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The measurement of IgM immunoglobulin
Journal of Biological Standardization (i982) 10, 1 0 5 - 1 0 7
The measurement immunoglobulin*
of IgM
A n i n f o r m a l m e e t i n g t t o o k p l a c e in W . H . O . , G e n e v a ( 2 2 - 2 3 O c t o b e r 1981) to d i s c u s s difficulties in t h e m e a s u r e m e n t o f I g M a n d t h e e x p r e s s i o n o f t h e results in International Units (i.u.). D r K l e i n e x p l a i n e d t h a t w h e n t h e M a n c i n i t e c h n i q u e was u s e d a n d t h e d a t a o f areas o f d i f f u s i o n ( D 2) w e r e p l o t t e d a g a i n s t m a s s c o n c e n t r a t i o n o f l g M (C) d i f f e r e n t s a m p l e s o f I g M (19S) g a v e t h r e e d i s t i n c t s t r a i g h t line r e g r e s s i o n s a n d w h e n t h e s e r u m s a m p l e s w e r e r e d u c e d to 7S all p o i n t s fell o n t h e s a m e line. ( K l e i n , d e B r u y n & R a d e n a , 1 9 7 3 ; d e B r u y n & K l e i n , 1976; d e B r u y n , K l e i n , N e u m a n n , S a n d k y u y l , V e r m e e r e n & B l a n s c h , in press. ) In w o r k i n g w i t h e i g h t d i f f e r e n t p r o c e s s e d sera u s e d as reference m a t e r i a l s a n d a t t e m p t i n g to find t h e w e i g h t o f l g M in 1-0 i . u . t h e m e a n c a l c u l a t e d by D r K l e i n e# al. ( 1 9 7 3 ) was 5"32 3/g (s.d. 0"99) c o m p a r e d w i t h t h e m e a n o f 8 - 4 7 tag/i, u. as p u b l i s h e d by R o w e , G r a b & A n d e r s o n ( 1 9 7 2 ) a n d H u m p h r e y & B a t t y ( 1 9 7 4 ) . O n t h e o t h e r h a n d , D r R e i m e r h a d c o o r d i n a t e d a r e c e n t s t u d y ( R e i m e r et a l . , 1982) c a r r i e d o u t by t h e C o l l e g e o f A m e r i c a n P a t h d l o g i s t s a n d t h e m e a n for t h e 26 l a b o r a t o r ies was 8 - 6 7 u g / i . u . It was clear t h a t t h e r e is a d i l e m m a in a t t e m p t i n g to e x p l a i n w h y t h e v a r i o u s sets o f d a t a g i v e d i f f e r e n t m e a n s . T h e role o f m a s s calibrators a n d m e t h o d s needs f u r t h e r s t u d y . T h e facts are as follows. (1) T h e r e is a n I n t e r n a t i o n a l R e f e r e n c e P r e p a r a t i o n ( I R P ) o f H u m a n S e r u m I m m u n o g l o b u l i n G , A a n d M for w h i c h t h e b i o l o g i c a l a c t i v i t y for each a n a l y t e has b e e n d e f i n e d as 100 i . u . p e r a m p o u l e . (2) T h e r e h a v e b e e n a n u m b e r o f reports w r i t t e n since 1 9 7 0 , w h e n t h e I R P ,,'as e s t a b l i s h e d , w h i c h s u g g e s t e d t h a t for t h o s e c l i n i c i a n s w h o w i s h to h a v e t h e l g M levels e x p r e s s e d o n a w e i g h t basis a m e a n v a l u e o f 8 - 4 7 ~lg/i.u. m a y be used. It has b e e n R e c e i v e d for p u b l i c a t i o n 10 N o v e m b e r 1981. t P a r t i c i p a n t s o f t h e m e e t i n g are s h o w n at t h e e n d o f t h e r e p o r t . 0 0 9 2 - 1 I57182/020105+03 $03.00/0
(~) 1982 The International Association o f Biological Standardization
105
W . H . O . REPORT e m p h a s i z e d , h o w e v e r , t h a t the biological activity s h o u l d always be expressed in i.u. a n d not on a w e i g h t basis. (3) T h e m e t h o d used for the d e f i n i t i o n o f activity o f the I R P was radial i m m u n o diffusion and in o r d e r to o b t a i n m o r e r e p r o d u c i b l e results it has been s u g g e s t e d t h a t r e d u c t i o n o f the s e r u m samples m a y be used (de B r u y n & K l e i n , I976). I f such a p r o c e d u r e is a d o p t e d it m u s t be r e m e m b e r e d t h a t by definition t h e r e are still 100 i.u. per ampoule. (4) T h e q u e s t i o n has been raised o f the need to show parallelism ( F i n n e y , 1964) after r e d u c t i o n b e t w e e n the I R P , national a n d c o m m e r c i a l reference materials and selected n o r m a l and a b n o r m a l sera. (5) W h e r e n e p h e l o m e t r y is used, t h e r e m a y be no need for the r e d u c t i o n o f the sera a n d as far as radio i m m u n o a s s a y is c o n c e r n e d , r e d u c t i o n s h o u l d not be used. (6) A n y calibration o f a national or w o r k i n g s t a n d a r d m u s t be in i.u. a n d not by c o m p a r i s o n on a w e i g h t basis. (7) T h e r e is no n e e d e i t h e r to w i t h d r a w or replace the I n t e r n a t i o n a l R e f e r e n c e P r e p a r a t i o n o f I m m u n o g l o b u l i n s G , M and A. W h e r e n e p h e l o m e t r y is used, however, this I R P is u n s u i t a b l e because o f t u r b i d i t y o f t h e r e c o n s t i t u t e d m a t e r i a l a n d o t h e r I n t e r n a t i o n a l a n d N a t i o n a l Reference Materials, c a l i b r a t e d in I n t e r n a t i o n a l U n i t s for t h e i r c o n t e n t o f I g M , m a y be used. Such preparations are: T h e I n t e r n a t i o n a l Reference P r e p a r a t i o n o f H u m a n S e r u m Proteins, f'or l m m u n o a s s a y . T h e First British S t a n d a r d for H u m a n S e r u m Proteins 7 4 / 5 2 0 . T h e U . S . N a t i o n a l R e f e r e n c e P r e p a r a t i o n for Specific H u m a n S e r u m P r o t e i n s ( R e i m e r , S m i t h & W e l l s , 1981). REFERENCES de Bruyn, A. M. & Klein, F. (1976). The validity of the radial immunodiffusion method for the quantitative determination of human IgM. Evaluation of a modified method. Journal of Immunological Methods 1 I, 311-320. de Bruyn, A. M., Klein, F., Neumann, H . , Sandkyuyl, L. A., Vermeeren, R. & Blanscb, de B. (1981). The absolute quantification of human IgM and IgG: standardization and normal values. Journal of Immunological Methods in press. Finney, D. J. (1964). Statistical Metl~ods in Biological Assay, 2nd edn. London: Griffin. Humphrey, 3- H. & Battey, Irent. (1974). An international reference preparation for 'human IgG, IgA and IgM. Clinical and Experimental Immunology 17, 708. Klein, F., de Bruyn, A. M. & Radena, H. (1973). Recognition of human IgM subgroups by quantitative measurement. Clinical and Experimental Immunology, 103-- 111. Reimer, C. B., Smith, S. 3. & Wells, T. W. (1981). Collaborative calibration of three established reference preparations for specific proteins in human sera as secondary standards for IgA, IgM and IgG. Journal of Biological Standardization 9, 393--400. Reimer, C. B., Smith, S . J . , Wells, T. W . , Nakamura, R. M., Keitges, P. W . , Ritchie, R. F., Williams, G. W . , Hanson, D . J . & Dorsey, D. B. (1982). Collaborative calibration o f the U.S. National and the College of American Pathologists" reference preparations for specific serum proteins. American Journal of Clinical Pathelogy 77, 12--18. Rowe, D, S., Grab, B. & Anderson, S. G. (1972). An international re.~rence preparation for human serum immunoglobulins G, A and M; content of i m m u n o g l o b u l i n by weight. Bulletin olc the World Health Organization 46, 6 7 - 7 9 . 106
M E A S U R E M E N T OF IsIM
Participants Mrs |. Batty, C o - C h a i r m a n , I . U . I . S . Standardization C o m m i t t e e . D r F. Klein, Vice-Chairman, I . U . I . S . Immunoglobulin S u b - C o m m i t t e e . Dr C. B. R e i m e r , Vice-Chairman, I . U . I . S . Standardization C o m m i t t e e . Dr F. Skvaril, C h a i r m a n , I . U . I . S . I m m u n o g l o b u l i n S u b - C o m m i t t e e .
Secretariat Dr F. T. Perkins, Chief, Biolo.gicals, W . H . O . , Geneva. Dr D. R o w e , Special P r o g r a m m e for Research and Training in Tropical Diseases, W . H . O . , Geneva.
107
The measurement immunoglobulin*
of IgM
A n i n f o r m a l m e e t i n g t t o o k p l a c e in W . H . O . , G e n e v a ( 2 2 - 2 3 O c t o b e r 1981) to d i s c u s s difficulties in t h e m e a s u r e m e n t o f I g M a n d t h e e x p r e s s i o n o f t h e results in International Units (i.u.). D r K l e i n e x p l a i n e d t h a t w h e n t h e M a n c i n i t e c h n i q u e was u s e d a n d t h e d a t a o f areas o f d i f f u s i o n ( D 2) w e r e p l o t t e d a g a i n s t m a s s c o n c e n t r a t i o n o f l g M (C) d i f f e r e n t s a m p l e s o f I g M (19S) g a v e t h r e e d i s t i n c t s t r a i g h t line r e g r e s s i o n s a n d w h e n t h e s e r u m s a m p l e s w e r e r e d u c e d to 7S all p o i n t s fell o n t h e s a m e line. ( K l e i n , d e B r u y n & R a d e n a , 1 9 7 3 ; d e B r u y n & K l e i n , 1976; d e B r u y n , K l e i n , N e u m a n n , S a n d k y u y l , V e r m e e r e n & B l a n s c h , in press. ) In w o r k i n g w i t h e i g h t d i f f e r e n t p r o c e s s e d sera u s e d as reference m a t e r i a l s a n d a t t e m p t i n g to find t h e w e i g h t o f l g M in 1-0 i . u . t h e m e a n c a l c u l a t e d by D r K l e i n e# al. ( 1 9 7 3 ) was 5"32 3/g (s.d. 0"99) c o m p a r e d w i t h t h e m e a n o f 8 - 4 7 tag/i, u. as p u b l i s h e d by R o w e , G r a b & A n d e r s o n ( 1 9 7 2 ) a n d H u m p h r e y & B a t t y ( 1 9 7 4 ) . O n t h e o t h e r h a n d , D r R e i m e r h a d c o o r d i n a t e d a r e c e n t s t u d y ( R e i m e r et a l . , 1982) c a r r i e d o u t by t h e C o l l e g e o f A m e r i c a n P a t h d l o g i s t s a n d t h e m e a n for t h e 26 l a b o r a t o r ies was 8 - 6 7 u g / i . u . It was clear t h a t t h e r e is a d i l e m m a in a t t e m p t i n g to e x p l a i n w h y t h e v a r i o u s sets o f d a t a g i v e d i f f e r e n t m e a n s . T h e role o f m a s s calibrators a n d m e t h o d s needs f u r t h e r s t u d y . T h e facts are as follows. (1) T h e r e is a n I n t e r n a t i o n a l R e f e r e n c e P r e p a r a t i o n ( I R P ) o f H u m a n S e r u m I m m u n o g l o b u l i n G , A a n d M for w h i c h t h e b i o l o g i c a l a c t i v i t y for each a n a l y t e has b e e n d e f i n e d as 100 i . u . p e r a m p o u l e . (2) T h e r e h a v e b e e n a n u m b e r o f reports w r i t t e n since 1 9 7 0 , w h e n t h e I R P ,,'as e s t a b l i s h e d , w h i c h s u g g e s t e d t h a t for t h o s e c l i n i c i a n s w h o w i s h to h a v e t h e l g M levels e x p r e s s e d o n a w e i g h t basis a m e a n v a l u e o f 8 - 4 7 ~lg/i.u. m a y be used. It has b e e n R e c e i v e d for p u b l i c a t i o n 10 N o v e m b e r 1981. t P a r t i c i p a n t s o f t h e m e e t i n g are s h o w n at t h e e n d o f t h e r e p o r t . 0 0 9 2 - 1 I57182/020105+03 $03.00/0
(~) 1982 The International Association o f Biological Standardization
105
W . H . O . REPORT e m p h a s i z e d , h o w e v e r , t h a t the biological activity s h o u l d always be expressed in i.u. a n d not on a w e i g h t basis. (3) T h e m e t h o d used for the d e f i n i t i o n o f activity o f the I R P was radial i m m u n o diffusion and in o r d e r to o b t a i n m o r e r e p r o d u c i b l e results it has been s u g g e s t e d t h a t r e d u c t i o n o f the s e r u m samples m a y be used (de B r u y n & K l e i n , I976). I f such a p r o c e d u r e is a d o p t e d it m u s t be r e m e m b e r e d t h a t by definition t h e r e are still 100 i.u. per ampoule. (4) T h e q u e s t i o n has been raised o f the need to show parallelism ( F i n n e y , 1964) after r e d u c t i o n b e t w e e n the I R P , national a n d c o m m e r c i a l reference materials and selected n o r m a l and a b n o r m a l sera. (5) W h e r e n e p h e l o m e t r y is used, t h e r e m a y be no need for the r e d u c t i o n o f the sera a n d as far as radio i m m u n o a s s a y is c o n c e r n e d , r e d u c t i o n s h o u l d not be used. (6) A n y calibration o f a national or w o r k i n g s t a n d a r d m u s t be in i.u. a n d not by c o m p a r i s o n on a w e i g h t basis. (7) T h e r e is no n e e d e i t h e r to w i t h d r a w or replace the I n t e r n a t i o n a l R e f e r e n c e P r e p a r a t i o n o f I m m u n o g l o b u l i n s G , M and A. W h e r e n e p h e l o m e t r y is used, however, this I R P is u n s u i t a b l e because o f t u r b i d i t y o f t h e r e c o n s t i t u t e d m a t e r i a l a n d o t h e r I n t e r n a t i o n a l a n d N a t i o n a l Reference Materials, c a l i b r a t e d in I n t e r n a t i o n a l U n i t s for t h e i r c o n t e n t o f I g M , m a y be used. Such preparations are: T h e I n t e r n a t i o n a l Reference P r e p a r a t i o n o f H u m a n S e r u m Proteins, f'or l m m u n o a s s a y . T h e First British S t a n d a r d for H u m a n S e r u m Proteins 7 4 / 5 2 0 . T h e U . S . N a t i o n a l R e f e r e n c e P r e p a r a t i o n for Specific H u m a n S e r u m P r o t e i n s ( R e i m e r , S m i t h & W e l l s , 1981). REFERENCES de Bruyn, A. M. & Klein, F. (1976). The validity of the radial immunodiffusion method for the quantitative determination of human IgM. Evaluation of a modified method. Journal of Immunological Methods 1 I, 311-320. de Bruyn, A. M., Klein, F., Neumann, H . , Sandkyuyl, L. A., Vermeeren, R. & Blanscb, de B. (1981). The absolute quantification of human IgM and IgG: standardization and normal values. Journal of Immunological Methods in press. Finney, D. J. (1964). Statistical Metl~ods in Biological Assay, 2nd edn. London: Griffin. Humphrey, 3- H. & Battey, Irent. (1974). An international reference preparation for 'human IgG, IgA and IgM. Clinical and Experimental Immunology 17, 708. Klein, F., de Bruyn, A. M. & Radena, H. (1973). Recognition of human IgM subgroups by quantitative measurement. Clinical and Experimental Immunology, 103-- 111. Reimer, C. B., Smith, S. 3. & Wells, T. W. (1981). Collaborative calibration of three established reference preparations for specific proteins in human sera as secondary standards for IgA, IgM and IgG. Journal of Biological Standardization 9, 393--400. Reimer, C. B., Smith, S . J . , Wells, T. W . , Nakamura, R. M., Keitges, P. W . , Ritchie, R. F., Williams, G. W . , Hanson, D . J . & Dorsey, D. B. (1982). Collaborative calibration o f the U.S. National and the College of American Pathologists" reference preparations for specific serum proteins. American Journal of Clinical Pathelogy 77, 12--18. Rowe, D, S., Grab, B. & Anderson, S. G. (1972). An international re.~rence preparation for human serum immunoglobulins G, A and M; content of i m m u n o g l o b u l i n by weight. Bulletin olc the World Health Organization 46, 6 7 - 7 9 . 106
M E A S U R E M E N T OF IsIM
Participants Mrs |. Batty, C o - C h a i r m a n , I . U . I . S . Standardization C o m m i t t e e . D r F. Klein, Vice-Chairman, I . U . I . S . Immunoglobulin S u b - C o m m i t t e e . Dr C. B. R e i m e r , Vice-Chairman, I . U . I . S . Standardization C o m m i t t e e . Dr F. Skvaril, C h a i r m a n , I . U . I . S . I m m u n o g l o b u l i n S u b - C o m m i t t e e .
Secretariat Dr F. T. Perkins, Chief, Biolo.gicals, W . H . O . , Geneva. Dr D. R o w e , Special P r o g r a m m e for Research and Training in Tropical Diseases, W . H . O . , Geneva.
107