Journal o f Immunological Methods, 24 (1978) 383--387 © Elsevier/North-Holland Biomedical Press
383
Short communication A RAPID SCREENING METHOD FOR THE DETECTION OF MONOSPECIFIC ANTIBODIES AGAINST HEMOGLOBINS
AFTAB A. ANSARI and HEINRICH V. MALLING Laboratory o f Biochemical Genetics, National Institute o f Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A.
(Received 6 ,July 1978, accepted 28 August 1978)
A simple procedure for the detection of monospecific antibody against C57BL/6 mouse hemoglobin that would not cross-react with DBA/2 mouse hemoglobin is described. The horse antiserum against C57BL/6 mouse hemoglobin is absorbed with DBA/2 mouse hemoglobin. The absorbed serum is then allowed to react with small amounts of DBA/2 hemoglobin-Sepharose and C57BL/6 hemoglobin-Sepharose in separate tubes, followed by reaction with fluorescein isothiocyanate-conjugated goat antihorse IgG. A strong fluorescence in the C57BL/6 immunoabsorbent and little or no fluorescence in the DBA/2 immunoabsorbent show the presence of monospecific antibody against C57BL/6 hemoglobin. The method has general applicability as antibodies against other hemoglobins and proteins can be detected. INTRODUCTION I m m u n o f l u o r e s c e n c e m e t h o d s p e r m i t the d e t e c t i o n o f variant or a b n o r m a l h e m o g l o b i n s at the cellular level. F l u o r e s c e n t anti-Hb* a n t i b o d i e s have been utilized in the investigation o f the switch f r o m e m b r y o n i c to adult Hb d u r i n g m e t a m o r p h o s i s in a m p h i b i a n s (Maniatis and I n g r a m , 1 9 7 1 ; Benba.ssat, 1 9 7 4 ; J u r d and MacLean, 1 9 7 4 ) as well as in the investigation o f c o n d i t i o n s associated with c o n t i n u a t i o n o f fetal Hb synthesis in the h u m a n adult ( B o y e r et al., 1 9 7 5 ; W o o d et al., 1 9 7 5 ; S t a m a t o y a n n o p o u l o s et al., 1 9 7 5 ; N u t e et al., 1976). T h e i m m u n o f l u o r e s c e n c e t e c h n i q u e s have also been useful in the d e t e c t i o n , in single h u m a n cells, o f Hb S ( P a p a y a n n o p o u l o u et al., 1 9 7 6 ) and Hb C ( P a p a y a n n o p o u l o u et al., 1 9 7 7 ) and, t h e r e f o r e , also m a y be applied to the s t u d y o f s o m a t i c m u t a t i o n s as well as for prenatal diagnosis o f ~-chain a b n o r m a l i t i e s {Boyer et al., 1974). All these applications o f the i m m u n o f l u o r e s c e n c e m e t h o d s require a n t i b o d i e s t h a t w o u l d react with o n e f o r m o f h e m o g l o b i n w i t h o u t s h o w i n g a n y cross-reaction with o t h e r forms, t h e so-called m o n o s p e c i f i c antibodies. However, an anti-Hb serum w o u l d crossreact with m o s t o f the Hb variants because o f the shared antigenic d e t e r m i -
* Abbreviations: C57, C57BL/6; D2, DBA/2; Hb, hemoglobin; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline, pH 7.2; rbc, red blood cell.
384 nants (Ansari et al., 1978). This situation calls for the purification of the monospecific antibody from the serum. Several investigators have reported production of antibodies specific for m u t a n t hemoglobins (Boerma and Huisman, 1964; Reichlin, 1974; Rowley et al., 1974; Shukla and Headings, 1974; Wood et al., 1975; Stamatoyannopoulos et al., 1975; Nute et al., 1976; Papayannopoulou et al., 1976, 1977). In principle, antiserum against one hemoglobin (mutant) is absorbed with another hemoglobin (normal) to remove the cross-reacting antibodies. The monospecific antibody is then purified by passing the absorbed serum through an immunoabsorbent prepared from the immunizing (mutant) hemoglobin. However, sometimes all these lengthy procedures of absorption and purification are carried out just to find out at the end that the serum did not contain any monospecific antibody. At present no method exists to detect the presence of monospecific antibody in a serum. This paper reports such a method. The method is exemplified by a system in which C57BL/6 and DBA/2 mice Hb, that differ in their fl-chains (Gilman, 1974; Russell and McFarland, 1974) were used. EXPERIMENTAL lmmunoabsorbents. Purified C57Hb and D2Hb were separately coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the directions of the manufacturer. A coupling ratio of 10 mg protein/ml gel was obtained. The immunoabsorbents were stored at 4°C in PBS containing 0.02% sodium azide and were always washed thoroughly with the same buffer before use. Horse anti-mouse hemoglobin antiserum. Horse (No. 88) was immunized against C57Hb as described earlier (Ansari et al., 1978). Goat anti-horse IgG. Goat anti-horse IgG antiserum (Lot No. 10404) was purchased from Cappel Laboratories. The gamma globulin fraction was precipitated from the serum by 18% Na2SO~ and then labeled with FITC (Sigma) (Nairn, 1969). The conjugate was purified by Sephadex G-25 chromatography followed by DEAE-cellulose chromatography (Nairn, 1969). The fraction having 1.9 mol FITC/mol protein was used in these studies. Detection o f the monospecific antibody. Twenty-five microliters of the antiserum that has been absorbed on 'opposite' Hb immunoabsorbent (antiC57Hb serum absorbed on D2Hb immunoabsorbent) are added to 100 ~l of 1 : 4 suspension, in PBS, of C57Hb immunoabsorbent and D2Hb immunoabsorbent in separate tubes. The tubes are rocked at 37°C for 30 rain. The beads are washed 4 times, each with 4 ml PBS, with thorough shaking. The washed beads are then incubated with the FITC conjugate of goat anti-horse IgG for 30 min at 37°C and then again washed 4 times with PBS before observing for FITC fluorescence in a fluorescence microscope. Fluorescence measurements. Leitz Orthoplan microscope and MPV2
385 photometer were used for the measurement ties of the beads.
of relative fluorescence intensi-
RESULTS AND DISCUSSION Table 1 shows the fluorescence intensity of the tester beads. Three kinds of tester beads were used: plain Sepharose-4B, D2Hb-Sepharose-4B and C57Hb-Sepharose-4B. These beads were reacted with horse anti-C57Hb s e r u m a n d t h e n w i t h F I T C - l a b e l e d g o a t a n t i - h o r s e I g G as d e s c r i b e d in t h e Experimental section. As expected, the unabsorbed horse anti-C57Hb antis e r u m gives s t r o n g f l u o r e s c e n c e w i t h D 2 a n d C 5 7 b e a d s ; h o w e v e r , i t a l s o r e a c t s , a l t h o u g h t o a l e s s e r e x t e n t , w i t h p l a i n S e p h a r o s e - 4 B b e a d s { T a b l e 1, A ) . T h e F I T C - l a b e l e d g o a t a n t i - h o r s e I g G a n t i b o d y u s e d in t h e s e s t u d i e s w a s f o u n d t o give l i t t l e o r n o f l u o r e s c e n c e w h e n i n c u b a t e d a l o n g w i t h p l a i n Sepharose or D2 or C57 immunoabsorbents. This means that immunoglobulin o r i m m u n o g l o b u l i n - l i k e m o l e c u l e s f r o m t h e h o r s e s e r u m b i n d t o t h e p l a i n Sepharose beads. Small amounts of immunoglobulins could bind nons p e c i f i c a l l y w i t h gel f i l t r a t i o n m e d i a b e c a u s e o f c h a r g e i n t e r a c t i o n s . H o w ever, t h e b i n d i n g o f h o r s e s e r u m t o t h e p l a i n S e p h a r o s e d o e s n o t s e e m t o b e t o t a l l y n o n - s p e c i f i c . T h i s c o n t e n t i o n is s u p p o r t e d b y t h e o b s e r v a t i o n t h a t t h e h o r s e s e r u m , a f t e r a b s o r p t i o n w i t h an e q u a l v o l u m e o f p l a i n S e p h a r o s e , d o e s n o t give a n y f l u o r e s c e n c e in t h e f l u o r e s c e n t b e a d t e s t w i t h p l a i n S e p h a r o s e , w h i l e it c o n t i n u e s t o give f l u o r e s c e n c e w i t h D 2 H b a n d C 5 7 H b i m m u n o a b s o r b e n t s ( T a b l e 1, B). I t is l i k e l y t h a t t h e h o r s e s e r u m h a s a ' n a t u r a l ' a n t i TABLE 1 FLUORESCENCE MEASUREMENTS ON THE TESTER BEADS The tester beads were allowed to react with unabsorbed or absorbed horse anti-C57Hb antiserum and then with FITC-labeled goat anti-horse IgG as described under the Experimental section. Fluorescence of these beads was then measured through a Leitz MPV2 photometer hooked to Orthoplan fluorescence microscope. Serum absorbed with
Tester beads a
Fluorescence intensity b
A
Unabsorbed
P D2 C57
13.0 _+_ 1.6 52.6 + 8.8 60.0 +_ 17.1
B
Plain Sepharose-4B
P D2 C57
1.1 _+ 1.8 43.2 t: 1.5 45.1 + 1.0
C
DBA/2Hb-Sepharose-4B
P D2 C57
0 + 1.3 10.9 _+ 2.7 51.5 _+ 7.4
a Tester beads: P, plain Sepharose-4B; D2, DBA/2 hemoglobin-Sepharose-4B; C57, C57BL/6 hemoglobin-Sepharose-4 B. b Fluorescense intensity is expressed as average of more than 10 measurements ¢ S.E.
386 b o d y t h a t interacts with the p o l y s a c c h a r i d e o f Sepharose. Most o f this natural a n t i b o d y is a b s o r b e d o u t on plain Sepharose-4B while m o s t o f the anti-Hb a n t i b o d y is left o u t in the serum as is evident from the high fluorescence intensities o f the D2 and C57 beads {Table 1, B). When the antiC 5 7 H b serum is absorbed with an equal v o l u m e o f D 2 H b - S e p h a r o s e (see Table 1, C) all o f the natural a n t i b o d y interacting with Sepharose, as well as m o s t o f the anti-Itb a n t i b o d y cross-reacting with D 2 t I b are r e m o v e d , as is evident from the very little or no f l u o r e s c e n c e o b t a i n e d with the plain and D2 tester beads. T h e fluorescence intensity o f the C57 tester beads is very high showing the presence o f m o n o s p e c i f i c a n t i b o d y against C 5 7 H b in the a n t i - C 5 7 H b antiserum t h a t has been absorbed on D 2 H b - S e p h a r o s e . E x p e r i m e n t s with several i m m u n e as well as normal horse scra showed t h a t all o f t h e m had natural agglutinins against m o u s e and h u m a n rbc and all o f t h e m gaw; a positive fluorescence in the bead fluorescence test with plain, D2 and C57 Sepharosc beads. T h e h e m a g g l u t i n a t i o n titer o f these scra was r a t h e r high, 4 0 9 6 , and did n o t change significantly over a period o f onc year during the course o f the i m m u n i z a t i o n with D2 or C57 Hb. A f t e r a b s o r p t i o n o f the sera with plain or D 2 ] t b - S e p h a r o s e , the titer d r o p p e d to 512 suggesting t h a t at least part o f the agglutinins present in the sera also bind to Sepharose. At present no t e c h n i q u e exists t h a t has been designed especially for the d e t e c t i o n o f m o n o s p e c i f i c antibodies. O f course, any suitable t e c h n i q u e , like h e m a g g l u t i n a t i o n , c o m p l e m e n t fixation, p r e c i p i t a t i o n analysis, or radioi m m u n o a s s a y could possibly be a d a p t e d to suit this especialized purpose. We observed t h a t horse sera {normal or i m m u n i z e d with m o u s e tlb) have natural agglutinins against m o u s e and h u m a n rbc, which makes it practically very unsuitable to a d a p t passive h e m a g g l u t i n a t i o n test to d e t e c t the m o n o s p e c i f i c a n t i b o d y in the p r e s e n t case. C o m p l e m e n t fixation t e c h n i q u e m a y also n o t be suitable for this p u r p o s e because all the antibodies do n o t fix complem e n t . P r e c i p i t a t i o n assay is especially unsuited for d e t e c t i o n o f m o n o specific antibodies because m o s t o f the m o n o s p e c i f i c antibodies are nonprecipitating. R a d i o i m m u n o a s s a y seems to be a good alternative. T h e bead f l u o r e s c e n c e t e c h n i q u e for the d e t e c t i o n o f m o n o s p e c i f i c a n t i b o d y is rapid, inexpensive and reliable because it d e p e n d s u p o n the d i r e c t i n t e r a c t i o n o f the antigen {coupled to Sepharose) with the a n t i b o d y . T h e t e c h n i q u e can be used for the d e t e c t i o n o f m o n o s p e c i f i c a n t i b o d y , n o t o n l y against h e m o g l o b i n s b u t against o t h e r antigens as well. ACKNOWLEDGEMENT We wish to t h a n k Dr. J.K. Spitznagel, D e p a r t m e n t o f Bacteriology and I m m u n o l o g y , University o f N o r t h Carolina, Chapel Hill, NC, for the use o f the Leitz m i c r o s c o p e and p h o t o m e t e r .
387 REFERENCES Ansari, A.A., L.M. Bahuguna, M. Jenison and H.V. Mailing, 1978, Immunochemistry (in press). Benbassat, d., 197.1, J. Cell. Sci. 16, 143. Boerma, F.W. and T.H.J. Huisman, 1964, J. Lab. Clin. Med. 6 3 , 2 6 4 . Boyer, S.H., M.L. Boyer, A.N. Noyes and T.K. Belding, 1974, Ann. N.Y. Acad. Sei. 241, 699. Boyer, S.H., T.K. Belding, L. Margolet and A.N. Noyes, 1975, Science 188,361. Gilman, J.G., 197,t, Ann. N.Y. Acad. Sci. 241, ,t16. Jurd, R.D. and N. MacLean, 1974, J. Microsc. 100,213. Maniatis, G.M. and V.M. Ingrain, 1971, J. Cell Biol. 49, 390. Nairn, R.C., 1969, in: Fluorescent Protein Tracing, ed. R.C. Nairn (Livingston, Edinburgh) p. 303. Nute, P.E., W.G. Wood, G. Stamatoyannopoulos, C. Olweny and P.J. Failkow, 1976, Brit. J. Haematol. 32, 55. Papayannopoulou, Th., T.C. McGuire, G. Lim, E. Garzel, P.E. Nute and G. Stamatoyannopoulos, 1976, Brit. J. Haematol. 3,1, 25. Papayannopoulou, Th., G. Lira, T.C. MeGuire, V. Ahem, P.E. Nute and G. Stamatoyannopoulo~, 1977, Am. J. Hematol. 2, 105. Reichlin, M., 1974, Immunochemistry 11, 21. Rowley, P.T., R.A. Doherty, C. Rosenerans and E. Cernichiari, 197,t, Blood 43,607. Russell, E.S. and E.C. MeFarland, 1974, Ann. N.Y. Acad. Sci. 241, 25. Shukla, S.B. and V.E. Headings, 1974, Immunochemistry 11,741. Stamatoyannopoulos, G., W.G. Wood, Th. Papayannopoulou and P.E. Nute, 1975, Blood 46,683. Wood, W.G., G. Stamatoyannopoulos, G. Lira and P.E. Nute, 1975, Blood 4 6 , 6 7 1 .
383
Short communication A RAPID SCREENING METHOD FOR THE DETECTION OF MONOSPECIFIC ANTIBODIES AGAINST HEMOGLOBINS
AFTAB A. ANSARI and HEINRICH V. MALLING Laboratory o f Biochemical Genetics, National Institute o f Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A.
(Received 6 ,July 1978, accepted 28 August 1978)
A simple procedure for the detection of monospecific antibody against C57BL/6 mouse hemoglobin that would not cross-react with DBA/2 mouse hemoglobin is described. The horse antiserum against C57BL/6 mouse hemoglobin is absorbed with DBA/2 mouse hemoglobin. The absorbed serum is then allowed to react with small amounts of DBA/2 hemoglobin-Sepharose and C57BL/6 hemoglobin-Sepharose in separate tubes, followed by reaction with fluorescein isothiocyanate-conjugated goat antihorse IgG. A strong fluorescence in the C57BL/6 immunoabsorbent and little or no fluorescence in the DBA/2 immunoabsorbent show the presence of monospecific antibody against C57BL/6 hemoglobin. The method has general applicability as antibodies against other hemoglobins and proteins can be detected. INTRODUCTION I m m u n o f l u o r e s c e n c e m e t h o d s p e r m i t the d e t e c t i o n o f variant or a b n o r m a l h e m o g l o b i n s at the cellular level. F l u o r e s c e n t anti-Hb* a n t i b o d i e s have been utilized in the investigation o f the switch f r o m e m b r y o n i c to adult Hb d u r i n g m e t a m o r p h o s i s in a m p h i b i a n s (Maniatis and I n g r a m , 1 9 7 1 ; Benba.ssat, 1 9 7 4 ; J u r d and MacLean, 1 9 7 4 ) as well as in the investigation o f c o n d i t i o n s associated with c o n t i n u a t i o n o f fetal Hb synthesis in the h u m a n adult ( B o y e r et al., 1 9 7 5 ; W o o d et al., 1 9 7 5 ; S t a m a t o y a n n o p o u l o s et al., 1 9 7 5 ; N u t e et al., 1976). T h e i m m u n o f l u o r e s c e n c e t e c h n i q u e s have also been useful in the d e t e c t i o n , in single h u m a n cells, o f Hb S ( P a p a y a n n o p o u l o u et al., 1 9 7 6 ) and Hb C ( P a p a y a n n o p o u l o u et al., 1 9 7 7 ) and, t h e r e f o r e , also m a y be applied to the s t u d y o f s o m a t i c m u t a t i o n s as well as for prenatal diagnosis o f ~-chain a b n o r m a l i t i e s {Boyer et al., 1974). All these applications o f the i m m u n o f l u o r e s c e n c e m e t h o d s require a n t i b o d i e s t h a t w o u l d react with o n e f o r m o f h e m o g l o b i n w i t h o u t s h o w i n g a n y cross-reaction with o t h e r forms, t h e so-called m o n o s p e c i f i c antibodies. However, an anti-Hb serum w o u l d crossreact with m o s t o f the Hb variants because o f the shared antigenic d e t e r m i -
* Abbreviations: C57, C57BL/6; D2, DBA/2; Hb, hemoglobin; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline, pH 7.2; rbc, red blood cell.
384 nants (Ansari et al., 1978). This situation calls for the purification of the monospecific antibody from the serum. Several investigators have reported production of antibodies specific for m u t a n t hemoglobins (Boerma and Huisman, 1964; Reichlin, 1974; Rowley et al., 1974; Shukla and Headings, 1974; Wood et al., 1975; Stamatoyannopoulos et al., 1975; Nute et al., 1976; Papayannopoulou et al., 1976, 1977). In principle, antiserum against one hemoglobin (mutant) is absorbed with another hemoglobin (normal) to remove the cross-reacting antibodies. The monospecific antibody is then purified by passing the absorbed serum through an immunoabsorbent prepared from the immunizing (mutant) hemoglobin. However, sometimes all these lengthy procedures of absorption and purification are carried out just to find out at the end that the serum did not contain any monospecific antibody. At present no method exists to detect the presence of monospecific antibody in a serum. This paper reports such a method. The method is exemplified by a system in which C57BL/6 and DBA/2 mice Hb, that differ in their fl-chains (Gilman, 1974; Russell and McFarland, 1974) were used. EXPERIMENTAL lmmunoabsorbents. Purified C57Hb and D2Hb were separately coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the directions of the manufacturer. A coupling ratio of 10 mg protein/ml gel was obtained. The immunoabsorbents were stored at 4°C in PBS containing 0.02% sodium azide and were always washed thoroughly with the same buffer before use. Horse anti-mouse hemoglobin antiserum. Horse (No. 88) was immunized against C57Hb as described earlier (Ansari et al., 1978). Goat anti-horse IgG. Goat anti-horse IgG antiserum (Lot No. 10404) was purchased from Cappel Laboratories. The gamma globulin fraction was precipitated from the serum by 18% Na2SO~ and then labeled with FITC (Sigma) (Nairn, 1969). The conjugate was purified by Sephadex G-25 chromatography followed by DEAE-cellulose chromatography (Nairn, 1969). The fraction having 1.9 mol FITC/mol protein was used in these studies. Detection o f the monospecific antibody. Twenty-five microliters of the antiserum that has been absorbed on 'opposite' Hb immunoabsorbent (antiC57Hb serum absorbed on D2Hb immunoabsorbent) are added to 100 ~l of 1 : 4 suspension, in PBS, of C57Hb immunoabsorbent and D2Hb immunoabsorbent in separate tubes. The tubes are rocked at 37°C for 30 rain. The beads are washed 4 times, each with 4 ml PBS, with thorough shaking. The washed beads are then incubated with the FITC conjugate of goat anti-horse IgG for 30 min at 37°C and then again washed 4 times with PBS before observing for FITC fluorescence in a fluorescence microscope. Fluorescence measurements. Leitz Orthoplan microscope and MPV2
385 photometer were used for the measurement ties of the beads.
of relative fluorescence intensi-
RESULTS AND DISCUSSION Table 1 shows the fluorescence intensity of the tester beads. Three kinds of tester beads were used: plain Sepharose-4B, D2Hb-Sepharose-4B and C57Hb-Sepharose-4B. These beads were reacted with horse anti-C57Hb s e r u m a n d t h e n w i t h F I T C - l a b e l e d g o a t a n t i - h o r s e I g G as d e s c r i b e d in t h e Experimental section. As expected, the unabsorbed horse anti-C57Hb antis e r u m gives s t r o n g f l u o r e s c e n c e w i t h D 2 a n d C 5 7 b e a d s ; h o w e v e r , i t a l s o r e a c t s , a l t h o u g h t o a l e s s e r e x t e n t , w i t h p l a i n S e p h a r o s e - 4 B b e a d s { T a b l e 1, A ) . T h e F I T C - l a b e l e d g o a t a n t i - h o r s e I g G a n t i b o d y u s e d in t h e s e s t u d i e s w a s f o u n d t o give l i t t l e o r n o f l u o r e s c e n c e w h e n i n c u b a t e d a l o n g w i t h p l a i n Sepharose or D2 or C57 immunoabsorbents. This means that immunoglobulin o r i m m u n o g l o b u l i n - l i k e m o l e c u l e s f r o m t h e h o r s e s e r u m b i n d t o t h e p l a i n Sepharose beads. Small amounts of immunoglobulins could bind nons p e c i f i c a l l y w i t h gel f i l t r a t i o n m e d i a b e c a u s e o f c h a r g e i n t e r a c t i o n s . H o w ever, t h e b i n d i n g o f h o r s e s e r u m t o t h e p l a i n S e p h a r o s e d o e s n o t s e e m t o b e t o t a l l y n o n - s p e c i f i c . T h i s c o n t e n t i o n is s u p p o r t e d b y t h e o b s e r v a t i o n t h a t t h e h o r s e s e r u m , a f t e r a b s o r p t i o n w i t h an e q u a l v o l u m e o f p l a i n S e p h a r o s e , d o e s n o t give a n y f l u o r e s c e n c e in t h e f l u o r e s c e n t b e a d t e s t w i t h p l a i n S e p h a r o s e , w h i l e it c o n t i n u e s t o give f l u o r e s c e n c e w i t h D 2 H b a n d C 5 7 H b i m m u n o a b s o r b e n t s ( T a b l e 1, B). I t is l i k e l y t h a t t h e h o r s e s e r u m h a s a ' n a t u r a l ' a n t i TABLE 1 FLUORESCENCE MEASUREMENTS ON THE TESTER BEADS The tester beads were allowed to react with unabsorbed or absorbed horse anti-C57Hb antiserum and then with FITC-labeled goat anti-horse IgG as described under the Experimental section. Fluorescence of these beads was then measured through a Leitz MPV2 photometer hooked to Orthoplan fluorescence microscope. Serum absorbed with
Tester beads a
Fluorescence intensity b
A
Unabsorbed
P D2 C57
13.0 _+_ 1.6 52.6 + 8.8 60.0 +_ 17.1
B
Plain Sepharose-4B
P D2 C57
1.1 _+ 1.8 43.2 t: 1.5 45.1 + 1.0
C
DBA/2Hb-Sepharose-4B
P D2 C57
0 + 1.3 10.9 _+ 2.7 51.5 _+ 7.4
a Tester beads: P, plain Sepharose-4B; D2, DBA/2 hemoglobin-Sepharose-4B; C57, C57BL/6 hemoglobin-Sepharose-4 B. b Fluorescense intensity is expressed as average of more than 10 measurements ¢ S.E.
386 b o d y t h a t interacts with the p o l y s a c c h a r i d e o f Sepharose. Most o f this natural a n t i b o d y is a b s o r b e d o u t on plain Sepharose-4B while m o s t o f the anti-Hb a n t i b o d y is left o u t in the serum as is evident from the high fluorescence intensities o f the D2 and C57 beads {Table 1, B). When the antiC 5 7 H b serum is absorbed with an equal v o l u m e o f D 2 H b - S e p h a r o s e (see Table 1, C) all o f the natural a n t i b o d y interacting with Sepharose, as well as m o s t o f the anti-Itb a n t i b o d y cross-reacting with D 2 t I b are r e m o v e d , as is evident from the very little or no f l u o r e s c e n c e o b t a i n e d with the plain and D2 tester beads. T h e fluorescence intensity o f the C57 tester beads is very high showing the presence o f m o n o s p e c i f i c a n t i b o d y against C 5 7 H b in the a n t i - C 5 7 H b antiserum t h a t has been absorbed on D 2 H b - S e p h a r o s e . E x p e r i m e n t s with several i m m u n e as well as normal horse scra showed t h a t all o f t h e m had natural agglutinins against m o u s e and h u m a n rbc and all o f t h e m gaw; a positive fluorescence in the bead fluorescence test with plain, D2 and C57 Sepharosc beads. T h e h e m a g g l u t i n a t i o n titer o f these scra was r a t h e r high, 4 0 9 6 , and did n o t change significantly over a period o f onc year during the course o f the i m m u n i z a t i o n with D2 or C57 Hb. A f t e r a b s o r p t i o n o f the sera with plain or D 2 ] t b - S e p h a r o s e , the titer d r o p p e d to 512 suggesting t h a t at least part o f the agglutinins present in the sera also bind to Sepharose. At present no t e c h n i q u e exists t h a t has been designed especially for the d e t e c t i o n o f m o n o s p e c i f i c antibodies. O f course, any suitable t e c h n i q u e , like h e m a g g l u t i n a t i o n , c o m p l e m e n t fixation, p r e c i p i t a t i o n analysis, or radioi m m u n o a s s a y could possibly be a d a p t e d to suit this especialized purpose. We observed t h a t horse sera {normal or i m m u n i z e d with m o u s e tlb) have natural agglutinins against m o u s e and h u m a n rbc, which makes it practically very unsuitable to a d a p t passive h e m a g g l u t i n a t i o n test to d e t e c t the m o n o s p e c i f i c a n t i b o d y in the p r e s e n t case. C o m p l e m e n t fixation t e c h n i q u e m a y also n o t be suitable for this p u r p o s e because all the antibodies do n o t fix complem e n t . P r e c i p i t a t i o n assay is especially unsuited for d e t e c t i o n o f m o n o specific antibodies because m o s t o f the m o n o s p e c i f i c antibodies are nonprecipitating. R a d i o i m m u n o a s s a y seems to be a good alternative. T h e bead f l u o r e s c e n c e t e c h n i q u e for the d e t e c t i o n o f m o n o s p e c i f i c a n t i b o d y is rapid, inexpensive and reliable because it d e p e n d s u p o n the d i r e c t i n t e r a c t i o n o f the antigen {coupled to Sepharose) with the a n t i b o d y . T h e t e c h n i q u e can be used for the d e t e c t i o n o f m o n o s p e c i f i c a n t i b o d y , n o t o n l y against h e m o g l o b i n s b u t against o t h e r antigens as well. ACKNOWLEDGEMENT We wish to t h a n k Dr. J.K. Spitznagel, D e p a r t m e n t o f Bacteriology and I m m u n o l o g y , University o f N o r t h Carolina, Chapel Hill, NC, for the use o f the Leitz m i c r o s c o p e and p h o t o m e t e r .
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