Clinica Chimica Acta, 188 (1990) 147-160 Elsevier

147

CCA 04702

Quantitative determination of &M-rheumatoid factor by enzyme immunoassay - Standardization using a serum from a rheumatoid arthritis patient Hiroshi Oka, Shunsei Hirohata, Tetsufurni Inoue, Sachiko Iwamoto and Terumasa Miyamoto Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Bunkyo-Ku, Tokyo (Japan) (Received

30 June 1989; revision received 2 January

Key words: IgM rheumatoid

factor;

1990; accepted

Enzymeimmunoassay;

5 January

Rheumatoid

1990)

arthritis

Summary

A method to standardize the quantitation of IgM-rheumatoid factor (RF) by enzymeimmunoassay (EIA) is presented. Serially diluted sera from rheumatoid arthritis patients were added to immobilized human IgG, and bound IgM-RF was detected by addition of horseradish peroxidase labeled anti-human IgM (HRPOaM). The concentration of IgM-RF which produced half of the maximal absorbance at 492 nm given by a saturating concentration of IgM-RF in the EIA plate, was defined as 1 U/ml. The IgM-RF values of test samples were measured as the dilution of the sample which provided half-maximal absorbance. The IgM-RF values determined by this method coincided with those determined by referring to a standard curve made from a serum containing known amounts of IgM-RF. Differences in IgM-RF values, which were caused by varying preparations of horseradish peroxidase anti-IgM (HRPOaM) were corrected for the binding capacity of each preparation to various concentrations of human IgM adherent to the plate. The IgM-RF values determined by this method correlated well with the RF values determined by latex photometric immunoassay (r = 0.956, p < 0.001). IgM-RF values determined by EIA were converted into WHO-units by an empirical formula described. The data observed suggest that the method here reported can standardize IgM-RF values obtained by EIA.

Correspondence to: Shunsei Hirohata, of Tokyo School of Medicine, Hongo

0009-8981/90/$03.50

M.D., Department of Medicine and Physical 7-3-1, Bunkyo-ku, Tokyo 113, Japan.

0 1990 Elsevier Science Publishers

B.V. (Biomedical

Division)

Therapy,

University

148

Introduction Rheumatoid factors (RF) are autoantibodies directed against the Fc portion of IgG, which are frequently detected in patients with rheumatoid arthritis (RA) and other autoimmune diseases [l]. Conventional methods for measurement of RF, such as the sheep red cell agglutination assay, are unable to detect small amounts of RF and to quantitate RF immunoglobulin isotypes, although these have already been standardized by other techniques [2,3]. Development of radioimmunoassay (RIA) and enzymeimmunoassay (EIA) has made this possible [4,5], since these techniques are sensitive enough to detect small amounts of IgM-RF, &A-RF, and even IgG-RF [6]. However, standardization of RF isotype by EIA has been technically difficult. Some investigators have expressed IgM-RF values as absorbance values [5,7], resulting in serious differences under varying experimental conditions or between repeated assays in the same laboratory. Other investigators have used monoclonal IgM-RF or purified IgM-RF as standards [4,8]. However, monoclonal IgM-RF and polyclonal IgM-RF may have different characteristics. Moreover, purified IgM-RF may not be 100% pure. Finally, monoclonal IgM-RF is not widely available and may also vary in different preparations. Therefore, it would be desirable to be able to standardize IgM-RF measurements without special materials. In the present study, an attempt was made to standardize the quantitative measurement of IgM-RF by EIA, using sera from RA patients as standards. The data suggest that the EIA method here described can provide standardized IgM-RF values without the use of special agents. Materials and methods Patients and samples Serum samples were obtained from the venous blood of 31 rheumatoid arthritis patients with definite or classical RA according to American Rheumatism Association criteria [9] and 20 healthy individuals. The patients consisted of 9 males, aged 17-57 (mean 46.8 yr), and 22 females, aged 18-65 (mean 47.6 yr). Twenty-one out of the 31 RA patients were seropositive and 11 were seronegative by laser nephelometry [lo]. The 20 healthy individuals consisted of 13 males, aged 18-62 (mean 43.2 yr) and 7 females, aged 22-58 (mean 43.2 yr). All these healthy individuals were seronegative. WHO RF-standard sera Three RF standard sera of known Hitachi Chemical Co., Ltd., Ibaraki, Pharmaceutical Co., Ibaraki, Japan.

WHO RF titer Hoechst Japan,

were kindly provided by Ltd., Tokyo, and Nissui

Preparation of IgG and antisera Human IgG was purchased from Green Cross Company, Osaka, Japan, and it was further purified by DEAE-cellulose chromatography in 0.005 mol/l phosphate buffer (pH 8.0). Human myeloma proteins of IgG(k) and IgG(1) types were purified

149

from the sera of multiple myeloma patients by DEAE-cellulose chromatography in 0.005 mol/l phosphate buffer (pH 8.0). Immunoelectrophoretically purified rabbit IgG was purchased from Miles Lab., Ltd., Elkhart, IN, USA. Chromatographically purified goat IgG was purchased from Cappel Laboratories. West Chester, PA, USA. Two different preparations of peroxidase conjugated F(ab’), goat anti-human IgM (Mu-chain specific) were purchased from Cappel Laboratories and Tago, Burlingame, CA, USA. Rabbit anti-human IgM (Mu-chain specific) serum was purchased from Behring Institute, Marburg, FRG. From this, the F(ab’), fragment was obtained by digestion with pepsin and gel filtration through Sephadex G-100 [ll]. Horseradish peroxidase (HRPO) (Sigma, Type VI) was conjugated to the F(ab’), fragment according to the method of Nakane and Kawaoi [12]. These conjugates were divided into small portions and kept frozen at - 70 o C until use. These three different preparations of HRPO conjugated F(ab’), anti-human IgM (HRPOaM) were checked for their specificity by EIA as described [13]. All three were found to be IgM specific, showing no reactivity with either IgA or IgG. Procedure

of EIA for IgM-RF

detection

Wells of a 96-well microtier plate (l-220-24, Dynatech Lab., Inc., VA, USA) were first coated with 50 ~1 of a 20 pg/ml solution of human IgG in phosphate-buffered saline, pH 7.2 (PBS). After incubation at 4” C overnight, each well was washed three times with cold PBS, then filled with 100 ~1 of PBS containing 1% bovine serum albumin (BSA) (Miles Lab., Ltd.) and kept at room temperature for 3 h in order to saturate any remaining binding sites on the polyvinyl surface. After three washes with cold PBS, 50 ~1 of samples containing IgM-RF, appropriately diluted with PBS containing 1% BSA (PBS-BSA), were added to the wells, and then incubated 37 o C for 1 h. After 3 washes with cold PBS, 50 ~1 of solution of HRPOaM appropriately diluted with PBS-BSA was added, and incubated overnight at 4°C. After 3 washes with cold PBS, each well received 100 ~1 of substrate solution containing 40 mg of o-phenylenediamine and 10 ~1 of 30% H,O, in 100 ml of 0.05 mol/l citrate-phosphate buffer, pH 4.8. After incubation for 30 min at 37 o C, the reaction was stopped by addition of 50 ~1 of 6 N H,SO,, and the absorbance at 492 nm (OD 492) was read in duplicate in a 2-wave length microplate photometer (MTP-12, Corona Electric Co., Ltd., Ibaraki, Japan). Standardization

of IgM-RF

values

Sera from 25 patients with RA were diluted serially and assayed for IgM-RF by EIA described above. The absorbance at 492 nm (OD 492) was measured for each dilution. The concentration of IgM-RF, which provided half of the maximal OD492 given by the saturating concentration of IgM-RF on the EIA plate, was defined as 1 U/ml. To confirm maximal absorbance, each plate included a serum sample with a very high titer of RF. From the serum dilution of test samples which provided 1 U/ml of IgM-RF, the IgM-RF values were calculated. Optionally, IgM-RF values of the samples were determined by referring to a standard curve made from a serum containing known amounts of IgM-RF determined by the above described method. The IgM-RF values determined by these two methods were compared.

150

Results

Determination of IgM-RF concentration by serial dilution of sera Serum samples were serially diluted and assayed for IgM-RF by EIA. Figure 1 shows relation between the dilution and OD492. The concentration of IgM-RF which provided half of the maximal OD 492 given by saturating concentration of IgM-RF was defined as 1 U/ml. In this way, individual IgM-RF values for different sera could be expressed as the dilution which provided half-maximal OD492. Thus, the IgM-RF values of sample A, sample B, and sample C were 13 500 U/ml, 1600 U/ml and 700 U/ml, respectively, as can be seen in Fig. 1.

Effects of different concentrations or preparations of IgG adsorbed to a solid phase The effects of different concentration of human IgG adsorbed to the plates on IgM-RF values were first examined. As shown in Table I, when human polyclonal IgG was used at a concentration of > 2 pg/ml for coating the wells, the IgM-RF values were constant, irrespective of the concentration of the IgG. However, when the concentration of the IgG was < 2 pg/ml. IgM-RF values decreased with the concentration of the IgG. On the other hand, when human myeloma IgG(k) or IgG(l) was used for coating, the IgM-RF values were the same as those obtained using a plate coated with human polyclonal IgG. However, the IgM-RF values obtained with plates coated with goat IgG or rabbit IgG were lower than those obtained with plates coated with human IgG, polyclonal or monoclonal. In this regard, goat IgG was least capable of binding human IgM-RF. These results indicate that when human IgG was used for coating at a concentration of greater than 2 pg/ml, the IgM-RF values were constant.

04

Sample

A

d-6

Sample

B

0-n

Sample

C

2 ; 0 05-

0 0-L lo*

10’

lOA

1 10=

Fig. 1. Relationship between serum dilution and OD492 in IgM-RF assay. Serially diluted sera were incubated in wells coated with human IgG. Bound IgM-RF was detected by HRPO conjugated anti-human IgM. The concentration of IgM-RF which provided half of the maximal OD492 given by the saturating concentration of IgM-RF was defined as 1 U/ml (shown by closed circles).

151 TABLE

I

Effects of the concentrations

and the species of IgG for coating

on IgM-RF

values

Species

Cone

IgM-RF

of IgG

@g/ml)

Exp. 1

Exp. 2

Exp. 3

Exp. 4

100 20 10 1 100 20 10 100 20 10 100 20 10 100 20 10

nd. nd. 11500 7200 n.d. n.d. n.d. nd. n.d. n.d. nd. nd. n.d. n.d. n.d. n.d.

n.d. n.d. 13000 10000 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. nd. n.d. nd.

12500 13500 12000 n.d. 12500 nd. 11500 13000 n.d. 12000 10500 n.d. 9400 4500 n.d. 5000

n.d. 11500 11000 n.d. n.d. 13000 nd. n.d. 14500 n.d. n.d. 10500 n.d. n.d. 4500 n.d.

Human IgG

polyclonal

Human myeloma Protein IgG(k) Human Protein

myloma IgG(1)

Rabbit IgG

polyclonal

Goat polyclonal IgG

The same sample containing

13500 U/ml

(U/ml)

was used throughout

the study.

n.d., not done.

Effects of different concentration and preparation of HRPO conjugated antibodies The concentration of HRPOaM did not influence the IgM-RF values when the same preparation was used. However, when the effects of different preparations of HRPOaM on IgM-RF values were compared, serious differences were noted, as shown in Fig. 2A. Sample A in fig. 1 was assayed for IgM-RF on three different plates with different preparations of HRPOaM. The optimal dilution of each preparation was initially determined. The IgM-RF values for sample A, determined on plates with HRPOaM from Cappel, Tago, and Behring Institute were 13 500 U/ml, 18 500 U/ml, and 2500 U/ml, respectively. It has been reported that the affinity of enzyme conjugated antibody for antigen might be different, depending upon the methods or conditions of conjugation [14]. Moreover, each preparation of HRPOaM utilized a different source of antibody. Therefore, it was most likely that the differences in IgM-RF values noted above might be explained by the differences in the affinity for IgM of the individual preparations of HRPOaM. To explore this possibility, the capacities of each preparation of HRPOaM to bind to known amounts of IgM adsorbed to the plate were examined. As shown in Fig. 2B, each preparation of HRPOaM was shown to have a different IgM binding capacity from those of the other preparations. Thus, the concentration of IgM which provided the half maximal 0D492 on the plates treated with HRPOaM from Cappel, Tago, and Behring Institute, were 100 ng/ml, 52 ng/ml, and 200 ng/ml, respectively. Of greater importance, the regression analysis revealed a linear relationship between IgM-RF and IgM values at half maximal OD492 (Fig. 3). Therefore, the difference in IgM-RF values due to differences in HRPOaM might be corrected by examining

152 15

o--O HRPO-antl-p(goat,cappel)

A

a-~HRPO-antI-~(goot,togo) 0-n

HRPO-anti-4

(rabblt

)

2 2 0 0.5-

0-l IO2

IO4

IO’ Serum

dllut

10

IO5

Ion

50

100 IgM

500

1000

(ngiml)

Fig. 2. A. The effects of different preparations of enzyme labeled antibody on IgM-RF values. The IgM-RF values obtained using three different preparations of HRPO conjugated anti-human IgM were 13500 U/ml (Cappel), 18500 U/ml (Tago), and 2500 U/ml (Behring Institute), respectively. B. The capacity of three different preparations of HRPO conjugated anti-human IgM to bind different concentrations of IgM adherent to the plate. The concentrations of IgM, which provided the half-maximal OD492 for each preparation of HRPO conjugated anti-human IgM are shown by closed circles.

the binding capacity of each labeled antibody to various concentrations of IgM adherent to a plate, as shown in Fig. 3. Of course, the problem of differences due to HRPOaM preparations was also overcome by using a given standard serum, such as the WHO RF standard.

Yz-90

x+20500 rz-0989 p.Co.001

150

100 IgM

at

half

-max

200

(ng/ml)

013492

Fig. 3. Relationship between IgM-RF values of a serum from an RA patient and IgM values at half-maximal OD 492 for three different preparations of HRPO conjugated anti-human IgM. The IgM-RF values were calculated by multiplying 1 U/ml by the dilution which provided half-maximal OD 492.

153 (uiml)

zo-

1.5. & i ul *

S & k c

l.O-

0.5-

0.5

I

I

1.0

1.5

I

2.0 (U/ml)

Added

IgM-RF

Fig. 4. Anaiytical recovery of IgM-RF. Linear plots were obtained between added IBM-RF and increased IgM-RF. The recovery rate ranged from 95.6% to 98.9%.

Recovery and linearity

Varying amo~ts of a diluted serum with known quantities of IgM-RF were added to a diluted sample with an IgM-RF concentration of 1.25 U/ml. The concentrations of IgM-RF in these mixtures were measured and compared with the expected IgM-RF values, As shown in Fig. 4, the results of this experiment yielded linearplots of added IgM-RF versus increase in IgM-RF. The recovery ranged from 95.6% to 98.9%. Precision

Intra-assay precision was examined using 5 different dilutions of a serum from an RA patient added to 10 wells of the same plate for each dilution. The intra-assay coefficient of variation (CV) ranged from 4.7% to 8.4%. To estimate the inter-assay precision, three reference samples ~ont~ning different ~n~ntrations of IgM-RF were prepared. These reference samples were assayed more than five times on different days by the above described method. The inter-assay CV ranged from 5.2% to 8.2%. Comparison of IgM-RF values determined photometric irnrn~n~a~say (APIA)

by ElA and RF values determined

by latex

IgM-RF values determined by EIA were compared with RF values determined by latex photometric immunoassay (LPIA) with autoanalyzer type 705. Hitachi Chemical Co., Ibaraki, Japan [15]. As shown in Fig. SA, the IgM-RF values determined by EIA were well correlated with the RF values determined by LPIA

154

IgM-RF(EIA)

(x103U/ml)

9B i

8-

?

7-

“$’ ”

6-

.

.

/

5? B

4-

Y:O.l6x-4.13

. /

.

r-0.998 p 0.1 U/ml IgM-RF were always higher than the mean OD 492 value of 10 blanks plus their 3-fold standard deviation. By referring to this standard curve, the IgM-RF values of the 24 sera from RA patients were determined. Each serum was appropriately prediluted so that the value of OD 492 might fall on the steep portion of the standard curve. As summarized in Table II, most serum samples showed higher IgM-RF values in the

TABLE II Determination of IgM-RF in 24 RF sera using enzymeimmunoassay Patient no.

IgG used for coating Human IgG

Rabbit IgG

IgM-RF

W/ml)

&M-RF

w/mU

Aa

Bb

A/B

C”

Db

C/D

A/C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

3700 4 800 5 050 4400 2200 700 2 600 1600 62 750 140 280 3600 52 220 355 190 34 310 140 152 100 1000 1750

3300 4446 5288 4330 2440 702 2700 1600 60 725 166 260 3600 72 215 320 174 30 266 140 168 106 1040 1884

1.11 1.08 0.95 1.02 0.90 1.00 0.96 1.00 1.03 1.03 0.88 1.08 1.00 0.72 1.02 1.11 1.09 1.13 1.17 1.00 0.90 0.94 0.96 0.93

1450 3000 3100 500 1650 220 840 1500 50 350 140 185 2400 68 280 60 66 25 100 70 110 70 420 1050

1493 3093 3 148 475 1562 275 752 1400 47 338 162 177 2021 60 280 54 60 22 100 64 114 66 455 1160

0.97 0.97 0.98 1.05 1.06 0.80 1.12 1.07 1.06 1.04 0.86 1.05 1.19 1.13 1 .oo 1.11 1.10 1.13 1.00 1.09 0.96 1.06 0.92 0.91

2.55 1.60 1.63 8.80 1.33 3.18 3.10 1.07 1.24 2.14 1.00 1.51 1.50 0.76 0.79 5.92 3.47 0.76 3.10 2.00 1.38 1.43 2.38 1.67

Mean f SEM

1424k 337

1419* 333

1.00* 0.02

724 f 188

1.02* 0.02

2.29 f 0.36

738 * 191

a A, C; IgM-RF values determined by half of the maximal absorbance method. b B, D; IgM-RF values determined by standard curve method.

156

plates coated with human IgG than in the plates coated with rabbit IgG, although the differences among the sera appeared to be somewhat variable. However, the IgM-RF values determined using human and rabbit IgG almost coincided with each other. In addition, when the other serum samples with high titers of RF were used as standards, the IgM-RF values of the 24 samples were the same (data not shown). Comparison of WHO RF-standard sera for IgM-RF

content

Since it is difficult for different laboratories to obtain the original WHO RF-standard serum, most laboratories use the standard sera calibrated to WHO units. We have examined three different RF-standard sera for IgM-RF by the EIA half-maximal OD 492 method. The RF-standard sera from Hitachi, Hoechst, and Nissui were found to contain 6.5, 10.8, and 32.6 EIA-units IgM-RF/WHO IU RF, respectively.

Id IO

P

.

t * ; :

5 3

*

10

.

:: i

c”

i . .

IO

*

Seronegatcve Normal

N I 20

Seroposltlve RA

NslO

N=21

Fig. 6. Comparison of IgM-RF values of sera from 20 healthy individuals with those of sera from 31 RA patients (21 seropositive, 10 seronegative). Normal range of IgM-RF determined by the mean + 2 SD of the IgM-RF values in 20 healthy individuals is indicated by the shaded bars.

157

~~rn~ar~s~~ of &M-RF patients

values

of sera

from he~~~hy indiuid~als and sera from RA

To establish the control values of @M-RF in healthy individuals, sera from 20 healthy individuals were studied. As can be seen in Fig. 6, the IgM-RF values of sera from normal healthy individuals ranged from 8 U/ml to 66 U/ml (23.7 it 3.4 U/ml, mean i SEM). By contrast, the IgM-RF values of sera from 21 seropositive RA patients ranged from 70 U/ml to 13 500 U/ml (2 198.3 i: 657.4 U/ml, mean + SEM). Of note, the IgM-RF values of sera from 10 seronegative RA patients ranged 34 U/ml to 190 U/ml (87.8 f 17.4 U/ml, mean f SEM). Six of the 10 patients showed the IgM-RF values exceeding the mean + 2 SD of the IgM-RF values of 20 healthy individuals. Discussion

The current studies described a method to standardize IgM-RF measurement by EIA. IgM-RF was expressed as the dilution of the RA sera which provided half-maximal absorbance at 492 nm (OD 492). IgM-RF values calculated in this manner were found to be signific~tly correlated with the RF values determined by LPIA. Moreover, the IgM-RF values thus obtained almost coincided with those determined by referring to a standard curve made from a serum with known amounts of IgM-RF. In addition, IgM-RF values were the same when another serum was used as a standard. These results indicate that the method proposed can provide standard measurements of IgM-RF in circumstances where a RF reference serum is available. For standardization, the nature and concentration of the IgG used for coating as well as the HRPOaM employed appeared to be factors which affected the magnitude of the IgM-RF values measured. However, the human IgG utilized as to IgG for coating, monoclonal or polyclonal, did not affect the IgM-RF values when added at the concentration of > 2 pg/ml. When rabbit IgG or goat IgG was used for coating, however, the IgM-RF values were decreased. This may be due to the differences in the structure of the C2 and C3 domains of the Fc portion of IgG, to which RF bind [16-181. The differences in the IgM-RF values attributable to the differences in HRPOaM, are probably caused by the differences in the affinity of the antibodies for human IgM. These could be corrected by examining the capacity of HRPOaM to bind to known amounts of IgM adherent to a plate. The linear relationship observed between the IgM value at the half-maximal OD 492 and the IgM-RF value of RA sera determined using different preparations of HRPOaM supported the utilization of this type of correction. It should be pointed out that the problem of differences due to different HRPOaM preparations can be overcome if a standard serum, such as WHO RF-standard, is available. However, since it is almost impossible for every laboratory to possess the original WHO RF-standard serum, most laboratories use standard sera calibrated against the original WHO RF-standard serum. This procedure may result in significant errors. In fact, the amounts of IgM-RF in the 3 different RF-standard sera available in Japan varied significantly when compared

158

on the basis of their WHO IU RF content, as shown in the current study. There may be several reasons for these differences. First, the original WHO RF concentration was determined by the semiquantitative passive hemagglutination technique. Second, the deter~nation of the RF IU was performed, not by measurement of the RF activity of the standard serum, but by the weight of the serum used. Finally, the methods of calibration in various laboratories were variable. Therefore, the EIA method utilized in the current study for standardization of IgM-RF values appeared more suitable than using a given WHO RF-standard serum. The existence of &A-RF and IgG-RF as well as IgM-RF has been well documented 15-71. However, their actual amounts in sera from RA patients have not yet been well quantitated, and their interactions with one another have been almost ignored [5-71. In the current studies, almost 100% recovery of added IgM-RF was accomplished. It is likely, therefore, that the presence of IgA-RF and IgG-RF may not affect the quantitation of IgM-RF by the method described. The proposed method detected IgM-RF in patients with seronegative RA which could not be detected by the sensitive laser nephelometry method [lo]. Moreover, the IgM-RF values in seronegative RA patients were significant higher than those in normal individuals. Therefore, the proposed method may provide a sensitive tool for the detection of IgM-RF.

The authors thank Dr. Morris Ziff, The University of Texas Southwestern Medical Center, Dallas TX, for his advice, on the manuscript. References 1 Egeland T, Munthe E. Rheumatoid Factors. Clin Rheum Dis 1983;9:135-160. 2 Singer JM, Plotz CM. The latex fixation test. 1. Application to the serologic diagnosis of rheumatoid arthritis. Am J Med 1956;21:888-892. 3 Cathcart EW, O’Sullivan JB. Standardization of the sheep red cell agglutination test. The use of pooled reference sera and hema~lutination trays. Arthritis Rheum 1965;8:530-537. 4 Koopman WJ, Schrohenloher RE. A seneitive radioimmunoassay for quantitation of IgM rheumatoid factor. Arthritis Rheum 1980;23:302-308. 5 Faith A, Pontesilli 0, Unger A, Panayi GS, Johns P. ELISA assays for IgM and IgG rheumatoid factors. J Immunol Method 1982;55:169-177. 6 Teitsson I, Valdimarsson H. Use of monoclonal antibodies and F(ab’), enzyme conjugates in ELISA for IgM, IgA and IgG rheumatoid factors. J Immunol Method 1984;71:149-161. 7 Scott DL, Dawes PT, Collins M, Stone R. ELISA assays for IgM and IgG rheumatoid factors their clinical correlations during therapy with slow-acting anti-rheumatic drugs. Ciin Rheum 1987;6:358368. 8 Robbins DL, Kenny T, Wutke A, Benisek W. Determination of the affinity of monoclonal 19s IgM rheumatoid factor for IgG by modified immunoassay (ELISA). J Immunol Method 1988;110:111-116. 9 Ropes MW, Bennet GA, Cobb S, Jacox R, Jessar RA. Revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 1958;9:1?5-176. 10 Jones CE, Roussear RJ, Maxwell KW. Quantitation of rheumatoid factor activity by nephelomet~. Am J Clin Path01 1979;72:432-436.

159 11 Nisonoff A. Enzymatic digestion of rabbit r-globulin and antibody and chromatography of digestion products. Methods Med Res 1964;10:134-140. 12 Nakane PK, Kawaoi A. Peroxidase-labeled antibody-a new method of conjugation. J Histochem Cytochem 1974;22:1084-1091. 13 Hirohata S, Inoue T, Yamada A, Hirose S, Miyamoto T. Quantitative determination of immunoglobulins in the cerbrospinal fluid based on their light chain properties using a solid-phase enzyme immunoassay. Clin Chim Acta 1984;142:11-20. 14 Johnson GD, Holborow EJ, Dorling J. Immunofluorescence and immunoenzyme techniques. Immunoenzyme Method. In: Weir DM, ed. Handbook of experimental immunology, Vol. 1. Immunochemistry. Oxford: Blackwell Scientific Publications. 15 Inoue T, Kisaki T, Miyamoto T, et al. Quantitative measurement of rheumatoid factor by latex photometric immunoassay, and its clinical significance in rheumatoid arthritis. Ryumachi 1988;28:346-353 (in Japanese). 16 Natving JB, Gaarder PI, Turner MW. IgG antigens of the C2 and C3 homology regions interacting with rheumatoid factors. Clin Exp Immunol 1972;12:177-184. 17 Pope RM, Mcduffy SJ. IgG rheumatoid factor: analysis of various species of IgG for detection by radioimmunoassay. J Lab Clin Med 1981;94:842-853. 18 Bourgois A, Fougereau M, Rocca-Serra J. Determination of the primary structure of a mouse IgG2a immunoglobulin: amino-acid sequence of the Fc fragment. Implications for the evolution of immunoglobulin structure and function. Eur J Biochem 1974;43:423-435.

Quantitative determination of IgM-rheumatoid factor by enzyme immunoassay--standardization using a serum from a rheumatoid arthritis patient.

A method to standardize the quantitation of IgM-rheumatoid factor (RF) by enzymeimmunoassay (EIA) is presented. Serially diluted sera from rheumatoid ...
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