International
InternationalOl~hopaedics(SICOT)(1992) 16:272-276
Orthopaedics © Springer-Verlag1992
The diagnostic significance of anti-Type II collagen antibody assay in rheumatoid arthritis K. Fujii, M. Tsuji, A. Kitamura, and K. Murota Departmentof OrthopaedicSurgery,The Jikei UniversitySchoolof Medicine,3-25-8 Nishi-Shinbashi,Minato-ku,Tokyo, 105, Japan
Summary. An improved enzyme-linked immunosorbent assay (ELISA) for the detection of anticollagen antibodies in human serum has been developed. With the use of this method, antibodies against native human Type H collagen were detected in 22.7% of sera from 480 patients with rheumatoid arthritis (RA). The antibodies were found to be collagen type specific, showing no reaction with human Type I and Type III collagens. The antibodies appeared in high incidence during the early phase of the disease, and RA patients with involvement of a single joint, monoarticular arthritis, were often positive for anti-Type II collagen antibodies. In most of these patients, antiType II collagen antibodies preceded the appearance of rheumatoid factors. The antibodies were all negative in sera from patients with gout, osteoarthritis (OA) and non-arthritic diseases. Thus, anti-Type II collagen antibody assay may have diagnostic significance for RA patients, especially those in whom laboratory and clinical findings provide only minimal help in diagnosis. R~sum~. En vue de ddtecter des anticorps anti-collagone dans le s6rum humain, une technique amdlior6e du test ELISA a ~t6 raise au point. Par cette technique on a d6tect6 dans 22.7% des 6chantillons rassemblds chez 480 patients atteints d' arthrite rhumatome (AR), des anticorps contre le collagOne Type II d'origine humaine. Ces anticorps agissent sur un type spdcifique de collagkne et restent inactifs vis gt vis des collagOnes naturels d'origine humaine de Type I et III. Les anticorps apparaissent avec une grande fr6quence pendant la premiOre phase de la maladie et les patients atteints d'une seule localisa-
Reprint requests to: K. Fujii
tion d'AR, c'est ~t dire d'une monoarthrite, sont souvent positifs aux anticorps anti-collagOne Type II. Chez la plupart de ces patients les anticorps anti-collagbne Type H ont pr~c6di l' apparition des facteurs rhumato'Mes. D'autre part, on a constat6 que ces anticorps ne sont pas prdsents dans le sdrum des patients atteints de goutte, d'ost~o-arthrose et de maladies non arthrosiques. Par consdquent la recherche d' anticorps anti-collagOne Type H pourrait jouer un role important dans le diagnostic d'AR, en particulier chez les malades pour lesquels l'examen clinique et les examens de laboratoire ne fournissent que peu d'616ments en faveur de ce diagnostic.
Introduction The antigenicity and the potential role of collagen as an autoantigen in connective tissue diseases were initially pointed out by Steffen and his collaborators during 1965-1969 [20]. However, the genetically distinct types of collagens had not been taken into consideration. Interest in the pathological role of collagen in rheumatoid arthritis (RA) was stimulated by Type II collagen-induced arthritis (CIA) developed in rats by Trentham et al. in 1977 [27], following the isolation of Type II collagen from hyaline cartilage by Miller [ 18]. In CIA developed in rodents, antibodies against autologous Type II collagen were shown to be critically involved in the pathogenesis [21 ], and it has been proposed that a major immunogenic and arthritogenic epitope on Type II collagen resides in the region of the molecule represented by cyanogen bromide peptide-ll (CB-11) in DBA/1 mice immunized with chick Type II collagen [24]. These results implied that the arthritogenic epitope is a
K. Fujii et al.: Anticollagen antibodies and rheumatoid arthritis c o m m o n antigen determinant on T y p e 11 collagen molecules regardless o f the species o f animal. Although a number of immunological studies have been performed to demonstrate the existence of antibodies against collagens in sera and joint fluids from RA patients by a variety of methods such as passive haemagglutination tests [1, 17], radioimmunoassay [16, 2] immunoftuorescence [3] and an enzyme-linked immunosorbent assay [5, 28], wide variations were observed between the data concerning the specificity and the incidence of antibodies. Furthermore, relationships have not been defined between the antibody titres and/or incidence and the clinical features o f RA. Therefore, the significance of the formation of antibodies against collagens in R A remained unknown. In order to clarify the role of autoimmunity to collagen in the pathogenesis o f RA, the need arose for a certain serological assay for anti-collagen antibodies. W e recently developed an improved E L I S A to diminish non-specific background interference and provide higher sensitivity [10]. Based on this assay system, it was found that anti-collagen antibodies in R A patients are specific to T y p e II collagen and have no cross-reactivity with Type I and T y p e 11I collagens [26]. Also, anti-Type II collagen antibodies were found in the articular cartilage of R A patients, and are most likely to contribute to the pathogenesis o f joint destruction [26]. In the present study, we have extended the previous observations and examined the sera from patients with various stages of R A for the incidence o f anti-Type II collagen antibodies, and have looked at the circulating antibodies in RA patients with involvement of a single joint at the onset. The data show the clinical importance o f anti-Type 11 collagen antibody assay in patients with a very early stage of atypical onset of R A who present a difficult diagnostic problem because of the absence of rheumatoid factors.
273
Table 1. Incidence of antibody against Type II collagen in sera from patients with rheumatoid arthritis ARA criteria
No. of patients
Total no. of sera positive for Type II collagen
Probable Definite Classic Total
35 69 376 480
25 (71.4%) 26 (37.7%) 58 (15.4%) 109 (22.7%)
Serum samples were diluted 1 : 50 with buffered normal rabbit serum and assayed for reactivity with human Type II collagen in an ELISA. Reactivity was confirmed by inhibition studies, and those inhibited 80% by human Type II collagen were listed. ARA: American Rheumatism Association
Preparation of collagens Human collagens used in this study consisted of three genetically distinct types. Type I, Type II and Type III collagens were prepared from skin, articular cartilage and placenta, respectively, following solubilization by limited pepsin digestion and fractionation by differential salt precipitation [7, 9, 19].
Assay for anti-collagen antibodies The detection of anti-collagen antibodies was performed by ELISA using buffered normal rabbit serum (NRS) as a blocking agent to diminish non-specific binding of human IgG to the plastic plates as previously described [10]. The ELISA plates (Serocluster U plates; Costar, Cambridge, MA) were coated with 100 [tl of collagen (5 [tg/ml) dissolved in phosphate buffer, pH 7.5. Serum samples were diluted 1:50 in buffered NRS and added to the plates. The second antibody consisted of peroxidase-conjugated rabbit anti-human IgG (Fc specific; Cooper Biochemical, Cochranville, PA) diluted 1:2000 with 25% NRS. Inhibition studies were performed by diluting sera with buffered NRS containing each antigen at a concentration of 50 ~tg/ml. The reactivity of serum with Type II collagen was more than 80% inhibited by the antigen. Antibody levels were calculated by reference to the optical density values of a standard serum from an arthritic cynomolgus monkey that had been immunized with chick Type II collagen [10, 25]. Results are given in arbitrary units, as defined by the reference serum diluted 1 : 128,000 [26].
Results Patients and methods
Subjects Serum samples were obtained from patients with probable (35), definite (69) and classic (376) RA, and stored at-20 ° C until use. The diagnosis and anatomical staging of the disease were established according to the American Rheumatism Association (ARA) criteria [15]. Of the 480 patients, 89 were classified as stage I, 168 as stage II, 150 as stage III and 73 as stage IV. Serum was also obtained from 16 patients with the polyarticular type of juvenile rheumatoid arthritis (JRA), 50 patients with OA, 38 patients with gout and 45 patients with other, non-rheumatic disease. One hundred and fifty normal control subjects provided serum samples.
O f the 480 R A sera assayed, 109 (22.7%) were found to react with native human T y p e II collagen (Table 1). All the sera confirmed to be positive for anti-Type II collagen antibodies were negative for anti-human T y p e I and anti-human T y p e III collagen antibodies. Interestingly, the antibodies were found to appear in a high incidence (43/57 cases, 75.4%) in patients who were diagnosed as having early RA. O f these patients with early RA, 31 were classified as probable and 26 as definite RA. The incidence of antiT y p e II collagen antibodies in stage I was relatively higher than that in stages II, III and IV (Fig. 1).
274
K. Fujii et al.: Anticollagen antibodies and rheumatoid arthritis
¢-. 09
co
Discussion
I00
o
09
80
09
60
55.1
O
40
21.4
46 09
20
T T'T*
¢-09
o~
IX.
0
Normal
11
10.7
11.0
III
IV
Stage of RA Fig. 1. Incidence of antibodies against Type II collagen in sera from normal control subjects (150) and patients with RA. Of the 480 RA patients, 89 were classified as stage I, 168 as stage II, 150 as stage I n and 73 as stage IV. Serum samples were diluted 1 : 50 with buffered normal rabbit serum and assayed for reactivity with human Type II collagen in an ELISA. Reactivity was confirmed by inhibition studies, and those inhibited 80% by human Type II collagen were listed. Asterisks (*) indicate a significant difference relative to the normal sera (p
InternationalOl~hopaedics(SICOT)(1992) 16:272-276
Orthopaedics © Springer-Verlag1992
The diagnostic significance of anti-Type II collagen antibody assay in rheumatoid arthritis K. Fujii, M. Tsuji, A. Kitamura, and K. Murota Departmentof OrthopaedicSurgery,The Jikei UniversitySchoolof Medicine,3-25-8 Nishi-Shinbashi,Minato-ku,Tokyo, 105, Japan
Summary. An improved enzyme-linked immunosorbent assay (ELISA) for the detection of anticollagen antibodies in human serum has been developed. With the use of this method, antibodies against native human Type H collagen were detected in 22.7% of sera from 480 patients with rheumatoid arthritis (RA). The antibodies were found to be collagen type specific, showing no reaction with human Type I and Type III collagens. The antibodies appeared in high incidence during the early phase of the disease, and RA patients with involvement of a single joint, monoarticular arthritis, were often positive for anti-Type II collagen antibodies. In most of these patients, antiType II collagen antibodies preceded the appearance of rheumatoid factors. The antibodies were all negative in sera from patients with gout, osteoarthritis (OA) and non-arthritic diseases. Thus, anti-Type II collagen antibody assay may have diagnostic significance for RA patients, especially those in whom laboratory and clinical findings provide only minimal help in diagnosis. R~sum~. En vue de ddtecter des anticorps anti-collagone dans le s6rum humain, une technique amdlior6e du test ELISA a ~t6 raise au point. Par cette technique on a d6tect6 dans 22.7% des 6chantillons rassemblds chez 480 patients atteints d' arthrite rhumatome (AR), des anticorps contre le collagOne Type II d'origine humaine. Ces anticorps agissent sur un type spdcifique de collagkne et restent inactifs vis gt vis des collagOnes naturels d'origine humaine de Type I et III. Les anticorps apparaissent avec une grande fr6quence pendant la premiOre phase de la maladie et les patients atteints d'une seule localisa-
Reprint requests to: K. Fujii
tion d'AR, c'est ~t dire d'une monoarthrite, sont souvent positifs aux anticorps anti-collagOne Type II. Chez la plupart de ces patients les anticorps anti-collagbne Type H ont pr~c6di l' apparition des facteurs rhumato'Mes. D'autre part, on a constat6 que ces anticorps ne sont pas prdsents dans le sdrum des patients atteints de goutte, d'ost~o-arthrose et de maladies non arthrosiques. Par consdquent la recherche d' anticorps anti-collagOne Type H pourrait jouer un role important dans le diagnostic d'AR, en particulier chez les malades pour lesquels l'examen clinique et les examens de laboratoire ne fournissent que peu d'616ments en faveur de ce diagnostic.
Introduction The antigenicity and the potential role of collagen as an autoantigen in connective tissue diseases were initially pointed out by Steffen and his collaborators during 1965-1969 [20]. However, the genetically distinct types of collagens had not been taken into consideration. Interest in the pathological role of collagen in rheumatoid arthritis (RA) was stimulated by Type II collagen-induced arthritis (CIA) developed in rats by Trentham et al. in 1977 [27], following the isolation of Type II collagen from hyaline cartilage by Miller [ 18]. In CIA developed in rodents, antibodies against autologous Type II collagen were shown to be critically involved in the pathogenesis [21 ], and it has been proposed that a major immunogenic and arthritogenic epitope on Type II collagen resides in the region of the molecule represented by cyanogen bromide peptide-ll (CB-11) in DBA/1 mice immunized with chick Type II collagen [24]. These results implied that the arthritogenic epitope is a
K. Fujii et al.: Anticollagen antibodies and rheumatoid arthritis c o m m o n antigen determinant on T y p e 11 collagen molecules regardless o f the species o f animal. Although a number of immunological studies have been performed to demonstrate the existence of antibodies against collagens in sera and joint fluids from RA patients by a variety of methods such as passive haemagglutination tests [1, 17], radioimmunoassay [16, 2] immunoftuorescence [3] and an enzyme-linked immunosorbent assay [5, 28], wide variations were observed between the data concerning the specificity and the incidence of antibodies. Furthermore, relationships have not been defined between the antibody titres and/or incidence and the clinical features o f RA. Therefore, the significance of the formation of antibodies against collagens in R A remained unknown. In order to clarify the role of autoimmunity to collagen in the pathogenesis o f RA, the need arose for a certain serological assay for anti-collagen antibodies. W e recently developed an improved E L I S A to diminish non-specific background interference and provide higher sensitivity [10]. Based on this assay system, it was found that anti-collagen antibodies in R A patients are specific to T y p e II collagen and have no cross-reactivity with Type I and T y p e 11I collagens [26]. Also, anti-Type II collagen antibodies were found in the articular cartilage of R A patients, and are most likely to contribute to the pathogenesis o f joint destruction [26]. In the present study, we have extended the previous observations and examined the sera from patients with various stages of R A for the incidence o f anti-Type II collagen antibodies, and have looked at the circulating antibodies in RA patients with involvement of a single joint at the onset. The data show the clinical importance o f anti-Type 11 collagen antibody assay in patients with a very early stage of atypical onset of R A who present a difficult diagnostic problem because of the absence of rheumatoid factors.
273
Table 1. Incidence of antibody against Type II collagen in sera from patients with rheumatoid arthritis ARA criteria
No. of patients
Total no. of sera positive for Type II collagen
Probable Definite Classic Total
35 69 376 480
25 (71.4%) 26 (37.7%) 58 (15.4%) 109 (22.7%)
Serum samples were diluted 1 : 50 with buffered normal rabbit serum and assayed for reactivity with human Type II collagen in an ELISA. Reactivity was confirmed by inhibition studies, and those inhibited 80% by human Type II collagen were listed. ARA: American Rheumatism Association
Preparation of collagens Human collagens used in this study consisted of three genetically distinct types. Type I, Type II and Type III collagens were prepared from skin, articular cartilage and placenta, respectively, following solubilization by limited pepsin digestion and fractionation by differential salt precipitation [7, 9, 19].
Assay for anti-collagen antibodies The detection of anti-collagen antibodies was performed by ELISA using buffered normal rabbit serum (NRS) as a blocking agent to diminish non-specific binding of human IgG to the plastic plates as previously described [10]. The ELISA plates (Serocluster U plates; Costar, Cambridge, MA) were coated with 100 [tl of collagen (5 [tg/ml) dissolved in phosphate buffer, pH 7.5. Serum samples were diluted 1:50 in buffered NRS and added to the plates. The second antibody consisted of peroxidase-conjugated rabbit anti-human IgG (Fc specific; Cooper Biochemical, Cochranville, PA) diluted 1:2000 with 25% NRS. Inhibition studies were performed by diluting sera with buffered NRS containing each antigen at a concentration of 50 ~tg/ml. The reactivity of serum with Type II collagen was more than 80% inhibited by the antigen. Antibody levels were calculated by reference to the optical density values of a standard serum from an arthritic cynomolgus monkey that had been immunized with chick Type II collagen [10, 25]. Results are given in arbitrary units, as defined by the reference serum diluted 1 : 128,000 [26].
Results Patients and methods
Subjects Serum samples were obtained from patients with probable (35), definite (69) and classic (376) RA, and stored at-20 ° C until use. The diagnosis and anatomical staging of the disease were established according to the American Rheumatism Association (ARA) criteria [15]. Of the 480 patients, 89 were classified as stage I, 168 as stage II, 150 as stage III and 73 as stage IV. Serum was also obtained from 16 patients with the polyarticular type of juvenile rheumatoid arthritis (JRA), 50 patients with OA, 38 patients with gout and 45 patients with other, non-rheumatic disease. One hundred and fifty normal control subjects provided serum samples.
O f the 480 R A sera assayed, 109 (22.7%) were found to react with native human T y p e II collagen (Table 1). All the sera confirmed to be positive for anti-Type II collagen antibodies were negative for anti-human T y p e I and anti-human T y p e III collagen antibodies. Interestingly, the antibodies were found to appear in a high incidence (43/57 cases, 75.4%) in patients who were diagnosed as having early RA. O f these patients with early RA, 31 were classified as probable and 26 as definite RA. The incidence of antiT y p e II collagen antibodies in stage I was relatively higher than that in stages II, III and IV (Fig. 1).
274
K. Fujii et al.: Anticollagen antibodies and rheumatoid arthritis
¢-. 09
co
Discussion
I00
o
09
80
09
60
55.1
O
40
21.4
46 09
20
T T'T*
¢-09
o~
IX.
0
Normal
11
10.7
11.0
III
IV
Stage of RA Fig. 1. Incidence of antibodies against Type II collagen in sera from normal control subjects (150) and patients with RA. Of the 480 RA patients, 89 were classified as stage I, 168 as stage II, 150 as stage I n and 73 as stage IV. Serum samples were diluted 1 : 50 with buffered normal rabbit serum and assayed for reactivity with human Type II collagen in an ELISA. Reactivity was confirmed by inhibition studies, and those inhibited 80% by human Type II collagen were listed. Asterisks (*) indicate a significant difference relative to the normal sera (p
