- Email: [email protected]
Comparison of three conjugation procedures for the formation of tracers for use in enzyme immunoassays
Journal oflmmunologicaIMethods, 72 (1984) 261-268
261
Elsevier JIM03153
Comparison of Three Conjugation Procedures for the Formation of Tracers for Use in Enzyme Immunoassays D.G. Williams Department of Clinical Biochemistry and Metabolic Medicine, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, U.K.
(Received 4 January 1984, accepted 11 April 1984)
One of the requirements for enzyme immunoassay is the formation of a labelled component of the assay system which can be either the ligand or the binder. Three methods for conjugating alkaline phosphatase to human placental lactogen were investigated, involving water-soluble carbodiimide, glutaraldehyde, and the periodate oxidation technique. Of the 3, the periodate oxidation technique proved superior giving a conjugate with only slight changes in the Michaelis constant (Kin) and maximum velocity (Vmax) which could be used for an enzyme immunoassay for human placental lactogen in human plasma. Key words: enzyme immunoassay - conjugation techniques - glutaraldehyde - periodate oxidation
Introduction A requirement for enzyme tmmunoassay (EIA) is the formation of an enzymelabelled component of the assay system. This can be either the ligand or the binder. A substantial number of techniques exist for covalently linking proteins to proteins and these have been listed in a comprehensive review by Kennedy et al. (1976). The majority of such agents are bifunctional in that they contain 2 groups capable of reacting with functional groups on a protein (e.g., - N H z, - O H , - S H ) . As both the enzyme and the target protein are likely to contain the same functional groups, the reaction will produce enzyme-enzyme and protein-protein conjugates as well as the desired enzyme-protein conjugate. Aggregates may also occur in which the enzymic and immunological activities of each component are lost due to steric hindrance. The outcome of the reaction is governed by a number of factors such as the relative rates of reaction of the agent with both proteins and the relative size of each protein, as well as the number of available functional groups on each protein (Modesto and Pesce, 1971). The purpose of the present work was to compare 3 such methods for the formation of enzyme-labelled proteins for use in EIAs. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
262 Three agents were chosen: (i) water-soluble carbodiimide, which is widely used in protein linking work; (ii) glutaraldehyde which is used for the formation of enzyme labels; (iii) periodate oxidation, which has the potential of giving a better conjugate as it involves 2 separate reactions. The enzyme chosen as the marker was calf intestinal alkaline phosphatase which is widely used for this purpose, is easily assayed, and yields a highly coloured product. The protein was human placental lactogen (HPL), used as an index of foeto-placental function, and currently measured by radioimmunoassay (Letchworth et al., 1971). The intent was to develop an enzyme immunoassay for HPL in which the radiolabelled (1251) HPL would be replaced by an enzyme-labelled tracer. The outcome of this work has been reported by Williams (1978).
Materials and Methods
The following reagents were used: (1-ethyl-3-3-dimethyl amino propyl) carbodiimide, hydrochloride, 20 mg/ml; glutaraldehyde, 1.5% solution; fluorodinitrobenzene, 1% in ethanol; sodium periodate 0.05 M; ethylene glycol 0.16 M; carbonate buffer, 0.01 M, pH 9.5; ethanolamine, 2 M adjusted to pH 9.5; phosphate-buffered saline (PBS), pH 7.2; alkaline phosphatase, Grade I, calf intestinal (Boehringer); diethanolamine buffer, 1.01 M, pH 9.8; p-nitrophenyl phosphate 110 mmol/l; sodium hydrogen carbonate 0.3 M; phosphate buffer, pH 6.8, 0.1 M; human placental lactogen MRC standard 70/144. Assay reagents
Assay diluent, barbitone buffer pH 8.6 with human serum albumin, 0.5 mg/ml; guinea pig anti-HPL antiserum, prepared by standard procedures; lzSI-labelled HPL, prepared by the chloramine-T method; normal guinea pig serum; donkey anti-guinea pig serum; pooled male plasma for the dilution of tests and standards. All reagents were Analar grade and made up in deionized water unless otherwise stated. Method I - - Water soluble carbodiimide (Goodfriend et al., 1964)
This reagent links free amino and carboxyl groups. Human placental lactogen (1 mg) and 100/~1 of alkaline phosphatase suspension were dissolved in 1.0 ml water. One milligram of (1-ethyl-3-3-dimethyl amino propyl) carbodiimide hydrochloride in 100/~1 of water were added in 10/~1 aliquots and the reaction allowed to proceed overnight at 4°C. The reaction was terminated by the addition of 100 ~tl of glacial acetic acid. Method 2 - - Glutaraldehyde (Miyai et al., 1976)
This reagent links free amino groups. One hundred microlitres of the enzyme suspension were centrifuged and the supernatant discarded. The precipitate was dissolved in 150/~1 of 0.1 M phosphate buffer pH 6.8, and 100/~1 of this solution were mixed with 100/~1 buffer containin8 100 ~tg HPL. To this were added, in 2/zl
263
amounts, 12 /~1 of a 1.5% aqueous solution of glutaraldehyde and the reaction allowed to proceed at room temperature for 3 h. The mixture was then dialysed against 200 ml phosphate-buffered saline overnight. Method 3 - - Periodate oxidation (Kawaoi and Nakone, 1973) This method oxidizes carbohydrates to aldehydes which then link with free amino groups. One hundred microlitres of the enzyme suspension were centrifuged and the pellet dissolved in 100/~1 of 0.3 M sodium hydrogen carbonate solution and 10/.tl of a 1% ethanolic solution of fluorodinitrobenzene (FDNB) added. This was left at room temperature for 2 h to block any free amino groups. Sodium periodate solution (0.05 M, 100 ~1) was added to oxidize carbohydrate groups to aldehyde and the reaction allowed to proceed for 6 h. The reaction was then terminated by the addition of excess ethylene glycol (0.16 M, 100 ~1) and left for 1 h before overnight dialysis against 500 ml carbonate buffer (pH 9.5, 0.01 M). The following morning 100/.tl of HPL in 100 ~1 of carbonate buffer were added to the dialysate and the mixture left for 24 h to allow the aldehyde groups to form links with free amino groups on the HPL. Unreacted aldehyde was removed by addition of excess ethanolamine (2 M, pH 9.5, 20 ~1) and the mixture dialysed overnight against 500 ml PBS.
Results
The results given here are representative of several preparations that were carried out during the course of the study. Carbodiimide Extensive loss of enzyme activity ( > 95%) was found with this procedure, which was not investigated further. Glutaraldehyde The dialysed reaction mixture was passed through a Sephadex G-100 column (1.5 cm diameter, 60 cm 3 volume) equilibrated with PBS. Fractions were collected (2.0 ml) and assayed for enzyme activity by taking 10 #1 of eluate and adding it to 1.0 ml of diethanolamine buffer pH 9.8 containing 0.505 mmol Mg2+/1. The reaction was started by addition of paranitrophenyl phosphate, 110 mmol/1. The absorbance at 405 nm was measured after 30 min, the reaction being allowed to proceed to completion at room temperature. Fractions that contained activity were assessed for their suitability as tracers by inclusion in the following assay. The assay system was derived from the radioimmunoassay of Letchworth et al. (1971) previously established as a reference assay. The only modifications to the published method were the use of enzyme-labelled H P L and a second antibody separation system as the alcohol used in the original would be likely to denature the enzyme. To 250 #1 of assay diluent (barbitone buffer, pH 8.6, 0.06 M with human serum albumin 0.5 m g / m l ) were added 50/.tl of serum, 10/xl of enzyme conjugate
264
and 100 #1 of antiserum (final dilution 1 : 5000). This was left at room temperature for 2 h and then 0.1 ml each of normal guinea pig serum (1 : 100 dilution) or donkey anti-guinea pig serum (1 : 10 dilution) were added. After a further 2 h incubation the tubes were spun and drained and the enzyme activity measured by resuspending the precipitate in 1.0 ml of diethanolamine buffer and adding paranitrophenyl phosphate. The absorbance at 405 nm was measured after 30 min at room temperature. For each label 4 sets of tubes were used, each tube being assayed in duplicate. The first tube contained a pooled pregnancy serum from the early second trimester with an H P L concentration of 1.5/~g/ml, the second, the early second trimester pool diluted 1:1, i.e., 0.75 ~tg/ml, the third contained no HPL, and the fourth no antiserum as a check on non-specific binding. The sera were diluted 1 : 11 in pooled male plasma. It had previously been shown that the radioimmunoassay could easily distinguish between these 3 levels so that if the enzyme label was to be of any use it should also be able to do this. Despite repeated attempts, it was not found possible to establish an assay capable of accurately differentiating between the 2 pools of serum. A dose-response curve when carried out with a full set of 8 standards (10 to 0.08 m g / m l in doubling dilutions) was very flat with absorbance differences of only 0.15-0.2 between the highest and lowest standards. Furthermore the reproducibility of duplicates was very poor, with sometimes as much as 0.1 A difference between the points. Non-specific binding was also very variable. Washing the precipitate and adding more tracer did not significantly increase the sensitivity or precision of the assay. Periodate oxidation The reaction mixture was purified by passing it through the Sephadex G-100 column. The elution pattern was similar to that obtained with the glutaraldehyde conjugates. Fractions containing enzyme activity were checked in the same way as
TABL E I DOSE-RESPONSE CURVES F O R F R A C T I O N S 11-15 ( P E R I O D A T E LABEL) Mean absorbances (duplicate assay) at 405 nm. HPL ~tg / m l
10 5 2.5 1.25 0.63 0.31 0.16 0.08 0 NSB a
Fraction nos. 11
12
13
14
15
0.970 1.00 1.08 1.19 1.31 1.48 1.75 2.01 2.12 0.86
1.10 1.27 1.46 1.67 2.20 2.43 2.75 2.82 2.80 0.88
1.44 1.54 2.06 2.32 2.60 2.78 2.81 2.85 2.88 1.180
1.76 1.71 1.87 2.04 2.64 2.75 2.82 2.88 2.88 1.28
1.25 1.25 1.21 1.42 1.50 2.17 2.77 2.33 2.50 0.90
a NSB = Non-specific binding.
265 A~.os 3-2
2'~ 2'4' 2'0' 1"6" 1"2"
0 "~,'
0;1
14)
10:0
HPL (~g/mt)
Fig. 1. Dose-response curve for 3 fractions (11-13) from periodate conjugation procedure, x fraction 11; A a, fraction 12; • • , fraction 13.
X,
the glutaraldehyde conjugates and 5 fractions were found to give encouraging results, with absorbance differences of about 1.4 A between the highest and lowest standards. The labels were then tested by using them to construct a dose-response curve for a series of HPL standards made up in pooled male plasma. 2.5/~1 of label were taken for each tube. Table I shows the results were obtained. All fractions gave satisfactory dose-response curves. The non-specific binding on fraction 14 was very high, and the curve for 15 was rather flat so these were not considered further. The dose-response curves for 11-13 are shown in Fig. 1. By using 1 #1 of label, (fraction
1.3
A~.os
1.'2 1.1 1.0
0.9 0.8 0.';
0 '1
1"0 HPL
(pg/ml)
Fig. 2. Dose-response curve for fraction 12 with 1 /xl of label.
10-0
266 TABLE II DOSE-RESPONSE CURVE FOR FRACTION 12 (1 ttl LABEL) H PL/Lg/ml
A 405 (mean of duplicates)
10 5 2.5 1.25 0.63 0.31 0.16 0.08 0 NSB Pool 1 Pool 2
0.74o o.735 0.743 0.762 0.815 0.872 1.030 1.12 1.545 0.638 1.50 0.80
12) a d o s e - r e s p o n s e curve with lower final a b s o r b a n c e s m o r e suitable for m e a s u r e m e n t was o b t a i n e d , as shown in Fig. 2 a n d T a b l e II. The pools were sera of k n o w n value (assayed b y r a d i o i m m u n o a s s a y ) diluted 1 : 1 1 in p o o l e d male p l a s m a before assay. As can be seen, the results are in g o o d agreement. As is d e s c r i b e d elsewhere (Williams, 1978) the m a n u a l assay was a d a p t e d to given a s e m i - a u t o m a t e d assay with g o o d c o r r e l a t i o n with the reference r a d i o i m m u n o a s s a y ( r = 0.96). F i n a l l y the effect of labelling on the Michaelis c o n s t a n t ( K m) a n d the m a x i m u m velocity (Vmax) of the p r e p a r a t i o n was investigated, as significant alterations w o u l d have r e d u c e d the e n z y m e ' s c a p a b i l i t y as a marker. It was not possible to do this for the g l u t a r a l d e h y d e label as the level of enzyme activity was not sufficiently high to o b t a i n suitable measurements. E n z y m e activity at different s u b s t r a t e c o n c e n t r a t i o n s was m e a s u r e d using the assay c o n d i t i o n s o u t l i n e d b y the S c a n d i n a v i a n Society for Clinical E n z y m o l o g y (1974) with, d i e t h a n o l a m i n e buffer a n d p - n i t r o p h e n y l p h o s p h a t e starter. All measurem e n t s were m a d e in d u p l i c a t e on a Pye U n i c a m SP8000 s p e c t r o p h o t o m e t e r with a t h e r m o s t a t t e d curvette h o l d e r set at 37°C. The results shown in T a b l e III were
TABLE III LINEWEAVER-BURK DATA FOR FREE ENZYME AND CONJUGATE Substrate concentration (mmol/1)
1 ~
1 ff (free enzyme)
1 T (label)
10 4 2 1 0.5
0.1 0.25 0.5 1 2
23.68 27.50 36.60 40.88 51.45
18.47 22.27 24.66 32.60 61.50
267 I/V 1~0
I2 0
/
~reeEnzyme
/ too 8O 6O 4
2 <
_
-i
-2
-1
0
1
2
3
4
5
6
1/S
Fig. 3, Double reciprocal (Lineweaver-Burk) plot for free enzyme and conjugate. Intercept on x-axis = K m. Intercept on y-axis = Vm.~.
obtained. A linear regression was calculated for the data giving the lines of best fit and these are shown in Fig. 3. The following values were also calculated:
K m (mmol) Vmax (AA
per minute x 10 3)
Conjugate
Free enzyme
1.00 596
0.82 428
As can be seen from Fig. 3, there is little difference in the K m (intercept on the x-axis) and only a small difference in the Vmax (intercept on the y-axis).
Conclusion
In the formation of radioiodinated compounds, the chloramine-T method of Hunter and Greenwood (1962) is widely used, and indeed, there are very few other techniques in use for this purpose. The position is not the same, however, in enzyme immunoassay where many linking techniques may be used, and not one technique has emerged. The purpose of this study was to investigate 3 protein-protein conjugation methods, success being judged by the establishment of a working enzyme immunoassay for HPL. Only the periodate oxidation technique worked sufficiently well to generate a good working label. In addition, the K m and Vmax were not significantly affected by the labelling procedure, resulting in a conjugate whose enzyme properties are very similar to the free enzyme. This is important as only small amounts of tracer need to be used in the assay as the free enzyme is highly active and rapidly forms product.
268 By c o m p a r i s o n , the g l u t a r a l d e h y d e conjugate was p o o r giving a much less active p r o d u c t as j u d g e d by the flat d o s e - r e s p o n s e curve, and significant differences b e t w e e n duplicates. The reason for the p o o r yield of label m a y be due to a n u m b e r of factors. Firstly, H P L has a much lower m o l e c u l a r weight (22,000) than calf intestinal alkaline p h o s p h a t a s e (approx. 100,000) and m a y be m o r e reactive than the enzyme, so that m o r e H P L - H P L conjugate is likely to be formed. Secondly, alkaline p h o s p h a t a s e is k n o w n to p o l y m e r i z e as e x t e n d e d chains with g l u t a r a l d e h y d e , as well as suffer loss of catalytic activity ( F o r d et al., 1978). This m a y also result in loss of i m m u n o l o g i c a l activity. Thus the p e r i o d a t e o x i d a t i o n technique holds c o n s i d e r a b l e p r o m i s e as a labelling m e t h o d . Larger batches m a y be p r e p a r e d if required, a n d no special a p p a r a t u s is required. T h e technique is a p p l i c a b l e to a wide variety of proteins, p r o v i d e d they have a free a m i n o group, or alternately, the sequence of the reaction could be reversed if the p r o t e i n c o n t a i n e d available c a r b o h y d r a t e residues. T h e technique has been used to form conjugates between horseradish p e r o x i d a s e a n d h u m a n p l a c e n t a l lactogen a n d h u m a n growth h o r m o n e which were used to establish working assays.
References Ford, D.J., R. Rodin and A.J. Pesce, 1978, Immunochemistry 15, 237. Goodfriend, T.L., L. Levine and G.D. Fasman, 1964, Science 144, 1344. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194, 495. Kawaoi, A. and P.K. Nakane, 1973, Fed. Proc. 32, 840. Kennedy, J.H., L.J. Kricka and P. Wilding, 1976, Clin. Chim. Acta 80, 1. Letchworth, A.T., R. Boardman, C. Bristow, J. Landon and T. Chard, 1971, J. Obstet. Gynaecol. Br. Commonw. 78, 535. Miyai, K., K. lshibashi and Y. Kumahara, 1976, Clin. Chim. Acta 67, 263. Modesto, R.R. and A.J. Pesce, 1971, Biochim. Biophys. Acta 229, 384. Scandinavian Society for Clinical Chemistry and Clinical Physiology; Committee on Enzymes, 1979, Scand. J. Clin. Lab. Invest. 33, 291. Williams, D.G., 1978, in: Enzyme Labelled Immunoassay of Hormones and Drugs, ed. S.B. Pal (Walter de Gruyter, Berlin) p. 129.
261
Elsevier JIM03153
Comparison of Three Conjugation Procedures for the Formation of Tracers for Use in Enzyme Immunoassays D.G. Williams Department of Clinical Biochemistry and Metabolic Medicine, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, U.K.
(Received 4 January 1984, accepted 11 April 1984)
One of the requirements for enzyme immunoassay is the formation of a labelled component of the assay system which can be either the ligand or the binder. Three methods for conjugating alkaline phosphatase to human placental lactogen were investigated, involving water-soluble carbodiimide, glutaraldehyde, and the periodate oxidation technique. Of the 3, the periodate oxidation technique proved superior giving a conjugate with only slight changes in the Michaelis constant (Kin) and maximum velocity (Vmax) which could be used for an enzyme immunoassay for human placental lactogen in human plasma. Key words: enzyme immunoassay - conjugation techniques - glutaraldehyde - periodate oxidation
Introduction A requirement for enzyme tmmunoassay (EIA) is the formation of an enzymelabelled component of the assay system. This can be either the ligand or the binder. A substantial number of techniques exist for covalently linking proteins to proteins and these have been listed in a comprehensive review by Kennedy et al. (1976). The majority of such agents are bifunctional in that they contain 2 groups capable of reacting with functional groups on a protein (e.g., - N H z, - O H , - S H ) . As both the enzyme and the target protein are likely to contain the same functional groups, the reaction will produce enzyme-enzyme and protein-protein conjugates as well as the desired enzyme-protein conjugate. Aggregates may also occur in which the enzymic and immunological activities of each component are lost due to steric hindrance. The outcome of the reaction is governed by a number of factors such as the relative rates of reaction of the agent with both proteins and the relative size of each protein, as well as the number of available functional groups on each protein (Modesto and Pesce, 1971). The purpose of the present work was to compare 3 such methods for the formation of enzyme-labelled proteins for use in EIAs. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
262 Three agents were chosen: (i) water-soluble carbodiimide, which is widely used in protein linking work; (ii) glutaraldehyde which is used for the formation of enzyme labels; (iii) periodate oxidation, which has the potential of giving a better conjugate as it involves 2 separate reactions. The enzyme chosen as the marker was calf intestinal alkaline phosphatase which is widely used for this purpose, is easily assayed, and yields a highly coloured product. The protein was human placental lactogen (HPL), used as an index of foeto-placental function, and currently measured by radioimmunoassay (Letchworth et al., 1971). The intent was to develop an enzyme immunoassay for HPL in which the radiolabelled (1251) HPL would be replaced by an enzyme-labelled tracer. The outcome of this work has been reported by Williams (1978).
Materials and Methods
The following reagents were used: (1-ethyl-3-3-dimethyl amino propyl) carbodiimide, hydrochloride, 20 mg/ml; glutaraldehyde, 1.5% solution; fluorodinitrobenzene, 1% in ethanol; sodium periodate 0.05 M; ethylene glycol 0.16 M; carbonate buffer, 0.01 M, pH 9.5; ethanolamine, 2 M adjusted to pH 9.5; phosphate-buffered saline (PBS), pH 7.2; alkaline phosphatase, Grade I, calf intestinal (Boehringer); diethanolamine buffer, 1.01 M, pH 9.8; p-nitrophenyl phosphate 110 mmol/l; sodium hydrogen carbonate 0.3 M; phosphate buffer, pH 6.8, 0.1 M; human placental lactogen MRC standard 70/144. Assay reagents
Assay diluent, barbitone buffer pH 8.6 with human serum albumin, 0.5 mg/ml; guinea pig anti-HPL antiserum, prepared by standard procedures; lzSI-labelled HPL, prepared by the chloramine-T method; normal guinea pig serum; donkey anti-guinea pig serum; pooled male plasma for the dilution of tests and standards. All reagents were Analar grade and made up in deionized water unless otherwise stated. Method I - - Water soluble carbodiimide (Goodfriend et al., 1964)
This reagent links free amino and carboxyl groups. Human placental lactogen (1 mg) and 100/~1 of alkaline phosphatase suspension were dissolved in 1.0 ml water. One milligram of (1-ethyl-3-3-dimethyl amino propyl) carbodiimide hydrochloride in 100/~1 of water were added in 10/~1 aliquots and the reaction allowed to proceed overnight at 4°C. The reaction was terminated by the addition of 100 ~tl of glacial acetic acid. Method 2 - - Glutaraldehyde (Miyai et al., 1976)
This reagent links free amino groups. One hundred microlitres of the enzyme suspension were centrifuged and the supernatant discarded. The precipitate was dissolved in 150/~1 of 0.1 M phosphate buffer pH 6.8, and 100/~1 of this solution were mixed with 100/~1 buffer containin8 100 ~tg HPL. To this were added, in 2/zl
263
amounts, 12 /~1 of a 1.5% aqueous solution of glutaraldehyde and the reaction allowed to proceed at room temperature for 3 h. The mixture was then dialysed against 200 ml phosphate-buffered saline overnight. Method 3 - - Periodate oxidation (Kawaoi and Nakone, 1973) This method oxidizes carbohydrates to aldehydes which then link with free amino groups. One hundred microlitres of the enzyme suspension were centrifuged and the pellet dissolved in 100/~1 of 0.3 M sodium hydrogen carbonate solution and 10/.tl of a 1% ethanolic solution of fluorodinitrobenzene (FDNB) added. This was left at room temperature for 2 h to block any free amino groups. Sodium periodate solution (0.05 M, 100 ~1) was added to oxidize carbohydrate groups to aldehyde and the reaction allowed to proceed for 6 h. The reaction was then terminated by the addition of excess ethylene glycol (0.16 M, 100 ~1) and left for 1 h before overnight dialysis against 500 ml carbonate buffer (pH 9.5, 0.01 M). The following morning 100/.tl of HPL in 100 ~1 of carbonate buffer were added to the dialysate and the mixture left for 24 h to allow the aldehyde groups to form links with free amino groups on the HPL. Unreacted aldehyde was removed by addition of excess ethanolamine (2 M, pH 9.5, 20 ~1) and the mixture dialysed overnight against 500 ml PBS.
Results
The results given here are representative of several preparations that were carried out during the course of the study. Carbodiimide Extensive loss of enzyme activity ( > 95%) was found with this procedure, which was not investigated further. Glutaraldehyde The dialysed reaction mixture was passed through a Sephadex G-100 column (1.5 cm diameter, 60 cm 3 volume) equilibrated with PBS. Fractions were collected (2.0 ml) and assayed for enzyme activity by taking 10 #1 of eluate and adding it to 1.0 ml of diethanolamine buffer pH 9.8 containing 0.505 mmol Mg2+/1. The reaction was started by addition of paranitrophenyl phosphate, 110 mmol/1. The absorbance at 405 nm was measured after 30 min, the reaction being allowed to proceed to completion at room temperature. Fractions that contained activity were assessed for their suitability as tracers by inclusion in the following assay. The assay system was derived from the radioimmunoassay of Letchworth et al. (1971) previously established as a reference assay. The only modifications to the published method were the use of enzyme-labelled H P L and a second antibody separation system as the alcohol used in the original would be likely to denature the enzyme. To 250 #1 of assay diluent (barbitone buffer, pH 8.6, 0.06 M with human serum albumin 0.5 m g / m l ) were added 50/.tl of serum, 10/xl of enzyme conjugate
264
and 100 #1 of antiserum (final dilution 1 : 5000). This was left at room temperature for 2 h and then 0.1 ml each of normal guinea pig serum (1 : 100 dilution) or donkey anti-guinea pig serum (1 : 10 dilution) were added. After a further 2 h incubation the tubes were spun and drained and the enzyme activity measured by resuspending the precipitate in 1.0 ml of diethanolamine buffer and adding paranitrophenyl phosphate. The absorbance at 405 nm was measured after 30 min at room temperature. For each label 4 sets of tubes were used, each tube being assayed in duplicate. The first tube contained a pooled pregnancy serum from the early second trimester with an H P L concentration of 1.5/~g/ml, the second, the early second trimester pool diluted 1:1, i.e., 0.75 ~tg/ml, the third contained no HPL, and the fourth no antiserum as a check on non-specific binding. The sera were diluted 1 : 11 in pooled male plasma. It had previously been shown that the radioimmunoassay could easily distinguish between these 3 levels so that if the enzyme label was to be of any use it should also be able to do this. Despite repeated attempts, it was not found possible to establish an assay capable of accurately differentiating between the 2 pools of serum. A dose-response curve when carried out with a full set of 8 standards (10 to 0.08 m g / m l in doubling dilutions) was very flat with absorbance differences of only 0.15-0.2 between the highest and lowest standards. Furthermore the reproducibility of duplicates was very poor, with sometimes as much as 0.1 A difference between the points. Non-specific binding was also very variable. Washing the precipitate and adding more tracer did not significantly increase the sensitivity or precision of the assay. Periodate oxidation The reaction mixture was purified by passing it through the Sephadex G-100 column. The elution pattern was similar to that obtained with the glutaraldehyde conjugates. Fractions containing enzyme activity were checked in the same way as
TABL E I DOSE-RESPONSE CURVES F O R F R A C T I O N S 11-15 ( P E R I O D A T E LABEL) Mean absorbances (duplicate assay) at 405 nm. HPL ~tg / m l
10 5 2.5 1.25 0.63 0.31 0.16 0.08 0 NSB a
Fraction nos. 11
12
13
14
15
0.970 1.00 1.08 1.19 1.31 1.48 1.75 2.01 2.12 0.86
1.10 1.27 1.46 1.67 2.20 2.43 2.75 2.82 2.80 0.88
1.44 1.54 2.06 2.32 2.60 2.78 2.81 2.85 2.88 1.180
1.76 1.71 1.87 2.04 2.64 2.75 2.82 2.88 2.88 1.28
1.25 1.25 1.21 1.42 1.50 2.17 2.77 2.33 2.50 0.90
a NSB = Non-specific binding.
265 A~.os 3-2
2'~ 2'4' 2'0' 1"6" 1"2"
0 "~,'
0;1
14)
10:0
HPL (~g/mt)
Fig. 1. Dose-response curve for 3 fractions (11-13) from periodate conjugation procedure, x fraction 11; A a, fraction 12; • • , fraction 13.
X,
the glutaraldehyde conjugates and 5 fractions were found to give encouraging results, with absorbance differences of about 1.4 A between the highest and lowest standards. The labels were then tested by using them to construct a dose-response curve for a series of HPL standards made up in pooled male plasma. 2.5/~1 of label were taken for each tube. Table I shows the results were obtained. All fractions gave satisfactory dose-response curves. The non-specific binding on fraction 14 was very high, and the curve for 15 was rather flat so these were not considered further. The dose-response curves for 11-13 are shown in Fig. 1. By using 1 #1 of label, (fraction
1.3
A~.os
1.'2 1.1 1.0
0.9 0.8 0.';
0 '1
1"0 HPL
(pg/ml)
Fig. 2. Dose-response curve for fraction 12 with 1 /xl of label.
10-0
266 TABLE II DOSE-RESPONSE CURVE FOR FRACTION 12 (1 ttl LABEL) H PL/Lg/ml
A 405 (mean of duplicates)
10 5 2.5 1.25 0.63 0.31 0.16 0.08 0 NSB Pool 1 Pool 2
0.74o o.735 0.743 0.762 0.815 0.872 1.030 1.12 1.545 0.638 1.50 0.80
12) a d o s e - r e s p o n s e curve with lower final a b s o r b a n c e s m o r e suitable for m e a s u r e m e n t was o b t a i n e d , as shown in Fig. 2 a n d T a b l e II. The pools were sera of k n o w n value (assayed b y r a d i o i m m u n o a s s a y ) diluted 1 : 1 1 in p o o l e d male p l a s m a before assay. As can be seen, the results are in g o o d agreement. As is d e s c r i b e d elsewhere (Williams, 1978) the m a n u a l assay was a d a p t e d to given a s e m i - a u t o m a t e d assay with g o o d c o r r e l a t i o n with the reference r a d i o i m m u n o a s s a y ( r = 0.96). F i n a l l y the effect of labelling on the Michaelis c o n s t a n t ( K m) a n d the m a x i m u m velocity (Vmax) of the p r e p a r a t i o n was investigated, as significant alterations w o u l d have r e d u c e d the e n z y m e ' s c a p a b i l i t y as a marker. It was not possible to do this for the g l u t a r a l d e h y d e label as the level of enzyme activity was not sufficiently high to o b t a i n suitable measurements. E n z y m e activity at different s u b s t r a t e c o n c e n t r a t i o n s was m e a s u r e d using the assay c o n d i t i o n s o u t l i n e d b y the S c a n d i n a v i a n Society for Clinical E n z y m o l o g y (1974) with, d i e t h a n o l a m i n e buffer a n d p - n i t r o p h e n y l p h o s p h a t e starter. All measurem e n t s were m a d e in d u p l i c a t e on a Pye U n i c a m SP8000 s p e c t r o p h o t o m e t e r with a t h e r m o s t a t t e d curvette h o l d e r set at 37°C. The results shown in T a b l e III were
TABLE III LINEWEAVER-BURK DATA FOR FREE ENZYME AND CONJUGATE Substrate concentration (mmol/1)
1 ~
1 ff (free enzyme)
1 T (label)
10 4 2 1 0.5
0.1 0.25 0.5 1 2
23.68 27.50 36.60 40.88 51.45
18.47 22.27 24.66 32.60 61.50
267 I/V 1~0
I2 0
/
~reeEnzyme
/ too 8O 6O 4
2 <
_
-i
-2
-1
0
1
2
3
4
5
6
1/S
Fig. 3, Double reciprocal (Lineweaver-Burk) plot for free enzyme and conjugate. Intercept on x-axis = K m. Intercept on y-axis = Vm.~.
obtained. A linear regression was calculated for the data giving the lines of best fit and these are shown in Fig. 3. The following values were also calculated:
K m (mmol) Vmax (AA
per minute x 10 3)
Conjugate
Free enzyme
1.00 596
0.82 428
As can be seen from Fig. 3, there is little difference in the K m (intercept on the x-axis) and only a small difference in the Vmax (intercept on the y-axis).
Conclusion
In the formation of radioiodinated compounds, the chloramine-T method of Hunter and Greenwood (1962) is widely used, and indeed, there are very few other techniques in use for this purpose. The position is not the same, however, in enzyme immunoassay where many linking techniques may be used, and not one technique has emerged. The purpose of this study was to investigate 3 protein-protein conjugation methods, success being judged by the establishment of a working enzyme immunoassay for HPL. Only the periodate oxidation technique worked sufficiently well to generate a good working label. In addition, the K m and Vmax were not significantly affected by the labelling procedure, resulting in a conjugate whose enzyme properties are very similar to the free enzyme. This is important as only small amounts of tracer need to be used in the assay as the free enzyme is highly active and rapidly forms product.
268 By c o m p a r i s o n , the g l u t a r a l d e h y d e conjugate was p o o r giving a much less active p r o d u c t as j u d g e d by the flat d o s e - r e s p o n s e curve, and significant differences b e t w e e n duplicates. The reason for the p o o r yield of label m a y be due to a n u m b e r of factors. Firstly, H P L has a much lower m o l e c u l a r weight (22,000) than calf intestinal alkaline p h o s p h a t a s e (approx. 100,000) and m a y be m o r e reactive than the enzyme, so that m o r e H P L - H P L conjugate is likely to be formed. Secondly, alkaline p h o s p h a t a s e is k n o w n to p o l y m e r i z e as e x t e n d e d chains with g l u t a r a l d e h y d e , as well as suffer loss of catalytic activity ( F o r d et al., 1978). This m a y also result in loss of i m m u n o l o g i c a l activity. Thus the p e r i o d a t e o x i d a t i o n technique holds c o n s i d e r a b l e p r o m i s e as a labelling m e t h o d . Larger batches m a y be p r e p a r e d if required, a n d no special a p p a r a t u s is required. T h e technique is a p p l i c a b l e to a wide variety of proteins, p r o v i d e d they have a free a m i n o group, or alternately, the sequence of the reaction could be reversed if the p r o t e i n c o n t a i n e d available c a r b o h y d r a t e residues. T h e technique has been used to form conjugates between horseradish p e r o x i d a s e a n d h u m a n p l a c e n t a l lactogen a n d h u m a n growth h o r m o n e which were used to establish working assays.
References Ford, D.J., R. Rodin and A.J. Pesce, 1978, Immunochemistry 15, 237. Goodfriend, T.L., L. Levine and G.D. Fasman, 1964, Science 144, 1344. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194, 495. Kawaoi, A. and P.K. Nakane, 1973, Fed. Proc. 32, 840. Kennedy, J.H., L.J. Kricka and P. Wilding, 1976, Clin. Chim. Acta 80, 1. Letchworth, A.T., R. Boardman, C. Bristow, J. Landon and T. Chard, 1971, J. Obstet. Gynaecol. Br. Commonw. 78, 535. Miyai, K., K. lshibashi and Y. Kumahara, 1976, Clin. Chim. Acta 67, 263. Modesto, R.R. and A.J. Pesce, 1971, Biochim. Biophys. Acta 229, 384. Scandinavian Society for Clinical Chemistry and Clinical Physiology; Committee on Enzymes, 1979, Scand. J. Clin. Lab. Invest. 33, 291. Williams, D.G., 1978, in: Enzyme Labelled Immunoassay of Hormones and Drugs, ed. S.B. Pal (Walter de Gruyter, Berlin) p. 129.