IMMUNOLOGICAL INVESTIGATIONS, 17(6&7), 5 6 1 - 5 6 5 (1988)

ENZYME IMMUNOASSAY FOR IgG RHEUMATOID FACTOR COMBItUNC WITE HOMOLWOUS IgG

M. Miyata and F. Milgroa

S ta te University of N e w York a t Buffalo, Buffa1.0, NY 14214 Department of Microbiology, School of Medicine

ABSTRACT

An enzyme immunoassay (EIA) was developed t o de tec t , in human sera, IgG rheumatoid fac tor (RF) combining with human I&. Wells of the EIA p l a t e were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies t o F(abr)2 of human IgG followed by rabbi t anti-goat I& conjugated with a lka l ine phosphatase and f i n a l l y by the substrate. Using this procedure, RF was detected in 35g-852 of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was a l so described fo r detection, i n rabbi t sera, of an RF combining with rabbi t 1s.

~ T R O D U C T I O N

In recent years there has been considerable in t e re s t in studies on IgG rheumatoid fac tors (RF) and t h e i r possxble r o l e i n the pathogenesis

of rheumatoid arthritis has been reviewed (1,2). The pathogenic ro l e of

IgG RF i n glomerulonephritides became obvious from several s tud ies conducted

i n the 1970s and 1980s ( 3 - 5 ) , a l so immune deposits containing t h i s fac tor

were demonstrated in rejected human renal a l log ra f t s (6,7). Significantly,

IgG RF has a propensity f o r self-association. Pope et al. (8) proposed

tha t each molecule of I& RF may a c t as bivalent antibody and bivalent

antigen, and in t h i s way these molecules may aggregate in to large complexes.

Most investigators have used enzyme imnunoassay (EIA) t o de tec t I&

RF. In this laboratory, human IgG RF was detected using, as an antigen,

an immune complex composed OP human serum albumin that had combined with

its corresponding antibodies of goat or ig in (9) . I t may be safe ly stated

tha t detection by EIA of an IgG RF combiniilg with heterologous IgG is a

r e l a t ive ly simple procedure, since the antiglobulin reagent can readi ly

distinguish IgG RF from the I& antigen when they originate from d i f f e ren t

species. On the other hand, detection of IgG RF directed against homologous

IgG, e.g., human RF which combines with hunlan lgC, presents considerable

d i f f i cu l ty , since the antiglobulin reagent i s incapable of dist inguishing

between IgG used a s an antigen and the IgG RF'. To awercome this d i f f i c u l b ,

w e developed an EIA i n which the Fc fragment of IgG was used as the antigen

561

( 'opyreht 0 1988 hy Marcel Dekkrr, lric.

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562 MIYATA AND MILGROM

and RF was detected by means of an anti-F(ab1)2 reagent. A similar approach was taken by Carson et al. (lo) in their studies performed by means of a radioinnnunoassay.

MATERIALS AND HETBODS EIA was conducted in 96-we11 plates of Dynatech, Alexandria, VA. The

wells were coated with the Fc fragment of human IgG (Cappel, Malvern, PA) used as a 2 &ml solution in carbonate-bicarbonate buffer, pH 9.6. Each well was charged with 100 pl of this preparation and the plates were incubated in an incubator at 37°C for 3 hrs. Then the wells were emptied and washed three times with phosphate buffered saline, pH 7.3 (Pl3S) with the addition of 0.05% Tween-20 (PTN). Subsequently the wells were charged with 150 fl of blocking buffer consisting of PBS, with the addition of 1% bovine serum albwain and 0.055 'Pween-20. The plates were incubated for 1 hr at 37OC. Then the wells were washed three times with PTN and charged with 100 pl of human serum studied for RF diluted 1:lOO in the blocking buffer. After 1 hr incubation at 37OC, the wells were washed in PTN and charged with 100 pl of goat antiserum to F(ab')2 fragment of human IgG (Cappel), diluted 1:1600 in blocking buffer. This particular antiserum needed absorption with the Fc fragment of human IgG to produce F(abl)p specific reactions. This was achieved by adding the Fc fragment to the blocking buffer used for dilution of anti-F(abr)2 serum at a final concentration of 25 &ml. The plates were incubated for 1 hr at 37°C and washed three times with PTN. Then the wells were charged with 100 fl of alkaline phosphatase-conjugated rabbit antibody to goat IgG, a preparation of Sigma (St. Louis, HO), diluted 1:lOOO in blocking buffer. In order to obtain specific results we had to add human IgG to this conjugate. This was achieved by adding Fraction I1 of human serum (Sigma) at a final concentration of 0.5 mg/ml to the blocking buffer used to dilute the antiserum. The plates were again incubated for 1 hr at 37OC and washed three times with PTN. This was followed by the addition of p-nitrophenyl phosphate (Sigma) in a substrate buffer at a concentration of 1 m g / m l . The results were expressed as optical density (OD) read by a spectrophotometer.

An analogous test was developed for the detection in rabbit sera of an RF combining with rabbit I&. The Fc fragment of rabbit IgG was a prepara- tion of Cappel; it was used at a concentration of 25 pg/ml. Rabbit sera examined for RF were diluted 1:20. Goat antiserum to F(ab')g fragment of rabbit IgG was also a preparation of Cappel and it was used at a 1:1600 dilution. This antiserum did not have to be absorbed with Fc fragment. Rabbit anti-goat IgG conjugated with alkaline phosphatase was the same prepar- ation of Sigma that was used for the detection of RF in human sera. No absorption of this reagent was necessary.

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563 LNZYME IMMUNOASSAY FOR IgC RHEUMATOID FACTOR

RESULTS We employed the outlined procedure fo r preliminLary studies on IgC RF

in normal human sera and human sera from several pathological conditions

(Table I) . We accepted OD of 0.50 as an a rb i t ra ry "dividing line" between

posit ive and negative resu l t s . It may be noted in the tab le tha t the mean

ODtSD f o r normal sera was 0.45. According t o t h i s c r i te r ion , the frequency

of posit ive results with pathological sera was between 35% and 855, whereas

with normal sera only 7% were positive. Sltgnifiwultly, sera t h a t gave

posit ive r e su l t s u i t h homologous IgC by means of t h e described procedure

were not always the same a s those tha t gave posit ive r e su l t s i n R I A with

heterologous I&, e.g., rabbi t IgG. This was most s t r i k i n g in studying

20 human transplantation sera of which 17 w e r e po&itive in the reaction

v i th homologous IgG (Table I), but none in the reaction with rabbi t IgC.

We a l so conducted a preliminary study u s i n g the procedure f o r detection,

in normal rabbit sera, of IgC RF combining with rabbi t I&. I n studying

78 sera, the mean OD of 0.19 with S D of 0.21 w a s noted. This procedure

i s used a t present f o r the detection of RF i n sera of rabbi t s submitted

to prolonged immunization with various antigens a s well a s i n sera of rabbi t

recipients of a l lograf t s .

DISCUSSION Application of the Fc fragment of IgG a s antigen and a n t i - F ( a b ' ) ~ a s

reagent fo r the detection of binding of RF permitted the development of

an EIA for human and rabbi t IgC RF combining v i t l i homologous I&. The commer-

c i a l reagent t o human F(ab1)2 contained t races of ac t iv i ty against the Fc

fragment, which could be readi ly abrogated by adding a s l i t t l e a s 25 pg/ml

of Fc t o the buffer. The reagent f o r rabbit F(abt )2 did not have any traces

of anti-Fc ac t iv i ty tha t could be detected ty E I A and, accordingly, did

not require any treatment. Alkaline phosphatase-conjwpted rabbi t antibodies

t o goat IgC produced very s l igh t cross-reactions with human I&; thit; cross-

reaction could be readily abolished by the addittion of Fraction I1 of human

serum t o the blocking buffer. There was obviously no cross-reaction of

t h i s reagent with rabbi t I& and i ts neutralization u a s unnecessary i n the

tests f o r rabbit RF. The addition of the f lu id phase of human Fc or human

Fraction I1 t o the reagents had no s igni f icant influence on detection of

the binding of IgG RF, as evidenced by the f ac t t ha t i f these reagents were

added in amounts four times higher, ident ica l results were obtained.

As reviewed i n our previous paper (91, I@ RF have been found by many investigators not only in RA, but a l so i n other diseaees and in normal human

beings, and, therefore, these fac tors a re of l i m t e d diagnostic value. S t i l l ,

because of t h e i r broadly recognized pathological significance, studies of

these fac tors a re of considerable interest. 1:n the present study, IgC RF combining with human IgG were found in 35-851 of pathological human sera and the difference between this frequency and the frequency of posit ive

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TABLE I

EIA

for I&

RF Combining with Fc Fragment

of Human I&

MIS

BA

Leprosy

Syphilis

SLE

Rl?Ilal

Benal

disease

allograft

rejection

Total

100

66

52

71

37

47

20

Num

ber of 6era

OD >0.50

7 55

41

47

22

17

17

Mean OD

0.30

1.11

0.71

0.60

0.65

0.43

1.02

Sta

nd

ard

deviation

0.15

0.42

0.40

0.29

0.37

0.28

0.40

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ENZYME IMMUNOASSAY FOR I g G RHEUMATOID FACTOR

results of 7% in normal sera is of high statistical significance. Especially interesting was the high frequency, 855, of TgG RF l o patients who rejected their renal grafts. Studies on 1gG RF to homologous IgG are now continued on human and rabbit allograft recipients.

5 6 5

ACKNOWLEDGEMENTS This work was supported by Research Grant DKl’1317 from the National

Institute of Diabetes, Digestive and Kidney D’iseases, and by a graa.nt from the Richard W. and Mae Stone Goode Estate.

1.

2.

3.

4.

5.

6.

7.

8. 9. 10.

REFERENCES

D.A. Carson, in Textbook of Rheumatology, W.N. Kelly, E.13. Harris, S.

Ruddy and C.B. Sledge, eds., W.B. Saunders, London, (1985) pp. 664-679. M. Mannik, in Arthritis and Allied Conditions. A Textbook of Rheumatology, D.J. McCarty, ed. Lea & Febiger, Philadelphia, (1985) pp. 660-667. R.M. McIntosh, C. Kulvinskas, and D.B. Kaufm, l i l t . Archs Allergy Appl. Immmol. kJ, 700-715 (1971). V. Agnello, D. Koffler, J.W. Eisenberg, R.J. Winchester and H.G. Kunkel, J. Exp. X e d . -134, 228s (1971). E. Penner, B. Albini, I. Glurich, G.A. Andres, and F. Kilgrom, Int. Archs Allergy Appl. Inrmunol. 67, 245-253 (1982). T. Sugisaki, K. Kano, J.R. Brentjens, S. Anthone, H. Anthone, G.A. Andres and F. Milgmm, Transplantation, 3, 90-94 (1982)., W.K. Wu, B.K. Noble, S.M. Mauer and F. MilgTom, Imunol. Invest. 2, 145-150 (1985). R.M. Pope, D.C. Teller and M. Mannik, J. Imanunol. 115, 365-373 (1975). T. Palosuo and F. Milgrom, J. Immunol. Methods x,, 171-181 (1981). D.A. Carson, S. Laurence, M.A. Catalono, J.H. Vaughan and G. Abraham, J. Immunol. At, 295-300 (1977).

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