Int. Archs Allergy appl. Immun.
51: 757-762 (1976)
Visualization Principles in Thin-Layer Immunoassays (TIA) on Plastic Surfaces1 H ans E lw in g , L ars-A ke N ilsson and Ö rjan O uchteri .ony Institute of Medical Microbiology. University of Göteborg, Göteborg
Abstract. Macromolecules may adsorb firmly as a monolayer to plastic sur faces and still retain their property to react specifically with antibodies. In the present communication four different principles for visualization of such antigenantibody interactions on plastic surfaces are described. In addition, a diffusion-ingel method for quantitation of antibodies is presented. The described methods of fer simple, sensitive and accurate means of assaying antigen-antibody reactions.
Many proteins as well as other macromolecules of antigenic nature have been shown to adsorb to hydrophobic plastic surface as a monolay er of molecules [1] usually retaining antigenic reactivity [2-4], Conse quently antigen coated plastic surfaces will act as immunosorbents when exposed to immune sera containing corresponding antibodies. Under such circumstances antigen-antibody complexes are formed on the plas tic surface. This immunoreaction will, in the following, be referred to as the primary reaction. Four different techniques for the visualization of such primary reactions are described in the present communication. In addition, a diffusion-in-gel method for quantitation of antibodies is pre sented where the recording in the assay is based on the aforementioned visualization principles. In the elaboration of the visualization techniques and the antibody quantitation method, we have employed a serological model system consisting of bovine serum albumin (BSA) and a rabbit anti-BSA im> This work supported by the Swedish Cancer Society and the Ellen, Walter and Lennart Hesselman Foundation.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 7:32:26 PM
Received: July 14. 1976.
758
E i .w ing /N ilsson/O uchteri ony
Ai^P
Cl
b
. A ...,.»
o o oo o
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•w
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• Condensation drops
Antigen-coated plastic surface A Antibody Kl Anti-immunoglobulin
•••
Plastic particles Sensitized chicken erythrocyte
Fig. /. Schematical illustration of the four visualization principles, a Water VCS visualization technique, b Serologically reinforced VCS visualization tech nique. c PAS visualization technique, d MHA visualization technique.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 7:32:26 PM
mune serum containing 1.6 mg specific antibody/ml as determined by quantitative precipitation [5]. The following series of experiments were performed. Antigen coating of plastic surfaces was performed by pouring a solu tion of BSA (100,//g/ml in 0.15 m NaCl) into polystyrole Petri dishes (Nunc AS, Roskilde, Denmark). The dishes were kept at room tempera ture for 30 min. After rinsing with 0.15 m NaCl, melted agar at a con centration of l°/o was poured into the dishes forming, after congelation, a 2.5-mm thick agar layer. Wells of 3 mm diameter were then punched in the gel and filled with anti-BSA in twofold serial dilutions if not oth erwise stated. Wells filled with normal rabbit serum were used as a con trol. After diffusion for 48 h at room temperature the agar gel was rinsed off, and the plates subjected to the following visualization proce dures. The water vapour condensation on surface (VCS) visualization tech nique. The Petri dish, rinsed from gel, was washed with distilled water and dried. The dish was then placed upside down over a container filled with water at 60 °C. After 1 min of exposure to water vapour the dish was removed and covered. A condensation pattern consisting of large confluent water drops was registrable over the circular areas where a primary antigen-antibody reaction had taken place. This hydrophilic pat tern contrasted to that over areas coated only with antigen where exclu-
Visualization of TIA on Plastic Surfaces
759
Fig. 2. Photographic recordings of visualized antigen-antibody reactions em ploying the diffusion-in-gel technique. Diffusion time 20 h. a VCS technique, b PAS technique, c MHA technique. Plastic surface coated with BSA, lOOug/ml. Application of antiserum by diffusion-in-gel procedure. Antiserum in a and b: left - undiluted, middle - diluted 1:10, c: left - 1:10, middle 1:100. Undiluted normal rabbit serum applied as controls in the holes to the right.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 7:32:26 PM
sively small drops of water were formed indicating a distinctly less hy drophilic surface. A schematical illustration of the VCS technique is giv en in figure la, and a photographic recording in figure 2a. The serologically reinforced VCS visualization technique. A goat an tiserum against rabbit immunoglobulin was poured into the Petri dish rinsed from gel. The antiserum was removed after 60 min at room tem perature. After washing and drying the Petri dish was subjected to the same visualization procedure as given above. The previously described two types of condensation patterns were recorded, the diameter of the corresponding hydrophilic areas being, however, larger. A schematical illustration of the reinforced VCS technique is given in figure lb. The particle adsorption on surface (PAS) visualization technique. For this visualization technique a suspension of plastic particles was em-
760
E i .w in g /N ilsson/O uchtt.ri.ony
Table l. Comparison of results obtained by the four visualization techniques employing the diffusion-in-gel technique (for explanation see text) Visualization technique
Slope of regression line
r
Relative sensitivity*1
Minimal demonstrable antibody concentration /ig/ml
VCS VCS (reinforced) PAS MHA
33.08 32.26 35.38 36.14
0.998 0.994 0.999 0.997
1 11.I 2.8 290
3.1 0.39 1.6 0.024
ployed. Acrylic particles (AB Bofors, Nobel Pharma, Sweden, approx, diameter 0.3-0.5 /
51: 757-762 (1976)
Visualization Principles in Thin-Layer Immunoassays (TIA) on Plastic Surfaces1 H ans E lw in g , L ars-A ke N ilsson and Ö rjan O uchteri .ony Institute of Medical Microbiology. University of Göteborg, Göteborg
Abstract. Macromolecules may adsorb firmly as a monolayer to plastic sur faces and still retain their property to react specifically with antibodies. In the present communication four different principles for visualization of such antigenantibody interactions on plastic surfaces are described. In addition, a diffusion-ingel method for quantitation of antibodies is presented. The described methods of fer simple, sensitive and accurate means of assaying antigen-antibody reactions.
Many proteins as well as other macromolecules of antigenic nature have been shown to adsorb to hydrophobic plastic surface as a monolay er of molecules [1] usually retaining antigenic reactivity [2-4], Conse quently antigen coated plastic surfaces will act as immunosorbents when exposed to immune sera containing corresponding antibodies. Under such circumstances antigen-antibody complexes are formed on the plas tic surface. This immunoreaction will, in the following, be referred to as the primary reaction. Four different techniques for the visualization of such primary reactions are described in the present communication. In addition, a diffusion-in-gel method for quantitation of antibodies is pre sented where the recording in the assay is based on the aforementioned visualization principles. In the elaboration of the visualization techniques and the antibody quantitation method, we have employed a serological model system consisting of bovine serum albumin (BSA) and a rabbit anti-BSA im> This work supported by the Swedish Cancer Society and the Ellen, Walter and Lennart Hesselman Foundation.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 7:32:26 PM
Received: July 14. 1976.
758
E i .w ing /N ilsson/O uchteri ony
Ai^P
Cl
b
. A ...,.»
o o oo o
MM
,>>,»«,4 »««,««,4 ».«»,«
•w
M M MM MM
MMKM KA K A » « * ,« » . * M . M . W M * M
MM ooc
m
• Condensation drops
Antigen-coated plastic surface A Antibody Kl Anti-immunoglobulin
•••
Plastic particles Sensitized chicken erythrocyte
Fig. /. Schematical illustration of the four visualization principles, a Water VCS visualization technique, b Serologically reinforced VCS visualization tech nique. c PAS visualization technique, d MHA visualization technique.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 7:32:26 PM
mune serum containing 1.6 mg specific antibody/ml as determined by quantitative precipitation [5]. The following series of experiments were performed. Antigen coating of plastic surfaces was performed by pouring a solu tion of BSA (100,//g/ml in 0.15 m NaCl) into polystyrole Petri dishes (Nunc AS, Roskilde, Denmark). The dishes were kept at room tempera ture for 30 min. After rinsing with 0.15 m NaCl, melted agar at a con centration of l°/o was poured into the dishes forming, after congelation, a 2.5-mm thick agar layer. Wells of 3 mm diameter were then punched in the gel and filled with anti-BSA in twofold serial dilutions if not oth erwise stated. Wells filled with normal rabbit serum were used as a con trol. After diffusion for 48 h at room temperature the agar gel was rinsed off, and the plates subjected to the following visualization proce dures. The water vapour condensation on surface (VCS) visualization tech nique. The Petri dish, rinsed from gel, was washed with distilled water and dried. The dish was then placed upside down over a container filled with water at 60 °C. After 1 min of exposure to water vapour the dish was removed and covered. A condensation pattern consisting of large confluent water drops was registrable over the circular areas where a primary antigen-antibody reaction had taken place. This hydrophilic pat tern contrasted to that over areas coated only with antigen where exclu-
Visualization of TIA on Plastic Surfaces
759
Fig. 2. Photographic recordings of visualized antigen-antibody reactions em ploying the diffusion-in-gel technique. Diffusion time 20 h. a VCS technique, b PAS technique, c MHA technique. Plastic surface coated with BSA, lOOug/ml. Application of antiserum by diffusion-in-gel procedure. Antiserum in a and b: left - undiluted, middle - diluted 1:10, c: left - 1:10, middle 1:100. Undiluted normal rabbit serum applied as controls in the holes to the right.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 7:32:26 PM
sively small drops of water were formed indicating a distinctly less hy drophilic surface. A schematical illustration of the VCS technique is giv en in figure la, and a photographic recording in figure 2a. The serologically reinforced VCS visualization technique. A goat an tiserum against rabbit immunoglobulin was poured into the Petri dish rinsed from gel. The antiserum was removed after 60 min at room tem perature. After washing and drying the Petri dish was subjected to the same visualization procedure as given above. The previously described two types of condensation patterns were recorded, the diameter of the corresponding hydrophilic areas being, however, larger. A schematical illustration of the reinforced VCS technique is given in figure lb. The particle adsorption on surface (PAS) visualization technique. For this visualization technique a suspension of plastic particles was em-
760
E i .w in g /N ilsson/O uchtt.ri.ony
Table l. Comparison of results obtained by the four visualization techniques employing the diffusion-in-gel technique (for explanation see text) Visualization technique
Slope of regression line
r
Relative sensitivity*1
Minimal demonstrable antibody concentration /ig/ml
VCS VCS (reinforced) PAS MHA
33.08 32.26 35.38 36.14
0.998 0.994 0.999 0.997
1 11.I 2.8 290
3.1 0.39 1.6 0.024
ployed. Acrylic particles (AB Bofors, Nobel Pharma, Sweden, approx, diameter 0.3-0.5 /
