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Biotin labelling of chicken antibodies and their subsequent use in ELISA and immunohistochemistry
Comp. lmmun. MicrobioL infect. Dis. Vol. 16, No. 2, pp. 145-152, 1993 Printed in Great Britain. All rights reserved
0147-9571/93 $6.00+0.00 Copyright © 1993 Pergamon Press Ltd
BIOTIN LABELLING OF CHICKEN ANTIBODIES AND THEIR SUBSEQUENT USE IN ELISA AND IMMUNOHISTOCHEMISTRY MATTS OLOVSSON I* a n d ANDERS LARSSON 2 ~Department of Human Anatomy, University of Uppsala, S-751 23 Uppsala and 2Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden
(Received for publication 19 January 1993) Abstract--Avian antibodies have many advantages to mammalian antibodies due to the phylogenetic differences between birds and mammals, resulting in an increased sensitivity and a decreased background in many immunological assays. Since the avidin-biotin system is an efficient detection system for antibodies with a high sensitivity, we wanted to investigate the activity and unspecific binding of optimally biotin labelled chicken antibodies in ELISA and immunohistochemistry. We report on the conditions for biotinylation of chicken antibodies and that optimally biotinylated antibodies show a high activity and a low background in both ELISA and immunohistochemistry.
Key words: Chicken antibody, biotin, ELISA, immunohistochemistry. R6stun~-Les anticorps de volailles ont plusieurs avantages sur les anticorps de mammif6res, dfi aux diff6rences phylog6n6tiques entre les oiseaux et les mammif6res qui r6sultent en une sensibilit6 croissante et un bruit de fond r6duit dans de nombreux dosages immunologiques. Le syst6me avidine-biotine pour la d6tection des anticorps 6tant d'une plus grande sensibilit6, nous avons voulu faire l'analyse de l'activit6 et des liaisons non sp6cifiques des anticorps de volailles marqu6s optimalement avec la biotine en flux et en immunohistochimie. Nous montrons ainsi que dans les conditions de biotinylation, les anticorps de poulet et les anticorps optimalement biotinyl6s pr6sentent une grande activit6 et un bruit de fond bas en ELISA et en immunohistochimie.
Mots-clefs: anticorps de volailles, biotine, ELISA, immunohistochemie.
INTRODUCTION
Traditionally, the species used for immunization have been of mammalian origin, but recently there has been an increasing use of chicken antibodies. Chicken antibodies have several advantages to mammalian antibodies due to the phylogenetic difference between mammalian and avian species [1]. Due to this difference chicken antibodies will recognize more epitopes on mammalian proteins than antisera raised in mammalian species will do [2]. This will usually result in an enhancement of the signal. Chicken IgG2 do not bind to mammalian or bacterial Fc-receptors such as protein A or G [3-5]. They do not react with rheumatoid factors or other anti-IgG antibodies present in patient sera [6, 7]. Also, they do not activate the mammalian complement system [8] or mammalian platelets [5]. These and other responses are known to give false positive reactions or increased background in *Author for correspondence. Abbreviations used: ABC: avidin biotin complex; ALP: alkaline phosphatase; BSA: bovine serum albumin; DMSO: dimethylsulphoxide; ELISA: enzyme-linked immunosorbent assay; FCS: foetal calf serum; NaCITween: 0.15 M sodium chloride containing 0.05% Tween 20 and 0.02% sodium azide; PBS: 20 mM sodium phosphate buffer containing 0.15 M sodium chloride, pH 7.2; PBS-azide: PBS containing 0.02% sodium azide. 145
146
MATTS OLOVSSON and ANDERSLARSSON
ELISA, RIA, flow-cytometry and immunohistochemistry when working with mammalian antibodies. Chicken antibodies is a simple way of reducing these problems. The avidin-biotin complex has found a widespread use in immunology [9]. The high affinity (Kd = 10 J5 M-~) of avidin for biotin is an important factor in amplifying the sensitivity of many immunoassays [10]. The combination of optimally biotinylated chicken antibodies and the avidin-biotin system could be suitable in many assays since there are theoretical possibilities for a high activity and a low background level. Protocols for biotinylation of mammalian proteins have been used also for biotinylation of chicken antibodies [6, 11, 12], but the optimal conditions for biotinylation of chicken antibodies has not been investigated. We have thus studied the conditions for biotinylation of chicken antibodies and showed that optimally biotinylated antibodies have a high activity in both ELISA and immunohistochemistry and that background problems could be reduced. MATERIAL AND METHODS
Antibodies Chicken anti-human C3, anti-human IgG and anti-mouse IgM were provided by Immunsystem AB (Uppsala, Sweden). The preparations were IgG fractions with c. 5-10% of the antibodies specific for human C3 and 10-15% specific for human IgG. The anti-mouse IgM antibodies were afffinity-purified on mouse IgM-Sepharose as previously described [8]. N-Hydroxysuccinimidobiotin (NHS-biotin) was obtained from Sigma Chemical Company (St Louis, Mo), and alkaline phosphatase labelled Vectastain ABCkits were obtained from Vector Laboratories Inc. (Burlingame, Calif.).
Variation of biotin-antibody ratio 1 ml of chicken antibody 1 mg/ml in 0.1 M NaHCO3 pH 9.5 was mixed with 120 ktl of NHS-biotin in 2-fold dilution steps in DMSO. The highest concentration of NHS-biotin was 5 mg/ml. The mixture was incubated for 5 h at room temperature with continuous stirring, and then dialysed against PBS-azide. After dialysis the samples were diluted to equal volumes with PBS-azide and assayed by ELISA against human C3, human IgG and chicken IgG.
Variation of labelling time 1 ml of anti-human IgG 1 mg/ml in 0.1 M NaHCO3 pH 9.5 was mixed with 120#1 of 2.5 or 1.25 NHS-biotin/ml of DMSO. The mixtures were incubated 0.5, 1, 3 and 5 h at room temperature with continuous stirring, and then dialysed against PBS-azide. After dialysis the samples were diluted to equal volumes with PBS-azide and assayed by ELISA.
ELISA procedure Flat-bottom micro ELISA plates (Dynatech M 129 A) were coated with 20/2g of human C3 or human IgG/ml of 0.1 M NaHCO 3, pH 9.5, 2h at 37°C or overnight at 4°C. Uncoated sites were blocked with 0.1 mg of chicken IgG/ml of the same buffer by incubation 2 h at 37°C or overnight at 4°C. The plates were washed three times with NaCI-Tween. 200 ~tl of the different anti-human IgG-biotin conjugates were added in various dilutions and the plates were incubated at 37°C for 2h and washed with NaC1-Tween as before. 200 pl of Vectastain ABC-kit diluted 1 : 1000 was added and the plates were incubated for another 2 h before being washed as above. 200 ~1 of 1 mg/ml
Biotin labelling of chicken antibodies
147
p-nitrophenyl phosphate in 1 M diethanolamine, 0.5 mM MgCI2, pH 9.8, was added to the wells and the plates were incubated for 30 min at room temperature in the dark. The reactions was stopped with 50 pl of 5 M NaOH and the plates were read in a Titertek Multiscan (Flow Laboratories Ltd, Scotland).
lmmunohistochemistry Specimens were obtained from mice of the NMRI-strain (Anticimex, Sweden) and fixed with 4% paraformaldehyde in PBS. In vitro egg-cylinders were obtained by culturing blastocysts on an agarose surface (Olovsson and Nilsson, submitted) and fixed by immersion. Implantation sites, full term placenta, adult liver and spleen were fixed by perfusion. Paraffin embedding was performed according to standard procedure after dehydration in a graded series of ethanol. Sections (7/~m) were cut and placed on sialinized slides [13] to which they were allowed to set over night at 40°C. Paraffin was removed with xylol over night before rehydration in a graded series of ethanol and an equilibrization in PBS for 10min. Staining was performed with the Super Sensitive biotin-streptavidin alkaline phosphatase kit (BioGenex, San Ramon, Calif.). Reagents were diluted in PBS supplemented with 1% BSA. Endogenous avidin binding activity was blocked by incubating the sections with streptavidin (0.01 mg/ml) for 20 min and, after rinsing, with biotin (0.001 mg/ml) for 20 min [14]. Streptavidin and biotin were obtained from Boehringer Mannheim Scandinavia AB (Bromma, Sweden). Unspecific protein binding was blocked by an incubation with 1% BSA for 30 min. Primary antibody was a mouse anti-mouse antibody of the IgM class that label mouse trophoblast cells. Sections were incubated over night with the primary antibody diluted 1:2. Secondary antibodies: the biotinylated chicken anti-mouse IgM was diluted 1:200, 1 : 1000 respectively 1:5000 and the goat anti-mouse Ig was diluted according to instructions in the kit, 1:10, 1:40 and 1:100. The label was diluted 1:100 for the chicken antibody and according to instructions in the kit to match the dilution of the goat antibody. Alkaline substrate kit I (Vector Laboratories Inc., Burlingame, Calif.) was used according to instructions. Endogenous alkaline phosphatase activity in the specimens was blocked with levamisol according to the instructions in the substrate kit. Reaction was stopped with distilled water when negative controls showed a tendency to become stained. Control staining was performed by omitting the primary antibody and by using an irrelevant mouse IgM primary antibody. RESULTS
Variation of biotin-antibody ratio A positive correlation between activity and the concentration of biotin was found (Table 1), but with the highest biotin concentration there was an increased background. The optimal ratio between specific and unspecific activity was with 2.5 and 1.25 mg biotin/ml. As 2.5 mg biotin/ml gave a higher activity this seems the best biotin:chicken IgG ratio. This corresponds to 0.3 mg of NHS-biotin: mg chicken antibody. No decay in activity could be observed for biotinylated chicken antibodies when stored at +4°C for 5 yr. The biotinylated anti-human C3 had a high activity in ELISA. As only 5-10% of the antibodies are specific to C3, C. 5-10 ng ofjspecific antibody in the well is sufficient to give an A405 of 1.0 with the most biotinylated antibody. CIMID 16/2~-C
148
MATTS OLOVSSON a n d ANDERS LARSSON Table 1. Effect of variation of biotin-antibody ratio on activity and background of the biotinylated chicken anti-human IgG in the ELISA Dilution of NHS-biotin
Specific activity
Unspecific activity
Specific:unspecific ratio
Undiluted Diluted 1:2 Diluted 1:4 Diluted 1 : 8 Diluted 1: 16
9150 6650 3750 2800 1150
133 46 28 29 22
69 145 134 97 52
Specific activity is the dilution of the biotinylated antibody that gives an A405= 1.0 against human C3. Unspecific activity is the dilution of the biotinylated antibody that gives an A405= 0.1 against chicken IgG.
Similar results were obtained with biotinylated anti-human IgG (results not shown).
Variation of labelling time No increased activity was observed with increasing time, when comparing the 0.5 h incubation with the longer incubation times (results not shown). Thus, it is sufficient with a 30 min conjugation time.
Immunohistochemistry Background staining was present in sections of all tissues tested when chicken antibody was diluted 1:200, in spleen when diluted 1 : 1000 and in none of the tissue sections when diluted 1:5000. Background staining was present in sections of all tissues tested when the goat antibody was diluted 1:10 and in liver and spleen at the two higher dilutions recommended in the kit. Staining intensity showed to be similar when the chicken antibody was diluted 1:1000 [Fig. I(A) and (C)] and when the goat antibody was diluted 1:100 [Fig. 1 (B) and (D)]. DISCUSSION Chicken antibodies are evolutionary separated far from mammalian antibodies [1]. They have, for instance, a different way of producing antibody diversity and show no immunological similarity with mammalian IgG. This makes them well suited for detection of mammalian proteins. Due to the phylogenetic distance they will recognize more epitopes on the antigen as foreign. Studies with fl2-microglobulin showed that 60% more chicken IgG than mammalian antibody was bound to the fl2-microglobulin and 3-5 times more chicken IgG then mammalian IgG was bound to rabbit IgG [2]. This will give an amplification in the assay. Chicken antibodies also give access to a different antibody repertoire [15], increasing the chances of obtaining antibodies to conserved antigens. The lack of reactivity with rheumatoid factors or Fc-receptors will reduce background in most assays. If mammalian antibodies are used when studying living blood cells there will be a formation of immune complexes between human plasma proteins and mammalian antibodies which induce platelet activation, a reaction known to give false results. By using chicken antibodies these problems can be avoided [5]. Chicken antibodies can be produced in large scale from egg yolk [16], which is better for the welfare of the animal than the traditional bleeding of mammals. Thus, chicken antibodies have many advantages to traditional mammalian antibodies.
Fig. 1. Immunohistochemical staining of paraffin sections of in vitro egg-cylinders [(A) and (B)] and full term placentas [(C) and (D)]. (A) and (C): secondary antibody was a biotinylated chicken anti-mouse IgM diluted 1 : 1000. TC, trophoblast cells; EP, becoming embryo proper. (B) and (D): Secondary antibody was a biotinylated goat anti-mouse Ig diluted 1 : 100. Bars = 50/tm.
Biotin labelling of chicken antibodies
151
The biotin-avidin system is used in many immunological assays since the high binding constant between avidin and biotin gives an amplification of the signal [10]. Biotinylated chicken antibodies have previously been used when biotinylated under conditions optimized for mammalian antibodies [6, 11, 12]. The present results show that it is necessary to use more biotin to get the best signal:noise ratio compared to coupling corresponding mammalian antibodies. The optimal conditions for labelling was a mixture of 1 ml antibody 1 mg/ml and 120/~1 biotin 2.5 mg/ml. Incubation for 0.5 h is sufficient since a longer incubation time not resulted in a higher activity. The biotinylated chicken antibodies thus obtained had a high activity in ELISA with a low background reaction. Since avidin is located in the egg white and the antibodies are found in the egg yolk there is a small risk for contamination of the yolk by avidin while collecting the antibodies. An eventual contamination of avidin was probably removed during the purification procedure of chicken IgG because of the differences in molecular weight and isoelectric point. We observed no interference from avidin in the antibody preparations. Background staining caused by unspecific binding of the secondary antibody is a problem in immunohistochemistry that often can be reduced or eliminated by selecting a suitable antibody and by blocking undesired binding sites. In mammalian tissues there are more potential sites for unspecific binding of mammalian antibodies than of chicken antibodies. For instance, mammalian antibodies, but not chicken antibodies, bind to mammalian Fc-receptors [5] and are bound to mammalian anti-IgG [6, 7]. A reduced background staining when using chicken antibodies instead of mammalian antibodies have been observed in immunohistochemistry [2], ELISA [7] and flow cytometry [5]. To be able to compare the unspecific binding of an optimally biotinylated chicken antibody with a mammalian antibody, we tested mammalian antibodies from different species and found that antibodies developed in goats gave the lowest background staining. When the goat antibody was optimally diluted in vitro egg-cylinders and full term placentas did not show any background staining and these tissues therefore served as controls. As test material we used sections of day 7 implantation sites, adult liver and spleen since they showed background staining with the goat antibody. Our results showed that the background staining could be eliminated in liver and spleen sections when using the chicken antibody, but not when using the goat antibody. For the goat antibody, background staining ever persisted on liver and spleen sections at all dilutions recommended. The unspecific binding of the goat antibody to liver and spleen sections could be caused by a binding to Fc-receptors [17] to which chicken antibodies do not bind [5]. Also implantation sites contain cells that express FcTreceptors [18, 19], but in this case the unspecific staining could be eliminated by diluting the goat antibody which indicates that unspecific binding to Fc-receptors was not an important factor. Another factor of importance for unspecific staining caused by unspecific binding of the secondary antibody is the dilution at which the secondary antibody is used. In conclusion, we show that optimally biotinylated chicken antibodies have a high activity and give a low background reaction in both ELISA and immunohistochemistry and that background staining in immunohistochemistry can be reduced by using a chicken antibody instead of mammalian antibodies. Acknowledgements--Excellent technical assistance was given by Mrs Monica Ferm, Mrs Barbro Einarsson and Mrs Marianne Ljungkvist. This work was supported by the Swedish Medical Research Council (project Nos 9875 and 00070) and the Swedish Society for Medicine.
152
MATTS OLOVSSON and ANDERS LARSSON REFERENCES
I. H/idge D. and Ambrosius H. Evolution of low molecular weight immunoglobulins. IV. IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA. Molec. Immunol. 21, 699-707 (1984). 2. Horton J. J., Holden C. A., Ward P. J., MacDonald D. M. and Sanderson A. R. Exploitation of phylogenetic distance in cell surface immune labeling: studies with fl2-microglobulin. J. invest. Dermatol. 85, 96-99 (1984). 3. Katz D., Lehrer S. and Kohn A. Use of chicken and rabbit antibodies in a solid phase protein A radioimmunoassay for virus detection. J. virol. Met& 12, 59-70 (1985) 4. Guss B., Eliasson M., Olsson A., Uhl6n M., Frej A.-K., J6rnvall H., Flock J.-I. and Lindberg M. Structure of the IgG-binding regions of streptococcal protein G. EMBO J. 5, 1567 1575 (1986). 5. Lindahl T. L., Festin R. and Larsson A. Studies of fibrinogen binding to platelets by flow cytometry: an improved method for studies of platelet activation. Throm. Haemost. 68, 221-225 (1992). 6. Larsson A., Karlsson-Parra A. and Sj6quist J. Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors. Clin. Chem. 37, 411-414 (1991). 7. Larsson A. and Mellstedt H. Chicken antibodies: a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodies. Hybridoma 11, 33-39 (1992). 8. Larsson A. and Sj6quist J. Binding of complement components Clq, C3, C4 and C5 to a model immune complex in ELISA. J. immunol. Meth. 119, 103-109 (1989). 9. Wilchek M. and Bayer E. A. The avidin biotin complex in immunology, lmmunol. Today 325, 39 43 (1984). 10. Guesdon J.-L., Ternynck T. and Avrameas S. The use of avidin-biotin interaction of immunoenzymatic techniques. J. histochem. Cytochem. 27, l l31 1139 (1979). 11. Larsson A. and Sj6quist J. Chicken antibodies: a tool to avoid false positive results by rheumatoid factor in latex fixation tests. J. immunol. Meth. 10g, 205 208 (1988). 12. Zrein M., Weiss E. and VanRegenmortel M. H. V. Detection of rheumatoid factors of different isotypes by ELISA using biotinylated avian antibodies. Immunol. Inv. 17, 165-181 (1988). 13. Rentrop M., Knapp B, Winter H. and Schweizer J. Aminoalkylsilane-treated glass slides as support for in silu hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions. Histochem. J. 18, 271-276 (1986). 14. Wood G. S. and Warnke R. Suppression of endogenous avidin binding activity in tissues and its relevance to biotin-avidin detection systems. J. histochem. Cytochem. 29, 1196 1204 (1981). 15. Song C.-S., Yu J.-H., Bai D. H., Hester P. Y. and Kim K.-H. Antibodies of the ct-subunit of insulin receptor from eggs of immunized hens. J. Immunol. 135, 3354-3359 (1985). 16. Jensenius J. C., Andersen I., Hau J., Crone M. and Koch C. Eggs: conveniently packaged antibodies. Methods for purification of yolk IgG. J. immunol. Meth. 46, 63 68 0981). 17. Weir D. M. In Handbook of Experimental Immunology in Four Volumes. Volume 2: Cellular Immunology. (Edited by Weir D. M.), p.48. Blackwell, Calif. (1986). 18. Elson J., Jenkinsson E. J. and Billington W. D. Fc receptors on mouse placenta and yolk sac cells. Nature 255, 412-413 (1975). 19. Daki N. M., Stewart I. J. and Wild A. E. Receptors for IgG2b on cells of the mouse metrial gland. J. reprod. Immunol. 16, 249 260 (1989).
0147-9571/93 $6.00+0.00 Copyright © 1993 Pergamon Press Ltd
BIOTIN LABELLING OF CHICKEN ANTIBODIES AND THEIR SUBSEQUENT USE IN ELISA AND IMMUNOHISTOCHEMISTRY MATTS OLOVSSON I* a n d ANDERS LARSSON 2 ~Department of Human Anatomy, University of Uppsala, S-751 23 Uppsala and 2Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden
(Received for publication 19 January 1993) Abstract--Avian antibodies have many advantages to mammalian antibodies due to the phylogenetic differences between birds and mammals, resulting in an increased sensitivity and a decreased background in many immunological assays. Since the avidin-biotin system is an efficient detection system for antibodies with a high sensitivity, we wanted to investigate the activity and unspecific binding of optimally biotin labelled chicken antibodies in ELISA and immunohistochemistry. We report on the conditions for biotinylation of chicken antibodies and that optimally biotinylated antibodies show a high activity and a low background in both ELISA and immunohistochemistry.
Key words: Chicken antibody, biotin, ELISA, immunohistochemistry. R6stun~-Les anticorps de volailles ont plusieurs avantages sur les anticorps de mammif6res, dfi aux diff6rences phylog6n6tiques entre les oiseaux et les mammif6res qui r6sultent en une sensibilit6 croissante et un bruit de fond r6duit dans de nombreux dosages immunologiques. Le syst6me avidine-biotine pour la d6tection des anticorps 6tant d'une plus grande sensibilit6, nous avons voulu faire l'analyse de l'activit6 et des liaisons non sp6cifiques des anticorps de volailles marqu6s optimalement avec la biotine en flux et en immunohistochimie. Nous montrons ainsi que dans les conditions de biotinylation, les anticorps de poulet et les anticorps optimalement biotinyl6s pr6sentent une grande activit6 et un bruit de fond bas en ELISA et en immunohistochimie.
Mots-clefs: anticorps de volailles, biotine, ELISA, immunohistochemie.
INTRODUCTION
Traditionally, the species used for immunization have been of mammalian origin, but recently there has been an increasing use of chicken antibodies. Chicken antibodies have several advantages to mammalian antibodies due to the phylogenetic difference between mammalian and avian species [1]. Due to this difference chicken antibodies will recognize more epitopes on mammalian proteins than antisera raised in mammalian species will do [2]. This will usually result in an enhancement of the signal. Chicken IgG2 do not bind to mammalian or bacterial Fc-receptors such as protein A or G [3-5]. They do not react with rheumatoid factors or other anti-IgG antibodies present in patient sera [6, 7]. Also, they do not activate the mammalian complement system [8] or mammalian platelets [5]. These and other responses are known to give false positive reactions or increased background in *Author for correspondence. Abbreviations used: ABC: avidin biotin complex; ALP: alkaline phosphatase; BSA: bovine serum albumin; DMSO: dimethylsulphoxide; ELISA: enzyme-linked immunosorbent assay; FCS: foetal calf serum; NaCITween: 0.15 M sodium chloride containing 0.05% Tween 20 and 0.02% sodium azide; PBS: 20 mM sodium phosphate buffer containing 0.15 M sodium chloride, pH 7.2; PBS-azide: PBS containing 0.02% sodium azide. 145
146
MATTS OLOVSSON and ANDERSLARSSON
ELISA, RIA, flow-cytometry and immunohistochemistry when working with mammalian antibodies. Chicken antibodies is a simple way of reducing these problems. The avidin-biotin complex has found a widespread use in immunology [9]. The high affinity (Kd = 10 J5 M-~) of avidin for biotin is an important factor in amplifying the sensitivity of many immunoassays [10]. The combination of optimally biotinylated chicken antibodies and the avidin-biotin system could be suitable in many assays since there are theoretical possibilities for a high activity and a low background level. Protocols for biotinylation of mammalian proteins have been used also for biotinylation of chicken antibodies [6, 11, 12], but the optimal conditions for biotinylation of chicken antibodies has not been investigated. We have thus studied the conditions for biotinylation of chicken antibodies and showed that optimally biotinylated antibodies have a high activity in both ELISA and immunohistochemistry and that background problems could be reduced. MATERIAL AND METHODS
Antibodies Chicken anti-human C3, anti-human IgG and anti-mouse IgM were provided by Immunsystem AB (Uppsala, Sweden). The preparations were IgG fractions with c. 5-10% of the antibodies specific for human C3 and 10-15% specific for human IgG. The anti-mouse IgM antibodies were afffinity-purified on mouse IgM-Sepharose as previously described [8]. N-Hydroxysuccinimidobiotin (NHS-biotin) was obtained from Sigma Chemical Company (St Louis, Mo), and alkaline phosphatase labelled Vectastain ABCkits were obtained from Vector Laboratories Inc. (Burlingame, Calif.).
Variation of biotin-antibody ratio 1 ml of chicken antibody 1 mg/ml in 0.1 M NaHCO3 pH 9.5 was mixed with 120 ktl of NHS-biotin in 2-fold dilution steps in DMSO. The highest concentration of NHS-biotin was 5 mg/ml. The mixture was incubated for 5 h at room temperature with continuous stirring, and then dialysed against PBS-azide. After dialysis the samples were diluted to equal volumes with PBS-azide and assayed by ELISA against human C3, human IgG and chicken IgG.
Variation of labelling time 1 ml of anti-human IgG 1 mg/ml in 0.1 M NaHCO3 pH 9.5 was mixed with 120#1 of 2.5 or 1.25 NHS-biotin/ml of DMSO. The mixtures were incubated 0.5, 1, 3 and 5 h at room temperature with continuous stirring, and then dialysed against PBS-azide. After dialysis the samples were diluted to equal volumes with PBS-azide and assayed by ELISA.
ELISA procedure Flat-bottom micro ELISA plates (Dynatech M 129 A) were coated with 20/2g of human C3 or human IgG/ml of 0.1 M NaHCO 3, pH 9.5, 2h at 37°C or overnight at 4°C. Uncoated sites were blocked with 0.1 mg of chicken IgG/ml of the same buffer by incubation 2 h at 37°C or overnight at 4°C. The plates were washed three times with NaCI-Tween. 200 ~tl of the different anti-human IgG-biotin conjugates were added in various dilutions and the plates were incubated at 37°C for 2h and washed with NaC1-Tween as before. 200 pl of Vectastain ABC-kit diluted 1 : 1000 was added and the plates were incubated for another 2 h before being washed as above. 200 ~1 of 1 mg/ml
Biotin labelling of chicken antibodies
147
p-nitrophenyl phosphate in 1 M diethanolamine, 0.5 mM MgCI2, pH 9.8, was added to the wells and the plates were incubated for 30 min at room temperature in the dark. The reactions was stopped with 50 pl of 5 M NaOH and the plates were read in a Titertek Multiscan (Flow Laboratories Ltd, Scotland).
lmmunohistochemistry Specimens were obtained from mice of the NMRI-strain (Anticimex, Sweden) and fixed with 4% paraformaldehyde in PBS. In vitro egg-cylinders were obtained by culturing blastocysts on an agarose surface (Olovsson and Nilsson, submitted) and fixed by immersion. Implantation sites, full term placenta, adult liver and spleen were fixed by perfusion. Paraffin embedding was performed according to standard procedure after dehydration in a graded series of ethanol. Sections (7/~m) were cut and placed on sialinized slides [13] to which they were allowed to set over night at 40°C. Paraffin was removed with xylol over night before rehydration in a graded series of ethanol and an equilibrization in PBS for 10min. Staining was performed with the Super Sensitive biotin-streptavidin alkaline phosphatase kit (BioGenex, San Ramon, Calif.). Reagents were diluted in PBS supplemented with 1% BSA. Endogenous avidin binding activity was blocked by incubating the sections with streptavidin (0.01 mg/ml) for 20 min and, after rinsing, with biotin (0.001 mg/ml) for 20 min [14]. Streptavidin and biotin were obtained from Boehringer Mannheim Scandinavia AB (Bromma, Sweden). Unspecific protein binding was blocked by an incubation with 1% BSA for 30 min. Primary antibody was a mouse anti-mouse antibody of the IgM class that label mouse trophoblast cells. Sections were incubated over night with the primary antibody diluted 1:2. Secondary antibodies: the biotinylated chicken anti-mouse IgM was diluted 1:200, 1 : 1000 respectively 1:5000 and the goat anti-mouse Ig was diluted according to instructions in the kit, 1:10, 1:40 and 1:100. The label was diluted 1:100 for the chicken antibody and according to instructions in the kit to match the dilution of the goat antibody. Alkaline substrate kit I (Vector Laboratories Inc., Burlingame, Calif.) was used according to instructions. Endogenous alkaline phosphatase activity in the specimens was blocked with levamisol according to the instructions in the substrate kit. Reaction was stopped with distilled water when negative controls showed a tendency to become stained. Control staining was performed by omitting the primary antibody and by using an irrelevant mouse IgM primary antibody. RESULTS
Variation of biotin-antibody ratio A positive correlation between activity and the concentration of biotin was found (Table 1), but with the highest biotin concentration there was an increased background. The optimal ratio between specific and unspecific activity was with 2.5 and 1.25 mg biotin/ml. As 2.5 mg biotin/ml gave a higher activity this seems the best biotin:chicken IgG ratio. This corresponds to 0.3 mg of NHS-biotin: mg chicken antibody. No decay in activity could be observed for biotinylated chicken antibodies when stored at +4°C for 5 yr. The biotinylated anti-human C3 had a high activity in ELISA. As only 5-10% of the antibodies are specific to C3, C. 5-10 ng ofjspecific antibody in the well is sufficient to give an A405 of 1.0 with the most biotinylated antibody. CIMID 16/2~-C
148
MATTS OLOVSSON a n d ANDERS LARSSON Table 1. Effect of variation of biotin-antibody ratio on activity and background of the biotinylated chicken anti-human IgG in the ELISA Dilution of NHS-biotin
Specific activity
Unspecific activity
Specific:unspecific ratio
Undiluted Diluted 1:2 Diluted 1:4 Diluted 1 : 8 Diluted 1: 16
9150 6650 3750 2800 1150
133 46 28 29 22
69 145 134 97 52
Specific activity is the dilution of the biotinylated antibody that gives an A405= 1.0 against human C3. Unspecific activity is the dilution of the biotinylated antibody that gives an A405= 0.1 against chicken IgG.
Similar results were obtained with biotinylated anti-human IgG (results not shown).
Variation of labelling time No increased activity was observed with increasing time, when comparing the 0.5 h incubation with the longer incubation times (results not shown). Thus, it is sufficient with a 30 min conjugation time.
Immunohistochemistry Background staining was present in sections of all tissues tested when chicken antibody was diluted 1:200, in spleen when diluted 1 : 1000 and in none of the tissue sections when diluted 1:5000. Background staining was present in sections of all tissues tested when the goat antibody was diluted 1:10 and in liver and spleen at the two higher dilutions recommended in the kit. Staining intensity showed to be similar when the chicken antibody was diluted 1:1000 [Fig. I(A) and (C)] and when the goat antibody was diluted 1:100 [Fig. 1 (B) and (D)]. DISCUSSION Chicken antibodies are evolutionary separated far from mammalian antibodies [1]. They have, for instance, a different way of producing antibody diversity and show no immunological similarity with mammalian IgG. This makes them well suited for detection of mammalian proteins. Due to the phylogenetic distance they will recognize more epitopes on the antigen as foreign. Studies with fl2-microglobulin showed that 60% more chicken IgG than mammalian antibody was bound to the fl2-microglobulin and 3-5 times more chicken IgG then mammalian IgG was bound to rabbit IgG [2]. This will give an amplification in the assay. Chicken antibodies also give access to a different antibody repertoire [15], increasing the chances of obtaining antibodies to conserved antigens. The lack of reactivity with rheumatoid factors or Fc-receptors will reduce background in most assays. If mammalian antibodies are used when studying living blood cells there will be a formation of immune complexes between human plasma proteins and mammalian antibodies which induce platelet activation, a reaction known to give false results. By using chicken antibodies these problems can be avoided [5]. Chicken antibodies can be produced in large scale from egg yolk [16], which is better for the welfare of the animal than the traditional bleeding of mammals. Thus, chicken antibodies have many advantages to traditional mammalian antibodies.
Fig. 1. Immunohistochemical staining of paraffin sections of in vitro egg-cylinders [(A) and (B)] and full term placentas [(C) and (D)]. (A) and (C): secondary antibody was a biotinylated chicken anti-mouse IgM diluted 1 : 1000. TC, trophoblast cells; EP, becoming embryo proper. (B) and (D): Secondary antibody was a biotinylated goat anti-mouse Ig diluted 1 : 100. Bars = 50/tm.
Biotin labelling of chicken antibodies
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The biotin-avidin system is used in many immunological assays since the high binding constant between avidin and biotin gives an amplification of the signal [10]. Biotinylated chicken antibodies have previously been used when biotinylated under conditions optimized for mammalian antibodies [6, 11, 12]. The present results show that it is necessary to use more biotin to get the best signal:noise ratio compared to coupling corresponding mammalian antibodies. The optimal conditions for labelling was a mixture of 1 ml antibody 1 mg/ml and 120/~1 biotin 2.5 mg/ml. Incubation for 0.5 h is sufficient since a longer incubation time not resulted in a higher activity. The biotinylated chicken antibodies thus obtained had a high activity in ELISA with a low background reaction. Since avidin is located in the egg white and the antibodies are found in the egg yolk there is a small risk for contamination of the yolk by avidin while collecting the antibodies. An eventual contamination of avidin was probably removed during the purification procedure of chicken IgG because of the differences in molecular weight and isoelectric point. We observed no interference from avidin in the antibody preparations. Background staining caused by unspecific binding of the secondary antibody is a problem in immunohistochemistry that often can be reduced or eliminated by selecting a suitable antibody and by blocking undesired binding sites. In mammalian tissues there are more potential sites for unspecific binding of mammalian antibodies than of chicken antibodies. For instance, mammalian antibodies, but not chicken antibodies, bind to mammalian Fc-receptors [5] and are bound to mammalian anti-IgG [6, 7]. A reduced background staining when using chicken antibodies instead of mammalian antibodies have been observed in immunohistochemistry [2], ELISA [7] and flow cytometry [5]. To be able to compare the unspecific binding of an optimally biotinylated chicken antibody with a mammalian antibody, we tested mammalian antibodies from different species and found that antibodies developed in goats gave the lowest background staining. When the goat antibody was optimally diluted in vitro egg-cylinders and full term placentas did not show any background staining and these tissues therefore served as controls. As test material we used sections of day 7 implantation sites, adult liver and spleen since they showed background staining with the goat antibody. Our results showed that the background staining could be eliminated in liver and spleen sections when using the chicken antibody, but not when using the goat antibody. For the goat antibody, background staining ever persisted on liver and spleen sections at all dilutions recommended. The unspecific binding of the goat antibody to liver and spleen sections could be caused by a binding to Fc-receptors [17] to which chicken antibodies do not bind [5]. Also implantation sites contain cells that express FcTreceptors [18, 19], but in this case the unspecific staining could be eliminated by diluting the goat antibody which indicates that unspecific binding to Fc-receptors was not an important factor. Another factor of importance for unspecific staining caused by unspecific binding of the secondary antibody is the dilution at which the secondary antibody is used. In conclusion, we show that optimally biotinylated chicken antibodies have a high activity and give a low background reaction in both ELISA and immunohistochemistry and that background staining in immunohistochemistry can be reduced by using a chicken antibody instead of mammalian antibodies. Acknowledgements--Excellent technical assistance was given by Mrs Monica Ferm, Mrs Barbro Einarsson and Mrs Marianne Ljungkvist. This work was supported by the Swedish Medical Research Council (project Nos 9875 and 00070) and the Swedish Society for Medicine.
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