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Comparison of monoclonal and polyclonal antibodies in a two-site binding enzyme immunoassay for alphafetoprotein (AFP)
Clinica Chimica Acta, 135 (1983) 13-22 Elsevier
13
CCA 02684
Comparison of monoclonal and polyclonal antibodies in a two-site binding enzyme immunoassay for alphafetoprotein ( AFP) T. Porstmann
*, Baerbel Porstmann, Elsa Nugel, B. Micheel, U. Karsten H. Fiebach
and
Department of Clinical Immunology and Department of Clinical Biochemistry, Faculty of Medicine (CharitP), Humboldt-University, Schumannstrasse 20/21, 1040 Berlin (GDR) and Department of Experimental and Clinical Immunology, Central Institute of Cancer Research, Academ_v of Sciences of the GDR (Received
February
14th; revision July 5th, 1983)
A two-site binding enzyme immunoassay for the detection of alphafetoprotein (AFP) was developed by using either a combination of two monoclonal antibodies or of one monoclonal antibody and polyclonal antibodies. The conjugation of the monoclonal antibodies to peroxidase by the periodate method yielded a somewhat higher sensitivity in the enzyme immunoassay (EIA), when compared to conjugates produced by the glutaraldehyde method. The detection limit was 10 yg AFP per litre when using only monoclonal antibodies in the assay. Simultaneous incubation of the sample and the monoclonal labelled antibody should be avoided unless using at least two different dilutions of the sample for investigation.
Introduction Two-site binding assays as introduced by Miles and Hales [ 1] do not require purified antigen, but need a relatively large quantity of antibodies both for solid phase adsorption and labelling. Mon~lonal antibodies [2] will overcome this limitation and their application in immunoassays will allow two-site binding assays to become the prevailing assay procedure in the future. Therefore we compared monoclonal and polyclonal antibodies in an enzyme immunoassay (EIA) for human ar-1-fetoprotein (AFP) in respect of sensitivity and checked the possibility of simultaneous incubation of the various reactants in an one-step assay as proposed by Uotila et al [3] using monoclonal antibodies in a two-site binding assay. * Correspondence should be addressed to Dr. T. Porstmann, Abt. IUinische Immunologic, Bereich Medizin (Charit&) der Humboldt-Universit;, Schumannstrasse 20/21, 1040 Berlin, Postfach 170, GDR.
~9-898~/83/$03.~
0 1983 Elsevier Science Publishers
B.V.
14
Material and methods Production and purification of antibodies Polyclonal antibodies were raised in sheep by immunisation with purified AFP [4]. The IgG fraction (Pol-1), purified by negative affinity chromatography on human serum proteins coupled to CNBr-Sepharose (Pharmacia, Uppsala, Sweden), was employed for solid phase coating only, and the monospecific anti-AFP antibodies (Pol-2), purified by positive affinity chromatography on AFP-Sepharose, used both for coating and labelling. Monoclonal antibodies were isolated from ascitic fluid by ammonium sulphate precipitation at a final concentration of 1.83 mol/l. Antibodies from clone 9 (Mon-9) and from clone 38 (Mon-38) reacting with different epitopes [5] were selected for the experiments. Coupling procedures and conjugate purification Pol-2, Mon-9 and Mon-38 were conjugated to horse radish peroxidase (HRP) of a purity number (PN) of 2.8 [6] by the periodate method (PI-method) of Wilson and Nakane [7] and by the two-step glutaraldehyde method (GA-method) of Avrameas and Ternynck [8] applying a molar ratio of IgG to HRP of 1 : 8. GA-conjugates were purified by combination of affinity chromatography and gel filtration, and PI-conjugates by gel filtration only [9]. Protein content of the purified conjugates was determined by the method of Lowry et al [IO]. ~e~erm~na~iun of enzyme activity Enzymatic activities of native HRP and of the different conjugates were estimated with the Multistat III Microcentrifugalanalyzer (IL, Milano, Italy). Ten ~1 of the diluted sample were mixed with 200 ~1 of substrate solution, containing 2 mmol/l ABTS (Boehringer, Mannheim, FRG) and 0.2 mmol/l H,O, dissolved in 0.1 mol/l phosphate buffer, pH 7.0. The increase in absorbance at 405 nm during the first 20 s of the reaction at a temperature of 37’C, was used for the calculation of specific enzyme activity, expressed as U/mg protein. One unit was defined as the degradation of 1 mmol H,O, .I-' - s- ‘, using a coefficient of absorption for ABTS of 18.4 cm2/~mol [I 11. Enzyme immunoassay Polystyrene tubes (VEB Polyplast, Halberstadt, GDR) were coated at 4°C overnight [12] using anti-AFP antibodies in the following concentrations: Pol-1 5 mg/l, Pol-2, Mon-9 and Mon-38 2.5 mg/l. AFP standard serum (Lot No. 042107 B, Behringwerke, Marburg, FRG) and the different conjugates were dissolved in PBS, containing 50 ml/l horse serum and 30 g/l polyethylene glycol 6000 (Fluka AG, Buchs, Switzerland). The enzyme reaction with ABTS was performed as described before [ 131. The volume for solid phase coating, of the standard and of the conjugate, was 200 ~1, and that of substrate and stopping solution 500 ~1. Between all the reaction steps tubes were washed with 3 X I ml PBS, containing 0.05% Tween
IS
20 (Serva, determined
Heidelberg, FRG). The detection iimit and within-run as previously described for other EIA procedures 1141.
precision
were
Successive incubation technique Coated tubes were incubated with different concentrations of AFP standard at 37°C for 2 h, followed by washing and incubation with identical concentrations of conjugates (1 mg/l) at 37°C for another 2 h. After washing the enzyme reaction was started.
Simultaneous incubation technique Equal volumes of different concentrations of AFP standard and identical concentrations of conjugates (2 mg,/l) were mixed and 200 ,ul immediately transferred into the coated tubes for a 2-h incubation at 37°C followed by washing and subsequent substrate reaction.
Competition experiments To detect antibodies in the polyclonal antiserum, which were directed against the same epitopes of the AFP molecule as the monoclonal antibodies, binding of Pol-2-HRP conjugate was inhibited by increasing amounts of 0.7 to 15 mg Mon-9 or Mon-38 IgG per mg conjugate and litre. In another assay the binding of the monoclonal IgG-HRP conjugates was inhibited by uniabelled Pal-2, using the same concentration ratios. For all experiments 80 pg AFP/l were used. Results
Characterisation of the conjugates As successfully done with polyclonal IgG from different species the HRP-labelling of monoclonal murine IgG is also possible by glutaraldehyde and periodate. The latter method yielded high quantities of conjugates with a f-fold higher molar ratio of HRP: IgG as concluded from the PN values and the specific activities (Table I). In one conjugate molecule produced by the PI-method at least three antibody molecules must be involved, because they show a molecular mass greater than 600000 dalton, whereas GA-linked conjugates had a molecular mass of about 2~000 dalton (unpublished results). Using identical reaction conditions, insertion of enzyme was about twice as high in the murine antibodies compared with the antibodies from the sheep when the PI-method was applied, but no differences occurred from GA-linking. The monoclonal antibody-HRP conjugates produced by the PI-method lost to a great extent their ability to react with Concanavalin-A, whereas GA-linked conjugates did not, and a complete separation of unlabelled monoclonal antibodies by Con A-Sepharose was possible as shown in Table I.
Enzyme immunoassay Combining (PI-method),
Mon-9 in solid-phase-supported and Mon-38 in enzyme-labelled form a detection limit of 10 pg AFP/l was obtained. It shifted to 20 pg
I
Yield of conjugate in relation to total amount of protein (IgG + HRP) applied for coupling 66.0%
17.2%
2.5 mg 0.5 246 U/mg
13.5 mg 0.9 432 U/mg
Quantity of conjugate purity number specific activity
1.5 mg
GA-method
5.5 mg 0.6 mg
HRP
not det.
PI-method
IgG 2a)
antibodies
using monoclonal
Mon-9 (subclass
Monoclonal
procedures
2.8 mg 4.2 mg
of uncoupled
Quantity
IgG
coupling
monomeric form dimeric form
of uncoupled
of the different
Quantity
Comparison
TABLE antibodies
66.0%’
13.5 mg 1.0 536 U/mg
2.4 mg 4.3 mg
not det.
PI-method
Mon-38 (subclass
and polyclonal
25.5%
3.1 mg 0.4 256 U/mg
6.2 mg 0.9 mg
0.8 mg
GA-method
IgG 1)
55%
13.1%
1.9 mg 0.4 212 U/mg
11.2 mg 0.6 263 U/mg
1.8 mg
GA-method
4.8 mg 1.2 mg
antibodies
5.6 mg 3.8 mg
not det.
PI-method
Polyclonal
17
28 24 22 2.0 1.8 E
1.6
g
1.4
; 12 x L 1.0 II a 0.8
I
0.6 O.r, 0.2 2
4682)
20 406OIXI2M3400
Blankdues
ConcentmtionofAFP(,ug/l) Fig. 1. Comparison of standard different coupling procedures.
curves obtained
with identical
solid phase
HRP-conjugate
coupling procedure
Mow9
Pal-2
PI-method
Mon-9
POI-2
GA-method
A-r
Pol-l
Man-9
PI-method
A-A
Pal-
Mon-9
GA-method
70 O-
0
1
L
I
Mow9
Man-38
PI-method
LJ
LI
Man-9
Man-38
GA-method
Standard curves (la) were plotted after subtracting actual measured absorbances. The mean of the blank recorded from IO-fold determinations of a AFP-negative
quantities
of conjugates
produced
the corresponding mean blank values values and their standard deviation serum (see also Fig. 2).
AFP/l accompanied by a flattening of the standard of Mon-38-HRP conjugates were used, produced by applying Mon-38 for solid phase coating and Mon-9 virtually no signal was found over the whole range
by the
from the
(1b) were
curve, when identical quantities the GA-method (Fig. 1). When or Pol-2 as enzyme conjugates, of AFP concentrations investi-
18
Q6
t
113*Oa .0.6 -0.4 .0.2 2
468x3
20 4Oosoo2am
xlOO2alO4m
EUankwdues
Concentrution of AFP&/l) Fig. 2. Standard curves (2a) obtained by simultaneous (closed symbols) incubation of the reactants and corresponding blank values (2b).
+
+-
solid phase
HRP-conjugate
Pal-I
Pal- 1
l
l
Pol-1
Mon-9
h
1
Pol-1
Mon-38
Mon-9
Mom38
A-A
and successive
(open symbols)
indicating that Mon-38 was unable to bind sufficient antigen in the chosen reaction conditions. The highest sensitivity with a detection limit of 1 pg AFP/l among the different combinations was found using Mon-9 for solid phase coating and Pol-2 in the enzyme-labelled from (Fig. 1). The use of Pol-2 instead of Pol-1 or Mon-9 for solid phase coating resulted in a slightly lower sensitivity with a detection limit of 6 pg AFP/l, whereas the working range was limited sooner when Pol-1 was
gated,
19
adsorbed to the solid phase. This is indicated by an increasing inaccuracy in the upper and lower parts of the standard curves (Table II). In all cases in which monoclonal antibodies in the enzyme-conjugated form were used, higher blank values occurred (Figs. 1, 2). Successive incubations of the
0 5
0
~~~~~~~
10 15 20 Of#d&d uniabeued Ont’iAFFJ (mgll
1
Fig. 3. Inhibition of conjugate binding to constant quantities of AFP by increasing amounts of unlabelled antibodies. HRP-twnjugate
unlabefled antibody
i!
i.3
P&l
Pal- 1
L
ti
P&I
Man-38
P&l
Mon-9
A-------&
d&m9
P&l
o-o
Man-38
Pof- 1
+------
*
20
TABLE
II
Intra-assay variance of the EIA with different technique for quantification of AFP
combinations
of antibodies
in the successive
Solidphase
Conjugate
pot-2
Mon-9
11.1 31.5 122.6
1.62 3.31 17.04
14.6 10.5 13.9
Pal- 1
Mon-9
10.3 32.9 127.9
0.93 4.47 29.16
9.1 13.6 22.8
Mom9
Pal-2
9.6 35.3 122.0
0.83 3.46 17.32
8.1 9.8 14.2
Mon-9
Mon-38
11.5 33.4 125.2
4.21 24.79
AFP standard
(350 mg/l)
was dissolved
Mean
SD
(pg/f)
incubation
cv (W)
1.92
in AFP free serum to concentrations
16.7 12.6 19.8 of 10, 35 and 140 pg/l.
reactants resulted in high AFP concentrations, both with monoclonal and polyclonal antibodies at different combinations in a plateau of the standard curve in each case. In contrast, simultaneous application of the reactants revealed a strong high dose ‘hook’ effect setting in at about 200 pg AFP/l (Fig. 2). This effect occurred with poiyclonal as well as with monoclonal antibodies in all combinations. Competition experiments Whereas the monoclonal antibodies pletely prevented from antigen-binding inhibition of the binding of polyclonal antibodies (Fig. 3).
did not inhibit each other, they were comby added polyclonal antibodies. Only partial antibodies was provoked by the monoclonal
Discussion The combination of two monoclonal antibodies in the EIA resulted in sufficient sensitivity for recording pathologically elevated AFP serum levels, assuming a cut-off value of about 25 pg AFP per litre [IS]. The chosen reaction conditions were the same for monoclonal and polyclonal antibodies and no attempts were made to optimise the assay by looking for optimal reaction conditions for the monoclonal antibodies. Further enhancement of assay sensitivity could also be achieved by elevation of incubation temperature or extending the reaction time. Increasing the concentration of monoclonal antibody enzyme conjugates to get higher sensitivity, however, is limited by the high blank values, especially in the case of PI-conjugates. Enhancement of assay sensitivity is obtained by the combination of monoclonal and
21
polyclonai antibodies. In accord with the findings of Brock et al [ 161the best results were obtained by use of monoclonal antibodies for solid phase coating and poiyclonal antibodies in the enzyme-labelled form. The multiple attachment to the AFP molecule of the PI-conjugates due to linkage of polyclonal antibodies directed against different AFP epitopes in one conjugate molecule may be one reason for producing greater sensitivity. Therefore it is also impossible to determine by the competition method described the exact quantity of antibodies in Pal-2, which reveal the same specificity as Mon-9 or Mon-38. Even by completely blocking these antibody activities in the Pol-2-HRP conjugate by the monoclonal antibodies, such a conjugate molecule can only react with AFP via an antibody of different epitope specificity. The homogeneity of monoclonal antibody Mon-9, however, results in higher antigen binding capacity per tube, when used for solid phase coating instead of an identical quantity of Pol-I, in which anti-AFP antibodies amount to 19% (unpublished results). But if monospecific anti AFP IgG were isolated by positive affinity chromatography antibodies of the highest affinity were lost. The standard curve deviated either sooner or sensitivity will be lost. Therefore monoclonal antibodies are superior to polyclonal when applied to solid phase adsorption. We were able to show that simultaneous incubation of the reactants is not an advantage of monoclonal antibodies, as asserted by Sevier et al [ 171, because polyclonal antibodies resulted at least in the same sensitivity and measuring range. The strong ‘hook’ effect we observed by the simultaneous incubation technique always occurred if the antigen concentration exceeded a certain level. Above it, a constant amount of the conjugate is consumed by increasing quantities of antigen molecules, for which no further solid-phase-supported antibodies are available. Thus, the two-site binding assay as a reagent excess technique has changed to a solid phase competitive procedure between unlabelled and conjugate-labelled AFP for binding to the limited solid phase antibody. The simultaneous incubation technique, as first described by Uotila et al 131, and also applied by Brock et al [ 161 for quantification of AFP with monoclonal antibodies, is deficient for disregarding this fact. Very high AFP concentrations, such as in the case of hepatomas, can behave similarly to quite normal AFP levels. To prevent such a failure, the investigated sera generally have to be examined at least at two different dilutions if the simultaneous incubation technique is used. References 1 Miles LEM, Hales CN. Labelled antibodies and immunological assay systems. Nature 1968; 219: 186-189. 2 Kohler C, Milstein C. Continuous cultures of
22
different epitopes of human alpha fetoprotein (AP). Eur J Cancer Clin Oncol, in press. 6 Porstmann B, Porstmann T. Reinigung handelstiblicher Meerrettich-Peroxidase fur den Einsatz im Enzymimmunoassay. Z Med Labor-Diagn 1979; 20: 87-95. 7 Wilson MB, Nakane PK. In: Knapp W, Holubar K, Wick G, eds. Immunofluorescence and related staining techniques. Amsterdam: Elsevier/North Holland, 1978: 215-221. 8 Avrameas S, Ternynk T. Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 1971; 8: 1175-l 179. 9 Porstmann T, Porstmann B. Purification of horse radish peroxidase conjugated antibodies by affinity chromatography. Comparison of glutaraldehyde and periodate conjugation. Acta Biol Med Germ 1979; 38: 1039-1054. 10 Lowry OH, Rosebrough NJ, Farr AL, Randall R.J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 11 Werner W, Rey HG, Wielinger H. On the properties of a new chromogen for the determination of glucose in blood according to the GOD/POD-method. Z Anal Chem 1970; 252: 224-228. 12 Catt K, Tregear GW. Solid-phase radioimmunoassay in antibody coated tubes. Science 1967; 158: 1570-1572. 13 Porstmann B, Porstmann T, Nugel E. Comparison of chromogens for the determination of horseradish peroxidase as a marker in enzyme immunoassay. J Clin Chem Clin Biochem 1981; 19: 435-439. 14 Porstmann T, Porstmann B, Seifert R. Application of the peroxidase anti-peroxidase system as an universal reagent for the two-site binding enzyme immunoassay. Clin Chim Acta 1983; 129: 107- 117. 15 Ruoslahti E, SeppUl M. a-fetoprotein in cancer and fetal development. Adv Cancer Res 1979; 29: 275-346. 16 Brock DJH, Barron L, van Heyningen V. Enzyme linked immunospecific assays for human alphafetoprotein using monoclonal antibodies. Clin Chim Acta 1982; 122: 353-358. 17 Sevier ED, David GS, Martinis J, Desmond WJ, Bartholomew RM, Wang R. Monoclonal antibodies in clinical immunology. Clin Chem 1981; 27: l797- 1806.
13
CCA 02684
Comparison of monoclonal and polyclonal antibodies in a two-site binding enzyme immunoassay for alphafetoprotein ( AFP) T. Porstmann
*, Baerbel Porstmann, Elsa Nugel, B. Micheel, U. Karsten H. Fiebach
and
Department of Clinical Immunology and Department of Clinical Biochemistry, Faculty of Medicine (CharitP), Humboldt-University, Schumannstrasse 20/21, 1040 Berlin (GDR) and Department of Experimental and Clinical Immunology, Central Institute of Cancer Research, Academ_v of Sciences of the GDR (Received
February
14th; revision July 5th, 1983)
A two-site binding enzyme immunoassay for the detection of alphafetoprotein (AFP) was developed by using either a combination of two monoclonal antibodies or of one monoclonal antibody and polyclonal antibodies. The conjugation of the monoclonal antibodies to peroxidase by the periodate method yielded a somewhat higher sensitivity in the enzyme immunoassay (EIA), when compared to conjugates produced by the glutaraldehyde method. The detection limit was 10 yg AFP per litre when using only monoclonal antibodies in the assay. Simultaneous incubation of the sample and the monoclonal labelled antibody should be avoided unless using at least two different dilutions of the sample for investigation.
Introduction Two-site binding assays as introduced by Miles and Hales [ 1] do not require purified antigen, but need a relatively large quantity of antibodies both for solid phase adsorption and labelling. Mon~lonal antibodies [2] will overcome this limitation and their application in immunoassays will allow two-site binding assays to become the prevailing assay procedure in the future. Therefore we compared monoclonal and polyclonal antibodies in an enzyme immunoassay (EIA) for human ar-1-fetoprotein (AFP) in respect of sensitivity and checked the possibility of simultaneous incubation of the various reactants in an one-step assay as proposed by Uotila et al [3] using monoclonal antibodies in a two-site binding assay. * Correspondence should be addressed to Dr. T. Porstmann, Abt. IUinische Immunologic, Bereich Medizin (Charit&) der Humboldt-Universit;, Schumannstrasse 20/21, 1040 Berlin, Postfach 170, GDR.
~9-898~/83/$03.~
0 1983 Elsevier Science Publishers
B.V.
14
Material and methods Production and purification of antibodies Polyclonal antibodies were raised in sheep by immunisation with purified AFP [4]. The IgG fraction (Pol-1), purified by negative affinity chromatography on human serum proteins coupled to CNBr-Sepharose (Pharmacia, Uppsala, Sweden), was employed for solid phase coating only, and the monospecific anti-AFP antibodies (Pol-2), purified by positive affinity chromatography on AFP-Sepharose, used both for coating and labelling. Monoclonal antibodies were isolated from ascitic fluid by ammonium sulphate precipitation at a final concentration of 1.83 mol/l. Antibodies from clone 9 (Mon-9) and from clone 38 (Mon-38) reacting with different epitopes [5] were selected for the experiments. Coupling procedures and conjugate purification Pol-2, Mon-9 and Mon-38 were conjugated to horse radish peroxidase (HRP) of a purity number (PN) of 2.8 [6] by the periodate method (PI-method) of Wilson and Nakane [7] and by the two-step glutaraldehyde method (GA-method) of Avrameas and Ternynck [8] applying a molar ratio of IgG to HRP of 1 : 8. GA-conjugates were purified by combination of affinity chromatography and gel filtration, and PI-conjugates by gel filtration only [9]. Protein content of the purified conjugates was determined by the method of Lowry et al [IO]. ~e~erm~na~iun of enzyme activity Enzymatic activities of native HRP and of the different conjugates were estimated with the Multistat III Microcentrifugalanalyzer (IL, Milano, Italy). Ten ~1 of the diluted sample were mixed with 200 ~1 of substrate solution, containing 2 mmol/l ABTS (Boehringer, Mannheim, FRG) and 0.2 mmol/l H,O, dissolved in 0.1 mol/l phosphate buffer, pH 7.0. The increase in absorbance at 405 nm during the first 20 s of the reaction at a temperature of 37’C, was used for the calculation of specific enzyme activity, expressed as U/mg protein. One unit was defined as the degradation of 1 mmol H,O, .I-' - s- ‘, using a coefficient of absorption for ABTS of 18.4 cm2/~mol [I 11. Enzyme immunoassay Polystyrene tubes (VEB Polyplast, Halberstadt, GDR) were coated at 4°C overnight [12] using anti-AFP antibodies in the following concentrations: Pol-1 5 mg/l, Pol-2, Mon-9 and Mon-38 2.5 mg/l. AFP standard serum (Lot No. 042107 B, Behringwerke, Marburg, FRG) and the different conjugates were dissolved in PBS, containing 50 ml/l horse serum and 30 g/l polyethylene glycol 6000 (Fluka AG, Buchs, Switzerland). The enzyme reaction with ABTS was performed as described before [ 131. The volume for solid phase coating, of the standard and of the conjugate, was 200 ~1, and that of substrate and stopping solution 500 ~1. Between all the reaction steps tubes were washed with 3 X I ml PBS, containing 0.05% Tween
IS
20 (Serva, determined
Heidelberg, FRG). The detection iimit and within-run as previously described for other EIA procedures 1141.
precision
were
Successive incubation technique Coated tubes were incubated with different concentrations of AFP standard at 37°C for 2 h, followed by washing and incubation with identical concentrations of conjugates (1 mg/l) at 37°C for another 2 h. After washing the enzyme reaction was started.
Simultaneous incubation technique Equal volumes of different concentrations of AFP standard and identical concentrations of conjugates (2 mg,/l) were mixed and 200 ,ul immediately transferred into the coated tubes for a 2-h incubation at 37°C followed by washing and subsequent substrate reaction.
Competition experiments To detect antibodies in the polyclonal antiserum, which were directed against the same epitopes of the AFP molecule as the monoclonal antibodies, binding of Pol-2-HRP conjugate was inhibited by increasing amounts of 0.7 to 15 mg Mon-9 or Mon-38 IgG per mg conjugate and litre. In another assay the binding of the monoclonal IgG-HRP conjugates was inhibited by uniabelled Pal-2, using the same concentration ratios. For all experiments 80 pg AFP/l were used. Results
Characterisation of the conjugates As successfully done with polyclonal IgG from different species the HRP-labelling of monoclonal murine IgG is also possible by glutaraldehyde and periodate. The latter method yielded high quantities of conjugates with a f-fold higher molar ratio of HRP: IgG as concluded from the PN values and the specific activities (Table I). In one conjugate molecule produced by the PI-method at least three antibody molecules must be involved, because they show a molecular mass greater than 600000 dalton, whereas GA-linked conjugates had a molecular mass of about 2~000 dalton (unpublished results). Using identical reaction conditions, insertion of enzyme was about twice as high in the murine antibodies compared with the antibodies from the sheep when the PI-method was applied, but no differences occurred from GA-linking. The monoclonal antibody-HRP conjugates produced by the PI-method lost to a great extent their ability to react with Concanavalin-A, whereas GA-linked conjugates did not, and a complete separation of unlabelled monoclonal antibodies by Con A-Sepharose was possible as shown in Table I.
Enzyme immunoassay Combining (PI-method),
Mon-9 in solid-phase-supported and Mon-38 in enzyme-labelled form a detection limit of 10 pg AFP/l was obtained. It shifted to 20 pg
I
Yield of conjugate in relation to total amount of protein (IgG + HRP) applied for coupling 66.0%
17.2%
2.5 mg 0.5 246 U/mg
13.5 mg 0.9 432 U/mg
Quantity of conjugate purity number specific activity
1.5 mg
GA-method
5.5 mg 0.6 mg
HRP
not det.
PI-method
IgG 2a)
antibodies
using monoclonal
Mon-9 (subclass
Monoclonal
procedures
2.8 mg 4.2 mg
of uncoupled
Quantity
IgG
coupling
monomeric form dimeric form
of uncoupled
of the different
Quantity
Comparison
TABLE antibodies
66.0%’
13.5 mg 1.0 536 U/mg
2.4 mg 4.3 mg
not det.
PI-method
Mon-38 (subclass
and polyclonal
25.5%
3.1 mg 0.4 256 U/mg
6.2 mg 0.9 mg
0.8 mg
GA-method
IgG 1)
55%
13.1%
1.9 mg 0.4 212 U/mg
11.2 mg 0.6 263 U/mg
1.8 mg
GA-method
4.8 mg 1.2 mg
antibodies
5.6 mg 3.8 mg
not det.
PI-method
Polyclonal
17
28 24 22 2.0 1.8 E
1.6
g
1.4
; 12 x L 1.0 II a 0.8
I
0.6 O.r, 0.2 2
4682)
20 406OIXI2M3400
Blankdues
ConcentmtionofAFP(,ug/l) Fig. 1. Comparison of standard different coupling procedures.
curves obtained
with identical
solid phase
HRP-conjugate
coupling procedure
Mow9
Pal-2
PI-method
Mon-9
POI-2
GA-method
A-r
Pol-l
Man-9
PI-method
A-A
Pal-
Mon-9
GA-method
70 O-
0
1
L
I
Mow9
Man-38
PI-method
LJ
LI
Man-9
Man-38
GA-method
Standard curves (la) were plotted after subtracting actual measured absorbances. The mean of the blank recorded from IO-fold determinations of a AFP-negative
quantities
of conjugates
produced
the corresponding mean blank values values and their standard deviation serum (see also Fig. 2).
AFP/l accompanied by a flattening of the standard of Mon-38-HRP conjugates were used, produced by applying Mon-38 for solid phase coating and Mon-9 virtually no signal was found over the whole range
by the
from the
(1b) were
curve, when identical quantities the GA-method (Fig. 1). When or Pol-2 as enzyme conjugates, of AFP concentrations investi-
18
Q6
t
113*Oa .0.6 -0.4 .0.2 2
468x3
20 4Oosoo2am
xlOO2alO4m
EUankwdues
Concentrution of AFP&/l) Fig. 2. Standard curves (2a) obtained by simultaneous (closed symbols) incubation of the reactants and corresponding blank values (2b).
+
+-
solid phase
HRP-conjugate
Pal-I
Pal- 1
l
l
Pol-1
Mon-9
h
1
Pol-1
Mon-38
Mon-9
Mom38
A-A
and successive
(open symbols)
indicating that Mon-38 was unable to bind sufficient antigen in the chosen reaction conditions. The highest sensitivity with a detection limit of 1 pg AFP/l among the different combinations was found using Mon-9 for solid phase coating and Pol-2 in the enzyme-labelled from (Fig. 1). The use of Pol-2 instead of Pol-1 or Mon-9 for solid phase coating resulted in a slightly lower sensitivity with a detection limit of 6 pg AFP/l, whereas the working range was limited sooner when Pol-1 was
gated,
19
adsorbed to the solid phase. This is indicated by an increasing inaccuracy in the upper and lower parts of the standard curves (Table II). In all cases in which monoclonal antibodies in the enzyme-conjugated form were used, higher blank values occurred (Figs. 1, 2). Successive incubations of the
0 5
0
~~~~~~~
10 15 20 Of#d&d uniabeued Ont’iAFFJ (mgll
1
Fig. 3. Inhibition of conjugate binding to constant quantities of AFP by increasing amounts of unlabelled antibodies. HRP-twnjugate
unlabefled antibody
i!
i.3
P&l
Pal- 1
L
ti
P&I
Man-38
P&l
Mon-9
A-------&
d&m9
P&l
o-o
Man-38
Pof- 1
+------
*
20
TABLE
II
Intra-assay variance of the EIA with different technique for quantification of AFP
combinations
of antibodies
in the successive
Solidphase
Conjugate
pot-2
Mon-9
11.1 31.5 122.6
1.62 3.31 17.04
14.6 10.5 13.9
Pal- 1
Mon-9
10.3 32.9 127.9
0.93 4.47 29.16
9.1 13.6 22.8
Mom9
Pal-2
9.6 35.3 122.0
0.83 3.46 17.32
8.1 9.8 14.2
Mon-9
Mon-38
11.5 33.4 125.2
4.21 24.79
AFP standard
(350 mg/l)
was dissolved
Mean
SD
(pg/f)
incubation
cv (W)
1.92
in AFP free serum to concentrations
16.7 12.6 19.8 of 10, 35 and 140 pg/l.
reactants resulted in high AFP concentrations, both with monoclonal and polyclonal antibodies at different combinations in a plateau of the standard curve in each case. In contrast, simultaneous application of the reactants revealed a strong high dose ‘hook’ effect setting in at about 200 pg AFP/l (Fig. 2). This effect occurred with poiyclonal as well as with monoclonal antibodies in all combinations. Competition experiments Whereas the monoclonal antibodies pletely prevented from antigen-binding inhibition of the binding of polyclonal antibodies (Fig. 3).
did not inhibit each other, they were comby added polyclonal antibodies. Only partial antibodies was provoked by the monoclonal
Discussion The combination of two monoclonal antibodies in the EIA resulted in sufficient sensitivity for recording pathologically elevated AFP serum levels, assuming a cut-off value of about 25 pg AFP per litre [IS]. The chosen reaction conditions were the same for monoclonal and polyclonal antibodies and no attempts were made to optimise the assay by looking for optimal reaction conditions for the monoclonal antibodies. Further enhancement of assay sensitivity could also be achieved by elevation of incubation temperature or extending the reaction time. Increasing the concentration of monoclonal antibody enzyme conjugates to get higher sensitivity, however, is limited by the high blank values, especially in the case of PI-conjugates. Enhancement of assay sensitivity is obtained by the combination of monoclonal and
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polyclonai antibodies. In accord with the findings of Brock et al [ 161the best results were obtained by use of monoclonal antibodies for solid phase coating and poiyclonal antibodies in the enzyme-labelled form. The multiple attachment to the AFP molecule of the PI-conjugates due to linkage of polyclonal antibodies directed against different AFP epitopes in one conjugate molecule may be one reason for producing greater sensitivity. Therefore it is also impossible to determine by the competition method described the exact quantity of antibodies in Pal-2, which reveal the same specificity as Mon-9 or Mon-38. Even by completely blocking these antibody activities in the Pol-2-HRP conjugate by the monoclonal antibodies, such a conjugate molecule can only react with AFP via an antibody of different epitope specificity. The homogeneity of monoclonal antibody Mon-9, however, results in higher antigen binding capacity per tube, when used for solid phase coating instead of an identical quantity of Pol-I, in which anti-AFP antibodies amount to 19% (unpublished results). But if monospecific anti AFP IgG were isolated by positive affinity chromatography antibodies of the highest affinity were lost. The standard curve deviated either sooner or sensitivity will be lost. Therefore monoclonal antibodies are superior to polyclonal when applied to solid phase adsorption. We were able to show that simultaneous incubation of the reactants is not an advantage of monoclonal antibodies, as asserted by Sevier et al [ 171, because polyclonal antibodies resulted at least in the same sensitivity and measuring range. The strong ‘hook’ effect we observed by the simultaneous incubation technique always occurred if the antigen concentration exceeded a certain level. Above it, a constant amount of the conjugate is consumed by increasing quantities of antigen molecules, for which no further solid-phase-supported antibodies are available. Thus, the two-site binding assay as a reagent excess technique has changed to a solid phase competitive procedure between unlabelled and conjugate-labelled AFP for binding to the limited solid phase antibody. The simultaneous incubation technique, as first described by Uotila et al 131, and also applied by Brock et al [ 161 for quantification of AFP with monoclonal antibodies, is deficient for disregarding this fact. Very high AFP concentrations, such as in the case of hepatomas, can behave similarly to quite normal AFP levels. To prevent such a failure, the investigated sera generally have to be examined at least at two different dilutions if the simultaneous incubation technique is used. References 1 Miles LEM, Hales CN. Labelled antibodies and immunological assay systems. Nature 1968; 219: 186-189. 2 Kohler C, Milstein C. Continuous cultures of
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different epitopes of human alpha fetoprotein (AP). Eur J Cancer Clin Oncol, in press. 6 Porstmann B, Porstmann T. Reinigung handelstiblicher Meerrettich-Peroxidase fur den Einsatz im Enzymimmunoassay. Z Med Labor-Diagn 1979; 20: 87-95. 7 Wilson MB, Nakane PK. In: Knapp W, Holubar K, Wick G, eds. Immunofluorescence and related staining techniques. Amsterdam: Elsevier/North Holland, 1978: 215-221. 8 Avrameas S, Ternynk T. Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 1971; 8: 1175-l 179. 9 Porstmann T, Porstmann B. Purification of horse radish peroxidase conjugated antibodies by affinity chromatography. Comparison of glutaraldehyde and periodate conjugation. Acta Biol Med Germ 1979; 38: 1039-1054. 10 Lowry OH, Rosebrough NJ, Farr AL, Randall R.J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 11 Werner W, Rey HG, Wielinger H. On the properties of a new chromogen for the determination of glucose in blood according to the GOD/POD-method. Z Anal Chem 1970; 252: 224-228. 12 Catt K, Tregear GW. Solid-phase radioimmunoassay in antibody coated tubes. Science 1967; 158: 1570-1572. 13 Porstmann B, Porstmann T, Nugel E. Comparison of chromogens for the determination of horseradish peroxidase as a marker in enzyme immunoassay. J Clin Chem Clin Biochem 1981; 19: 435-439. 14 Porstmann T, Porstmann B, Seifert R. Application of the peroxidase anti-peroxidase system as an universal reagent for the two-site binding enzyme immunoassay. Clin Chim Acta 1983; 129: 107- 117. 15 Ruoslahti E, SeppUl M. a-fetoprotein in cancer and fetal development. Adv Cancer Res 1979; 29: 275-346. 16 Brock DJH, Barron L, van Heyningen V. Enzyme linked immunospecific assays for human alphafetoprotein using monoclonal antibodies. Clin Chim Acta 1982; 122: 353-358. 17 Sevier ED, David GS, Martinis J, Desmond WJ, Bartholomew RM, Wang R. Monoclonal antibodies in clinical immunology. Clin Chem 1981; 27: l797- 1806.