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Novel and rapid immunofiltration assays with enhanced chemiluminescence detection
Journal of Immunological Methods, 135 (1990) 289-291 Elsevier
289
JIM 05798
L e t t e r to the editors
Novel and rapid immunofiltration assays with enhanced chemiluminescence detection J.K. H o r t o n , J.E. James, S. S w i n b u r n e a n d M.J. O ' S u l l i v a n Amersham International pIe, Forest Farm, Whitchurch, Cardiff, CF4 7YT Wales, U.K. (Received 1 June 1990, revised received 20 August 1990, accepted 14 September 1990)
Dear Editors, The development of solid-phase immunoassays using nitrocellulose as a support for either antigen or antibody have provided rapid alternative methods to both ELISA and RIA (IJsselmuiden et al., 1989). Here we describe rapid immunofiltration assays using enhanced chemiluminescence (Whitehead et al., 1983; Thorpe et al., 1985), for the detection of antibodies raised in mouse and rabbit and for the detection of total human IgG, IgM and IgA. The assays were carried out on membrane-bottomed 96-well microtitre plates (Amersham International plc, Amersham, Bucks, U.K.) which have a typical 96-well format featuring open-bottomed wells individually sealed with a nitrocellulose filter. The membrane-bottomed plates are designed to be used with a single or multiple (ten plate) vacuum manifold (Amersham). The vacuum can be generated with a simple water pump or low cost electrical pump. In order to detect specific mouse and rabbit antibodies, the membrane-bottomed plates were coated with antigen (200 #l/well; see figure legends) and incubated for 15 rain at room temperature. After drawing the solution through the membrane-bottomed plate by vacuum, the filter was washed ( x 3) by adding 200 #1 of phosphatebuffered saline (PBS) p H 7.4, containing 0.5% ( v / v ) Tween 20 (PBS-T)/well under vacuum. Any remaining active sites on the membrane were
Correspondence to: J.K. Horton, Amersham International pic, Forest Farm, Whitchurch, Cardiff, CF4 7YT Wales, U.K.
blocked by incubating with 1% ( w / v ) dried milk powder dissolved in PBS (200 #l/well) for 15 rain at room temperature. The wells were washed as before and samples (100 #l/well) containing the appropriate antibody added and incubated for 15 min at room temperature. The wells were washed to remove unbound material as above and horseradish peroxidase-labelled detecting antibody (100 gl/well) in PBS was then added. Following an incubation for 15 rain the plates were washed as before, and activity of the bound enzyme conjugate revealed by the addition (50 /zl/well) of enhanced chemiluminescence signal reagent (Amersham). Chemiluminescence was measured immediately in a microtitre plate luminometer (Amersham). Representative dilution curves for the detection of monoclonal antibodies to human chorionic gonadotrophin (hCG) (Hotton et al., 1989) and rabbit polyclonal antibodies to cyclic AMP (from a commercial cAMP immunoassay kit, Amersham) are shown in Figs. 1 and 2. The monoclonal antibodies to h C G diluted 1/2000 and the rabbit polyclonal anti-cAMP antibodies diluted 1/200,000 still exhibited activities greater than background values. Control experiments involved the use of monoclonal antibodies and rabbit hyperimmune antisera to irrelevant proteins. A further refinement of the technique was the quantitative detection of human immunoglobufins. Membrane-bottomed plates were coated with polyvalent goat anti-human immunoglubulin (IgG fraction, 10 g g / m l in PBS, 200 /~l/well, Sigma), and incubated for 30 min at room temperature. The filter was washed with PBS-T and blocked as
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
290 350
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Fig. 1. Enhanced chemiluminescencedetection of antibodies to hCG. Membrane-bottomed plates were coated with hCG (10 /~g/ml in PBS), washed and blocked before application of anti-hCG (1/100-1/32130 in PBS). The plates were washed and diluted (1/500) peroxidase-labeUed sheep anti-mouse Ig (Amersham) added. The plates were washed and activity of the bound enzyme measured (in relative units) by chemiluminescence. The data shows the mean (+ 1 SD) of quadruplicate determinations. Non-specific binding is represented on the ordinate as the amount of light emitted in the absence of antibodies to hCG.
described previously. The m e m b r a n e was washed as before, h u m a n i m m u n o g l o b u l i n standards ( 0 100 n g / w e l l ) (IgG, IgA, Sigma, IgM, Calbiochem) a d d e d a n d i n c u b a t e d for 60 m i n at r o o m temperature. The wells were washed with PBS-T a n d 100 /~1 of diluted (in PBS) peroxidase-labelled goat a n t i - h u m a n I g G ( 1 / 2 5 0 , Sigma), peroxidaselabelled goat a n t i - h u m a n IgM ( 1 / 5 0 0 , Sigma) a n d peroxidase-labelled goat a n t i - h u m a n IgA ( 1 / 5 0 0 , Sigma) added, as appropriate. T h e labelled antibodies were i n c u b a t e d for 60 m i n at r o o m temperature, the wells washed with PBS-T a n d enzyme activity measured b y c h e m i l u m i n e s c e n c e as before. Representative dose-response curves for the detection of h u m a n IgG, IgM a n d IgA are shown in Fig. 3. F r o m these curves, as little as 5 n g of h u m a n i m m u n o g l o b u l i n s can be detected. I n conclusion, the i m m u n o f i l t r a t i o n assay procedure described here is simple a n d rapid to perform a n d has several key advantages over conventional techniques such as E L I S A or ILIA. I n contrast to microtitre plate based ELISA, using the single or ten station v a c u u m manifolds, a n y n u m ber of m u r i n e or r a b b i t a n t i b o d y samples, between 1 a n d 960, c a n be processed in a b o u t 90 rain, reducing b o t h workload a n d cost per test. Further-
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Fig. 2. Enhanced chemiluminescencedetection of antibodies to cyclic AMP. Cyclic AMP (0.25 #mol; 10 Fmol/ml) was conjugated to mouse IgG (0.025/tmol) through an activated N-hydroxysuccinimide ester (Erlanger, 1973). The ester was prepared by reaction of the carboxylic acid group of 2',O-monosuccinyladenosine-3',5'-cyclic monophosphate (Sigma) with N-hydroxysuccinimide (Sigma) in the presence of 1-ethyl-3(3dimethylaminopropyl) carbodiimide hydrochloride (Sigma). Membrane-bottomed plates were coated with the cyclic AMP conjugate (0.025/~mol/ml in PBS); washed and blocked before application of anti-cyclic-AMP (1/8000-1/500,000 in PBS). The plates were washed and diluted (1/500) peroxidase-labelled donkey anti-rabbit Ig (Amersham) added. The plates were washed and activity of the bound enzyme measured in relative units by chemiluminescence. The data shows the mean (+ 1 SD) of quadruplicate determinations. Non-specific binding is represented on the ordinate as the amount of light emitted in the absence of antibody. 60
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Fig. 3. Enhanced chemiluminescence detection of human immunoglobulins. Standard curves are shown for human IgG (O); human lgA (~); and human IgM (c=). The data shows the mean (_ 1 SD) of quadruplicate determinations. Experimental detail is outlined in the text. Non-specific binding values for IgG (0), lgA (,,,) and IgM (c=) are shown on the ordinate as light emission in the absence of the respective immunoglobulin.
291
more the technique has all of the advantages of enhanced chemiluminescence assays. For example, the rapid signal generation compares very favourably with the 30-60 min needed for many enzyme immunoassays. In addition, the technique has none of the problems of radioactive waste disposal and does not suffer from short isotopic half-life. Finally, the procedure can be readily adapted for the quantitative detection of molecules of biological interest, such as human immunoglobulins.
References Erlanger, B.F. (1973) Principles and methods for the preparation of drug protein conjugates for immunological studies. Pharmacol. Rev. 25, 271-280.
Horton, J.K., Evans, O.M., Swarm, K. and Swinburne, S. (1989) A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres. J. Immunol. Methods 124, 225-230. IJsselmuiden, O.E., Herbrink, P., Meddens, M.J.M., Tank, B., Stolz, E. and Van Eijk, R.V.W. (1989) Optimizing the solid-phase immunofiltration assay. J. Immunol. Methods 119, 35-43. Thorpe, G.H.G., Kricka, L.J., Mosely, S.B. and Whitehead, T.P. (1985) Phenols as enhancers of the chemiluminescent horseradish peroxidase reaction, applications in luminescence monitored enzyme immunoassays. Clin. Chem. 31, 1335-1341. Whitehead, T.P., Thorpe, G.H.G., Carter, T.J.N., Groucatt, C. and Kricka, L.J. (1983) Enhanced luminescence procedure for sensitive determination of peroxidase-labelled conjugates in immunoassay. Nature 305, 158-159.
289
JIM 05798
L e t t e r to the editors
Novel and rapid immunofiltration assays with enhanced chemiluminescence detection J.K. H o r t o n , J.E. James, S. S w i n b u r n e a n d M.J. O ' S u l l i v a n Amersham International pIe, Forest Farm, Whitchurch, Cardiff, CF4 7YT Wales, U.K. (Received 1 June 1990, revised received 20 August 1990, accepted 14 September 1990)
Dear Editors, The development of solid-phase immunoassays using nitrocellulose as a support for either antigen or antibody have provided rapid alternative methods to both ELISA and RIA (IJsselmuiden et al., 1989). Here we describe rapid immunofiltration assays using enhanced chemiluminescence (Whitehead et al., 1983; Thorpe et al., 1985), for the detection of antibodies raised in mouse and rabbit and for the detection of total human IgG, IgM and IgA. The assays were carried out on membrane-bottomed 96-well microtitre plates (Amersham International plc, Amersham, Bucks, U.K.) which have a typical 96-well format featuring open-bottomed wells individually sealed with a nitrocellulose filter. The membrane-bottomed plates are designed to be used with a single or multiple (ten plate) vacuum manifold (Amersham). The vacuum can be generated with a simple water pump or low cost electrical pump. In order to detect specific mouse and rabbit antibodies, the membrane-bottomed plates were coated with antigen (200 #l/well; see figure legends) and incubated for 15 rain at room temperature. After drawing the solution through the membrane-bottomed plate by vacuum, the filter was washed ( x 3) by adding 200 #1 of phosphatebuffered saline (PBS) p H 7.4, containing 0.5% ( v / v ) Tween 20 (PBS-T)/well under vacuum. Any remaining active sites on the membrane were
Correspondence to: J.K. Horton, Amersham International pic, Forest Farm, Whitchurch, Cardiff, CF4 7YT Wales, U.K.
blocked by incubating with 1% ( w / v ) dried milk powder dissolved in PBS (200 #l/well) for 15 rain at room temperature. The wells were washed as before and samples (100 #l/well) containing the appropriate antibody added and incubated for 15 min at room temperature. The wells were washed to remove unbound material as above and horseradish peroxidase-labelled detecting antibody (100 gl/well) in PBS was then added. Following an incubation for 15 rain the plates were washed as before, and activity of the bound enzyme conjugate revealed by the addition (50 /zl/well) of enhanced chemiluminescence signal reagent (Amersham). Chemiluminescence was measured immediately in a microtitre plate luminometer (Amersham). Representative dilution curves for the detection of monoclonal antibodies to human chorionic gonadotrophin (hCG) (Hotton et al., 1989) and rabbit polyclonal antibodies to cyclic AMP (from a commercial cAMP immunoassay kit, Amersham) are shown in Figs. 1 and 2. The monoclonal antibodies to h C G diluted 1/2000 and the rabbit polyclonal anti-cAMP antibodies diluted 1/200,000 still exhibited activities greater than background values. Control experiments involved the use of monoclonal antibodies and rabbit hyperimmune antisera to irrelevant proteins. A further refinement of the technique was the quantitative detection of human immunoglobufins. Membrane-bottomed plates were coated with polyvalent goat anti-human immunoglubulin (IgG fraction, 10 g g / m l in PBS, 200 /~l/well, Sigma), and incubated for 30 min at room temperature. The filter was washed with PBS-T and blocked as
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
290 350
50
II-
I
I
I I
I
I
i
I
I I II
I
I
I
,
,
, ,,
I
280 (:3 uJ I-I--
~ 3o
210
U.J
I--r 0
10
0
/I
I 100
,
,
,
i
~ ,,~t
L
1000
b
,
L
,
,,
10000
140
.-I
70
ANTIBODY DILUTION
Fig. 1. Enhanced chemiluminescencedetection of antibodies to hCG. Membrane-bottomed plates were coated with hCG (10 /~g/ml in PBS), washed and blocked before application of anti-hCG (1/100-1/32130 in PBS). The plates were washed and diluted (1/500) peroxidase-labeUed sheep anti-mouse Ig (Amersham) added. The plates were washed and activity of the bound enzyme measured (in relative units) by chemiluminescence. The data shows the mean (+ 1 SD) of quadruplicate determinations. Non-specific binding is represented on the ordinate as the amount of light emitted in the absence of antibodies to hCG.
described previously. The m e m b r a n e was washed as before, h u m a n i m m u n o g l o b u l i n standards ( 0 100 n g / w e l l ) (IgG, IgA, Sigma, IgM, Calbiochem) a d d e d a n d i n c u b a t e d for 60 m i n at r o o m temperature. The wells were washed with PBS-T a n d 100 /~1 of diluted (in PBS) peroxidase-labelled goat a n t i - h u m a n I g G ( 1 / 2 5 0 , Sigma), peroxidaselabelled goat a n t i - h u m a n IgM ( 1 / 5 0 0 , Sigma) a n d peroxidase-labelled goat a n t i - h u m a n IgA ( 1 / 5 0 0 , Sigma) added, as appropriate. T h e labelled antibodies were i n c u b a t e d for 60 m i n at r o o m temperature, the wells washed with PBS-T a n d enzyme activity measured b y c h e m i l u m i n e s c e n c e as before. Representative dose-response curves for the detection of h u m a n IgG, IgM a n d IgA are shown in Fig. 3. F r o m these curves, as little as 5 n g of h u m a n i m m u n o g l o b u l i n s can be detected. I n conclusion, the i m m u n o f i l t r a t i o n assay procedure described here is simple a n d rapid to perform a n d has several key advantages over conventional techniques such as E L I S A or ILIA. I n contrast to microtitre plate based ELISA, using the single or ten station v a c u u m manifolds, a n y n u m ber of m u r i n e or r a b b i t a n t i b o d y samples, between 1 a n d 960, c a n be processed in a b o u t 90 rain, reducing b o t h workload a n d cost per test. Further-
0
~
i
0
, , .... i
1000
,
, t,,,,]
104 ANTIBODY
105
10 °
DILUTION
Fig. 2. Enhanced chemiluminescencedetection of antibodies to cyclic AMP. Cyclic AMP (0.25 #mol; 10 Fmol/ml) was conjugated to mouse IgG (0.025/tmol) through an activated N-hydroxysuccinimide ester (Erlanger, 1973). The ester was prepared by reaction of the carboxylic acid group of 2',O-monosuccinyladenosine-3',5'-cyclic monophosphate (Sigma) with N-hydroxysuccinimide (Sigma) in the presence of 1-ethyl-3(3dimethylaminopropyl) carbodiimide hydrochloride (Sigma). Membrane-bottomed plates were coated with the cyclic AMP conjugate (0.025/~mol/ml in PBS); washed and blocked before application of anti-cyclic-AMP (1/8000-1/500,000 in PBS). The plates were washed and diluted (1/500) peroxidase-labelled donkey anti-rabbit Ig (Amersham) added. The plates were washed and activity of the bound enzyme measured in relative units by chemiluminescence. The data shows the mean (+ 1 SD) of quadruplicate determinations. Non-specific binding is represented on the ordinate as the amount of light emitted in the absence of antibody. 60
/j
p..
I.-
36
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24
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i
1
. . . . . . . .
i
10
. . . . . . . .
i
. . . . . . . .
1OO
,
1000
[IMMUNOGLOBULINS] ng/ml
Fig. 3. Enhanced chemiluminescence detection of human immunoglobulins. Standard curves are shown for human IgG (O); human lgA (~); and human IgM (c=). The data shows the mean (_ 1 SD) of quadruplicate determinations. Experimental detail is outlined in the text. Non-specific binding values for IgG (0), lgA (,,,) and IgM (c=) are shown on the ordinate as light emission in the absence of the respective immunoglobulin.
291
more the technique has all of the advantages of enhanced chemiluminescence assays. For example, the rapid signal generation compares very favourably with the 30-60 min needed for many enzyme immunoassays. In addition, the technique has none of the problems of radioactive waste disposal and does not suffer from short isotopic half-life. Finally, the procedure can be readily adapted for the quantitative detection of molecules of biological interest, such as human immunoglobulins.
References Erlanger, B.F. (1973) Principles and methods for the preparation of drug protein conjugates for immunological studies. Pharmacol. Rev. 25, 271-280.
Horton, J.K., Evans, O.M., Swarm, K. and Swinburne, S. (1989) A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres. J. Immunol. Methods 124, 225-230. IJsselmuiden, O.E., Herbrink, P., Meddens, M.J.M., Tank, B., Stolz, E. and Van Eijk, R.V.W. (1989) Optimizing the solid-phase immunofiltration assay. J. Immunol. Methods 119, 35-43. Thorpe, G.H.G., Kricka, L.J., Mosely, S.B. and Whitehead, T.P. (1985) Phenols as enhancers of the chemiluminescent horseradish peroxidase reaction, applications in luminescence monitored enzyme immunoassays. Clin. Chem. 31, 1335-1341. Whitehead, T.P., Thorpe, G.H.G., Carter, T.J.N., Groucatt, C. and Kricka, L.J. (1983) Enhanced luminescence procedure for sensitive determination of peroxidase-labelled conjugates in immunoassay. Nature 305, 158-159.