, M. and Koch, C. (1981) Eggs, conveniently packaged antibodies. Methods for purification of yolk IgG. J. Immunol. Methods 46, 63. Johnson, P.M. and Page Faulk, W. (1976) Rheumatoid factor, its nature, specificity and production in rheumatoid arthritis. Clin. Immunol. Immunopathol. 6, 416. K~ipyaho, K., Tanner, P. and Weber, T. (1989) Effect of complement binding on a solid-phase immunometric TSH assay. Scand. J. Clin. Lab. Invest. 49, 211. Lachmann, P.J. and Walport, M.J. (1987) Deficiency of the effector mechanisms of the immune response and autoimmunity. Ciba Found. Symp. 129, 149. Larsson, A. and Sj6quist, J. (1988) Quantification of circulating immune complexes by chicken anti-C4 microELISA.J. Clin. Lab. Immunol. 27, 39. Larsson, A. and Sj6quist, J. (1989) Binding of complement component Clq, C3, C4 and C5 to a model immune complex in ELISA.J. Immunol. Methods 119, 103. Larsson, A., Karlsson-Parra, A. and Sj6quist, J. (1991) Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors. Clin. Chem. 37, 411. Larsson, A. and Mellstedt, H. (1992) Chicken antibodies, a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodies. Hybridoma 1, 33. Lee, K., Ametani, A., Shimizu, M., Hana, H., Yamamoto, T. and Kaminogawa, S. (1991) Production and characterization of anti-human insulin antibodies in the hen's egg. Agric. Biol. Chem. 55, 2141.
83 L6sch, U., Schranner, I., Wanke, R. and Jurgens, L. (1986) The chicken egg, an antibody source. J. Vet. Med. 33, 609. Miller, G.W. and Nussenzweig, V. (1975) A new complement function, solubilization of antigen-antibody aggregates. Proc. Natl. Acad. Sci. USA 72, 418. Schifferli, J.A., Woo, P. and Peters, D.K. (1982) Complement-
mediated inhibition of immune precipitation. 1. Role of the classical and alternative pathways. Clin. Exp. Immunol. 47, 555. Weber, T.H., K~ipyaho, K.I. and Tanner, P. (1990) Endogenous interference in immunoassays in clinical chemistry. A review. Scand. J. Clin. Lab. Invest. 50, Suppl 201, 77.
