101
Journal of lmmunological Methods, 156 (1992) 101-105
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM06493
An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase D a n i e l U. R a b i n , R e b e c c a P a l m e r - C r o c k e r , D i a n e V. M i e r z a n d K w o k K. Y e u n g Miles Inc., Diagnostics Division, West Haven, CT, USA
(Received 27 April 1992,revised received 2 June 1992, accepted 8 June 1992)
A sandwich capture ELISA technique is presented for detection of recombinant proteins sharing a common affinity domain or reagents such as antibodies that bind to these proteins. An activated carrier protein (BSA) is modified with reduced glutathione (GT), forming an affinity capture reagent for glutathione-S-transferase (GST) and recombinant fusion proteins bearing the GST moiety. GT-BSA is immobilized on microtiter plates, and a sandwich is formed consisting of the recombinant fusion protein, reactive antibodies, and detection antibodies. An example is given that demonstrates that this format yields equivalent results to a conventional ELISA test with a panel of newly diagnosed diabetic sera reacting with an islet cell autoantigen. Key words: Glutathione-S-transferase;ELISA; Fusion protein capture
Introduction
The use of enzyme-linked immunosorbent assays (ELISA) in clinical and research laboratories has become widespread (Engvall and Perlmann, 1971, 1972; Kemeny and Challacombe, 1988). Such assays can be quantitative, rapid, dependable, and many samples can be tested in parallel. In the most common format an antigen is immobilized by adsorption to the wells of a microtiter plate, and then reacted with the analyte. The analyte is detected with a secondary reagent such as an enzyme-labelled antibody.
Correspondence to: D.U. Rabin, MRC, 400 Morgan Lane, West Haven, CT 06516, USA. Tel.: (203) 937-2895; Fax: (203) 937-5467. Abbreviations: BSA, bovine serum" albumin; GT, glutathione; PBS, phosphate-buffered saline; GST, glutathioneS-transferase; ICA, islet,cell antibodies.
In some situations ELISA measurement by direct immobilization of the antigen is not practical, usually due to the state of the antigen. For example, non-ionic detergents are useful for extraction of proteins and can help promote specificity in immunoreactions, but interfere with the efficient binding of proteins to the polystyrene supports commonly in use. With some proteins antigenic epitopes are lost when the proteins are bound directly to plastic. In addition, the amount of antigen that can be immobilized is limited by the binding capacity of such supports. This is usually not a problem, but can present difficulties when the antigen is Part of a complex mixture. Finally, when multiple antigens are being examined, it is convenient to use a single system of immobilization and detection. In this work we present a sandwich capture assay format as an alternative to the conventional ELISA format that may be useful in some of the situations outlined above.
102 The p G E X vector (Smith and Johnson, 1988) has proven useful as a high level expression system that yields a glutathione-S-transferase (GST) fusion protein. The GST enables a one-step purification by means of glutathione affinity chromatography. The method described in this study combines the advantages of the glutathione-GST affinity capture with the convenience of the microtiter-based ELISA format. Recombinant antigens used in this study have been isolated from a human islet A g t l l c D N A library. Characterization of the fl-galactosidase fusion proteins ICA12 and ICA512 indicates that they react specifically with sera from newly diagnosed type I diabetic patients (Rabin et al., 1992), and may be useful as part of a panel of antigens for diagnosis of this disorder. On the basis of DNA sequencing, both ICA12 (MW = 22 kDa) and ICA512 ( M W = 3 3 kDa) appear to be COOH-terminal fragments of larger proteins. ICA512 and ICA514 (MW = 36 kDa), which contains the putative full-length version of ICA12, are used to characterize the ELISA sandwich capture assay.
Materials
and methods
Glutathione-BSA coupling Reduced glutathione (2 mg, Sigma G-4251) was reacted with maleimide-activated BSA (2 mg, Pierce 77115 G) in 700 /~1 PBS (10 mM phosphate pH 7.5/137 mM NaCI/2.7 mM KC1) for 2 h at room temperature. Aliquots were frozen at - 70oC. Expression The EcoRI inserts from h gt11 clones ICA512 and ICA514 (a clone representing a larger version of ICA12 (Rabin et al., 1992)) were cloned into the p G E X vector and expressed as GST fusion proteins (Smith and Johnson, 1988). The recombinant proteins were purified, and ICA512 was cleaved with thrombin as described by the manufacturer (Pharmacia). ELISA GT-BSA was diluted in 0.1 M N a H C O 2 / NaeCO 2 pH 9.5. Wells of an Immulon-1 mi-
crotiter plate (Dynatech, Chantilly, VA) were coated with 50 /~l of solution overnight. The plates were blocked for 30 min with b l o t t o / T w e e n 20 (5% Carnation non-fat dry milk, 10 mM Tris pH 8.0, 150 mM NaCI, 0.05% Tween 20, and 0.05% sodium azide (Johnson et al., 1984)) for 1 h and then washed. Fusion proteins and antibodies were diluted in b l o t t o / T w e e n , and allowed to react for 60 min; on an orbital shaker. Between incubations the microtiter plates were washed 5 x with P B S / T w e e n . Ascites fluid from monoclonal antibody (mAb) 204.2 (raised against ICA12) was diluted 1/1000 and human sera were diluted 1/30. Bound antibodies were detected with alkaline phosphatase labelled second antibody (Sigma anti-human IgG A-0287 or Dako anti-mouse D314) at a 1/1000 dilution in b l o t t o / Tween for 60 min. The plate was developed with p-nitrophenylphosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgC1 z pH 9.8 and read at 405 nm. All incubations were at room temperature. For direct ELISA testing of ICA512, 30 ng of cleaved and purified ICA512 was deposited in each well of an Immulon-2 microtiter plate in TBS buffer (20 mM Tris pH 8.0, 150 mM NaCI). The plate was blocked with b l o t t o / T w e e n 20 and then reacted in duplicate with human sera.
Sera Diabetic sera were obtained from juvenile onset diabetic subjects and kindly provided by Dr. David Goldstein of the University of Missouri Medical Center, Columbia. The normal serum used in Fig. 2 was from a volunteer at the Miles Research Center. All sera were obtained with informed consent.
Results
The capture format shown in Fig. 1 utilizes a conjugate of glutathione and BSA as an affinity capture system. A one step reaction was performed to couple commercially available reagents. The experiments described here indicate that purification of the capture conjugate may not be necessary.
103
Capture of GST514 by GT-BSA 5"
DETECTION ANTIBODY
4" GST514,
ng 0
¢
a
ANTIBODY
o
10 30
2,
=
ANTIGEN (I
100 300
A
1.
1000
GST o
0
~10
•
T 30
.
GT-BSA,
Fig. 1. Sandwich capture format. Glutathione-BSA is immobilized on the solid support and binds the GST fusion protein. Binding of an antifusion protein antibody and detection with a labelled second antibody is shown.
A variety of cocktails are used by investigators to block unoccupied protein binding sites on E L I S A solid supports. Common recipes include BSA, Tween 20, both reagents, or a non-fat dry milk formulation (blotto). Fig. 2 shows a comparison of three of these blocking buffers: blotto, PBS, and B S A in PBS, all containing 0.05% Tween 20. Though the PBS and P B S / B S A conditions allowed slightly more sensitive reaction with
BLOCKING BUFFER 3
,q.
•
+
÷
[]
+
.
Human []
Blotto
PBS
+
serum +
PBS/BSA
Fig. 2. Selection of ELISA blocking buffer. Wells were coated with 100 ng of GT-BSA, incubated with 300 ng GST-514, mAb 204.2 and detection antibody. Only when both GST514 and GT-BSA are present is there a positive signal (solid bar). The open bar shows the signal obtained with a normal human serum. The error bars in this and subsequent figures indicate the standard deviation of duplicate measurements. The blocking buffers (x axis) all contained 0.05% Tween 20.
-,
•
100
","
300
,
",
1000
ng
Fig. 3. Titration of sandwich components. the mAb, they also showed higher background with a normal human serum. This serum had b e e n shown by W e s t e r n blotting to be non-reactive with ICA12. Fig. 3 shows a titration of G T - B S A and GST514 over the range o f . 0 - 1 0 0 0 ng. On the basis of this curve, 100 ng G T - B S A and 300 ng recombinant antigen were used as standard concentrations. To test whether the conditions derived for ICA514 were applicable to a n o t h e r fusion antigen, a panel of diabetic sera were tested with ICA512, an islet cell autoantigen whose sequence and antigen profile are u n r e l a t e d to ICA514 (data not shown). A series of optimization studies has shown that ICA512 gives the most sensitive and specific reactivity with diabetic sera when the fusion p r o t e i n is cleaved, purified, and bound at 30 n g / w e l l to Immulon-2 plates in a TBS buffer. In a separate experiment the uncleaved fusion protein was c a p t u r e d with G T - B S A and reacted with the same panel of 40 sera. The two E L I S A formats are juxtaposed in Fig. 4. This result indicates that the sandwich capture E L I S A format is a viable alternative to a conventional E L I S A configuration.
Discussion
A modification of the common E L I S A test has been d e m o n s t r a t e d that may be of use in some situations where the conventional format is not
104
ICA512 Capture and ELISA
2t
•
Capture
1
8
0
Diabetic
Sera
Fig. 4. Comparison of conventional ELISA with the sandwich capture format. A panel of 40 sera from newly diagnosed diabetic subjects was tested with the sandwich capture format (solid bars) and by conventional ELISA (open bars). The conventional ELISA data has been scaled by a factor of 0.45 for comparison purposes.
applicable. A recombinant fusion protein (GSTICA514) and a cognate m A b (mAb 204.2) were used to validate the system and optimize some of the p a r a m e t e r s such as the blocking buffer and the G T - B S A and antigen concentrations. To test the general applicability of the sandwich capture method, an unrelated antigen was tested with a panel of human diabetic sera. The capture method gives comparable results to a direct E L I S A method which had been independently optimized (Fig. 4). The signal obtained with the sandwich capture assay is somewhat lower than with the direct ELISA. It is possible that this may be a disadvantage with sensitive m e a s u r e m e n t s or with human serum samples. In addition, the capture method entails more reagents than direct ELISA, which could introduce non-specific reaction, especially with human sera. Fig. 3 demonstrates that it is possible to detect 30 ng of GST514 in a 5% solution of blotto (2.5
mg in 50 Izl). In such a mixture it would not be practical to detect this amount of antigen by Western blotting or by direct ELISA. The sandwich capture assay could thus be used to detect fusion proteins at low levels in mixtures such as cell lysates in the presence of nonionic detergents. With conformationally sensitive epitopes the sandwich capture format may offer an advantage, as the antigen is not bound directly to the solid support and may not suffer loss of immunoreactivity. The sandwich capture format also permits solution reaction of the fusion protein with the analyte prior to immobilization. This should permit faster reaction, as the immunoreaction is not limited by diffusion to the solid support. In cases where multiple recombinant clones are under analysis, crude fractions can be applied to the immobilized G T - B S A and probed with an
105 affinity r e a g e n t . F o r e x a m p l e , l a r g e n u m b e r s o f clones g e n e r a t e d by d e l e t i o n o r r a n d o m m u t a g e n esis c o u l d b e s c r e e n e d for e p i t o p e m u t a n t s . Bact e r i a l m i n i - p r e p s can b e lysed a n d d i r e c t l y app l i e d to G T - B S A c o a t e d wells, w a s h e d , a n d t h e n p r o b e d with specific r e a g e n t s . In s u m m a r y , we have d e m o n s t r a t e d t h a t a simple, c o n v e n i e n t affinity c a p t u r e m e t h o d for rec o m b i n a n t fusion p r o t e i n s c a n b e u s e d as an a l t e r n a t i v e to c o n v e n t i o n a l E L I S A . This techn i q u e m a y b e useful w h e n t h e n u m b e r o r state o f recombinant antigens makes conventional ELISA impractical.
Acknowledgement W e t h a n k S u s a n P l e a s i c for t h e I C A 5 1 2 d i r e c t E L I S A data.
References Engvall, E. and Perlmann, P. (1971) Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871-879. Engvatl, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA, III. Quantitation of specific antibodies by enzyme-labeled antiimmunoglobulin in antigen-coated tubes. J. Immunol. 109, 129-135. Johnson, D.A,, Gautsch, J.W., Sportsman, J.R. and Elder, J.H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1, 3-8. Kemeny, D.M. and Challacombe, S.J. (1988) ELISA and Other Solid Phase Immunoassays; Theoretical and Practical Aspects. Wiley, New York. Rabin, D.U., Pleasic, S.M., Palmer-Crocker, R. and Shapiro, J.A. (1992) Cloning and expression of IDDM-specific human autoantigens. Diabetes 41, 183-186. Smith, D.B. and Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67, 31-40.
Journal of lmmunological Methods, 156 (1992) 101-105
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM06493
An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase D a n i e l U. R a b i n , R e b e c c a P a l m e r - C r o c k e r , D i a n e V. M i e r z a n d K w o k K. Y e u n g Miles Inc., Diagnostics Division, West Haven, CT, USA
(Received 27 April 1992,revised received 2 June 1992, accepted 8 June 1992)
A sandwich capture ELISA technique is presented for detection of recombinant proteins sharing a common affinity domain or reagents such as antibodies that bind to these proteins. An activated carrier protein (BSA) is modified with reduced glutathione (GT), forming an affinity capture reagent for glutathione-S-transferase (GST) and recombinant fusion proteins bearing the GST moiety. GT-BSA is immobilized on microtiter plates, and a sandwich is formed consisting of the recombinant fusion protein, reactive antibodies, and detection antibodies. An example is given that demonstrates that this format yields equivalent results to a conventional ELISA test with a panel of newly diagnosed diabetic sera reacting with an islet cell autoantigen. Key words: Glutathione-S-transferase;ELISA; Fusion protein capture
Introduction
The use of enzyme-linked immunosorbent assays (ELISA) in clinical and research laboratories has become widespread (Engvall and Perlmann, 1971, 1972; Kemeny and Challacombe, 1988). Such assays can be quantitative, rapid, dependable, and many samples can be tested in parallel. In the most common format an antigen is immobilized by adsorption to the wells of a microtiter plate, and then reacted with the analyte. The analyte is detected with a secondary reagent such as an enzyme-labelled antibody.
Correspondence to: D.U. Rabin, MRC, 400 Morgan Lane, West Haven, CT 06516, USA. Tel.: (203) 937-2895; Fax: (203) 937-5467. Abbreviations: BSA, bovine serum" albumin; GT, glutathione; PBS, phosphate-buffered saline; GST, glutathioneS-transferase; ICA, islet,cell antibodies.
In some situations ELISA measurement by direct immobilization of the antigen is not practical, usually due to the state of the antigen. For example, non-ionic detergents are useful for extraction of proteins and can help promote specificity in immunoreactions, but interfere with the efficient binding of proteins to the polystyrene supports commonly in use. With some proteins antigenic epitopes are lost when the proteins are bound directly to plastic. In addition, the amount of antigen that can be immobilized is limited by the binding capacity of such supports. This is usually not a problem, but can present difficulties when the antigen is Part of a complex mixture. Finally, when multiple antigens are being examined, it is convenient to use a single system of immobilization and detection. In this work we present a sandwich capture assay format as an alternative to the conventional ELISA format that may be useful in some of the situations outlined above.
102 The p G E X vector (Smith and Johnson, 1988) has proven useful as a high level expression system that yields a glutathione-S-transferase (GST) fusion protein. The GST enables a one-step purification by means of glutathione affinity chromatography. The method described in this study combines the advantages of the glutathione-GST affinity capture with the convenience of the microtiter-based ELISA format. Recombinant antigens used in this study have been isolated from a human islet A g t l l c D N A library. Characterization of the fl-galactosidase fusion proteins ICA12 and ICA512 indicates that they react specifically with sera from newly diagnosed type I diabetic patients (Rabin et al., 1992), and may be useful as part of a panel of antigens for diagnosis of this disorder. On the basis of DNA sequencing, both ICA12 (MW = 22 kDa) and ICA512 ( M W = 3 3 kDa) appear to be COOH-terminal fragments of larger proteins. ICA512 and ICA514 (MW = 36 kDa), which contains the putative full-length version of ICA12, are used to characterize the ELISA sandwich capture assay.
Materials
and methods
Glutathione-BSA coupling Reduced glutathione (2 mg, Sigma G-4251) was reacted with maleimide-activated BSA (2 mg, Pierce 77115 G) in 700 /~1 PBS (10 mM phosphate pH 7.5/137 mM NaCI/2.7 mM KC1) for 2 h at room temperature. Aliquots were frozen at - 70oC. Expression The EcoRI inserts from h gt11 clones ICA512 and ICA514 (a clone representing a larger version of ICA12 (Rabin et al., 1992)) were cloned into the p G E X vector and expressed as GST fusion proteins (Smith and Johnson, 1988). The recombinant proteins were purified, and ICA512 was cleaved with thrombin as described by the manufacturer (Pharmacia). ELISA GT-BSA was diluted in 0.1 M N a H C O 2 / NaeCO 2 pH 9.5. Wells of an Immulon-1 mi-
crotiter plate (Dynatech, Chantilly, VA) were coated with 50 /~l of solution overnight. The plates were blocked for 30 min with b l o t t o / T w e e n 20 (5% Carnation non-fat dry milk, 10 mM Tris pH 8.0, 150 mM NaCI, 0.05% Tween 20, and 0.05% sodium azide (Johnson et al., 1984)) for 1 h and then washed. Fusion proteins and antibodies were diluted in b l o t t o / T w e e n , and allowed to react for 60 min; on an orbital shaker. Between incubations the microtiter plates were washed 5 x with P B S / T w e e n . Ascites fluid from monoclonal antibody (mAb) 204.2 (raised against ICA12) was diluted 1/1000 and human sera were diluted 1/30. Bound antibodies were detected with alkaline phosphatase labelled second antibody (Sigma anti-human IgG A-0287 or Dako anti-mouse D314) at a 1/1000 dilution in b l o t t o / Tween for 60 min. The plate was developed with p-nitrophenylphosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgC1 z pH 9.8 and read at 405 nm. All incubations were at room temperature. For direct ELISA testing of ICA512, 30 ng of cleaved and purified ICA512 was deposited in each well of an Immulon-2 microtiter plate in TBS buffer (20 mM Tris pH 8.0, 150 mM NaCI). The plate was blocked with b l o t t o / T w e e n 20 and then reacted in duplicate with human sera.
Sera Diabetic sera were obtained from juvenile onset diabetic subjects and kindly provided by Dr. David Goldstein of the University of Missouri Medical Center, Columbia. The normal serum used in Fig. 2 was from a volunteer at the Miles Research Center. All sera were obtained with informed consent.
Results
The capture format shown in Fig. 1 utilizes a conjugate of glutathione and BSA as an affinity capture system. A one step reaction was performed to couple commercially available reagents. The experiments described here indicate that purification of the capture conjugate may not be necessary.
103
Capture of GST514 by GT-BSA 5"
DETECTION ANTIBODY
4" GST514,
ng 0
¢
a
ANTIBODY
o
10 30
2,
=
ANTIGEN (I
100 300
A
1.
1000
GST o
0
~10
•
T 30
.
GT-BSA,
Fig. 1. Sandwich capture format. Glutathione-BSA is immobilized on the solid support and binds the GST fusion protein. Binding of an antifusion protein antibody and detection with a labelled second antibody is shown.
A variety of cocktails are used by investigators to block unoccupied protein binding sites on E L I S A solid supports. Common recipes include BSA, Tween 20, both reagents, or a non-fat dry milk formulation (blotto). Fig. 2 shows a comparison of three of these blocking buffers: blotto, PBS, and B S A in PBS, all containing 0.05% Tween 20. Though the PBS and P B S / B S A conditions allowed slightly more sensitive reaction with
BLOCKING BUFFER 3
,q.
•
+
÷
[]
+
.
Human []
Blotto
PBS
+
serum +
PBS/BSA
Fig. 2. Selection of ELISA blocking buffer. Wells were coated with 100 ng of GT-BSA, incubated with 300 ng GST-514, mAb 204.2 and detection antibody. Only when both GST514 and GT-BSA are present is there a positive signal (solid bar). The open bar shows the signal obtained with a normal human serum. The error bars in this and subsequent figures indicate the standard deviation of duplicate measurements. The blocking buffers (x axis) all contained 0.05% Tween 20.
-,
•
100
","
300
,
",
1000
ng
Fig. 3. Titration of sandwich components. the mAb, they also showed higher background with a normal human serum. This serum had b e e n shown by W e s t e r n blotting to be non-reactive with ICA12. Fig. 3 shows a titration of G T - B S A and GST514 over the range o f . 0 - 1 0 0 0 ng. On the basis of this curve, 100 ng G T - B S A and 300 ng recombinant antigen were used as standard concentrations. To test whether the conditions derived for ICA514 were applicable to a n o t h e r fusion antigen, a panel of diabetic sera were tested with ICA512, an islet cell autoantigen whose sequence and antigen profile are u n r e l a t e d to ICA514 (data not shown). A series of optimization studies has shown that ICA512 gives the most sensitive and specific reactivity with diabetic sera when the fusion p r o t e i n is cleaved, purified, and bound at 30 n g / w e l l to Immulon-2 plates in a TBS buffer. In a separate experiment the uncleaved fusion protein was c a p t u r e d with G T - B S A and reacted with the same panel of 40 sera. The two E L I S A formats are juxtaposed in Fig. 4. This result indicates that the sandwich capture E L I S A format is a viable alternative to a conventional E L I S A configuration.
Discussion
A modification of the common E L I S A test has been d e m o n s t r a t e d that may be of use in some situations where the conventional format is not
104
ICA512 Capture and ELISA
2t
•
Capture
1
8
0
Diabetic
Sera
Fig. 4. Comparison of conventional ELISA with the sandwich capture format. A panel of 40 sera from newly diagnosed diabetic subjects was tested with the sandwich capture format (solid bars) and by conventional ELISA (open bars). The conventional ELISA data has been scaled by a factor of 0.45 for comparison purposes.
applicable. A recombinant fusion protein (GSTICA514) and a cognate m A b (mAb 204.2) were used to validate the system and optimize some of the p a r a m e t e r s such as the blocking buffer and the G T - B S A and antigen concentrations. To test the general applicability of the sandwich capture method, an unrelated antigen was tested with a panel of human diabetic sera. The capture method gives comparable results to a direct E L I S A method which had been independently optimized (Fig. 4). The signal obtained with the sandwich capture assay is somewhat lower than with the direct ELISA. It is possible that this may be a disadvantage with sensitive m e a s u r e m e n t s or with human serum samples. In addition, the capture method entails more reagents than direct ELISA, which could introduce non-specific reaction, especially with human sera. Fig. 3 demonstrates that it is possible to detect 30 ng of GST514 in a 5% solution of blotto (2.5
mg in 50 Izl). In such a mixture it would not be practical to detect this amount of antigen by Western blotting or by direct ELISA. The sandwich capture assay could thus be used to detect fusion proteins at low levels in mixtures such as cell lysates in the presence of nonionic detergents. With conformationally sensitive epitopes the sandwich capture format may offer an advantage, as the antigen is not bound directly to the solid support and may not suffer loss of immunoreactivity. The sandwich capture format also permits solution reaction of the fusion protein with the analyte prior to immobilization. This should permit faster reaction, as the immunoreaction is not limited by diffusion to the solid support. In cases where multiple recombinant clones are under analysis, crude fractions can be applied to the immobilized G T - B S A and probed with an
105 affinity r e a g e n t . F o r e x a m p l e , l a r g e n u m b e r s o f clones g e n e r a t e d by d e l e t i o n o r r a n d o m m u t a g e n esis c o u l d b e s c r e e n e d for e p i t o p e m u t a n t s . Bact e r i a l m i n i - p r e p s can b e lysed a n d d i r e c t l y app l i e d to G T - B S A c o a t e d wells, w a s h e d , a n d t h e n p r o b e d with specific r e a g e n t s . In s u m m a r y , we have d e m o n s t r a t e d t h a t a simple, c o n v e n i e n t affinity c a p t u r e m e t h o d for rec o m b i n a n t fusion p r o t e i n s c a n b e u s e d as an a l t e r n a t i v e to c o n v e n t i o n a l E L I S A . This techn i q u e m a y b e useful w h e n t h e n u m b e r o r state o f recombinant antigens makes conventional ELISA impractical.
Acknowledgement W e t h a n k S u s a n P l e a s i c for t h e I C A 5 1 2 d i r e c t E L I S A data.
References Engvall, E. and Perlmann, P. (1971) Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871-879. Engvatl, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA, III. Quantitation of specific antibodies by enzyme-labeled antiimmunoglobulin in antigen-coated tubes. J. Immunol. 109, 129-135. Johnson, D.A,, Gautsch, J.W., Sportsman, J.R. and Elder, J.H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1, 3-8. Kemeny, D.M. and Challacombe, S.J. (1988) ELISA and Other Solid Phase Immunoassays; Theoretical and Practical Aspects. Wiley, New York. Rabin, D.U., Pleasic, S.M., Palmer-Crocker, R. and Shapiro, J.A. (1992) Cloning and expression of IDDM-specific human autoantigens. Diabetes 41, 183-186. Smith, D.B. and Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67, 31-40.
