Veterinary Immunology and Immunopathology, 34 (1992) 139-147
139
Elsevier Science Publishers B.V., Amsterdam
Detection of IgM rheumatoid factor in canine serum using a standardized enzyme-linked immunosorbent assay O.L. Nielsen Department of Pharmacology and Pathobiology, The Royal Veterinaryand Agricultural University, Biilowsvej 13, DK-1870, Frederiksberg C, Denmark (Accepted 27 January 1992 )
ABSTRACT Nielsen, O.L., 1992. Detection of IgM rheumatoid factor in canine serum using a standardized enzyme-linked immunosorbentassay. Vet. Immunol. Immunopathol., 34:139-147. An ELISA measuring IgM rheumatoid factor (RF) in dog serum is presented. Dog sera and a human IgM RF standard, calibrated against the international World Health Organisation (WHO) standard, are compared. It is concluded that the human IgM RF standard may be used as reference serum in the canine assay, which makes it possible to compare results from different veterinary laboratories.
ABBREVIATIONS CV, coefficient of variance; ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; OD, optical density; OPD, orthophenylene diamine; RA, rheumatoid arthritis; RF, rheumatoid factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. INTRODUCTION
Rheumatoid arthritis (RA) is a chronic, recurrent, erosive, polyarthritis of man. It affects 1-2% of the population worldwide, with a prevalence in the female population three times that of the male. The inflammatory response in RA has a distinctive immunological component, and it is believed that "the presentation of a relevant antigen to an immunogenetically susceptible host" precedes the disease (Harris, 1990). The antigen has not been identified, but both viruses and bacteria have been implicated. The majority of paCorrespondence to."O.L. Nielsen, National Veterinary Laboratory, Hangovej 2, DK-8200,/krhus N, Denmark.
© 1992 Elsevier Science publishers B.V. All rights reserved 0165-2427/92/$05.00
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tients with RA carry the MHC class II antigens of HLA-DR4 a n d / o r HLADR 1. The central role of MHC class II antigens in communication between the cells of the i m m u n e system is well known, as is the role of autoimmunity in the self-perpetuation of RA (Pope and Talal, 1985 ). Approximately 70% of people with RA have elevated serum levels of IgM rheumatoid factor (RF). The canine counterpart of RA has been documented by several authors (e.g. Lewis and Borel, 1971; Bennett, 1987), as has the occurrence of RF, detected by various methods, in serum, synovial fluid and aqueous humor (Wood et al., 1980; Bernadina et al., 1988; Carter et al., 1989; Thoren-Tolling, 1990). In this article an indirect enzyme-linked immunosorbent assay (ELISA) for the measurement of IgM RF in dog serum is described, and the use of the h u m a n IgM RF standard serum as reference in the dog assay is verified. MATERIALSAND METHODS
Serum samples Sera from 226 dogs were collected and stored at - 20 ° C until used. Ninetysix dogs were without clinically detectable disease (sera from 13 of these dogs were kindly supplied by Dr. Hans Flemming Hojelse, H. Lundbeck, Copenhagen, Denmark), five suffered from unclassifiable polyarthritis and 125 were miscellaneous hospital admissions. The five sera were positive in a commercially available RF latex agglutination test (Synbiotics ®, Canine Rheumatoid Factor Test Kit, San Diego, CA), and were the kind gift of Dr. Kerstin ThorenTolling, Biovet, Sweden. The human IgM RF standard serum (code Jan. 1986, 107 IU ), was kindly donated by Dr. Mimi Hoier-Madsen, Autoimmunlaboratoriet, Statens Seruminstitut, Copenhagen, Denmark. The standard serum contains 107 IU m l - 1, when tested at a dilution of 1/ 100, but the serum prepa~a~ahally used was concentrated five-fold by redissolving 1 ml of freezedried serum to a total volume of 0.2 ml.
IgM-RF ELISA procedure The ELISA test was modified after Hoier-Madsen et al. (1986).
Buffers Carbonate buffer was comprised of 1.59 Na2CO3, 2.93 g NaHCO3, 0.20 g NAN3, and 20 ml phenol red 0.05% in 1000 ml distilled water, pH 9.6. Sodium chloride buffer was 29.2 g NaC1, 0.2 g KC1, 0.2 g KH2PO4, 1.15 g Na2HPO4- 2H20, and 10 ml Triton X- 100 in 1000 ml distilled water, pH 7.2. Citrate buffer consisted of 7.3 g C6H807" 1H20 (citric acid), and 11.86 g Na2HPO4" 2H20 in 1000 ml distilled water, pH 5.0.
ELISA DETECTION OF IgM RHEUMATOID FACTOR IN CANINE SERUM
141
Coating Polystyrene microtitre plates (Teknunc, MaxiSorp F96, cat. no. 439454, Roskilde, Denmark) were incubated with 50 #1 dog IgG (Sigma, code 1 4006, St. Louis, MO) at 100 #g ml-1 in carbonate buffer, at 4 °C overnight. The solution was removed, and areas without adsorbed IgG were blocked by applying 200 #1 sodium chloride buffer (containing Triton X- 100) at 4 ° C overnight. The buffer was meticulously removed. The plates were used immediately, or stored at - 20 ° C, for not more than 3 months before being utilized. The purity of IgG was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Testing The wells were washed four times with sodium chloride buffer. Standard serum (serum no. 152, four-fold dilution, starting at 1/40, arbitrarily addressed as 330 U, and ending at 1/2560), sodium chloride buffer, one negative and two positive controls ( t / 100) and samples to be tested ( 1/ 100) were applied in duplicate, 50 #1 in each well, and were incubated at 20°C for 1 h. All sera were diluted in sodium chloride buffer. The wells were washed four times as previously described, and incubated with 50/tl of the horseradish peroxidase conjugated, F ( a b ' ) z fragment of rabbit anti-human IgM (DAKO, code P 322, Glostrup, Denmark) 1/400, for 1 h at 20°C. The wells were washed six times. Orthophenylene diamine (OPD) ( 18 mg) were dissolved in 20 ml citrate buffer and 38 #1 H202 (30%) applied. Of this solution, 50 #1 were added to each well, and the color reaction allowed to evolve for 10-20 min. The reaction was stopped by adding 150 #1 of I mol sulfuric acid. Optical density (OD) was read at 492 nm in an ELISA reader.
Qualitative control of the ELISA procedure ?
To make sucre that the L I S A a e ~ a ~ d e t e c t s l g M RF in serum from dogs, the following procedures were performed.
Verification of the cross-reactivityof rabbit anti-human IgM towards canine IgM The reactivity of anti-human antibody preparations against various animal proteins have been tested by Hau et al. (1990). The reactivity of anti-human IgM (DAKO, code A091/A426, Glostrup, Denmark) with dog serum, using rocket immune electrophoresis, indicated strong cross-reaction. Protein A binds IgG, IgM (Goudswaard et al., 1978; Miele and Krakowha, 1981 ), and IgA (Miele and Krakowha, 1981 ) from dog serum. Using protein A column chromatography (Pharmacia, protein A sepharose, Uppsala, Sweden), IgG and IgM were isolated from the serum often healthy dogs. Approximately 50% of the total serum IgM was retained by protein A, judged by
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visually comparing the precipitates generated in crossed i m m u n e electrophoresis. The IgG- and IgM-containing fractions were subsequently separated by gel-filtration in an high-performance liquid chromatography (HPLC) set-up using a column with a cut-off equivalent to 500 kD (LKB Produkter, TSK G 3000 SW, 8 × 300 m m , Uppsala, Sweden). Proteins obtained in void volume, and proteins filtrated were, for each of the ten dogs, tested against goat antidog IgM (Nordic Immunological Laboratories, code GAD IgM (Fc), Netherlands), and rabbit anti-human IgM (DAKO, code A091, Glostrup, Denmark), using rocket i m m u n e electrophoresis.
Absorption of lgM RF to IgG-coated latex particles Latex particles were coated by standard methods (Singer and Plotz, 1956 ) with dog IgG (Sigma, code 1 4006, St. Louis, MO), and human serum albumin. Serial dilutions of the latex reagents were added to a positive and a negative serum, making a final serum dilution of 1/ 100. A Tris buffer was used. The mixture was incubated for 1 h at 37°C. The latex particles were spun down at 2000 × g 30 min-~. Finally the supernatant was tested in the ELISA. IgM RF activity in the IgM-containing and IgM-depletedfractions of a positive serum The IgM fraction of a positive serum (no. 152 ) was isolated using HPLC gel filtration. The column used had a cut-off corresponding to 500 kD (LKB, Produkter, TSK G 3000 SWG, 21.5 × 600 m m , Uppsala, Sweden). Proteins in void volume and filtrated proteins were concentrated on spin filters (Millipore, code UFC2 TTK, Bedford, MA), retaining molecules with molar weight exceeding 30 kD. The two fractions were then tested in the ELISA. The majority of the IgM RF activity was located in the IgM-containing fraction (results are not shown). Heat inactivation No effect of heating for 30 min at 56°C on the h u m a n IgM RF standard serum and positive dog serum no. 108 was observed. RESULTS A total of 226 sera were tested. Ninety-six sera from healthy dogs gave a cut-off (mean + two times the standard deviation) on 15.0 U. Three sera, no. 72 (mean 4.0 U ) , no. 108 (mean 26.0 U) and no. 152 (mean 128.6 U), were tested in 10 independent set-ups. The coefficient of variance (CV) was 9.0%, 6.5% and 16.4% respectively. Testing three sera, no. 74 (mean 3.3 U ) , no. 45 (mean 33.7 U) and no. 152 (mean 136.8 U) 10 times on the same plate gave CV of 2.1%, 6.3% and 7.9%. Sera from two out of the five dogs with oolyarthritis were positive ( > 15.0
143
ELISA DETECTION OF IgM RHEUMATOID FACTOR IN CANINE SERUM
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2 Fig. 1. rocket immune electrophoresis of serum fractions from three healthy dogs. The numbers 1-3 represent serum fractions (a and b) from the three dogs. a, IgM-containingfraction; b, IgGcontaining fraction. The gel in (A) contains anti-dog IgM; the gel in (B) contains anti-human lgM. t .00
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Fig. 2. effect on OD in ELISA by absorbing IgM RF into dog IgG-coated latex particles. In 2 (A) latex particles were coated with dog IgG, and in (B) latex particles were coated with human serum albumin. X-axis: dilutions of the latex reagent. 1/2 indicates a two-fold dilution; - , buffer without the addition of latex reagent. Y-axis: optical density (OD). +, positive serum no 208; A, negative serum no. 80; O, plain buffer. As the latex reagent was only available in a limited amount, the curves for plain buffer are incomplete.
U ) in the IgM RF ELISA (22.7 U and 25.1 U, respectively). The 125 miscellaneous admissions were allocated into the following 11 diagnostic groups (total number of dogs and the individual positive values of IgM RF are shown
144
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Fig. 3. titration curves for the human IgM RF standard, three IgM RF-positive dog sera and a serum pool from ten healthy dogs. (A) compares the human standard and the three positive dog sera. (B) compares one positive dog serum and the dog serum pool. +, human standard; Q, positive serum 152, from a dog with pododermatitis; O, positive serum 108; A, positive serum 208; A, negative serum pool.
in parentheses): tumors (25 dogs, 22.8 U), flea infestation ( 17 dogs, 31.3 U, 28.5 U, 21.4 U); dental calculus (16 dogs); osteoarthritis (15 dogs); otitis externa ( 13 dogs ); miscellaneous bacterial infections ( 11 dogs); endometritis (8 dogs); pododermatitis (8 dogs, 128.7 U, 26.0 U); cruciate ligament rupture (5 dogs, 18.7 U); gastro-intestinal disorders (5 dogs, 20.7 U); hot spot (three dogs). Figure 1 presents the results of the rocket immune electrophoresis on three of the ten sera tested. IgG from dogs, mainly migrating towards the cathode at the chosen test conditions, does not react with either anti-dog IgM or antihuman IgM. Dog IgM is precipitated by both anti-dog IgM and anti-human IgM. The difference in IgM concentration between the various dog sera can be read from the height of the rockets. It is seen that anti-dog IgM and antihuman IgM detect the same concentration difference between the individual sera. HPLC-filtrated proteins (fraction b ) contain small amounts of IgM (see Fig. 1 (A), sera nos. 1 and 3). Intermediate gel rocket immune electrophoresis on a pool of sera from the ten healthy dogs showed, taking the sensitivity of the assay into account, that anti-human IgM apparently precipitated all dog IgM. That is, no IgM was left to migrate into the gel containing anti-dog IgM. Figure 2 shows the effect of absorbing IgM RF into dog IgG-coated latex particles, followed by testing the supernatant in the ELISA set-up. By increas-
EL1SA DETECTION OF IgM RHEUMATOID FACTOR IN CANINE SERUM
145
ing the concentration of latex particles coated by IgG, but not by latex particles coated by h u m a n serum albumin, the OD is diminished. In Fig. 3 the titration curves of human IgM RF standard serum, three positive dog sera and a pool of sera from ten healthy dogs are presented. It is seen that all four curves have the typical sigmoid shape. The human standard has a higher activity than the three dog sera, of which serum no. 152 has the highest activity. Dog sera no. 108 and no. 208 have merging titration curves. The dog serum pool has the lowest activity.
DISCUSSION
The reliability of the latex agglutination test in measuring RF activity in dog serum has been questioned by several authors e.g. Bennett and Kirkham ( 1987 ) and Halliwell et al. ( 1989 ), who placed greater confidence on various modifications of the Rose-Waaler test. Bernadina et al. ( 1988 ) introduced a reliable gel precipitation test. An IgM-specific RF radioimmunoassay has been described by Carter et al. ( 1989 ), and an ELISA, also measuring IgM RF, has been described by Thoren-Tolling (1990). Neither of the two isotype-specific RF tests used reference sera, but instead recorded the OD values in determining the RF activity. The ELISA described in this paper seems to be a reliable assay for testing IgM RF in dog serum. Although cross-reactivity of anti-human IgM was shown with a batch of antiserum different from the one used in the ELISA, the strong cross-reactivity indicated is probably still valid. However, different batches of horseradish peroxidase-conjugated anti-human IgM may not cross-react with dog IgM to the same extent. This probably represents a minor problem when using an internal dog IgM RF standard, but might be significant when using the h u m a n standard. Testing the same dog serum using different conjugates would settle this problem. The binding of h u m a n RF to animal IgG has been verified by Butler and Vaughan (1964), although the binding of human RF to dog IgG was not investigated. By comparing titration curves of the human IgM RF standard and IgM RF positive and negative dog sera (see Fig. 3(A) and Fig. 3 ( B ) ) , it seems reasonable to assume that human IgM RF in fact binds to dog IgG, although simultaneous titration of h u m a n IgM RF and normal human serum was not performed. Using, as a reference serum, a four-fold dilution of the h u m a n IgM RF standard, starting at 1/40 (see Fig. 3 ( A ) ) , addressed as 1338 U, and ending at 1/2560, would provide an IgM isotype-specific RF test on dog serum, and a test which probably makes it possible to compare results from different veterinary laboratories.
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ACKNOWLEDGMENTS
I would like to thank Dr. Mimi Hoier-Madsen and the staff at Autoimmunlaboratoriet, Statens Seruminstitut, Copenhagen, Denmark for their support and advice in setting up this assay, and Dr. Agnete Ingild, DAKO, Copenhagen, Denmark for advice and criticism. Likewise, thanks are due to Dr. Kerstin Thoren-Tolling, Biovet, Sweden for many fruitful discussions on the subject. Finally I would like to thank Drs. Niels Lind-Andersen, Willy Gormsen, Jorgen Have and Per Theo Alex Johansen at Herlufmagle Veterinary Clinic and Dr. Stig Taber at Charlottenlund Veterinary Clinic for collecting most of the serum samples used.
REFERENCES Bennett, D., 1987. Immune-based inflammatory joint disease of the dog: canine rheumatoid arthritis. 1. Clinical, radiological and laboratory investigations. J. Small Anim. Pract., 28: 779-797. Bennett, D. and Kirkham, D., 1987. The laboratory identification of serum rheumatoid factor in the dog. J. Comp. Pathol., 97: 541-550. Bernadina, W.E., van Kol, P.J. and Willemse, A., 1988. Antibodies to immunoglobulin-G in dog sera, synovial fluids and aqueous humor: a comparative study of rheumatoid factor assays, suitable for routine application. Vet. Immunol. Immunopathol., 19:259-271. Butler, Jr., V.P. and Vaughan, J.H., 1964. Hemagglutination by rheumatoid factor of cells coated with animal gamma globulins. Proc. Soc. Exp. Biol. Med., 116: 585-593. Carter, S.D., Bell, S.C., Bari, A.S.M. and Bennett, D., 1989. Immune complexes and rheumatoid factors in canine arthritides. Ann. Rheum. Dis., 48: 986-991. Goudswaard, J., van der Donk, J.A., Noordzij, A., van Dam, R.H. and Vaerman, J.-P., 1978. Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol., 8 : 2 1 28. Halliwell, R.E.W., Werner, L.L., Baum, D.E., Newton, C.D., Wolfe, J.H. and Schumacher, H.R., 1989. Incidence and characterization of canine rheumatoid factor. Vet. I mmunol. Immunopathol., 21: 161-175. Harris, E.D., 1990. Rheumatoid arthritis. Pathophysiology and implications for therapy. Review. New Engl. J. Med., 322: 1277-1289. Hau, J., Nilsson, M., Skovgaard-Jensen, H.-J., de Souza, A., Eriksen, E. and Wandall, L.T., 1990. Analysis of animal serum proteins using antisera against human analogous proteins. A study of immunological cross-reaction between human and animal serum proteins. Scand. J. Lab. Anim. Sci., 17: 3-7. Hoier-Madsen, M., Nielsen, L.P. and Moiler, S., 1986. Bestemmelse af IgM-rheumafaktorer ved enzyme-linked immunosorbent assay (ELISA). (Determination of IgM rheumatoid factors by enzyme-linked immunosorbent assay (ELISA)). Ugeskr. Leg. (J. Danish Med. Assoc. ), 148: 2018-2021. Lewis, R.M. and Borel, Y., 1971. Canine rheumatoid arthritis. A case report. Arthritis Rheum., 14: 67-74. Miele, J.A. and Krakowha, S., 1981. Quantitative aspects of binding of canine serum immunoglobulins and Staphylococcus aureus protein A. Am. J. Vet. Res., 42: 2065-2067. Pope, R.M. and Talal, N., 1985. Autoimmunity in rheumatoid arthritis. In: J.M. Cruse and R.E.
ELISA DETECTION OF IgM RHEUMATOID FACTOR IN CANINE SERUM
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Lewis, Jr. (Editors), Concepts in Immunopathology, Vol. 1, Autoimmunity: Basic Concepts; Systemic and Selected Organ-Specific Diseases, Karger, Basel, Switzerland, pp. 219-250. Singer, J.M. and Plotz, C.M., 1956. The latex fixation test I. Application to the serologic diagnosis of rheumatoid arthritis. Am. J. Med., 21: 888-892. Thoren-Tolling, K., 1990. A comparative study of different methods for measurement of rheumatoid factor in dog serum. J. Vet. Med. A, 37: 430-438. Wood, D.D., Hurvitz, A.I. and Schultz, R.D., 1980. A latex test for canine rheumatoid factor. Vet. Immunol. Immunopathol., 1:103-111.
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Elsevier Science Publishers B.V., Amsterdam
Detection of IgM rheumatoid factor in canine serum using a standardized enzyme-linked immunosorbent assay O.L. Nielsen Department of Pharmacology and Pathobiology, The Royal Veterinaryand Agricultural University, Biilowsvej 13, DK-1870, Frederiksberg C, Denmark (Accepted 27 January 1992 )
ABSTRACT Nielsen, O.L., 1992. Detection of IgM rheumatoid factor in canine serum using a standardized enzyme-linked immunosorbentassay. Vet. Immunol. Immunopathol., 34:139-147. An ELISA measuring IgM rheumatoid factor (RF) in dog serum is presented. Dog sera and a human IgM RF standard, calibrated against the international World Health Organisation (WHO) standard, are compared. It is concluded that the human IgM RF standard may be used as reference serum in the canine assay, which makes it possible to compare results from different veterinary laboratories.
ABBREVIATIONS CV, coefficient of variance; ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; OD, optical density; OPD, orthophenylene diamine; RA, rheumatoid arthritis; RF, rheumatoid factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. INTRODUCTION
Rheumatoid arthritis (RA) is a chronic, recurrent, erosive, polyarthritis of man. It affects 1-2% of the population worldwide, with a prevalence in the female population three times that of the male. The inflammatory response in RA has a distinctive immunological component, and it is believed that "the presentation of a relevant antigen to an immunogenetically susceptible host" precedes the disease (Harris, 1990). The antigen has not been identified, but both viruses and bacteria have been implicated. The majority of paCorrespondence to."O.L. Nielsen, National Veterinary Laboratory, Hangovej 2, DK-8200,/krhus N, Denmark.
© 1992 Elsevier Science publishers B.V. All rights reserved 0165-2427/92/$05.00
140
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tients with RA carry the MHC class II antigens of HLA-DR4 a n d / o r HLADR 1. The central role of MHC class II antigens in communication between the cells of the i m m u n e system is well known, as is the role of autoimmunity in the self-perpetuation of RA (Pope and Talal, 1985 ). Approximately 70% of people with RA have elevated serum levels of IgM rheumatoid factor (RF). The canine counterpart of RA has been documented by several authors (e.g. Lewis and Borel, 1971; Bennett, 1987), as has the occurrence of RF, detected by various methods, in serum, synovial fluid and aqueous humor (Wood et al., 1980; Bernadina et al., 1988; Carter et al., 1989; Thoren-Tolling, 1990). In this article an indirect enzyme-linked immunosorbent assay (ELISA) for the measurement of IgM RF in dog serum is described, and the use of the h u m a n IgM RF standard serum as reference in the dog assay is verified. MATERIALSAND METHODS
Serum samples Sera from 226 dogs were collected and stored at - 20 ° C until used. Ninetysix dogs were without clinically detectable disease (sera from 13 of these dogs were kindly supplied by Dr. Hans Flemming Hojelse, H. Lundbeck, Copenhagen, Denmark), five suffered from unclassifiable polyarthritis and 125 were miscellaneous hospital admissions. The five sera were positive in a commercially available RF latex agglutination test (Synbiotics ®, Canine Rheumatoid Factor Test Kit, San Diego, CA), and were the kind gift of Dr. Kerstin ThorenTolling, Biovet, Sweden. The human IgM RF standard serum (code Jan. 1986, 107 IU ), was kindly donated by Dr. Mimi Hoier-Madsen, Autoimmunlaboratoriet, Statens Seruminstitut, Copenhagen, Denmark. The standard serum contains 107 IU m l - 1, when tested at a dilution of 1/ 100, but the serum prepa~a~ahally used was concentrated five-fold by redissolving 1 ml of freezedried serum to a total volume of 0.2 ml.
IgM-RF ELISA procedure The ELISA test was modified after Hoier-Madsen et al. (1986).
Buffers Carbonate buffer was comprised of 1.59 Na2CO3, 2.93 g NaHCO3, 0.20 g NAN3, and 20 ml phenol red 0.05% in 1000 ml distilled water, pH 9.6. Sodium chloride buffer was 29.2 g NaC1, 0.2 g KC1, 0.2 g KH2PO4, 1.15 g Na2HPO4- 2H20, and 10 ml Triton X- 100 in 1000 ml distilled water, pH 7.2. Citrate buffer consisted of 7.3 g C6H807" 1H20 (citric acid), and 11.86 g Na2HPO4" 2H20 in 1000 ml distilled water, pH 5.0.
ELISA DETECTION OF IgM RHEUMATOID FACTOR IN CANINE SERUM
141
Coating Polystyrene microtitre plates (Teknunc, MaxiSorp F96, cat. no. 439454, Roskilde, Denmark) were incubated with 50 #1 dog IgG (Sigma, code 1 4006, St. Louis, MO) at 100 #g ml-1 in carbonate buffer, at 4 °C overnight. The solution was removed, and areas without adsorbed IgG were blocked by applying 200 #1 sodium chloride buffer (containing Triton X- 100) at 4 ° C overnight. The buffer was meticulously removed. The plates were used immediately, or stored at - 20 ° C, for not more than 3 months before being utilized. The purity of IgG was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Testing The wells were washed four times with sodium chloride buffer. Standard serum (serum no. 152, four-fold dilution, starting at 1/40, arbitrarily addressed as 330 U, and ending at 1/2560), sodium chloride buffer, one negative and two positive controls ( t / 100) and samples to be tested ( 1/ 100) were applied in duplicate, 50 #1 in each well, and were incubated at 20°C for 1 h. All sera were diluted in sodium chloride buffer. The wells were washed four times as previously described, and incubated with 50/tl of the horseradish peroxidase conjugated, F ( a b ' ) z fragment of rabbit anti-human IgM (DAKO, code P 322, Glostrup, Denmark) 1/400, for 1 h at 20°C. The wells were washed six times. Orthophenylene diamine (OPD) ( 18 mg) were dissolved in 20 ml citrate buffer and 38 #1 H202 (30%) applied. Of this solution, 50 #1 were added to each well, and the color reaction allowed to evolve for 10-20 min. The reaction was stopped by adding 150 #1 of I mol sulfuric acid. Optical density (OD) was read at 492 nm in an ELISA reader.
Qualitative control of the ELISA procedure ?
To make sucre that the L I S A a e ~ a ~ d e t e c t s l g M RF in serum from dogs, the following procedures were performed.
Verification of the cross-reactivityof rabbit anti-human IgM towards canine IgM The reactivity of anti-human antibody preparations against various animal proteins have been tested by Hau et al. (1990). The reactivity of anti-human IgM (DAKO, code A091/A426, Glostrup, Denmark) with dog serum, using rocket immune electrophoresis, indicated strong cross-reaction. Protein A binds IgG, IgM (Goudswaard et al., 1978; Miele and Krakowha, 1981 ), and IgA (Miele and Krakowha, 1981 ) from dog serum. Using protein A column chromatography (Pharmacia, protein A sepharose, Uppsala, Sweden), IgG and IgM were isolated from the serum often healthy dogs. Approximately 50% of the total serum IgM was retained by protein A, judged by
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visually comparing the precipitates generated in crossed i m m u n e electrophoresis. The IgG- and IgM-containing fractions were subsequently separated by gel-filtration in an high-performance liquid chromatography (HPLC) set-up using a column with a cut-off equivalent to 500 kD (LKB Produkter, TSK G 3000 SW, 8 × 300 m m , Uppsala, Sweden). Proteins obtained in void volume, and proteins filtrated were, for each of the ten dogs, tested against goat antidog IgM (Nordic Immunological Laboratories, code GAD IgM (Fc), Netherlands), and rabbit anti-human IgM (DAKO, code A091, Glostrup, Denmark), using rocket i m m u n e electrophoresis.
Absorption of lgM RF to IgG-coated latex particles Latex particles were coated by standard methods (Singer and Plotz, 1956 ) with dog IgG (Sigma, code 1 4006, St. Louis, MO), and human serum albumin. Serial dilutions of the latex reagents were added to a positive and a negative serum, making a final serum dilution of 1/ 100. A Tris buffer was used. The mixture was incubated for 1 h at 37°C. The latex particles were spun down at 2000 × g 30 min-~. Finally the supernatant was tested in the ELISA. IgM RF activity in the IgM-containing and IgM-depletedfractions of a positive serum The IgM fraction of a positive serum (no. 152 ) was isolated using HPLC gel filtration. The column used had a cut-off corresponding to 500 kD (LKB, Produkter, TSK G 3000 SWG, 21.5 × 600 m m , Uppsala, Sweden). Proteins in void volume and filtrated proteins were concentrated on spin filters (Millipore, code UFC2 TTK, Bedford, MA), retaining molecules with molar weight exceeding 30 kD. The two fractions were then tested in the ELISA. The majority of the IgM RF activity was located in the IgM-containing fraction (results are not shown). Heat inactivation No effect of heating for 30 min at 56°C on the h u m a n IgM RF standard serum and positive dog serum no. 108 was observed. RESULTS A total of 226 sera were tested. Ninety-six sera from healthy dogs gave a cut-off (mean + two times the standard deviation) on 15.0 U. Three sera, no. 72 (mean 4.0 U ) , no. 108 (mean 26.0 U) and no. 152 (mean 128.6 U), were tested in 10 independent set-ups. The coefficient of variance (CV) was 9.0%, 6.5% and 16.4% respectively. Testing three sera, no. 74 (mean 3.3 U ) , no. 45 (mean 33.7 U) and no. 152 (mean 136.8 U) 10 times on the same plate gave CV of 2.1%, 6.3% and 7.9%. Sera from two out of the five dogs with oolyarthritis were positive ( > 15.0
143
ELISA DETECTION OF IgM RHEUMATOID FACTOR IN CANINE SERUM
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Fig. 2. effect on OD in ELISA by absorbing IgM RF into dog IgG-coated latex particles. In 2 (A) latex particles were coated with dog IgG, and in (B) latex particles were coated with human serum albumin. X-axis: dilutions of the latex reagent. 1/2 indicates a two-fold dilution; - , buffer without the addition of latex reagent. Y-axis: optical density (OD). +, positive serum no 208; A, negative serum no. 80; O, plain buffer. As the latex reagent was only available in a limited amount, the curves for plain buffer are incomplete.
U ) in the IgM RF ELISA (22.7 U and 25.1 U, respectively). The 125 miscellaneous admissions were allocated into the following 11 diagnostic groups (total number of dogs and the individual positive values of IgM RF are shown
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B
d) ©
0 1/ 1 0 2 4 0
1/20 Serum dilution
1/5
,., 0 1/10240
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--
_
1120
115
Serum dilution
Fig. 3. titration curves for the human IgM RF standard, three IgM RF-positive dog sera and a serum pool from ten healthy dogs. (A) compares the human standard and the three positive dog sera. (B) compares one positive dog serum and the dog serum pool. +, human standard; Q, positive serum 152, from a dog with pododermatitis; O, positive serum 108; A, positive serum 208; A, negative serum pool.
in parentheses): tumors (25 dogs, 22.8 U), flea infestation ( 17 dogs, 31.3 U, 28.5 U, 21.4 U); dental calculus (16 dogs); osteoarthritis (15 dogs); otitis externa ( 13 dogs ); miscellaneous bacterial infections ( 11 dogs); endometritis (8 dogs); pododermatitis (8 dogs, 128.7 U, 26.0 U); cruciate ligament rupture (5 dogs, 18.7 U); gastro-intestinal disorders (5 dogs, 20.7 U); hot spot (three dogs). Figure 1 presents the results of the rocket immune electrophoresis on three of the ten sera tested. IgG from dogs, mainly migrating towards the cathode at the chosen test conditions, does not react with either anti-dog IgM or antihuman IgM. Dog IgM is precipitated by both anti-dog IgM and anti-human IgM. The difference in IgM concentration between the various dog sera can be read from the height of the rockets. It is seen that anti-dog IgM and antihuman IgM detect the same concentration difference between the individual sera. HPLC-filtrated proteins (fraction b ) contain small amounts of IgM (see Fig. 1 (A), sera nos. 1 and 3). Intermediate gel rocket immune electrophoresis on a pool of sera from the ten healthy dogs showed, taking the sensitivity of the assay into account, that anti-human IgM apparently precipitated all dog IgM. That is, no IgM was left to migrate into the gel containing anti-dog IgM. Figure 2 shows the effect of absorbing IgM RF into dog IgG-coated latex particles, followed by testing the supernatant in the ELISA set-up. By increas-
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ing the concentration of latex particles coated by IgG, but not by latex particles coated by h u m a n serum albumin, the OD is diminished. In Fig. 3 the titration curves of human IgM RF standard serum, three positive dog sera and a pool of sera from ten healthy dogs are presented. It is seen that all four curves have the typical sigmoid shape. The human standard has a higher activity than the three dog sera, of which serum no. 152 has the highest activity. Dog sera no. 108 and no. 208 have merging titration curves. The dog serum pool has the lowest activity.
DISCUSSION
The reliability of the latex agglutination test in measuring RF activity in dog serum has been questioned by several authors e.g. Bennett and Kirkham ( 1987 ) and Halliwell et al. ( 1989 ), who placed greater confidence on various modifications of the Rose-Waaler test. Bernadina et al. ( 1988 ) introduced a reliable gel precipitation test. An IgM-specific RF radioimmunoassay has been described by Carter et al. ( 1989 ), and an ELISA, also measuring IgM RF, has been described by Thoren-Tolling (1990). Neither of the two isotype-specific RF tests used reference sera, but instead recorded the OD values in determining the RF activity. The ELISA described in this paper seems to be a reliable assay for testing IgM RF in dog serum. Although cross-reactivity of anti-human IgM was shown with a batch of antiserum different from the one used in the ELISA, the strong cross-reactivity indicated is probably still valid. However, different batches of horseradish peroxidase-conjugated anti-human IgM may not cross-react with dog IgM to the same extent. This probably represents a minor problem when using an internal dog IgM RF standard, but might be significant when using the h u m a n standard. Testing the same dog serum using different conjugates would settle this problem. The binding of h u m a n RF to animal IgG has been verified by Butler and Vaughan (1964), although the binding of human RF to dog IgG was not investigated. By comparing titration curves of the human IgM RF standard and IgM RF positive and negative dog sera (see Fig. 3(A) and Fig. 3 ( B ) ) , it seems reasonable to assume that human IgM RF in fact binds to dog IgG, although simultaneous titration of h u m a n IgM RF and normal human serum was not performed. Using, as a reference serum, a four-fold dilution of the h u m a n IgM RF standard, starting at 1/40 (see Fig. 3 ( A ) ) , addressed as 1338 U, and ending at 1/2560, would provide an IgM isotype-specific RF test on dog serum, and a test which probably makes it possible to compare results from different veterinary laboratories.
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ACKNOWLEDGMENTS
I would like to thank Dr. Mimi Hoier-Madsen and the staff at Autoimmunlaboratoriet, Statens Seruminstitut, Copenhagen, Denmark for their support and advice in setting up this assay, and Dr. Agnete Ingild, DAKO, Copenhagen, Denmark for advice and criticism. Likewise, thanks are due to Dr. Kerstin Thoren-Tolling, Biovet, Sweden for many fruitful discussions on the subject. Finally I would like to thank Drs. Niels Lind-Andersen, Willy Gormsen, Jorgen Have and Per Theo Alex Johansen at Herlufmagle Veterinary Clinic and Dr. Stig Taber at Charlottenlund Veterinary Clinic for collecting most of the serum samples used.
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Lewis, Jr. (Editors), Concepts in Immunopathology, Vol. 1, Autoimmunity: Basic Concepts; Systemic and Selected Organ-Specific Diseases, Karger, Basel, Switzerland, pp. 219-250. Singer, J.M. and Plotz, C.M., 1956. The latex fixation test I. Application to the serologic diagnosis of rheumatoid arthritis. Am. J. Med., 21: 888-892. Thoren-Tolling, K., 1990. A comparative study of different methods for measurement of rheumatoid factor in dog serum. J. Vet. Med. A, 37: 430-438. Wood, D.D., Hurvitz, A.I. and Schultz, R.D., 1980. A latex test for canine rheumatoid factor. Vet. Immunol. Immunopathol., 1:103-111.
