Journal o f Immunological Methods, 11 (1976) 15--23
15
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
DEVELOPMENT OF AN ENZYME IMMUNOASSAY FOR HUMAN PLACENTAL LACTOGEN USING LABELLED ANTIBODIES
H.M. BARBOUR * Technicon Methods and Standards Laboratory, 54 Bartholomew Close, London EC1A 7HL, England
(Received 24 July 1975, accepted 22 October 1975) Synopsis An immunoassay for the determination of human placental lactogen (HPL) has been developed using specific antibodies labelled with horseradish peroxidase. A method is presented in which the enzyme : antibody conjugate is purified with an antigenic solid phase to ensure the production of conjugates with intact immunological activity. Serum components interfere with the assay but this problem can only be avoided at the expense of a precise and practical assay for the determination of HPL. INTRODUCTION R a d i o i m m u n o a s s a y s using r a d i o a c t i v e l y labelled antigens h a v e b e e n e x t e n sively e m p l o y e d in clinical practice. I m m u n o r a d i o m e t r i c assays involving labelled a n t i b o d i e s m a y p r o v e a d v a n t a g e o u s in certain c i r c u m s t a n c e s (Miles a n d Hales, 1 9 6 8 a , b ; A d d i s o n et al., 1 9 7 1 ) as it is easier to label a n t i b o d y t h a n c e r t a i n antigens such as drugs and steroids lacking reactive g r o u p s a n d it m a k e s possible the use o f labelled s e c o n d a n t i b o d y as a universal label. R e c e n t l y , e n z y m e labelled a n t i b o d i e s have b e e n applied t o s e r u m i m m u n o a s s a y s ( V a n W e e m e n and S c h u u r s , 1 9 7 4 ; Maiolini et al., 1 9 7 5 ) , as an ext e n s i o n o f t h e i r use in h i s t o c h e m i s t r y ( A v r a m e a s , 1 9 6 9 , 1 9 7 2 ) . This o f f e r s several a d v a n t a g e s as c o m p a r e d w i t h r a d i o i s o t o p e s including a greatly prol o n g e d shelf-life, little or n o h e a l t h h a z a r d a n d e n a b l i n g such assays t o be p e r f o r m e d b y l a b o r a t o r i e s w i t h o u t e x p e n s i v e i s o t o p e c o u n t i n g e q u i p m e n t . In s o m e assays e m p l o y i n g e n z y m e labelled antigens ( R u b e n s t e i n et al., 1 9 7 2 ) a n t i b o d y b i n d i n g r e d u c e s e n z y m e activity b y steric h i n d r a n c e t h e r e b y rem o v i n g t h e n e e d f o r a s e p a r a t i o n step. I t s e e m s i m p r o b a b l e t h a t a similar eff e c t will be o b t a i n e d w h e n t h e e n z y m e is used to label a m o l e c u l e as large as an a n t i b o d y . P e r o x i d a s e was c h o s e n as t h e e n z y m e used to label t h e a n t i b o d i e s since it is available c o m m e r c i a l l y in a highly p u r i f i e d and stable f o r m , retains activity * Present address: Department of Diagnostic Chemical Pathology St. Mary's Hospital, Pread St, London W2, England.
16 after covalent coupling by a variety of coupling methods and there are many sensitive assay methods to measure its activity, allowing choice when selecting a m e t h o d to optimise for automated analysis. MATERIALS AND METHODS
Preparation of immunoadsorbent This was prepared by adding 40 mg human placental lactogen (HPL) from Nutritional Biochemicals, U.S.A., to 250 mg of the diazotised m-nitrobenzylo x y m e t h y l pyridinium chloride derivative of cellulose, as described by Gurvich (1961) with modifications by Addison (1971). The immunoadsorbent was stored at 4°C in 0.05 M barbitone b u f f e r , pH 8.2, containing per ml, 5 mg NaC1, 5 mg bovine serum albumin (BSA) and (}.2 mg sodium azide.
Preparation of peroxidase labelled IgG Preparation of the antiserum IgG fraction A crude IgG fraction was prepared by mixing 1.8 g anhydrous Na2SO4 with 10 ml goat anti-HPL serum for 30 min at room temperature followed by centrifugation at 1875 g for 10 min. The precipitate was dissolved in 10 ml 0.15 M NaC1 and stored at --20°C.
Conjugation of peroxidase to antiserum IgG with glutaraldehyde The m e t h o d of Avrameas was followed using 10 mg peroxidase and 0.5 ml of the IgG fraction.
Conjugation of peroxidase with antiserum IgG using periodate oxidation The m e t h o d of Kawaoi and Nakane (1973) was followed. The remaining aldehyde groups after incubating the peroxidase-aldehyde with the IgG preparation were deactivated by incubation with 0.2 ml of 2 M ethanolamine adjusted to pH 9.5 for 7 h. The preparation was then dialysed against phosphate buffered saline pH 7.2. A peroxidase preparation, R.Z.1.0 from Worthington, Freehold, New Jersey was used for both methods.
Purification of peroxidase labelled anti-HPL IgG : peroxidase conjugates were incubated with HPL cellulose (1 mg immunoadsorbent added per 0.1 ml of the original antiserum) at 4°C for 24 h and the immunoadsorbent was then washed 8 times in phosphate buffered saline, 0.05 M pH 7.2. A m m o n i u m thiocyanate was used to elute labelled antibody from immunoadsorbent as described by Cambiaso et al. (1974) and the eluates were
17
stored at 4°C with 6 mg Nal21, 0.5 mg sodium azide and 5 mg lactalbumin being added per ml.
Immunoassay using peroxidase labelled anti-HPL Fifty pl of a 1 : 20 dilution of the 2 M a m m o n i u m thiocyanate fraction was incubated with 10 pl serum sample or HPL standard, prepared in male serum (10--100 ng) in 350 pl phosphate buffered saline pH 7.2, 0.1% Tween 20, at room temperature. One hundred pl of phosphate buffered saline containing 300 pg HPL cellulose was added and the tubes rotated at room temperature for one hour. After centrifugation either 300 pl of the supernatant was added to 2 ml of the enzyme reagent or 2 ml enzyme reagent was added to the immunoadsorbent which had been washed twice.
Assay for peroxidase Manual assay Peroxidase activity was determined by a modification of the glucose enzymic m e t h o d described by Trinder (1969), which was fully optimised for the determination of peroxidase activity. 10 to 200 ng peroxidase was added to 2 ml of 0.05 M phosphate buffer pH 7.3 preheated to 50°C, containing 1 X 10 -3 M H202, 1 X 10 -2 M phenol and 1.4 X 10 -3 M 4-aminophenazone. The rate of increase of optical density was measured at 515 nm.
Automated assay The manual determination was adapted to an automated assay (fig. 1). G
:
gLQSS t u b i n g
PE= polyethylenetubing
~[/30/hr'~mVmin
PE
I.I
,v sample
0.16
o I, phenol
1.00
I /, -am nophenazone ,o, , ,
i
~w(3sto,
oir
0.32
H2o
0.05
I
LJ P
1.00
50ram F/C, 52Onto Fig. 1. A u t o m a t e d m e t h o d f o r t h e d e t e r m i n a t i o n o f p e r o x i d a s e .
18 Since phenol is present in the enzyme reagent the a m o u n t of tygon tubing was kept to a minimum and was replaced with polyethylene tubing be[ore and after the proportionating pump. Glass transmission tubing was used into and out of the heating block to minimise adsorption of the coloured reaction product. RESULTS
Properties of the antigenic solid phase The uptake of HPL on diazotised cellulose was estimated as 14.2 mg HPL/ 250 mg cellulose by analysis of HPL in the washes and on the solid phase. The antigenic activity of the attached HPL was assessed by incubating HPL cellulose with antiserum overnight at 4 ° C. After eight washes the uptake of antibody was found to be 250 mg/g antigenic solid phase, estimated by the Folin--Ciocalteau reaction. On a mole ratio basis, HPL : antiHPL was 2 : 1. The non-specific adsorption characteristics of the immunoadsorbent were investigated using a human serum albumin (HSA) cellulose derivative (28 mg protein/250 mg cellulose), which was incubated with HPL, anti-HPL, nonimmune serum and the enzyme lysosome. HPL and anti-HPL were determined by radioimmunoassay before and after incubation and no uptake was demonstrated. The Folin--Ciocalteau reaction was performed before and after incubation with non-immune serum and no non-specific adsorption occurred. About a 26% uptake of the lysosome was demonstrated probably due to charge differences but this was reduced to 9% by the addition of 0.1% (w/v) Tween 20.
Enzyme labelling of antibody insolubolised on antigenic solid phase The conditions of pH required by many methods which have been described for conjugating enzymes to antibodies were found to be unsuitable for maintaining the antibody on immunoadsorbent, and bifunctional reagents such as glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride caused extensive cross-linking of the immunoadsorbent.
Enzyme labelling of antibody in free solution 1) The m e t h o d of Avrameas and Ternynck (1971) was chosen whereby excess glutaraldehyde is believed to activate a free lysine in peroxidase and unreacted glutaraldehyde is easily removed. The low recovery of enzyme activity of 4% was in agreement with other reports (Kawaoi and Nakane, 1973; Van Weemen, 1974). 2) The m e t h o d of Kawaoi and Nakane (1973) employs the oxidation of the peroxidase carbohydrate with periodate to generate an aldehyde derivative which may be coupled with another protein, resulting in a retention of enzyme activity of 95%. After coupling the peroxidase was shown to remain
19 B 6C
0.4
E
~o.3 OJ
E I
g ~ o.1 c
1
txl
i
1.0
1.5
2.0
Ammonium
2.5
3.0
N
1/5
ThiocyonQte eluotes
L
i
i
l
112501F'o001/lC]031/2Q30 Dilutions of ontibody preporotions
Fig. 2. A) Enzyme activity present in 10 pl samples of the peroxidase labelled antibody eluates fractionated from HPL cellulose by increasing" molarities of Ammoniuml2sthiocyanate. B) Comparison of the ability of the labelled antibody fractions to bind I-HPL. A, 1.0 M; ~, 1.5 M; e, 2.0 M; o, 2.5 M; o, 3.0 M, A m m o n i u m thiocyanate.
70 "5
60
c eJ o_
~ 5o _c 2
z,c
:z c o
2
3
x:l
g
o
o 2; 5;
~oo
i;o
2o0
2;oog~L
Fig. 3. An HPL standard curve using enzyme activity associated with the sample antigen in the supernatant.
20
activated since there was a covalent uptake of peroxidase conjugates on immunoadsorbent with extensive cross-linking. In order to deactivate the conjugate without introducing extra charged groups (lysine or glycine), sodium borohydride and ethanolamine deactivation were tried. The former gave low enzyme recoveries but ethanolamine treatment resulted in a 70% recovery of enzyme activity.
Purification o f labelled anti-HPL The method of Cambioso et al. (1974) was followed. The results appear in fig. 2. Since elution with 2.5 and 3 M a m m o n i u m thiocyanate gave a low recovery of conjugates, the fraction at 2 M was used and studied. Analysis of the enzyme to antibody ratio resulted in an average value of 0.8 to 1.0.
Assays Peroxidase assay The automated assay for th'e determination of peroxidase exhibited negligible drift, excellent precision and no carry-over between samples of varying activity at 30 analyses per hour. The automated m e t h o d had been adapted to obtain m a x i m u m sensitivity to result in a signal giving full scale deflection on
9eL ~0 o
g
70
J
cL 60 I
o
_~ 4o
o
~g ~ 2o o
2 oJ ck
o 0
10
20
&O
60
80
lOOng HPL
Fig. 4. A n HPL s t a n d a r d curve, e m p l o y i n g t h e m e a s u r e m e n t o f free a n t i b o d y b o u n d t o t h e solid phase antigen.
21 the recorder with a 7.5 ng peroxidase sample at a standard calibration setting of 1.0.
I m m unoassays 1) Analysis of the immunoassay supernatant. Incubation of varying duration for the reaction of labelled antibody with antigen, and the subsequent i m m u n o a d s o r b e n t reaction were studied. The results indicated t h a t two, one hour incubations gave sufficient sensitivity with a range of 48% between the blank of 13% and top standard of 61%. The curve appears in fig. 3. The coefficient of variation of 4 pg HPL per ml using a 10 pl sample was 5.2%. 2) Results with serum. It was discovered that sera affected the immunoassay supernatant analyses in two ways: a) Serum samples exhibited significant and variable peroxidase blanks. To establish that this was peroxidase activity the enzyme reagent was replaced with buffer but no response appeared on the recorder. There was a correlation between the degree of haemolysis and the resulting blank. Many of the sera exhibited peroxidase blanks far in excess of the peroxidase activity associated with the labelled antibody. Concluding t h a t the serum peroxidase activity was in part due to presence of haptoglobin--haemoglobin complexes (Burrows and Hosten, 1969), compounds were tested for their ability to break down the association to reduce peroxidase activity (Nyman, 1959) and also serum incubations were studied with anti-haptoglobin on solid phase. Both approaches were unsuccessful. b) There is also interference by serum components which compete with the phenol-aminophenazone chromogen system for peroxide free radicals. In assays determining blood glucose these interferences are usually removed but this is impractical in the case of an immunoassay. Physiological levels of ascorbate and glutathione reduced the peak height of a peroxidase sample, whereas uric acid and creatinine increased the signal. Analysis o f the enzyme activity bound to solid phase Since the analysis of the supernatant was presenting varied and numerous problems, analysis of the labelled antibody b o u n d to HPL cellulose was undertaken. In fig. 4 the precision is seen to be poor, with a coefficient of variation at 4 pg HPL per ml of 12%. It was also found t h a t the analytical time was greatly increased due to the introduction of more stages to prepare the solid phase for enzyme determination. DISCUSSION The diazotised cellulose derivative exhibits m a n y of the criteria required from an immunoadsorbent, including a low degree of non-specific adsorption and the ability to extract antibodies as a result of the high retention of antigenic activity found upon the a t t a c h m e n t of HPL. Immunoradiometric assays described in the literature regard the labelling
22 o f antibody on immunoadsorbent necessary for the protection of one antigen combining site (Miles and Hales, 1968a,b). In this present study attempts were made to reproduce these experiments, replacing 12sI with an enzyme but this approach has two disadvantages. Firstly, the use of bifunctional agents in methods analogous to Weston and Avrameas (1971) and use of carbodiimides results in extensive cross-linking of the antibody on immunoadsorbent before the enzyme may be attached. Secondly, the enzyme may be activated and suitably linked to the insolubilised antibody but this involves conflicting pH requirements. Reactions such as glutaraldehyde activated peroxidase (Avrameas and Ternynck, 1971), the enzyme coupled to one isocyanate group of toluene diisocyanate (Schick and Singer, 1961), and periodate oxidised peroxidase (Kawaoi and Nakane, 1973) need a pH of 9.3 for reaction, which would not be suitable for maintaining the antibody on immunoadsorbent for any prolonged reaction (Hughes-Jones et al., 1964). The alternative approach of labelling the antibody in solution was employed. Any destruction of the antigen combining site is accounted for by purification of the conjugates on antigenic solid phase, so that only peroxidase conjugates with intact anti-HPL activity will be produced. Elution of the labelled antibodies from immunoadsorbent was achieved using increasing concentrations of a m m o n i u m thiocyanate, as opposed to 0.001 N and 0.01 N HC1 which irreversibly deactivates peroxidase. The poor precision associated with the analysis of labelled antibody on solid phase may be attributed to an increase in the analytical time, some loss of the solid phase in the washes and the need for agitation of the immunoadsorbent in the enzyme reagent. An alternative m e t h o d would be to cleave the labelled antibody attached to the solid phase and assay the activity liberated, but a m m o n i u m thiocyanate did cause a significant blank reaction in the enzyme assay. Evidence has been presented here to suggest that peroxidase as a label is not suitable when the supernatant of a serum immunoassay is analysed. This by no means questions the utility of this enzyme for histochemical purposes where it is widely used. No doubt, in the latter example, it may be considered as a 'solid phase' analysis whereby interfering components in the cell preparation may be discarded by washing or minimised before the specimen slides are analysed. A further application of a peroxidase labelled antibody immunoassay may be found in the field of steroid assays where a separation technique is usually required. Although it is unlikely that peroxidase-like activity would affect urine h o r m o n e analyses, interferences by compounds which compete with the chromogen for peroxide free radicals may be greater in this case. The sensitivity of the immunoassay described, with a 10 min enzyme reaction, is satisfactory for the determination of HPL and it is estimated that an assay for ~ fetoprotein (5 nmol/1) could be achieved. This is based on the conditions used, whereas greater sensitivity is possible by increasing the enzyme incubation.
23 T h e relative ease o f p r e p a r a t i o n o f a p e r o x i d a s e labelled a n t i b o d y , its stability and l o w c o s t m a k e p e r o x i d a s e t h e p r e f e r r e d e n z y m e f o r i m m u n o a s s a y s a l t h o u g h t h e s e r u m b l a n k is a serious d r a w b a c k . T h e c i r c u m s t a n c e s leading to t h e last c o m m e n t m a y p r o m p t w o r k e r s in t h e e n z y m e i m m u n o a s s a y field t o add o n e m o r e criterion t o t h e list describing a suitable e n z y m e f o r labelling p u r p o s e s , and this is t h a t t h e e n z y m e s u b s t r a t e r e a c t i o n s h o u l d be u n i q u e f o r t h e e n z y m e , u n d e r t h e assay condit i o n s to b e studied. ACKNOWLEDGEMENTS I a m g r a t e f u l t o P r o f e s s o r J. L a n d o n and Mr. J. C r o o k a l l - G r e e n i n g f o r t h e i r v a l u a b l e assistance in p r e p a r i n g t h e script a n d also Mr. R. J a y a n d Miss M. Shields f o r t h e i r h e l p in t h e a u t o m a t i o n o f t h e p e r o x i d a s e assay.
REFERENCES Addison, G.M., 1971, Ph.D. thesis, Cambridge. Addison, G.M., C.N. Hales, J.S. Woodhead and J.L.H. O'Riordan, 1971, J. Endocrinol. 49, 521. Avrameas, S., 1969, Immunochem. 6, 43. Avrameas, S., 1972, Histochem. J. 4, 321. Avrameas, S. and T. Ternynck, 1971, Immunochem. 8, 1175. Burrows, S. and E.H. Hosten, 1969, Technicon Symposium. 'Automation in Analytical Chemistry' (New York, N.Y.) p. 401. Cambiaso, C.L., P.L. Masson, J.P. Vaerman and J.F. Heremans, 1974, J. Immunol. Methods 5,153. Gurvich, A.E., O.B. Kuzovleva and A.E. Tumanova, 1961, Biokhimiya 26,803. Hughes-Jones, N.C., B. Gardner and R. Telford, 1964, Immunology 7, 72. Kawaoi, A. and P.K. Nakane, 1973, Fed. Proc. 32,840. Maiolini, R., B. Ferrua and R. Masseyeff, 1975, J. Immunol. Methods 6, 355. Miles, L.E.M. and C.N. Hales, 1968a, Biochem. J. 108,611. Miles, L.E.M. and C.N. Hales, 1968b, Nature 219,186. Nyman, M., 1959, Scand. J. Clin. Lab. Invest. 11, Suppl. 39. Rubenstein, K.E., R.S. Schneider and E.F. Ullman, 1972, Biochem. Biophys. Res. Commun. 47, 4,846. Schick, A.F. and S.J. Singer, 1961, J. Biol. Chem. 236, 9, 2477. Trinder, P., 1969, Anal. Clin. Biochem. 6, 24. Van Weemen, B.K., 1974, Ph.D. thesis. Van Weemen, B.K. and A.H.W.M. Schuurs, 1974, Febs. Letts. 43, 2,215. Weston, P.D. and S. Avrameas, 1971, Biochem. Biophys. Res. Commun. 45, 6, 1574.
15
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
DEVELOPMENT OF AN ENZYME IMMUNOASSAY FOR HUMAN PLACENTAL LACTOGEN USING LABELLED ANTIBODIES
H.M. BARBOUR * Technicon Methods and Standards Laboratory, 54 Bartholomew Close, London EC1A 7HL, England
(Received 24 July 1975, accepted 22 October 1975) Synopsis An immunoassay for the determination of human placental lactogen (HPL) has been developed using specific antibodies labelled with horseradish peroxidase. A method is presented in which the enzyme : antibody conjugate is purified with an antigenic solid phase to ensure the production of conjugates with intact immunological activity. Serum components interfere with the assay but this problem can only be avoided at the expense of a precise and practical assay for the determination of HPL. INTRODUCTION R a d i o i m m u n o a s s a y s using r a d i o a c t i v e l y labelled antigens h a v e b e e n e x t e n sively e m p l o y e d in clinical practice. I m m u n o r a d i o m e t r i c assays involving labelled a n t i b o d i e s m a y p r o v e a d v a n t a g e o u s in certain c i r c u m s t a n c e s (Miles a n d Hales, 1 9 6 8 a , b ; A d d i s o n et al., 1 9 7 1 ) as it is easier to label a n t i b o d y t h a n c e r t a i n antigens such as drugs and steroids lacking reactive g r o u p s a n d it m a k e s possible the use o f labelled s e c o n d a n t i b o d y as a universal label. R e c e n t l y , e n z y m e labelled a n t i b o d i e s have b e e n applied t o s e r u m i m m u n o a s s a y s ( V a n W e e m e n and S c h u u r s , 1 9 7 4 ; Maiolini et al., 1 9 7 5 ) , as an ext e n s i o n o f t h e i r use in h i s t o c h e m i s t r y ( A v r a m e a s , 1 9 6 9 , 1 9 7 2 ) . This o f f e r s several a d v a n t a g e s as c o m p a r e d w i t h r a d i o i s o t o p e s including a greatly prol o n g e d shelf-life, little or n o h e a l t h h a z a r d a n d e n a b l i n g such assays t o be p e r f o r m e d b y l a b o r a t o r i e s w i t h o u t e x p e n s i v e i s o t o p e c o u n t i n g e q u i p m e n t . In s o m e assays e m p l o y i n g e n z y m e labelled antigens ( R u b e n s t e i n et al., 1 9 7 2 ) a n t i b o d y b i n d i n g r e d u c e s e n z y m e activity b y steric h i n d r a n c e t h e r e b y rem o v i n g t h e n e e d f o r a s e p a r a t i o n step. I t s e e m s i m p r o b a b l e t h a t a similar eff e c t will be o b t a i n e d w h e n t h e e n z y m e is used to label a m o l e c u l e as large as an a n t i b o d y . P e r o x i d a s e was c h o s e n as t h e e n z y m e used to label t h e a n t i b o d i e s since it is available c o m m e r c i a l l y in a highly p u r i f i e d and stable f o r m , retains activity * Present address: Department of Diagnostic Chemical Pathology St. Mary's Hospital, Pread St, London W2, England.
16 after covalent coupling by a variety of coupling methods and there are many sensitive assay methods to measure its activity, allowing choice when selecting a m e t h o d to optimise for automated analysis. MATERIALS AND METHODS
Preparation of immunoadsorbent This was prepared by adding 40 mg human placental lactogen (HPL) from Nutritional Biochemicals, U.S.A., to 250 mg of the diazotised m-nitrobenzylo x y m e t h y l pyridinium chloride derivative of cellulose, as described by Gurvich (1961) with modifications by Addison (1971). The immunoadsorbent was stored at 4°C in 0.05 M barbitone b u f f e r , pH 8.2, containing per ml, 5 mg NaC1, 5 mg bovine serum albumin (BSA) and (}.2 mg sodium azide.
Preparation of peroxidase labelled IgG Preparation of the antiserum IgG fraction A crude IgG fraction was prepared by mixing 1.8 g anhydrous Na2SO4 with 10 ml goat anti-HPL serum for 30 min at room temperature followed by centrifugation at 1875 g for 10 min. The precipitate was dissolved in 10 ml 0.15 M NaC1 and stored at --20°C.
Conjugation of peroxidase to antiserum IgG with glutaraldehyde The m e t h o d of Avrameas was followed using 10 mg peroxidase and 0.5 ml of the IgG fraction.
Conjugation of peroxidase with antiserum IgG using periodate oxidation The m e t h o d of Kawaoi and Nakane (1973) was followed. The remaining aldehyde groups after incubating the peroxidase-aldehyde with the IgG preparation were deactivated by incubation with 0.2 ml of 2 M ethanolamine adjusted to pH 9.5 for 7 h. The preparation was then dialysed against phosphate buffered saline pH 7.2. A peroxidase preparation, R.Z.1.0 from Worthington, Freehold, New Jersey was used for both methods.
Purification of peroxidase labelled anti-HPL IgG : peroxidase conjugates were incubated with HPL cellulose (1 mg immunoadsorbent added per 0.1 ml of the original antiserum) at 4°C for 24 h and the immunoadsorbent was then washed 8 times in phosphate buffered saline, 0.05 M pH 7.2. A m m o n i u m thiocyanate was used to elute labelled antibody from immunoadsorbent as described by Cambiaso et al. (1974) and the eluates were
17
stored at 4°C with 6 mg Nal21, 0.5 mg sodium azide and 5 mg lactalbumin being added per ml.
Immunoassay using peroxidase labelled anti-HPL Fifty pl of a 1 : 20 dilution of the 2 M a m m o n i u m thiocyanate fraction was incubated with 10 pl serum sample or HPL standard, prepared in male serum (10--100 ng) in 350 pl phosphate buffered saline pH 7.2, 0.1% Tween 20, at room temperature. One hundred pl of phosphate buffered saline containing 300 pg HPL cellulose was added and the tubes rotated at room temperature for one hour. After centrifugation either 300 pl of the supernatant was added to 2 ml of the enzyme reagent or 2 ml enzyme reagent was added to the immunoadsorbent which had been washed twice.
Assay for peroxidase Manual assay Peroxidase activity was determined by a modification of the glucose enzymic m e t h o d described by Trinder (1969), which was fully optimised for the determination of peroxidase activity. 10 to 200 ng peroxidase was added to 2 ml of 0.05 M phosphate buffer pH 7.3 preheated to 50°C, containing 1 X 10 -3 M H202, 1 X 10 -2 M phenol and 1.4 X 10 -3 M 4-aminophenazone. The rate of increase of optical density was measured at 515 nm.
Automated assay The manual determination was adapted to an automated assay (fig. 1). G
:
gLQSS t u b i n g
PE= polyethylenetubing
~[/30/hr'~mVmin
PE
I.I
,v sample
0.16
o I, phenol
1.00
I /, -am nophenazone ,o, , ,
i
~w(3sto,
oir
0.32
H2o
0.05
I
LJ P
1.00
50ram F/C, 52Onto Fig. 1. A u t o m a t e d m e t h o d f o r t h e d e t e r m i n a t i o n o f p e r o x i d a s e .
18 Since phenol is present in the enzyme reagent the a m o u n t of tygon tubing was kept to a minimum and was replaced with polyethylene tubing be[ore and after the proportionating pump. Glass transmission tubing was used into and out of the heating block to minimise adsorption of the coloured reaction product. RESULTS
Properties of the antigenic solid phase The uptake of HPL on diazotised cellulose was estimated as 14.2 mg HPL/ 250 mg cellulose by analysis of HPL in the washes and on the solid phase. The antigenic activity of the attached HPL was assessed by incubating HPL cellulose with antiserum overnight at 4 ° C. After eight washes the uptake of antibody was found to be 250 mg/g antigenic solid phase, estimated by the Folin--Ciocalteau reaction. On a mole ratio basis, HPL : antiHPL was 2 : 1. The non-specific adsorption characteristics of the immunoadsorbent were investigated using a human serum albumin (HSA) cellulose derivative (28 mg protein/250 mg cellulose), which was incubated with HPL, anti-HPL, nonimmune serum and the enzyme lysosome. HPL and anti-HPL were determined by radioimmunoassay before and after incubation and no uptake was demonstrated. The Folin--Ciocalteau reaction was performed before and after incubation with non-immune serum and no non-specific adsorption occurred. About a 26% uptake of the lysosome was demonstrated probably due to charge differences but this was reduced to 9% by the addition of 0.1% (w/v) Tween 20.
Enzyme labelling of antibody insolubolised on antigenic solid phase The conditions of pH required by many methods which have been described for conjugating enzymes to antibodies were found to be unsuitable for maintaining the antibody on immunoadsorbent, and bifunctional reagents such as glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride caused extensive cross-linking of the immunoadsorbent.
Enzyme labelling of antibody in free solution 1) The m e t h o d of Avrameas and Ternynck (1971) was chosen whereby excess glutaraldehyde is believed to activate a free lysine in peroxidase and unreacted glutaraldehyde is easily removed. The low recovery of enzyme activity of 4% was in agreement with other reports (Kawaoi and Nakane, 1973; Van Weemen, 1974). 2) The m e t h o d of Kawaoi and Nakane (1973) employs the oxidation of the peroxidase carbohydrate with periodate to generate an aldehyde derivative which may be coupled with another protein, resulting in a retention of enzyme activity of 95%. After coupling the peroxidase was shown to remain
19 B 6C
0.4
E
~o.3 OJ
E I
g ~ o.1 c
1
txl
i
1.0
1.5
2.0
Ammonium
2.5
3.0
N
1/5
ThiocyonQte eluotes
L
i
i
l
112501F'o001/lC]031/2Q30 Dilutions of ontibody preporotions
Fig. 2. A) Enzyme activity present in 10 pl samples of the peroxidase labelled antibody eluates fractionated from HPL cellulose by increasing" molarities of Ammoniuml2sthiocyanate. B) Comparison of the ability of the labelled antibody fractions to bind I-HPL. A, 1.0 M; ~, 1.5 M; e, 2.0 M; o, 2.5 M; o, 3.0 M, A m m o n i u m thiocyanate.
70 "5
60
c eJ o_
~ 5o _c 2
z,c
:z c o
2
3
x:l
g
o
o 2; 5;
~oo
i;o
2o0
2;oog~L
Fig. 3. An HPL standard curve using enzyme activity associated with the sample antigen in the supernatant.
20
activated since there was a covalent uptake of peroxidase conjugates on immunoadsorbent with extensive cross-linking. In order to deactivate the conjugate without introducing extra charged groups (lysine or glycine), sodium borohydride and ethanolamine deactivation were tried. The former gave low enzyme recoveries but ethanolamine treatment resulted in a 70% recovery of enzyme activity.
Purification o f labelled anti-HPL The method of Cambioso et al. (1974) was followed. The results appear in fig. 2. Since elution with 2.5 and 3 M a m m o n i u m thiocyanate gave a low recovery of conjugates, the fraction at 2 M was used and studied. Analysis of the enzyme to antibody ratio resulted in an average value of 0.8 to 1.0.
Assays Peroxidase assay The automated assay for th'e determination of peroxidase exhibited negligible drift, excellent precision and no carry-over between samples of varying activity at 30 analyses per hour. The automated m e t h o d had been adapted to obtain m a x i m u m sensitivity to result in a signal giving full scale deflection on
9eL ~0 o
g
70
J
cL 60 I
o
_~ 4o
o
~g ~ 2o o
2 oJ ck
o 0
10
20
&O
60
80
lOOng HPL
Fig. 4. A n HPL s t a n d a r d curve, e m p l o y i n g t h e m e a s u r e m e n t o f free a n t i b o d y b o u n d t o t h e solid phase antigen.
21 the recorder with a 7.5 ng peroxidase sample at a standard calibration setting of 1.0.
I m m unoassays 1) Analysis of the immunoassay supernatant. Incubation of varying duration for the reaction of labelled antibody with antigen, and the subsequent i m m u n o a d s o r b e n t reaction were studied. The results indicated t h a t two, one hour incubations gave sufficient sensitivity with a range of 48% between the blank of 13% and top standard of 61%. The curve appears in fig. 3. The coefficient of variation of 4 pg HPL per ml using a 10 pl sample was 5.2%. 2) Results with serum. It was discovered that sera affected the immunoassay supernatant analyses in two ways: a) Serum samples exhibited significant and variable peroxidase blanks. To establish that this was peroxidase activity the enzyme reagent was replaced with buffer but no response appeared on the recorder. There was a correlation between the degree of haemolysis and the resulting blank. Many of the sera exhibited peroxidase blanks far in excess of the peroxidase activity associated with the labelled antibody. Concluding t h a t the serum peroxidase activity was in part due to presence of haptoglobin--haemoglobin complexes (Burrows and Hosten, 1969), compounds were tested for their ability to break down the association to reduce peroxidase activity (Nyman, 1959) and also serum incubations were studied with anti-haptoglobin on solid phase. Both approaches were unsuccessful. b) There is also interference by serum components which compete with the phenol-aminophenazone chromogen system for peroxide free radicals. In assays determining blood glucose these interferences are usually removed but this is impractical in the case of an immunoassay. Physiological levels of ascorbate and glutathione reduced the peak height of a peroxidase sample, whereas uric acid and creatinine increased the signal. Analysis o f the enzyme activity bound to solid phase Since the analysis of the supernatant was presenting varied and numerous problems, analysis of the labelled antibody b o u n d to HPL cellulose was undertaken. In fig. 4 the precision is seen to be poor, with a coefficient of variation at 4 pg HPL per ml of 12%. It was also found t h a t the analytical time was greatly increased due to the introduction of more stages to prepare the solid phase for enzyme determination. DISCUSSION The diazotised cellulose derivative exhibits m a n y of the criteria required from an immunoadsorbent, including a low degree of non-specific adsorption and the ability to extract antibodies as a result of the high retention of antigenic activity found upon the a t t a c h m e n t of HPL. Immunoradiometric assays described in the literature regard the labelling
22 o f antibody on immunoadsorbent necessary for the protection of one antigen combining site (Miles and Hales, 1968a,b). In this present study attempts were made to reproduce these experiments, replacing 12sI with an enzyme but this approach has two disadvantages. Firstly, the use of bifunctional agents in methods analogous to Weston and Avrameas (1971) and use of carbodiimides results in extensive cross-linking of the antibody on immunoadsorbent before the enzyme may be attached. Secondly, the enzyme may be activated and suitably linked to the insolubilised antibody but this involves conflicting pH requirements. Reactions such as glutaraldehyde activated peroxidase (Avrameas and Ternynck, 1971), the enzyme coupled to one isocyanate group of toluene diisocyanate (Schick and Singer, 1961), and periodate oxidised peroxidase (Kawaoi and Nakane, 1973) need a pH of 9.3 for reaction, which would not be suitable for maintaining the antibody on immunoadsorbent for any prolonged reaction (Hughes-Jones et al., 1964). The alternative approach of labelling the antibody in solution was employed. Any destruction of the antigen combining site is accounted for by purification of the conjugates on antigenic solid phase, so that only peroxidase conjugates with intact anti-HPL activity will be produced. Elution of the labelled antibodies from immunoadsorbent was achieved using increasing concentrations of a m m o n i u m thiocyanate, as opposed to 0.001 N and 0.01 N HC1 which irreversibly deactivates peroxidase. The poor precision associated with the analysis of labelled antibody on solid phase may be attributed to an increase in the analytical time, some loss of the solid phase in the washes and the need for agitation of the immunoadsorbent in the enzyme reagent. An alternative m e t h o d would be to cleave the labelled antibody attached to the solid phase and assay the activity liberated, but a m m o n i u m thiocyanate did cause a significant blank reaction in the enzyme assay. Evidence has been presented here to suggest that peroxidase as a label is not suitable when the supernatant of a serum immunoassay is analysed. This by no means questions the utility of this enzyme for histochemical purposes where it is widely used. No doubt, in the latter example, it may be considered as a 'solid phase' analysis whereby interfering components in the cell preparation may be discarded by washing or minimised before the specimen slides are analysed. A further application of a peroxidase labelled antibody immunoassay may be found in the field of steroid assays where a separation technique is usually required. Although it is unlikely that peroxidase-like activity would affect urine h o r m o n e analyses, interferences by compounds which compete with the chromogen for peroxide free radicals may be greater in this case. The sensitivity of the immunoassay described, with a 10 min enzyme reaction, is satisfactory for the determination of HPL and it is estimated that an assay for ~ fetoprotein (5 nmol/1) could be achieved. This is based on the conditions used, whereas greater sensitivity is possible by increasing the enzyme incubation.
23 T h e relative ease o f p r e p a r a t i o n o f a p e r o x i d a s e labelled a n t i b o d y , its stability and l o w c o s t m a k e p e r o x i d a s e t h e p r e f e r r e d e n z y m e f o r i m m u n o a s s a y s a l t h o u g h t h e s e r u m b l a n k is a serious d r a w b a c k . T h e c i r c u m s t a n c e s leading to t h e last c o m m e n t m a y p r o m p t w o r k e r s in t h e e n z y m e i m m u n o a s s a y field t o add o n e m o r e criterion t o t h e list describing a suitable e n z y m e f o r labelling p u r p o s e s , and this is t h a t t h e e n z y m e s u b s t r a t e r e a c t i o n s h o u l d be u n i q u e f o r t h e e n z y m e , u n d e r t h e assay condit i o n s to b e studied. ACKNOWLEDGEMENTS I a m g r a t e f u l t o P r o f e s s o r J. L a n d o n and Mr. J. C r o o k a l l - G r e e n i n g f o r t h e i r v a l u a b l e assistance in p r e p a r i n g t h e script a n d also Mr. R. J a y a n d Miss M. Shields f o r t h e i r h e l p in t h e a u t o m a t i o n o f t h e p e r o x i d a s e assay.
REFERENCES Addison, G.M., 1971, Ph.D. thesis, Cambridge. Addison, G.M., C.N. Hales, J.S. Woodhead and J.L.H. O'Riordan, 1971, J. Endocrinol. 49, 521. Avrameas, S., 1969, Immunochem. 6, 43. Avrameas, S., 1972, Histochem. J. 4, 321. Avrameas, S. and T. Ternynck, 1971, Immunochem. 8, 1175. Burrows, S. and E.H. Hosten, 1969, Technicon Symposium. 'Automation in Analytical Chemistry' (New York, N.Y.) p. 401. Cambiaso, C.L., P.L. Masson, J.P. Vaerman and J.F. Heremans, 1974, J. Immunol. Methods 5,153. Gurvich, A.E., O.B. Kuzovleva and A.E. Tumanova, 1961, Biokhimiya 26,803. Hughes-Jones, N.C., B. Gardner and R. Telford, 1964, Immunology 7, 72. Kawaoi, A. and P.K. Nakane, 1973, Fed. Proc. 32,840. Maiolini, R., B. Ferrua and R. Masseyeff, 1975, J. Immunol. Methods 6, 355. Miles, L.E.M. and C.N. Hales, 1968a, Biochem. J. 108,611. Miles, L.E.M. and C.N. Hales, 1968b, Nature 219,186. Nyman, M., 1959, Scand. J. Clin. Lab. Invest. 11, Suppl. 39. Rubenstein, K.E., R.S. Schneider and E.F. Ullman, 1972, Biochem. Biophys. Res. Commun. 47, 4,846. Schick, A.F. and S.J. Singer, 1961, J. Biol. Chem. 236, 9, 2477. Trinder, P., 1969, Anal. Clin. Biochem. 6, 24. Van Weemen, B.K., 1974, Ph.D. thesis. Van Weemen, B.K. and A.H.W.M. Schuurs, 1974, Febs. Letts. 43, 2,215. Weston, P.D. and S. Avrameas, 1971, Biochem. Biophys. Res. Commun. 45, 6, 1574.
