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Chemiluminescence-linked immunoassay
Journal ofhnmunological Methods, 21 (1978) 179--184
179
© Elsevier/North-Holland Biomedical Press
CHEMILUMINESCENCE-LINKED IMMUNOASSAY
J.J. PRATT, M.G. WOLDRING and L. VILLERIUS Isotopenlaboratorium, Academisch Ziekenhuis, Groningen, The Netherlands
(Received 29 July 1977, accepted 15 November 1977) Covalent complexes of a chemilumineseent compound (a luminol derivative), protein and a steroidal hapten have been used as a labelled antigen in a way analogous to the radioactive label in radioimmunoassay. The antibody-bound complex emits light when treated with hydrogen peroxide and copper acetate at high pH. The chemiluminescent complex bound to antibody can be displaced by free steroid to produce a standard curve in a way analogous to radioimmunoassay. INTRODUCTION Since its d e v e l o p m e n t b y Y a l o w a n d Berson ( 1 9 6 0 ) , r a d i o i m m u n o a s s a y has f o u n d m a n y a p p l i c a t i o n s , especially in t h e clinical l a b o r a t o r y . T h e m e t h o d d e p e n d s on t h e c o m p e t i t i o n b e t w e e n antigen in t h e s a m p l e and r a d i o a c t i v e l y labelled antigen. A r a d i o a c t i v e label has the a d v a n t a g e t h a t v e r y small a m o u n t s o f label can easily be m e a s u r e d and t h a t m e a s u r e m e n t is free f r o m i n t e r f e r e n c e f r o m o t h e r m a t e r i a l s in t h e sample. Nevertheless, alternatives to t h e use o f r a d i o i s o t o p e s have b e e n d e v e l o p e d . As r e p l a c e m e n t s f o r r a d i o a c t i v e l y labelled ligands, antigens have b e e n c o u p l e d to e n z y m e s (Miles and Hales, 1 9 6 8 ; Engvall a n d P e r l m a n n , 1 9 7 1 ; Van W e e m a n and Schuurs, 1 9 7 1 ; R u b e n s t e i n et al., 1 9 7 2 ) , c o m p o u n d s giving an e l e c t r o n spin r e s o n a n c e signal ( L e u t e et al., 1972), f l u o r e s c e n t d y e s (Miles and Hales, 1 9 6 8 ; U l l m a n et al., 1 9 7 6 ) and b a c t e r i o p h a g e s ( H a i m o v i c h et al., 1970). We r e p o r t h e r e t h a t c h e m i l u m i n e s c e n t c o m p o u n d s can be c o u p l e d to antigen and t h a t t h e light e m i t t i n g c o m p l e x can be used as t h e t r a c e r in an i m m u n o a s s a y . Whilst this w o r k was in progress, S c h r o e d e r et al. ( 1 9 7 6 ) r e p o r t e d t h e use o f a c h e m i l u m i n e s c e n t g r o u p as t h e label in the c o m p e t i t i v e p r o t e i n b i n d i n g assay f o r b i o t i n using avidin as t h e binding p r o t e i n . T h e s e a u t h o r s s y n t h e s i s e d a simple, well d e f i n e d derivative o f i s o - l u m i n o l a n d biotin for use as the tracer. T h e s y n t h e t i c a p p r o a c h which we have used is q u i t e d i f f e r e n t a n d allows c h e m i l u m i n e s c e n c e to be used in c o n j u n c t i o n with a n t i b o d i e s as b i n d i n g p r o t e i n s . Because o f t h e i r stability, t h e c a p a c i t y o f d i f f e r e n t a n t i s e r a to r e a c t w i t h an i n d e f i n i t e l y large n u m b e r o f antigens, t h e i r high s p e c i f i c i t y and t h e i r high a f f i n i t y f o r antigens, a n t i b o d i e s are the m o s t widely used t y p e o f b i n d i n g p r o t e i n in this t y p e o f assay and are likely to r e m a i n so.
180 The c o m p o u n d described below can be coupled directly to proteins making it suitable for immunoassay of all proteins and peptides. If bot h the chemiluminescent c o m p o u n d and a hapten are coupled to the same protein molecule, the ternary c om pl ex can be used for immunoassay of the hapten. Such an immunoassay for testosterone (4-androsten-173-ol-3-one) is described below. The luminol derivative whose synthesis is described below can be considered as a universal labelling agent for antigens. This paper demonstrates the principle of chemiluminescence linked immunoassay. It is n o t intended as a r e c o m m e n d e d m e t h o d for the clinical laboratory. Such a m e t h o d requires that the accuracy of assays on biological samples be very t h o r o u g h l y tested and compared with conventional radioimmunoassays. MATERIALS AND METHODS Fifty mmoles of 4 - c a r b o x y m e t h y l aniline in 1 litre of 0.5 M hydrochloric acid at 0°C is diazotised by adding 60 mmoles of sodium nitrite. After 30 min at 0°C, excess nitrous acid is destroyed by adding 20 mmoles of urea. It is very i m p o r t a n t that excess nitrous acid is completely removed because it reacts with luminol to give a polymeric chemiluminescent c o m p o u n d and this gives rise to difficulties later on. After a further 15 min at 0°C, the diazotising mix tur e is added, slowly and with constant stirring, to 50 mmoles o f luminol ( 5 - a m i n o - l , 2 , 3 , 4 - t e t r a h y d r o p h t h a l a z i n e - l , 4 - d i o n e ) dissolved in 5 litres of a saturated solution of sodium bicarbonate. The pH is immediately readjusted to 9.0 with a saturated solution of sodium carbonate. The red solution is stirred overnight. The p r o d u c t must be separated from excess luminol or 4 - c a r b o x y m e t h y l aniline. We t o o k advantage of the fact that the pK a of the dissociation of the h yd r o g en - - n itr ogen bond in the phthalazinedione nucleus is about 7.0 and t h at the pK a of the aromatic amine group is about 3.0. At a pH of 6.0, luminol is uncharged and precipitates from aqueous solution. At a pH of 2.0, the p r o d u c t is uncharged and precipitates but excess 4 - c a r b o x y m e t h y l aniline is soluble at this pH owing to the presence of the free amino group. The process of acidifying the mixture is begun by adding 100 ml of orthophosphoric acid and 100 ml of glacial acetic acid. These acids provide the buffering capacity necessary for the fine adjustments of pH at 6.0 and 2.0. After addition of the phosphoric acid and acetic acid, the pH of the reaction mixture is reduced to 6.0 with c o n c e n t r a t e d HC1 and the suspension is allowed to stand overnight. The unreacted luminol precipitates and is filtered off. The pH o f the filtrate is reduced to 2.0 with HC1. The p r o d u c t precipitates and is filtered of f and carefully washed on the filter with 0.1 M HC1. The yield varied between 50 and 70%. The p r o d u c t has been provisionally assigned the structure: 8-(4'-carboxymeth y lp h en y l) - azo- 5- am i no- l , 2, 3, 4- t e t r ahydropht hal azi ne-l , 4-di one. The product, as isolated, has an Rr of 0.4 on silica gel thin-layer c h r o m a t o g r a p h y
181 eluted with 80% methanol in dichlormethane. Two minor impurities are always present, one more polar and the other less polar than the main product. No unreacted luminol is present. The quantum yield of chemiluminescence in 0.1 M borate buffer, pH 12.5, with an excess of hydrogen peroxide and with cobalt as the catalyst, is about 20% of that of luminol, i.e. about 0.004 quanta per molecule. Testosterone-7a-carboxyethyl thioether covalently coupled to bovine serum albumin using 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide as coupling agent was prepared according to the method of Weinstein et al. (1972). A solution of the conjugate containing 1 mg of protein/ml of physiological saline was emulsified in an equal volume of Freund's complete adjuvant. Each of 10 rabbits was injected with 2 ml of the water-in-oil emulsion divided over 20 intradermal sites. Injection were repeated every 4 weeks. Antisera obtained 20 weeks after the first injections were used for the experiments described in this paper. The labelled hapten is prepared by dissolving 50 mg of testosterone-Tacarboxyethyl thioether, 50 mg of 8-(4'-carboxymethylphenyl)-azo-5-amino1,2,3,4-tetrahydrophthalazine-l,4-dione and 100 mg of EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline; Aldrich Chemical Co.) in 6 ml of dimethyl sulphoxide. After about 5 min, this solution is added rapidly to 10 ml of 4% ovalbumin (Gallus gallus domesticus) in 0.02 M sodium phosphate buffer, pH 7.8 which has been precooled to 0°C. The reaction mixture is stirred for 24 h at 6°C. The precipitate is centrifuged off and the supernatant dialysed, first against 300 vol of 0.02 M sodium phosphate buffer in 35% dimethyl sulphoxide in water, adjusted to pH 7.8, and then against 3 X 300 vol of 0.02 M phosphate buffer in water, pH 7.8, containing 1% bovine serum albumin (crude fraction V). The conjugate was stored at --I96°C. The choice of coupling agent for the preparation of the labelled antigen is very important. Carbodiimides react with proteins to form stable conjugates. During immunisation, antibodies to carbodiimide residues are also produced. If a carbodiimide were to be used for synthesis of the labelled antigen, these antibodies would react with the carbodiimide residues on the labelled antigen and would not be displaced by the steroid. One hundred ng of the luminol--ovalbumin--testosterone conjugate, 0.1 ml of a suitable antiserum dilution and up to 10 ng of testosterone in a total volume of 0.5 ml are incubated overnight at 6°C. The a m o u n t of rabbit IgG per tube is adjusted to 40 pg by addition of normal rabbit serum and antibody-bound label is separated by precipitating with sheep antiserum specific for rabbit IgG. The tubes are centrifuged, the supernatants removed and the precipitates resuspended in 0~5 ml of buffer. All dilutions are m a d e in buffer containing sodium sulphate, 0.1 M, boric acid, 0.01 M, adjusted to pH 8.0 with sodium hydroxide. To the suspension in each tube is added 0.5 ml of 0.1 M sodium borate buffer, pH 13.0, and then 0.1 ml of a copper acetate--hydrogen peroxide
182
solution. The latter is prepared by mixing equal volumes of aqueous H202, 0.05% (w/v), and cupric acetate, 1 mg/ml, very rapidly in the dark a few hours before use. Both the borate buffer and the cupric acetate--H202 solution are mixed directly with the sample without allowing the reagents to run down the side of the test tube. If the reagents are not completely mixed before measurement of the luminescence, a high and irregular background is obtained owing to the additional mixing of the reagents which occurs when the measurements are being made. After addition of the cupric acetate-H202 solution, chemiluminescence is measured in a liquid scintillation counter (Nuclear Chicago Mark-l,150), operated out of coincidence with a very wide energy channel. Half an hour before the addition of the pH 13 borate buffer, the tubes containing the antibody-bound chemiluminescent material are pre-cooled to
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STEROID ADDED {ng } Fig. 1. Standard curve for the testosterone c h e m i l u m i n e s c e n c e - l i n k e d i m m u n o a s s a y . The graph is plotted w i t h a n t i b o d y - b o u n d c h e m i l u m i n e s c e n c e as the ordinate and standard a m o u n t s o f steroid as the abscissa.
183 10°C and dark adapted to minimise interference from phosphorescence of the glass. The addition of pH 13 borate buffer and all ot her operations immediately co n cer ne d with the m e a s ur e ment of chemiluminescence must be d o n e u n d er red safety light taking great care n o t to expose the photomultiplier even to this red light. The time between addition o f the pH 13 b u ff er and the peroxide must be exactly 15 sec for each tube. The time between addition of peroxide and the counting of the luminescence must also be 15 sec exactly for each tube. Because the rate of light p r o d u c t i o n is n o t constant, the timing of the addition of reagents is very important. All times are measured with a stopwatch. Fifteen sec is sufficient time to add the reagents and t hor oughl y mix the solution. The rate of change of light p r o d u c t i o n 15 sec after addition of the cupric acetate--H202 solution is fairly low so that manual addition o f reagents under stowatch cont rol is sufficiently accurate. It is convenient to measure light p r o d u c t i o n for 6 sec per sample. The background is typically 6000 counts/6 sec. The working range o f the liquid scintillation c o u n t e r used as a chemiluminescence m et er is from 10,000 to 300,000 co u n ts / 6 sec. RESULTS The testosterone standard curve is shown in Fig. 1. The total a m o u n t of chemiluminescent material per t ube ( a nt i b ody-bound plus free) was equivalent to 300,000 count s / 6 sec. As can be seen from the figure, oestradiol (oestra-l,3,5-triene-3,17~-diol) has no effect on the binding of the labelled antigen. Separate experiments showed that ot her steroids were w i t hout effect on the testosterone standard curve indicating that the effect of test o ster o n e is specific. Similar results have been obtained by coupling ot her steroids to the luminol--ovalbumin complex and incubating with the corresponding antiserum. The binding capacity of the labelled antigen for a n t i b o d y diminished in the course o f a few weeks even t hough the labelled antigen was stored under liquid nitrogen. The cause of this instability is currently under investigation. DISCUSSION The m e t h o d described above has obvious shortcomings. These arise from the use o f the liquid scintillation c o u n t e r for the m easurem ent of chemiluminescence. Apparatus specially designed for luminescence m easurem ent is commercially available and the use of such apparatus, where the reagents are mixed immediately in f r o n t of the photomultiplier, avoids these difficulties and should greatly improve counting efficiency. The m e t h o d has the advantage t hat the luminescent label can be coupled to protein so that the m e t h o d is applicable to immunoassay for proteins, peptides and all haptens w i t h o u t extensive modification. Chemiluminescence-
184 linked i m m u n o a s s a y ( C L I A ) shares the advantage o f e n z y m e - l i n k e d i m m u n o assay and fluorescence-linked i m m u n o a s s a y in t h a t radioactive isotopes and the increasingly stringent g o v e r n m e n t regulations governing their use are avoided. The m e t h o d shares the advantage o f r a d i o i m m u n o a s s a y in t h a t it ' c o u n t s ' single molecules o f the labelled antigen and t h a t the sensitivity o f t h e m e t h o d is limited o n l y b y the a f f i n i t y o f the antibodies. The m e t h o d shares a f u r t h e r advantage o f r a d i o i m m u n o a s s a y in t h a t n e i t h e r radioactive n o r c h e m i l u m i n e s c e n t materials o c c u r in m o s t biological samples to be analysed. REFERENCES Engvall, E. and P. Perlmann, 1971, Immunoehemistry 8, 871. Haimovich, J., E. Hurwitz, N. Novik and M. Sela, 1970, Biochim. Biophys. Acta 207,125. Leute, R.K., E.F. Ullman, A. Goldstein and L.E. Herzenberg, 1972, Nature New Biol. 236, 93. Miles, L.E.M. and C.N. Hales, 1968, Nature 219, 186. Rubenstein, K.E., R.S. Schneider and E.F. Ullman, 1972, Biochem. Biophys. Res. Commun. 47,846. Schroeder, H.R., P.O. Vogelhut, R.J. Carrico, R.C. Boguslaski and R.T. Buckler, 1976, Anal. Chem. 48, 1933. Ullman, E.F., M. Schwarzberg and K.E. Rubenstein, 1976, J. Biol. Chem. 251, 4172. Van Weeman, B.K. and A.H.W.M. Schuurs, 1971, FEBS Lett. 15,232. Weinstein, A., H.R. Lindner, A. Friedlander and S. Bauminger, 1972, Steroids 20,789. Yalow, R.S. and S.A. Berson, 1960, J. Clin. Invest. 39, 1157.
179
© Elsevier/North-Holland Biomedical Press
CHEMILUMINESCENCE-LINKED IMMUNOASSAY
J.J. PRATT, M.G. WOLDRING and L. VILLERIUS Isotopenlaboratorium, Academisch Ziekenhuis, Groningen, The Netherlands
(Received 29 July 1977, accepted 15 November 1977) Covalent complexes of a chemilumineseent compound (a luminol derivative), protein and a steroidal hapten have been used as a labelled antigen in a way analogous to the radioactive label in radioimmunoassay. The antibody-bound complex emits light when treated with hydrogen peroxide and copper acetate at high pH. The chemiluminescent complex bound to antibody can be displaced by free steroid to produce a standard curve in a way analogous to radioimmunoassay. INTRODUCTION Since its d e v e l o p m e n t b y Y a l o w a n d Berson ( 1 9 6 0 ) , r a d i o i m m u n o a s s a y has f o u n d m a n y a p p l i c a t i o n s , especially in t h e clinical l a b o r a t o r y . T h e m e t h o d d e p e n d s on t h e c o m p e t i t i o n b e t w e e n antigen in t h e s a m p l e and r a d i o a c t i v e l y labelled antigen. A r a d i o a c t i v e label has the a d v a n t a g e t h a t v e r y small a m o u n t s o f label can easily be m e a s u r e d and t h a t m e a s u r e m e n t is free f r o m i n t e r f e r e n c e f r o m o t h e r m a t e r i a l s in t h e sample. Nevertheless, alternatives to t h e use o f r a d i o i s o t o p e s have b e e n d e v e l o p e d . As r e p l a c e m e n t s f o r r a d i o a c t i v e l y labelled ligands, antigens have b e e n c o u p l e d to e n z y m e s (Miles and Hales, 1 9 6 8 ; Engvall a n d P e r l m a n n , 1 9 7 1 ; Van W e e m a n and Schuurs, 1 9 7 1 ; R u b e n s t e i n et al., 1 9 7 2 ) , c o m p o u n d s giving an e l e c t r o n spin r e s o n a n c e signal ( L e u t e et al., 1972), f l u o r e s c e n t d y e s (Miles and Hales, 1 9 6 8 ; U l l m a n et al., 1 9 7 6 ) and b a c t e r i o p h a g e s ( H a i m o v i c h et al., 1970). We r e p o r t h e r e t h a t c h e m i l u m i n e s c e n t c o m p o u n d s can be c o u p l e d to antigen and t h a t t h e light e m i t t i n g c o m p l e x can be used as t h e t r a c e r in an i m m u n o a s s a y . Whilst this w o r k was in progress, S c h r o e d e r et al. ( 1 9 7 6 ) r e p o r t e d t h e use o f a c h e m i l u m i n e s c e n t g r o u p as t h e label in the c o m p e t i t i v e p r o t e i n b i n d i n g assay f o r b i o t i n using avidin as t h e binding p r o t e i n . T h e s e a u t h o r s s y n t h e s i s e d a simple, well d e f i n e d derivative o f i s o - l u m i n o l a n d biotin for use as the tracer. T h e s y n t h e t i c a p p r o a c h which we have used is q u i t e d i f f e r e n t a n d allows c h e m i l u m i n e s c e n c e to be used in c o n j u n c t i o n with a n t i b o d i e s as b i n d i n g p r o t e i n s . Because o f t h e i r stability, t h e c a p a c i t y o f d i f f e r e n t a n t i s e r a to r e a c t w i t h an i n d e f i n i t e l y large n u m b e r o f antigens, t h e i r high s p e c i f i c i t y and t h e i r high a f f i n i t y f o r antigens, a n t i b o d i e s are the m o s t widely used t y p e o f b i n d i n g p r o t e i n in this t y p e o f assay and are likely to r e m a i n so.
180 The c o m p o u n d described below can be coupled directly to proteins making it suitable for immunoassay of all proteins and peptides. If bot h the chemiluminescent c o m p o u n d and a hapten are coupled to the same protein molecule, the ternary c om pl ex can be used for immunoassay of the hapten. Such an immunoassay for testosterone (4-androsten-173-ol-3-one) is described below. The luminol derivative whose synthesis is described below can be considered as a universal labelling agent for antigens. This paper demonstrates the principle of chemiluminescence linked immunoassay. It is n o t intended as a r e c o m m e n d e d m e t h o d for the clinical laboratory. Such a m e t h o d requires that the accuracy of assays on biological samples be very t h o r o u g h l y tested and compared with conventional radioimmunoassays. MATERIALS AND METHODS Fifty mmoles of 4 - c a r b o x y m e t h y l aniline in 1 litre of 0.5 M hydrochloric acid at 0°C is diazotised by adding 60 mmoles of sodium nitrite. After 30 min at 0°C, excess nitrous acid is destroyed by adding 20 mmoles of urea. It is very i m p o r t a n t that excess nitrous acid is completely removed because it reacts with luminol to give a polymeric chemiluminescent c o m p o u n d and this gives rise to difficulties later on. After a further 15 min at 0°C, the diazotising mix tur e is added, slowly and with constant stirring, to 50 mmoles o f luminol ( 5 - a m i n o - l , 2 , 3 , 4 - t e t r a h y d r o p h t h a l a z i n e - l , 4 - d i o n e ) dissolved in 5 litres of a saturated solution of sodium bicarbonate. The pH is immediately readjusted to 9.0 with a saturated solution of sodium carbonate. The red solution is stirred overnight. The p r o d u c t must be separated from excess luminol or 4 - c a r b o x y m e t h y l aniline. We t o o k advantage of the fact that the pK a of the dissociation of the h yd r o g en - - n itr ogen bond in the phthalazinedione nucleus is about 7.0 and t h at the pK a of the aromatic amine group is about 3.0. At a pH of 6.0, luminol is uncharged and precipitates from aqueous solution. At a pH of 2.0, the p r o d u c t is uncharged and precipitates but excess 4 - c a r b o x y m e t h y l aniline is soluble at this pH owing to the presence of the free amino group. The process of acidifying the mixture is begun by adding 100 ml of orthophosphoric acid and 100 ml of glacial acetic acid. These acids provide the buffering capacity necessary for the fine adjustments of pH at 6.0 and 2.0. After addition of the phosphoric acid and acetic acid, the pH of the reaction mixture is reduced to 6.0 with c o n c e n t r a t e d HC1 and the suspension is allowed to stand overnight. The unreacted luminol precipitates and is filtered off. The pH o f the filtrate is reduced to 2.0 with HC1. The p r o d u c t precipitates and is filtered of f and carefully washed on the filter with 0.1 M HC1. The yield varied between 50 and 70%. The p r o d u c t has been provisionally assigned the structure: 8-(4'-carboxymeth y lp h en y l) - azo- 5- am i no- l , 2, 3, 4- t e t r ahydropht hal azi ne-l , 4-di one. The product, as isolated, has an Rr of 0.4 on silica gel thin-layer c h r o m a t o g r a p h y
181 eluted with 80% methanol in dichlormethane. Two minor impurities are always present, one more polar and the other less polar than the main product. No unreacted luminol is present. The quantum yield of chemiluminescence in 0.1 M borate buffer, pH 12.5, with an excess of hydrogen peroxide and with cobalt as the catalyst, is about 20% of that of luminol, i.e. about 0.004 quanta per molecule. Testosterone-7a-carboxyethyl thioether covalently coupled to bovine serum albumin using 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide as coupling agent was prepared according to the method of Weinstein et al. (1972). A solution of the conjugate containing 1 mg of protein/ml of physiological saline was emulsified in an equal volume of Freund's complete adjuvant. Each of 10 rabbits was injected with 2 ml of the water-in-oil emulsion divided over 20 intradermal sites. Injection were repeated every 4 weeks. Antisera obtained 20 weeks after the first injections were used for the experiments described in this paper. The labelled hapten is prepared by dissolving 50 mg of testosterone-Tacarboxyethyl thioether, 50 mg of 8-(4'-carboxymethylphenyl)-azo-5-amino1,2,3,4-tetrahydrophthalazine-l,4-dione and 100 mg of EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline; Aldrich Chemical Co.) in 6 ml of dimethyl sulphoxide. After about 5 min, this solution is added rapidly to 10 ml of 4% ovalbumin (Gallus gallus domesticus) in 0.02 M sodium phosphate buffer, pH 7.8 which has been precooled to 0°C. The reaction mixture is stirred for 24 h at 6°C. The precipitate is centrifuged off and the supernatant dialysed, first against 300 vol of 0.02 M sodium phosphate buffer in 35% dimethyl sulphoxide in water, adjusted to pH 7.8, and then against 3 X 300 vol of 0.02 M phosphate buffer in water, pH 7.8, containing 1% bovine serum albumin (crude fraction V). The conjugate was stored at --I96°C. The choice of coupling agent for the preparation of the labelled antigen is very important. Carbodiimides react with proteins to form stable conjugates. During immunisation, antibodies to carbodiimide residues are also produced. If a carbodiimide were to be used for synthesis of the labelled antigen, these antibodies would react with the carbodiimide residues on the labelled antigen and would not be displaced by the steroid. One hundred ng of the luminol--ovalbumin--testosterone conjugate, 0.1 ml of a suitable antiserum dilution and up to 10 ng of testosterone in a total volume of 0.5 ml are incubated overnight at 6°C. The a m o u n t of rabbit IgG per tube is adjusted to 40 pg by addition of normal rabbit serum and antibody-bound label is separated by precipitating with sheep antiserum specific for rabbit IgG. The tubes are centrifuged, the supernatants removed and the precipitates resuspended in 0~5 ml of buffer. All dilutions are m a d e in buffer containing sodium sulphate, 0.1 M, boric acid, 0.01 M, adjusted to pH 8.0 with sodium hydroxide. To the suspension in each tube is added 0.5 ml of 0.1 M sodium borate buffer, pH 13.0, and then 0.1 ml of a copper acetate--hydrogen peroxide
182
solution. The latter is prepared by mixing equal volumes of aqueous H202, 0.05% (w/v), and cupric acetate, 1 mg/ml, very rapidly in the dark a few hours before use. Both the borate buffer and the cupric acetate--H202 solution are mixed directly with the sample without allowing the reagents to run down the side of the test tube. If the reagents are not completely mixed before measurement of the luminescence, a high and irregular background is obtained owing to the additional mixing of the reagents which occurs when the measurements are being made. After addition of the cupric acetate-H202 solution, chemiluminescence is measured in a liquid scintillation counter (Nuclear Chicago Mark-l,150), operated out of coincidence with a very wide energy channel. Half an hour before the addition of the pH 13 borate buffer, the tubes containing the antibody-bound chemiluminescent material are pre-cooled to
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:
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7
N-
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TESTOSTE~
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100
STEROID ADDED {ng } Fig. 1. Standard curve for the testosterone c h e m i l u m i n e s c e n c e - l i n k e d i m m u n o a s s a y . The graph is plotted w i t h a n t i b o d y - b o u n d c h e m i l u m i n e s c e n c e as the ordinate and standard a m o u n t s o f steroid as the abscissa.
183 10°C and dark adapted to minimise interference from phosphorescence of the glass. The addition of pH 13 borate buffer and all ot her operations immediately co n cer ne d with the m e a s ur e ment of chemiluminescence must be d o n e u n d er red safety light taking great care n o t to expose the photomultiplier even to this red light. The time between addition o f the pH 13 b u ff er and the peroxide must be exactly 15 sec for each tube. The time between addition of peroxide and the counting of the luminescence must also be 15 sec exactly for each tube. Because the rate of light p r o d u c t i o n is n o t constant, the timing of the addition of reagents is very important. All times are measured with a stopwatch. Fifteen sec is sufficient time to add the reagents and t hor oughl y mix the solution. The rate of change of light p r o d u c t i o n 15 sec after addition of the cupric acetate--H202 solution is fairly low so that manual addition o f reagents under stowatch cont rol is sufficiently accurate. It is convenient to measure light p r o d u c t i o n for 6 sec per sample. The background is typically 6000 counts/6 sec. The working range o f the liquid scintillation c o u n t e r used as a chemiluminescence m et er is from 10,000 to 300,000 co u n ts / 6 sec. RESULTS The testosterone standard curve is shown in Fig. 1. The total a m o u n t of chemiluminescent material per t ube ( a nt i b ody-bound plus free) was equivalent to 300,000 count s / 6 sec. As can be seen from the figure, oestradiol (oestra-l,3,5-triene-3,17~-diol) has no effect on the binding of the labelled antigen. Separate experiments showed that ot her steroids were w i t hout effect on the testosterone standard curve indicating that the effect of test o ster o n e is specific. Similar results have been obtained by coupling ot her steroids to the luminol--ovalbumin complex and incubating with the corresponding antiserum. The binding capacity of the labelled antigen for a n t i b o d y diminished in the course o f a few weeks even t hough the labelled antigen was stored under liquid nitrogen. The cause of this instability is currently under investigation. DISCUSSION The m e t h o d described above has obvious shortcomings. These arise from the use o f the liquid scintillation c o u n t e r for the m easurem ent of chemiluminescence. Apparatus specially designed for luminescence m easurem ent is commercially available and the use of such apparatus, where the reagents are mixed immediately in f r o n t of the photomultiplier, avoids these difficulties and should greatly improve counting efficiency. The m e t h o d has the advantage t hat the luminescent label can be coupled to protein so that the m e t h o d is applicable to immunoassay for proteins, peptides and all haptens w i t h o u t extensive modification. Chemiluminescence-
184 linked i m m u n o a s s a y ( C L I A ) shares the advantage o f e n z y m e - l i n k e d i m m u n o assay and fluorescence-linked i m m u n o a s s a y in t h a t radioactive isotopes and the increasingly stringent g o v e r n m e n t regulations governing their use are avoided. The m e t h o d shares the advantage o f r a d i o i m m u n o a s s a y in t h a t it ' c o u n t s ' single molecules o f the labelled antigen and t h a t the sensitivity o f t h e m e t h o d is limited o n l y b y the a f f i n i t y o f the antibodies. The m e t h o d shares a f u r t h e r advantage o f r a d i o i m m u n o a s s a y in t h a t n e i t h e r radioactive n o r c h e m i l u m i n e s c e n t materials o c c u r in m o s t biological samples to be analysed. REFERENCES Engvall, E. and P. Perlmann, 1971, Immunoehemistry 8, 871. Haimovich, J., E. Hurwitz, N. Novik and M. Sela, 1970, Biochim. Biophys. Acta 207,125. Leute, R.K., E.F. Ullman, A. Goldstein and L.E. Herzenberg, 1972, Nature New Biol. 236, 93. Miles, L.E.M. and C.N. Hales, 1968, Nature 219, 186. Rubenstein, K.E., R.S. Schneider and E.F. Ullman, 1972, Biochem. Biophys. Res. Commun. 47,846. Schroeder, H.R., P.O. Vogelhut, R.J. Carrico, R.C. Boguslaski and R.T. Buckler, 1976, Anal. Chem. 48, 1933. Ullman, E.F., M. Schwarzberg and K.E. Rubenstein, 1976, J. Biol. Chem. 251, 4172. Van Weeman, B.K. and A.H.W.M. Schuurs, 1971, FEBS Lett. 15,232. Weinstein, A., H.R. Lindner, A. Friedlander and S. Bauminger, 1972, Steroids 20,789. Yalow, R.S. and S.A. Berson, 1960, J. Clin. Invest. 39, 1157.