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Microplate enzyme-linked immunosorbent assay in the study of the structural relationship between myosin light chains
Journal o f Immunological Methods, 31 (1979) 93--100
93
© Elsevier/North-Holland Biomedical Press
MICROPLATE ENZYME-LINKED IMMUNOSORBENT ASSAY IN THE STUDY OF THE STRUCTURAL RELATIONSHIP BETWEEN MYOSIN LIGHT CHAINS
DONATELLA BIRAL 1 , LUCIANO DALLA LIBERA ALFREDO MARGRETH 1
1,
CLAUDIO FRANCESCHI 2 and
1 National Research Council Center for Muscle Biology and Physiopathology, Institute o f General Pathology, University o f Padova, and 2 Institute o f General Pathology, University o f Bologna, Italy
(Received 9 April 1979, accepted 14 July 1979)
A microplate enzyme-linked immunosorbent assay (microELISA) for the study of immunochemical relationships between rabbit myosin light chains is described. Purified individual fast-muscle myosin light chains (LCIF, LC2F and LC3F) and their respective antisera, obtained in chicken, were used. Optimal conditions for antigen concentration, antiserum dilution, substrate concentration, incubation time and reproducibility with time were established. The observed cross-reactivities between the different types of light chains associated with rabbit fast-muscle myosin confirm and extend previous results obtained by other authors using radioimmunoassay procedures. It was concluded that microELISA may be successfully employed also to the study of macromolecule cross-reactivities.
INTRODUCTION
E n z y m e i m m u n o a s s a y in solid p h a s e ( E L I S A ) w h i c h has b e e n d e v e l o p e d r e c e n t l y as a m i c r o m e t h o d f o r testing several antigens (Voller et al., 1 9 7 7 ) , is a likely s u b s t i t u t e f o r o t h e r i m m u n o a s s a y s , such as r a d i o i m m u n o a s s a y ( R I A ) , b e c a u s e o f its high sensitivity a n d ability t o d e t e c t n o n - p r e c i p i t a b l e i m m u n e c o m p l e x e s , as well as f o r g r e a t e r s a f e t y a n d l o w e r cost. In view o f t h e increasing use o f m i c r o E L I S A a n d o f o u r i n t e r e s t in struct u r a l r e l a t i o n s h i p s b e t w e e n d i f f e r e n t t y p e s o f light chains a s s o c i a t e d w i t h skeletal m u s c l e m y o s i n s (Dalla L i b e r a e t al., 1 9 7 8 ; M a r g r e t h et al., in press), we d e c i d e d t o s u b j e c t this m e t h o d to e x p e r i m e n t a l test, especially in r e l a t i o n to t h e p r o b l e m o f i m m u n o l o g i c a l i n t e r r e l a t e d n e s s b e t w e e n t h e light chains o f fast-muscle myosin. T h e light chains a s s o c i a t e d w i t h t h e m y o s i n o f v e r t e b r a t e fast muscles, are well c h a r a c t e r i z e d c h e m i c a l l y a n d f u n c t i o n a l l y a n d , b y t h e s e criteria, are Correspondence and reprint requests should be addressed to: Dr. C. Franceschi, Istituto di Patologia Genera]e, Via S. Giacomo 14, 40126 Bologna, Italy.
94 classified into two main classes (Gauthier et al., 1978), namely alkali-light chains (A1 or LC1F, mol. wt., a b o u t 25,000 and A2 or LC3F, mol. wt., a b o u t 16,000) which are released on exposure of myosin to alkaline pH, and the DTNB-light chain (LC2F, mol. wt. a b o u t 18,000}, which may be dissociated from myosin by the thiol reagent DTNB (5,5'dithiobis2-nitrobenzoic acid). Sequence studies have shown (Frank and Weeds, 1974) that the extent of homology is greatest between LC1F and LC3F, the main distinguishing feature of LC1F being related to an N-terminal sequence ('difference peptide'), n o t present in LC3F, which accounts for its higher molecular weight. However, Silberstein and L o w e y (1977) and Holt and L o w e y (1977) by using RIA instead of double-diffusion (which had produced negative results) found an unexpected cross-reactivity between L C I F and LC2F, in the presence of antibody against LC2F, as well as, surprisingly, with antibody specific for the N-terminal sequence which is uniquely present in LC1F. The results reported here, with the individual light chains dissociated and purified from the myosin of rabbit fast muscle as immunogens, show that microELISA can be used to advantage in the study of their serological crossreactivity, with results which confirm and extend the previous studies on the myosin of chicken fast muscle by RIA procedures. MATERIALS AND METHODS
Antigens and antisera Fast-twitch muscle myosin was prepared from rabbit adductor myosins according to Barany and Close (1971), with the modifications reported by Dalla Libera et al. (1978). Light chains were dissociated by treating myosin with 4 M urea and were fractionated by DEAE column chromatography (Whatman DE-52) and a linear phosphate gradient, as described by L o w e y and Holt (1972). The pooled fractions corresponding to the 3 separated peaks of L C I F , LC2F and LC3F were precipitated by adding solid (NH4)2SO4 to 80% and were stored in a solution containing 0.4 M KC1, 0.05 M K-phosphate buffer pH 7.4, after overnight dialysis against the same buffer. The purity of each light chain preparation was determined by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis according to Weber and Osborn (1969). Antibodies against L C I F , LC2F and LC3F were elicited in adult hens by intramuscular injections with 1.5 mg protein in 1 ml of phosphate buffer emulsified with an equal volume of complete Freund's adjuvant (Difco, Detroit), repeated 3 times at 2--3 week intervals, followed by 3 injections of 3 mg of antigen protein every 4 days. Animals were bled after 4--5 months from the beginning Of the immunization schedule. Each specific antiserum used in the present study was obtained from a single bleeding of individual animals.
Preparation of anti-chicken IgG-alkaline phosphatase conjugate Chicken IgG were precipitated from normal sera with Na2SO4 and were
95 purified by DEAE-column chromatography at pH 6.4 (Whatman DE-52) as reported by Benedict (1967). Specific antibodies against chicken IgG were isolated from commercial rabbit anti-chicken IgG sera (Miles) by adsorption on purified chicken IgG coupled to activated Sepharose 4-B CNBr (Pharmacia) and elution at pH 2.8 with a solution containing 0.2 M CH3COOH and 0.3 M KC1. Anti-chicken IgG antibodies were conjugated to alkaline phosphatase (type VII, Sigma) according to Engvall and Perlmann (1972). Conjugates were stored at 4°C after appropriate dilution with 5% bovine serum albumin (Sigma), 0.001 M MgC12, 0.02% NAN3, 0.05 M Tris--HC1 pH 8.0.
Alkaline phosphatase substrate A solution (0.1--3 mg/ml) of the disodium salt of p-nitrophenol phosphate (NPP) (Sigma) was prepared immediately before use in 1 mM MgC12, 0.05 M Na2CO3--NaHCO3 buffer pH 9.8.
MicroELISA procedure MicroELISA test was carried out using microELISA plates (M29E, Cooke Microtiter) essentially as described by Voller et al. (1976) using 7 wells for each determination. The phosphatase activity bound to the wells was measured by adding 0.2 ml of the substrate solution (NNP) and by incubating at 25°C. The reaction was stopped after 30--60 min by adding 0.05 ml of 2 N NaOH, and the absorbance was measured at 400 nm. RESULTS
Antigens Figure 1 shows the chromatography elution profile of myosin light chains and the extent of purification achieved with each of the 3 types of chains. As examined by SDS-gel electrophoresis, and with overcharged gels (25--30 pg protein per gel), there was a very low degree of cross-contamination between LC1F and LC3F, as well as some contamination of LC3F by LC2F, and, occasionally, by LC1F. These results are comparable with those obtained originally by Lowey and Holt (1972} with the same procedures.
Optimum concentrations o f antigens and specific antiserum Microplate wells were coated with antigen at different concentrations. In Fig. 2 a typical experiment is shown using LC1F as antigen, at a concentration of 0.05--100 pg/ml, and anti L C I F serum at 1 : 1000, or at 1 : 100.000 dilution. The highest absorbance values (0.5--0.8) were obtained at serum dilutions of 1 : 1000 and with antigen coating concentrations in the 1 t~g to 10 pg/ml range; but measurable absorption values were obtained at antigen concentrations as low as 0.1 pg/ml. Within this range of antigen concentrations higher
96
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Po4H
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1'0
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,'o FRACTION NUuSER
Fig. 1. P u r i f i c a t i o n b y D E A E - c h r o m a t o g r a p h y o f light c h a i n s f r o m fast muscle m y o s i n o f t h e rabbit. A b o u t 100 m g of t o t a l light c h a i n s was applied t o a c o l u m n (1.5 × 30 c m ) of D E A E cellulose ( W h a t m a n DE-52), e q u i l i b r a t e d w i t h 0.05 M Na p h o s p h a t e pH 6.0 cont a i n i n g 0.1 mM d i t h i o t h r e i t o l . T h e p r o t e i n s were e l u t e d b y a p p l y i n g a linear g r a d i e n t of Na p h o s p h a t e pH 6.0, as i n d i c a t e d in the figure. SDS (0.1%) p o l y a c r y l a m i d e gel electrop h o r e s i s o f t h e p o o l e d fractions, c o r r e s p o n d i n g to the 3 s e p a r a t e d light chains, are s h o w n a b o v e t h e respective a b s o r p t i o n peaks ( L C 1 F , L C 2 F , L C 3 F , f r o m left t o right). F o r e x p e r i m e n t a l details see Materials a n d M e t h o d s .
A 400/30 mln
A 4 0 0 / 3 0 rain
1.0-
14)"
0.5-
0,5-
0:01
o.1
10
160 0 .~ ~. pg/ml L C I F -log serum dilution Fig. 2. A n t i g e n t i t r a t i o n . Plate wells were c o a t e d w i t h s o l u t i o n s of a n t i g e n ( L C I F ) at d i f f e r e n t c o n c e n t r a t i o n s a n d were i n c u b a t e d w i t h d i f f e r e n t d i l u t i o n s of a n t i s e r u m (anti L C 1 F ) , i.e. e, 1 : 1 0 0 0 , ©, 1 : 1 0 0 , 0 0 0 .
Fig. 3. E f f e c t o f a n t i s e r u m dilution. M i c r o p l a t e wells c o a t e d w i t h L C I F as a n t i g e n (5 m l ) were i n c u b a t e d at d i f f e r e n t c o n c e n t r a t i o n s o f s e r u m ( a n t i - L C 1 F ) .
pg/
97 serum dilutions gave very low absorbance values. Figure 3 shows the absorbance values obtained by coating the microplate wells with a fixed antigen (LC1F) c o n c e n t r a t i o n (5 pg/ml) in the presence of varying serum dilutions. Maximum absorbance values (1.0) were again re co r d ed with 1 : 1000 serum dilution. Similar results were obtained with LC2F and LC3F (not shown in the Figure). The m a x i m u m colour d e v e l o p m e n t after 30 min incubation at 25°C was f o u n d in a range of substrate c o n c e n t r a t i o n bet w een 1.5 and 3.0 mg/ml. The time-course of colour d e v e l o p m e n t was f o u n d to be linear during 60 min incubation of serum dilutions 1 : 1000 and 1 : 10,000.
Reproducibility of the microELISA The reproducibility investigated in several enzyme-conjugate, and These results are shown
o f the results obtained with the m i croE L ISA was experiments, by using different batches of fresh of the conjugate after storage in the cold r o o m . in Table 1.
Serological cross-reactivity between myosin light chains F o r investigating by ELISA the immunological relationship between myosin light chains, each t y p e of chain (LC1F, LC2F, and L C3F) was reacted with specific antisera for L C I F and LC3F, respectively. On a c c o u n t o f the previous work of Holt and L o w e y (1977), it was presumed that antibodies against L C I F should comprise antibodies reactive with antigenic sequence determinants shared by the h o m o l o g o u s antigen and by LC3F, as well as an a n t i b o d y population specific for the N-terminal sequence ('difference peptide') of L C I F (see I n t r o d u c t i o n section). The latter a n t i b o d y p o p u latio n should, in turn, be cross-reactive with LC2F (Holt and L ow ey, 1977; Silberstein and L ow ey, 1977). The results in Fig. 4A show t hat antiserum against LC1F contained crossreactive a n t i b o d y and that cross-reactivity was indeed most extensive TABLE 1 EFFECT OF ENZYME-CONJUGATE STORAGE ON MICROELISA REPRODUCIBILITY The assay was performed with LC1F as antigen and anti-LC1F as serum. For details see Materials and Methods. Conjugate Preparation I Preparation II Preparation III
Absorbance values at 400 nm, 30 rain incubation at 25°C fresh stored for 2 months at 4°C fresh stored for 2 months at 4°C fresh
0.950--0.970 0.320--0.400 0.935--1.055--1.035 0.690--0.810--0.755 0.720
98 A 400/30 mtn A 4 0 ( / 3 0 min
A 1.0.
i
(15-
o
0 5'
4'
~
~
3
- l o g s e r u m dilution
- log serum dilution
Fig. 4. Serological cross-reactivity between myosin light chains. The microELISA test was performed with A, anti-LC1F serum; B, anti-LC3F serum. ©, LC1F as antigen; o, LC2F as antigen; A, LC3F as antigen.
between L C I F and LC2F. On the other hand, it was found that antiserum against LC3F (Fig. 4B) was unable to distinguish between the immunogen and L C I F , and that LC2F, surprisingly, was also highly cross-reactive. DISCUSSION The results reported here provide new evidence of the wide range of applicability of microELISA. They further show that, under carefully controlled experimental conditions, this immunoassay technique satisfies the sensitivity requirements making it adequate to detect immunological cross-reactivities between the different classes of myosin light chains (Silberstein and Lowey, 1977). As applied to the investigation of the immunological relationship between myosin light chains, it should be emphasized that microELISA is able to reveal the formation of immune complexes at relatively high (1 : 1000 to 1 : 10,000) dilutions of antiserum, and in the presence of amounts of antigen per well as low as 1/~g of protein. Spectrophotometric assay of coupled chromogen formation from NPP by the anti-IgG-bound phosphatase probe is inherently much less sensitive, by a b o u t two orders of magnitude, than detection of radioactive label from 12SI-light chains in the immune complex by RIA (Silberstein and Lowey, 1977). However, the a m o u n t of antigen required for microELISA is still low
99 enough to allow use of this technique in immunological studies of myosin from h u m a n muscle biopsies. With regard to the observed cross-reactivities between the different types of light chains associated with rabbit fast-muscle myosin, it is doubtful, on account of the previous thorough studies of Silberstein and Lowey (1977) on homologous light chains from fast-muscle myosin of the chicken, that the results can be explained simply by cross-contamination of the immunogens. As reported in the Results section, this was of such a low degree to make it unlikely as a serious source of artifactual error. On the other hand, the long immunization periods (4--5 months) used in the present investigation, as in the previous study by Silberstein and Lowey (1977), may have helped in raising a population of antibodies of relatively lower specificity and with a higher affinity and hence with increasing crossreactivities with respect to antibodies elicited in short periods. Although it cannot be excluded that the immune sera used in the present study may be polyspecific it is significant that our results are in complete agreement with the reports of Silberstein and Lowey (1977) and Holt and L o w e y (1977) that LC1F and LC2F of fast-muscle myosin share c o m m o n configurational determinants, which property would therefore appear to be c o m m o n to the homologous light chains of myosins from different species, such as the chicken and the rabbit (present study). Our own results further indicate that LC2F contains antigenic determinants cross-reacting with LC3F, and is thereby distinguished from those reacting with antibodies specific for the 'difference peptide' uniquely present in L C I F . Further work along these lines with purified specific antibodies to the several antigenic determinants present in myosin light chains may profitably be carried out with ELISA and may provide interesting new information on the structural relationship between these peptides at the primary and tertiary structure levels. ACKNOWLEDGEMENTS Grateful acknowledgement is made to Mr. Silvio Bellucco for skillful technical assistance, and to Miss Giovanna Roberti, biology student, for help in several phases of this work. We wish to thank Professor G. Vicari and Dr. P. Iacona of the Istituto Superiore di SanitY, Rome, for helpful suggestions in the setting up of the technique. This investigation was supported in part by a grant from the Muscular Dystrophy Association to Professor Massimo Aloisi, Director of the Institute of General Pathology of the University of Padova. REFERENCES Barany, M. and R.I. Close, 1971, J. Physiol. (Lond.) 213,455. Benedict, A.A., 1967, in: Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 229.
100 Dalla Libera, L., A. Margreth, I. Mussini, C. Cerri and G. Scarlato, 1978, Muscle and Nerve 1,280. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Frank, G. and A.G. Weeds, 1974, Europ. J. Biochem. 4 4 , 3 1 7 . Gauthier, G.F., S. Lowey and A.W. Hobbs, 1978, Nature (Lond.) 274, 25. Holt, J.C. and S. Lowey, 1977, Biochemistry 16, 4398. Lowey, S. and J.C. Holt, 1972, Cold Spring Harbor Symp. Quant. Biol. 37, 19. Margreth, A., L. Dalla Libera, D. Biral and R. Betto, in: Proceedings of the International Symposium on 'New Frontiers in Muscular Dystrophies', Maranello, Modena, Italy, April 1, 1978, In press. Silberstein, L. and S. Lowey, 1977, Biochemistry 16, 4403. Voller, A., D. Bidwell and A. Bartlett, 1976, in: Manual of Clinical Immunology, eds. N.R. Rose and H. Friedman (American Society for Microbiology, Washington, DC) p. 506. Voller, A., D.E. Bidwell and A. Bartlett, 1977, The Enzyme Linked Immunosorbent Assay (ELISA) (Flowline Publications, Guernsey). Weber, K. and M. Osborn, 1969, J. Biol. Chem. 244, 4406.
93
© Elsevier/North-Holland Biomedical Press
MICROPLATE ENZYME-LINKED IMMUNOSORBENT ASSAY IN THE STUDY OF THE STRUCTURAL RELATIONSHIP BETWEEN MYOSIN LIGHT CHAINS
DONATELLA BIRAL 1 , LUCIANO DALLA LIBERA ALFREDO MARGRETH 1
1,
CLAUDIO FRANCESCHI 2 and
1 National Research Council Center for Muscle Biology and Physiopathology, Institute o f General Pathology, University o f Padova, and 2 Institute o f General Pathology, University o f Bologna, Italy
(Received 9 April 1979, accepted 14 July 1979)
A microplate enzyme-linked immunosorbent assay (microELISA) for the study of immunochemical relationships between rabbit myosin light chains is described. Purified individual fast-muscle myosin light chains (LCIF, LC2F and LC3F) and their respective antisera, obtained in chicken, were used. Optimal conditions for antigen concentration, antiserum dilution, substrate concentration, incubation time and reproducibility with time were established. The observed cross-reactivities between the different types of light chains associated with rabbit fast-muscle myosin confirm and extend previous results obtained by other authors using radioimmunoassay procedures. It was concluded that microELISA may be successfully employed also to the study of macromolecule cross-reactivities.
INTRODUCTION
E n z y m e i m m u n o a s s a y in solid p h a s e ( E L I S A ) w h i c h has b e e n d e v e l o p e d r e c e n t l y as a m i c r o m e t h o d f o r testing several antigens (Voller et al., 1 9 7 7 ) , is a likely s u b s t i t u t e f o r o t h e r i m m u n o a s s a y s , such as r a d i o i m m u n o a s s a y ( R I A ) , b e c a u s e o f its high sensitivity a n d ability t o d e t e c t n o n - p r e c i p i t a b l e i m m u n e c o m p l e x e s , as well as f o r g r e a t e r s a f e t y a n d l o w e r cost. In view o f t h e increasing use o f m i c r o E L I S A a n d o f o u r i n t e r e s t in struct u r a l r e l a t i o n s h i p s b e t w e e n d i f f e r e n t t y p e s o f light chains a s s o c i a t e d w i t h skeletal m u s c l e m y o s i n s (Dalla L i b e r a e t al., 1 9 7 8 ; M a r g r e t h et al., in press), we d e c i d e d t o s u b j e c t this m e t h o d to e x p e r i m e n t a l test, especially in r e l a t i o n to t h e p r o b l e m o f i m m u n o l o g i c a l i n t e r r e l a t e d n e s s b e t w e e n t h e light chains o f fast-muscle myosin. T h e light chains a s s o c i a t e d w i t h t h e m y o s i n o f v e r t e b r a t e fast muscles, are well c h a r a c t e r i z e d c h e m i c a l l y a n d f u n c t i o n a l l y a n d , b y t h e s e criteria, are Correspondence and reprint requests should be addressed to: Dr. C. Franceschi, Istituto di Patologia Genera]e, Via S. Giacomo 14, 40126 Bologna, Italy.
94 classified into two main classes (Gauthier et al., 1978), namely alkali-light chains (A1 or LC1F, mol. wt., a b o u t 25,000 and A2 or LC3F, mol. wt., a b o u t 16,000) which are released on exposure of myosin to alkaline pH, and the DTNB-light chain (LC2F, mol. wt. a b o u t 18,000}, which may be dissociated from myosin by the thiol reagent DTNB (5,5'dithiobis2-nitrobenzoic acid). Sequence studies have shown (Frank and Weeds, 1974) that the extent of homology is greatest between LC1F and LC3F, the main distinguishing feature of LC1F being related to an N-terminal sequence ('difference peptide'), n o t present in LC3F, which accounts for its higher molecular weight. However, Silberstein and L o w e y (1977) and Holt and L o w e y (1977) by using RIA instead of double-diffusion (which had produced negative results) found an unexpected cross-reactivity between L C I F and LC2F, in the presence of antibody against LC2F, as well as, surprisingly, with antibody specific for the N-terminal sequence which is uniquely present in LC1F. The results reported here, with the individual light chains dissociated and purified from the myosin of rabbit fast muscle as immunogens, show that microELISA can be used to advantage in the study of their serological crossreactivity, with results which confirm and extend the previous studies on the myosin of chicken fast muscle by RIA procedures. MATERIALS AND METHODS
Antigens and antisera Fast-twitch muscle myosin was prepared from rabbit adductor myosins according to Barany and Close (1971), with the modifications reported by Dalla Libera et al. (1978). Light chains were dissociated by treating myosin with 4 M urea and were fractionated by DEAE column chromatography (Whatman DE-52) and a linear phosphate gradient, as described by L o w e y and Holt (1972). The pooled fractions corresponding to the 3 separated peaks of L C I F , LC2F and LC3F were precipitated by adding solid (NH4)2SO4 to 80% and were stored in a solution containing 0.4 M KC1, 0.05 M K-phosphate buffer pH 7.4, after overnight dialysis against the same buffer. The purity of each light chain preparation was determined by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis according to Weber and Osborn (1969). Antibodies against L C I F , LC2F and LC3F were elicited in adult hens by intramuscular injections with 1.5 mg protein in 1 ml of phosphate buffer emulsified with an equal volume of complete Freund's adjuvant (Difco, Detroit), repeated 3 times at 2--3 week intervals, followed by 3 injections of 3 mg of antigen protein every 4 days. Animals were bled after 4--5 months from the beginning Of the immunization schedule. Each specific antiserum used in the present study was obtained from a single bleeding of individual animals.
Preparation of anti-chicken IgG-alkaline phosphatase conjugate Chicken IgG were precipitated from normal sera with Na2SO4 and were
95 purified by DEAE-column chromatography at pH 6.4 (Whatman DE-52) as reported by Benedict (1967). Specific antibodies against chicken IgG were isolated from commercial rabbit anti-chicken IgG sera (Miles) by adsorption on purified chicken IgG coupled to activated Sepharose 4-B CNBr (Pharmacia) and elution at pH 2.8 with a solution containing 0.2 M CH3COOH and 0.3 M KC1. Anti-chicken IgG antibodies were conjugated to alkaline phosphatase (type VII, Sigma) according to Engvall and Perlmann (1972). Conjugates were stored at 4°C after appropriate dilution with 5% bovine serum albumin (Sigma), 0.001 M MgC12, 0.02% NAN3, 0.05 M Tris--HC1 pH 8.0.
Alkaline phosphatase substrate A solution (0.1--3 mg/ml) of the disodium salt of p-nitrophenol phosphate (NPP) (Sigma) was prepared immediately before use in 1 mM MgC12, 0.05 M Na2CO3--NaHCO3 buffer pH 9.8.
MicroELISA procedure MicroELISA test was carried out using microELISA plates (M29E, Cooke Microtiter) essentially as described by Voller et al. (1976) using 7 wells for each determination. The phosphatase activity bound to the wells was measured by adding 0.2 ml of the substrate solution (NNP) and by incubating at 25°C. The reaction was stopped after 30--60 min by adding 0.05 ml of 2 N NaOH, and the absorbance was measured at 400 nm. RESULTS
Antigens Figure 1 shows the chromatography elution profile of myosin light chains and the extent of purification achieved with each of the 3 types of chains. As examined by SDS-gel electrophoresis, and with overcharged gels (25--30 pg protein per gel), there was a very low degree of cross-contamination between LC1F and LC3F, as well as some contamination of LC3F by LC2F, and, occasionally, by LC1F. These results are comparable with those obtained originally by Lowey and Holt (1972} with the same procedures.
Optimum concentrations o f antigens and specific antiserum Microplate wells were coated with antigen at different concentrations. In Fig. 2 a typical experiment is shown using LC1F as antigen, at a concentration of 0.05--100 pg/ml, and anti L C I F serum at 1 : 1000, or at 1 : 100.000 dilution. The highest absorbance values (0.5--0.8) were obtained at serum dilutions of 1 : 1000 and with antigen coating concentrations in the 1 t~g to 10 pg/ml range; but measurable absorption values were obtained at antigen concentrations as low as 0.1 pg/ml. Within this range of antigen concentrations higher
96
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0.5
!
t
t
!
0.4.
0.3. 0.~ o.2-
_
........
- .....
Po4H
olo
0.1-
o"
1'0
2'0
3'o
,'o FRACTION NUuSER
Fig. 1. P u r i f i c a t i o n b y D E A E - c h r o m a t o g r a p h y o f light c h a i n s f r o m fast muscle m y o s i n o f t h e rabbit. A b o u t 100 m g of t o t a l light c h a i n s was applied t o a c o l u m n (1.5 × 30 c m ) of D E A E cellulose ( W h a t m a n DE-52), e q u i l i b r a t e d w i t h 0.05 M Na p h o s p h a t e pH 6.0 cont a i n i n g 0.1 mM d i t h i o t h r e i t o l . T h e p r o t e i n s were e l u t e d b y a p p l y i n g a linear g r a d i e n t of Na p h o s p h a t e pH 6.0, as i n d i c a t e d in the figure. SDS (0.1%) p o l y a c r y l a m i d e gel electrop h o r e s i s o f t h e p o o l e d fractions, c o r r e s p o n d i n g to the 3 s e p a r a t e d light chains, are s h o w n a b o v e t h e respective a b s o r p t i o n peaks ( L C 1 F , L C 2 F , L C 3 F , f r o m left t o right). F o r e x p e r i m e n t a l details see Materials a n d M e t h o d s .
A 400/30 mln
A 4 0 0 / 3 0 rain
1.0-
14)"
0.5-
0,5-
0:01
o.1
10
160 0 .~ ~. pg/ml L C I F -log serum dilution Fig. 2. A n t i g e n t i t r a t i o n . Plate wells were c o a t e d w i t h s o l u t i o n s of a n t i g e n ( L C I F ) at d i f f e r e n t c o n c e n t r a t i o n s a n d were i n c u b a t e d w i t h d i f f e r e n t d i l u t i o n s of a n t i s e r u m (anti L C 1 F ) , i.e. e, 1 : 1 0 0 0 , ©, 1 : 1 0 0 , 0 0 0 .
Fig. 3. E f f e c t o f a n t i s e r u m dilution. M i c r o p l a t e wells c o a t e d w i t h L C I F as a n t i g e n (5 m l ) were i n c u b a t e d at d i f f e r e n t c o n c e n t r a t i o n s o f s e r u m ( a n t i - L C 1 F ) .
pg/
97 serum dilutions gave very low absorbance values. Figure 3 shows the absorbance values obtained by coating the microplate wells with a fixed antigen (LC1F) c o n c e n t r a t i o n (5 pg/ml) in the presence of varying serum dilutions. Maximum absorbance values (1.0) were again re co r d ed with 1 : 1000 serum dilution. Similar results were obtained with LC2F and LC3F (not shown in the Figure). The m a x i m u m colour d e v e l o p m e n t after 30 min incubation at 25°C was f o u n d in a range of substrate c o n c e n t r a t i o n bet w een 1.5 and 3.0 mg/ml. The time-course of colour d e v e l o p m e n t was f o u n d to be linear during 60 min incubation of serum dilutions 1 : 1000 and 1 : 10,000.
Reproducibility of the microELISA The reproducibility investigated in several enzyme-conjugate, and These results are shown
o f the results obtained with the m i croE L ISA was experiments, by using different batches of fresh of the conjugate after storage in the cold r o o m . in Table 1.
Serological cross-reactivity between myosin light chains F o r investigating by ELISA the immunological relationship between myosin light chains, each t y p e of chain (LC1F, LC2F, and L C3F) was reacted with specific antisera for L C I F and LC3F, respectively. On a c c o u n t o f the previous work of Holt and L o w e y (1977), it was presumed that antibodies against L C I F should comprise antibodies reactive with antigenic sequence determinants shared by the h o m o l o g o u s antigen and by LC3F, as well as an a n t i b o d y population specific for the N-terminal sequence ('difference peptide') of L C I F (see I n t r o d u c t i o n section). The latter a n t i b o d y p o p u latio n should, in turn, be cross-reactive with LC2F (Holt and L ow ey, 1977; Silberstein and L ow ey, 1977). The results in Fig. 4A show t hat antiserum against LC1F contained crossreactive a n t i b o d y and that cross-reactivity was indeed most extensive TABLE 1 EFFECT OF ENZYME-CONJUGATE STORAGE ON MICROELISA REPRODUCIBILITY The assay was performed with LC1F as antigen and anti-LC1F as serum. For details see Materials and Methods. Conjugate Preparation I Preparation II Preparation III
Absorbance values at 400 nm, 30 rain incubation at 25°C fresh stored for 2 months at 4°C fresh stored for 2 months at 4°C fresh
0.950--0.970 0.320--0.400 0.935--1.055--1.035 0.690--0.810--0.755 0.720
98 A 400/30 mtn A 4 0 ( / 3 0 min
A 1.0.
i
(15-
o
0 5'
4'
~
~
3
- l o g s e r u m dilution
- log serum dilution
Fig. 4. Serological cross-reactivity between myosin light chains. The microELISA test was performed with A, anti-LC1F serum; B, anti-LC3F serum. ©, LC1F as antigen; o, LC2F as antigen; A, LC3F as antigen.
between L C I F and LC2F. On the other hand, it was found that antiserum against LC3F (Fig. 4B) was unable to distinguish between the immunogen and L C I F , and that LC2F, surprisingly, was also highly cross-reactive. DISCUSSION The results reported here provide new evidence of the wide range of applicability of microELISA. They further show that, under carefully controlled experimental conditions, this immunoassay technique satisfies the sensitivity requirements making it adequate to detect immunological cross-reactivities between the different classes of myosin light chains (Silberstein and Lowey, 1977). As applied to the investigation of the immunological relationship between myosin light chains, it should be emphasized that microELISA is able to reveal the formation of immune complexes at relatively high (1 : 1000 to 1 : 10,000) dilutions of antiserum, and in the presence of amounts of antigen per well as low as 1/~g of protein. Spectrophotometric assay of coupled chromogen formation from NPP by the anti-IgG-bound phosphatase probe is inherently much less sensitive, by a b o u t two orders of magnitude, than detection of radioactive label from 12SI-light chains in the immune complex by RIA (Silberstein and Lowey, 1977). However, the a m o u n t of antigen required for microELISA is still low
99 enough to allow use of this technique in immunological studies of myosin from h u m a n muscle biopsies. With regard to the observed cross-reactivities between the different types of light chains associated with rabbit fast-muscle myosin, it is doubtful, on account of the previous thorough studies of Silberstein and Lowey (1977) on homologous light chains from fast-muscle myosin of the chicken, that the results can be explained simply by cross-contamination of the immunogens. As reported in the Results section, this was of such a low degree to make it unlikely as a serious source of artifactual error. On the other hand, the long immunization periods (4--5 months) used in the present investigation, as in the previous study by Silberstein and Lowey (1977), may have helped in raising a population of antibodies of relatively lower specificity and with a higher affinity and hence with increasing crossreactivities with respect to antibodies elicited in short periods. Although it cannot be excluded that the immune sera used in the present study may be polyspecific it is significant that our results are in complete agreement with the reports of Silberstein and Lowey (1977) and Holt and L o w e y (1977) that LC1F and LC2F of fast-muscle myosin share c o m m o n configurational determinants, which property would therefore appear to be c o m m o n to the homologous light chains of myosins from different species, such as the chicken and the rabbit (present study). Our own results further indicate that LC2F contains antigenic determinants cross-reacting with LC3F, and is thereby distinguished from those reacting with antibodies specific for the 'difference peptide' uniquely present in L C I F . Further work along these lines with purified specific antibodies to the several antigenic determinants present in myosin light chains may profitably be carried out with ELISA and may provide interesting new information on the structural relationship between these peptides at the primary and tertiary structure levels. ACKNOWLEDGEMENTS Grateful acknowledgement is made to Mr. Silvio Bellucco for skillful technical assistance, and to Miss Giovanna Roberti, biology student, for help in several phases of this work. We wish to thank Professor G. Vicari and Dr. P. Iacona of the Istituto Superiore di SanitY, Rome, for helpful suggestions in the setting up of the technique. This investigation was supported in part by a grant from the Muscular Dystrophy Association to Professor Massimo Aloisi, Director of the Institute of General Pathology of the University of Padova. REFERENCES Barany, M. and R.I. Close, 1971, J. Physiol. (Lond.) 213,455. Benedict, A.A., 1967, in: Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 229.
100 Dalla Libera, L., A. Margreth, I. Mussini, C. Cerri and G. Scarlato, 1978, Muscle and Nerve 1,280. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Frank, G. and A.G. Weeds, 1974, Europ. J. Biochem. 4 4 , 3 1 7 . Gauthier, G.F., S. Lowey and A.W. Hobbs, 1978, Nature (Lond.) 274, 25. Holt, J.C. and S. Lowey, 1977, Biochemistry 16, 4398. Lowey, S. and J.C. Holt, 1972, Cold Spring Harbor Symp. Quant. Biol. 37, 19. Margreth, A., L. Dalla Libera, D. Biral and R. Betto, in: Proceedings of the International Symposium on 'New Frontiers in Muscular Dystrophies', Maranello, Modena, Italy, April 1, 1978, In press. Silberstein, L. and S. Lowey, 1977, Biochemistry 16, 4403. Voller, A., D. Bidwell and A. Bartlett, 1976, in: Manual of Clinical Immunology, eds. N.R. Rose and H. Friedman (American Society for Microbiology, Washington, DC) p. 506. Voller, A., D.E. Bidwell and A. Bartlett, 1977, The Enzyme Linked Immunosorbent Assay (ELISA) (Flowline Publications, Guernsey). Weber, K. and M. Osborn, 1969, J. Biol. Chem. 244, 4406.