MICROSCOPY RESEARCH AND TECHNIQUE 77:841–849 (2014)
New Sensitive Detection Method for Lectin Hemagglutination using Microscopy 1,2 LENKA MALINOVSKA, 1 AND MICHAELA WIMMEROVA 1,2,3* LENKA ADAMOVA, 1 2 3
Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotl arsk a 2, 611 37 Brno, Czech Republic Department of Biochemistry, Faculty of Science, Masaryk University, Kotl arsk a 2, 611 37 Brno, Czech Republic
KEY WORDS
hemagglutination; protein-carbohydrate interactions; blood group antigens; lectin
ABSTRACT The blood group system AB0 is determined by the composition of terminal oligosaccharides on red blood cells. Thanks to this structural feature, these groups can be recognized by saccharide-recognizing compounds. Lectins are proteins that are able to reversibly bind saccharide structures. They generally occur as multimers and are known as hemagglutination agents. Hemagglutination is a process in which blood cells are cross-linked via multivalent molecules. Apart from lectins, hemagglutination can also be caused by antibodies or viruses. A hemagglutination assay is commonly used for the detection of multivalent molecules that recognize blood cells, in order to search for their sugar specificity. It is traditionally performed on a microtiter plate, where the lectin solution is serially diluted and the lowest concentration of lectin causing agglutination is detected. This experimental set-up is utilized further for testing lectin specificity via a hemagglutination inhibition assay. We have developed a new way of detecting hemagglutination using microscopy, which was tested on purified lectins as well as cell lysates. Hemagglutination was performed on a microscope slide directly and detected using a microscope. Comparison with the standard hemagglutination assay using microtiter plates revealed that microscopic approach is faster and more robust and allows fast determination of lectin activities immediately in bacterial cytosols. Microsc. Res. Tech. 77:841–849, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Human blood groups were discovered in the last century and about 30 major human blood groups have been recognized to date. They are characterized by specific epitopes expressed on the surfaces of red blood cells and other cells as well. The best known blood group system is AB0, which was discovered by Landsteiner and completed by Jansky at the beginning of the 20th century (Roseman, 2001). The AB0 system is determined by the composition of the terminal oligosaccharide on red blood cells and epithelial cells, which are called the A, B, and H antigens, respectively. Antigen H is the terminal epitope of the blood group 0 and forms the basis of antigens A and B, which are present in blood group A and B, respectively. H represents Fuc-a-1,2-Gal disaccharide, A GalNAc-a-1,3-[Fuc-a1,2]Gal trisaccharide, B Gal-a-1,3[Fuc-a-1,2]Gal trisaccharide and blood group AB contains both trisaccharides (Imberty et al., 2003). The chemical structures of the antigens are depicted in Figure 1. Thanks to these structural features, the individual blood groups of the AB0 system can be recognized by various saccharide-recognizing compounds. Lectins are ubiquitous proteins able to bind saccharide compounds (Sharon, 2008). Since carbohydrates are universally present on the outer surfaces of cell membranes as a part of glycolipids and glycoproteins, lectins have great potential as markers and detectors of a membrane’s composition (Sharon and Lis, 1989). C V
2014 WILEY PERIODICALS, INC.
Well characterized lectins from opportunistic pathogenic bacteria were used in this study. The first one, RSL, originates from the plant pathogen Ralstonia solanacearum. It occurs in trimeric form while adopting a six-bladed beta-propeller architecture, similar to the fungal lectin AAL from Aleuria aurantia. It has six saccharide binding sites, two per 9 kDa monomer, which do not include any metal ions (Fig. 1a). The lectin preferably binds L-fucose with a Ka of 1.4 3 106 M21 and it also recognizes 2-fucosyllactose (Kostlanova et al., 2005). The second one, PA-IL, originates from the human pathogen Pseudomonas aeruginosa, and prefers to bind D-galactose, with a Ka of 3.4 3 104 M21 (Garber, 1992). It has a narrow specificity for DGal-containing molecules. It was discovered thanks to a strong agglutination of red blood cells (Glick and Garber, 1982) and it is highly cytotoxic to human respiratory tract epithelial cells (Bajolet-Laudinat et al., 1994). PA-IL is formed of four subunits consisting of 121 amino acids with a total molar weight of 51 kDa *Correspondence to: Michaela Wimmerova; CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czeche Republic. E-mail: [email protected] Received 7 February 2014; accepted in revised form 9 July 2014 REVIEW EDITOR: Dr. Peter Saggau Abbreviations: D-Gal, D-galactose; D-Glc, D-glucose; HA, hemagglutination assay; HI, hemagglutination inhibition assay; L-Fuc, L-fucose; RSL, Ralstonia solanacearum lectin; PA-IL, Pseudomonas aeruginosa lectin 1; RBC, suspension of red blood cells in PBS buffer; RBC_A/B/0, suspension of red blood cells group A/B/0 in PBS buffer. DOI 10.1002/jemt.22407 Published online 25 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
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ET AL. L. ADAMOVA
Fig. 1. Upper panel: (a) Architecture of trimeric six-bladed betapropeller structure of RSL lectin from Ralstonia solanacearum (PDB 2BT9) with Me-a-L-fucoside in binding sites. (b), Tetrameric arrangement of PA-IL lectin from Pseudomonas aeruginosa (PDB 1OKO) with D-galactose in binding sites. Lower panel: Trisaccharides deter-
mining blood group A (c), blood group B (d) and disaccharide determining blood group 0 (e). Terminal sugars recognized by lectins are highlighted by background circles (blue for PA-IL, red for RSL). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(Gilboa-Garber, 1972). Each of the monomers has one saccharide-binding site with one calcium ion that participates in binding (Cioci, 2003) (Fig. 1b). Terminal monosaccharides of red blood group antigens recognized by RSL and PA-IL are depicted in Figures 1c–1e. Lectins generally occur as multimers, and because of their multivalency they can form cross-links between cells (Sharon and Lis, 2004). One such example may be the ability to agglutinate blood cells, where the lectins interact with sugar moieties on the surface of the blood cells and essentially interconnect them, enabling the formation of multi-cellular aggregates. This process is called hemagglutination. Thanks to this particular property of lectins, hemagglutination assay has become a very important lectin detection method (Sharon and Lis, 2004). Hemagglutination assay (HA) was for a long time the main method used for the quick and easy detection of the presence of not only lectins but also other agglutinins (antigens or viruses) in a sample (Hierholzer et al., 1969), (Sajid et al., 2013). It is currently widely used for testing and detecting lectin activity (Manikandan and Ramar, 2012), (Wang et al., 1998). The method is usually performed by adding a sample solution (an
unknown sample or a lectin solution) to a suspension of red blood cells in a buffer. If there are agglutinationactive compounds present, the result of this mixing is a diffuse mat of agglutinated cells (compared to the relatively clearly defined sediment of non-agglutinated cells) that can be observed with the naked eye or by other optical methods. Hence, agglutination is a positive result in this setup, confirming the presence of agglutinins in the sample. To determine the specificity of a lectin or other agglutinin, a hemagglutination inhibition assay (HI) is used (Morokutti et al., 2013). HI measures the amount of ligand (generally a saccharide) needed to prevent the agglutination of red blood cells caused by agglutinin/lectin and can help determine the specific saccharide for the lectin of interest. In this setup, a potential ligand (saccharide) of the analyzed lectin is added, along with the lectin itself, to a red blood cell suspension. When there is a successful inhibition, addition of the ligand prevents the formation of the agglutinated diffuse mat. In this case, the lack of agglutination is a positive result, proving the inhibitory activity of the ligand used. Using various concentrations of various ligands, a semiquantitive evaluation of the affinity and specificity of a lectin can be determined. Microscopy Research and Technique
NEW SENSITIVE HEMAGGLUTINATION DETECTION METHOD
Hemagglutination techniques are generally performed in a microtiter plate, where a lectin (HA) or a saccharide (HI) is serially diluted into wells. A uniform amount of the erythrocyte suspension (HA) or the lectin followed by the erythrocytes (HI) is then appended to the wells. After approximately 1 hour, hemagglutination and or red blood cell sedimentation is evaluated. The minimal concentration of the lectin that is able to agglutinate (HA) and minimal concentration of the saccharide that is able to inhibit agglutination (HI) is determined. This approach provides a quick analysis of lectin activity or specificity with a relatively small volume of reagents. Sometimes the low amount of available sample requires using even smaller quantities, therefore there effort is made to reduce the volume of solutions needed for these experiments. We have developed a simple procedure to perform hemagglutination directly on a microscopy slide, with subsequent observation using a microscope. Instead of observing the formation of a diffuse mat versus clearly defined sediment, it is possible to directly observe the clustering of individual cells. This enables the use of smaller volumes of reagents and even highly diluted samples while still being able to identify the agglutination. The method was further optimized to detect the hemaglutination of red blood cells directly from the cell extracts instead of a purified lectin. The purification of lectins can be very complicated and timeconsuming, and the possibility of detecting lectin activity in the cell extract itself could help to save substantial amounts of time, otherwise wasted on purification of the inactive lectin. MATERIALS AND METHODS Lectin Production Escherichia coli BL21(DE3) cells with plasmids containing the gene for RSL [pRSETrsl (Kostlanova et al., 2005)] or PA-IL [pRSETpa1l (Blanchard et al., 2008)], respectively, were cultivated in LB medium with 100 mg/mL ampicilin. When the OD600 (optic density at 600 nm) reached 0.5, the expression of the lectin gene was induced by 0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG) and the cell culture was shaken at 30 C for 3 hours. Cells were harvested, resuspended in a buffer consisting of 20 mM Tris/HCl, 100 mM NaCl, and 100 mM CaCl2 (pH 7.5). Cells were disintegrated by sonication (Soniprep 150; Schoeller Instruments). The lectins were purified using affinity chromatography accordingly to previously used and described methods (Gilboa-Garber, 1972; Kostlanova et al., 2005). The proteins were dialyzed against water and lyophilized for long-term storage. For experiments, the lectins were dissolved in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) to the desired concentration. Preparing of Red Blood Cells (RBC) Anonymized human blood treated with natrium citrate (obtained from Transfusion and Tissue Department, The University Hospital Brno) was washed three times with PBS, diluted to 50% with PBS with 0.005% (w/v) natrium azide and stored at 4 C. The desired concentration of RBC suspension was obtained by further diluting the RBC with the PBS buffer. Microscopy Research and Technique
843
Hemagglutination on Microtiter Plate Each well in a microtiter plate was filled with 50 mL PBS. Total of 50 mL of a lectin (1 mg/mL) was pipetted into the first well and serially diluted except for the last well (control). Total of 50 ml of 2%, 10%, or 20% RBC was added to each well and mixed. The incubation lasted approximately 30 min at room temperature until the control had fully sedimented and the results were recorded. Hemagglutination on Microscope Glass Slide Three aliquots of RBC of different concentrations were prepared (2, 10, and 20%). Similarly, three lectin solutions were prepared (10 mg/mL, 100 mg/mL, 1 mg/ mL). For each combination of RBC and lectin concentration, the RBC sample was pipetted onto a glass microscopy slide and mixed in a 10 mL:10 mL ratio with the lectin solution. The mixture was incubated for 5 minutes and observed using a motorized inverted fluorescence microscope IX81 (Olympus). Images were captured with a DP72 microscope digital camera (Olympus). In the blank experiment, 10 mL of the PBS buffer was mixed with RBC instead of the lectin solution and the sample was processed in the same way. Hemagglutination Inhibition Test on the Microscope Glass Slide Total of 10 mL of the lectin solution (1 mg/mL) was mixed with 10 mL of the sugar (50 mM) specific for the lectin (L-Fuc for RSL, D-Gal for PA-IL) and incubated for 10 min at room temperature. Total of 10 mL of this mixture was mixed on a glass slide with 10 mL of 20% RBC and incubated as mentioned above. As a negative control, a saccharide that the lectin does not bind (DGal for RSL, D-Glc for PA-IL) was incubated with the lectin. As a blank experiment, the lectin was incubated with 10 mL of the PBS buffer. Cytosol-induced Hemagglutination Lectins were produced in E. coli as mentioned above. After sonication, the disrupted cells were centrifuged at 14,000 g for 1 h and the supernatant was directly used as a lectin solution for incubation with RBC. As a negative control, non-expressing E. coli XL1 cells were cultivated in LB medium to the mid-log phase (OD6005 0.5) and processed in the same way as the expressing cells. For the blank experiment, PBS was incubated with RBC instead of the lectin sample. Hemolysis on the Microscope Glass Slide Sample of 10% RBC_0 was pipetted onto a glass microscopy slide and mixed in a 10 mL:10 mL ratio with the PA-IL (500 mg/mL) solution. The mixture was incubated for 5, 10, and 30 minutes at room temperature and observed using a motorized inverted fluorescence microscope IX81 (Olympus). Images were captured with a DP72 microscope digital camera (Olympus). In the blank experiment, 10 mL of the PBS buffer was mixed with RBC instead of the lectin solution and the sample was processed in the same way. RESULTS Hemagglutination on Microtiter Plate Hemagglutination was performed with the lectins PA-IL (blood groups 0, B) and RSL (blood group A). In
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Fig. 2. Hemagglutination assay on microplate performed with RBC_0 and RBC_B and PA-IL (1 mg/mL), and RBC_A and RSL (1 mg/mL). Lectin concentration decreases from left to right by a ratio of 0.5 between two neighboring wells. Three different concentrations of RBC (2, 10, and 20%) suspension were used for the experiment.
Agglutinated red blood cells form a diffuse mat, whereas nonagglutinated red blood cells sediment and form a clear dot in the bottom of the well. Last wells represent control experiments in absence of lectins. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
both cases, the best results were obtained with the 2% RBC suspension (Fig. 2). When using higher concentrations of RBC, agglutination was not even observed. The strongest hemagglutination was visible using RSL and 2% RBC. The protein is able to agglutinate RBC_A at a concentration of about 30 mg/mL and above. PA-IL gives better results with RBC_B (agglutination ability
New Sensitive Detection Method for Lectin Hemagglutination using Microscopy 1,2 LENKA MALINOVSKA, 1 AND MICHAELA WIMMEROVA 1,2,3* LENKA ADAMOVA, 1 2 3
Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotl arsk a 2, 611 37 Brno, Czech Republic Department of Biochemistry, Faculty of Science, Masaryk University, Kotl arsk a 2, 611 37 Brno, Czech Republic
KEY WORDS
hemagglutination; protein-carbohydrate interactions; blood group antigens; lectin
ABSTRACT The blood group system AB0 is determined by the composition of terminal oligosaccharides on red blood cells. Thanks to this structural feature, these groups can be recognized by saccharide-recognizing compounds. Lectins are proteins that are able to reversibly bind saccharide structures. They generally occur as multimers and are known as hemagglutination agents. Hemagglutination is a process in which blood cells are cross-linked via multivalent molecules. Apart from lectins, hemagglutination can also be caused by antibodies or viruses. A hemagglutination assay is commonly used for the detection of multivalent molecules that recognize blood cells, in order to search for their sugar specificity. It is traditionally performed on a microtiter plate, where the lectin solution is serially diluted and the lowest concentration of lectin causing agglutination is detected. This experimental set-up is utilized further for testing lectin specificity via a hemagglutination inhibition assay. We have developed a new way of detecting hemagglutination using microscopy, which was tested on purified lectins as well as cell lysates. Hemagglutination was performed on a microscope slide directly and detected using a microscope. Comparison with the standard hemagglutination assay using microtiter plates revealed that microscopic approach is faster and more robust and allows fast determination of lectin activities immediately in bacterial cytosols. Microsc. Res. Tech. 77:841–849, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Human blood groups were discovered in the last century and about 30 major human blood groups have been recognized to date. They are characterized by specific epitopes expressed on the surfaces of red blood cells and other cells as well. The best known blood group system is AB0, which was discovered by Landsteiner and completed by Jansky at the beginning of the 20th century (Roseman, 2001). The AB0 system is determined by the composition of the terminal oligosaccharide on red blood cells and epithelial cells, which are called the A, B, and H antigens, respectively. Antigen H is the terminal epitope of the blood group 0 and forms the basis of antigens A and B, which are present in blood group A and B, respectively. H represents Fuc-a-1,2-Gal disaccharide, A GalNAc-a-1,3-[Fuc-a1,2]Gal trisaccharide, B Gal-a-1,3[Fuc-a-1,2]Gal trisaccharide and blood group AB contains both trisaccharides (Imberty et al., 2003). The chemical structures of the antigens are depicted in Figure 1. Thanks to these structural features, the individual blood groups of the AB0 system can be recognized by various saccharide-recognizing compounds. Lectins are ubiquitous proteins able to bind saccharide compounds (Sharon, 2008). Since carbohydrates are universally present on the outer surfaces of cell membranes as a part of glycolipids and glycoproteins, lectins have great potential as markers and detectors of a membrane’s composition (Sharon and Lis, 1989). C V
2014 WILEY PERIODICALS, INC.
Well characterized lectins from opportunistic pathogenic bacteria were used in this study. The first one, RSL, originates from the plant pathogen Ralstonia solanacearum. It occurs in trimeric form while adopting a six-bladed beta-propeller architecture, similar to the fungal lectin AAL from Aleuria aurantia. It has six saccharide binding sites, two per 9 kDa monomer, which do not include any metal ions (Fig. 1a). The lectin preferably binds L-fucose with a Ka of 1.4 3 106 M21 and it also recognizes 2-fucosyllactose (Kostlanova et al., 2005). The second one, PA-IL, originates from the human pathogen Pseudomonas aeruginosa, and prefers to bind D-galactose, with a Ka of 3.4 3 104 M21 (Garber, 1992). It has a narrow specificity for DGal-containing molecules. It was discovered thanks to a strong agglutination of red blood cells (Glick and Garber, 1982) and it is highly cytotoxic to human respiratory tract epithelial cells (Bajolet-Laudinat et al., 1994). PA-IL is formed of four subunits consisting of 121 amino acids with a total molar weight of 51 kDa *Correspondence to: Michaela Wimmerova; CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czeche Republic. E-mail: [email protected] Received 7 February 2014; accepted in revised form 9 July 2014 REVIEW EDITOR: Dr. Peter Saggau Abbreviations: D-Gal, D-galactose; D-Glc, D-glucose; HA, hemagglutination assay; HI, hemagglutination inhibition assay; L-Fuc, L-fucose; RSL, Ralstonia solanacearum lectin; PA-IL, Pseudomonas aeruginosa lectin 1; RBC, suspension of red blood cells in PBS buffer; RBC_A/B/0, suspension of red blood cells group A/B/0 in PBS buffer. DOI 10.1002/jemt.22407 Published online 25 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
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ET AL. L. ADAMOVA
Fig. 1. Upper panel: (a) Architecture of trimeric six-bladed betapropeller structure of RSL lectin from Ralstonia solanacearum (PDB 2BT9) with Me-a-L-fucoside in binding sites. (b), Tetrameric arrangement of PA-IL lectin from Pseudomonas aeruginosa (PDB 1OKO) with D-galactose in binding sites. Lower panel: Trisaccharides deter-
mining blood group A (c), blood group B (d) and disaccharide determining blood group 0 (e). Terminal sugars recognized by lectins are highlighted by background circles (blue for PA-IL, red for RSL). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(Gilboa-Garber, 1972). Each of the monomers has one saccharide-binding site with one calcium ion that participates in binding (Cioci, 2003) (Fig. 1b). Terminal monosaccharides of red blood group antigens recognized by RSL and PA-IL are depicted in Figures 1c–1e. Lectins generally occur as multimers, and because of their multivalency they can form cross-links between cells (Sharon and Lis, 2004). One such example may be the ability to agglutinate blood cells, where the lectins interact with sugar moieties on the surface of the blood cells and essentially interconnect them, enabling the formation of multi-cellular aggregates. This process is called hemagglutination. Thanks to this particular property of lectins, hemagglutination assay has become a very important lectin detection method (Sharon and Lis, 2004). Hemagglutination assay (HA) was for a long time the main method used for the quick and easy detection of the presence of not only lectins but also other agglutinins (antigens or viruses) in a sample (Hierholzer et al., 1969), (Sajid et al., 2013). It is currently widely used for testing and detecting lectin activity (Manikandan and Ramar, 2012), (Wang et al., 1998). The method is usually performed by adding a sample solution (an
unknown sample or a lectin solution) to a suspension of red blood cells in a buffer. If there are agglutinationactive compounds present, the result of this mixing is a diffuse mat of agglutinated cells (compared to the relatively clearly defined sediment of non-agglutinated cells) that can be observed with the naked eye or by other optical methods. Hence, agglutination is a positive result in this setup, confirming the presence of agglutinins in the sample. To determine the specificity of a lectin or other agglutinin, a hemagglutination inhibition assay (HI) is used (Morokutti et al., 2013). HI measures the amount of ligand (generally a saccharide) needed to prevent the agglutination of red blood cells caused by agglutinin/lectin and can help determine the specific saccharide for the lectin of interest. In this setup, a potential ligand (saccharide) of the analyzed lectin is added, along with the lectin itself, to a red blood cell suspension. When there is a successful inhibition, addition of the ligand prevents the formation of the agglutinated diffuse mat. In this case, the lack of agglutination is a positive result, proving the inhibitory activity of the ligand used. Using various concentrations of various ligands, a semiquantitive evaluation of the affinity and specificity of a lectin can be determined. Microscopy Research and Technique
NEW SENSITIVE HEMAGGLUTINATION DETECTION METHOD
Hemagglutination techniques are generally performed in a microtiter plate, where a lectin (HA) or a saccharide (HI) is serially diluted into wells. A uniform amount of the erythrocyte suspension (HA) or the lectin followed by the erythrocytes (HI) is then appended to the wells. After approximately 1 hour, hemagglutination and or red blood cell sedimentation is evaluated. The minimal concentration of the lectin that is able to agglutinate (HA) and minimal concentration of the saccharide that is able to inhibit agglutination (HI) is determined. This approach provides a quick analysis of lectin activity or specificity with a relatively small volume of reagents. Sometimes the low amount of available sample requires using even smaller quantities, therefore there effort is made to reduce the volume of solutions needed for these experiments. We have developed a simple procedure to perform hemagglutination directly on a microscopy slide, with subsequent observation using a microscope. Instead of observing the formation of a diffuse mat versus clearly defined sediment, it is possible to directly observe the clustering of individual cells. This enables the use of smaller volumes of reagents and even highly diluted samples while still being able to identify the agglutination. The method was further optimized to detect the hemaglutination of red blood cells directly from the cell extracts instead of a purified lectin. The purification of lectins can be very complicated and timeconsuming, and the possibility of detecting lectin activity in the cell extract itself could help to save substantial amounts of time, otherwise wasted on purification of the inactive lectin. MATERIALS AND METHODS Lectin Production Escherichia coli BL21(DE3) cells with plasmids containing the gene for RSL [pRSETrsl (Kostlanova et al., 2005)] or PA-IL [pRSETpa1l (Blanchard et al., 2008)], respectively, were cultivated in LB medium with 100 mg/mL ampicilin. When the OD600 (optic density at 600 nm) reached 0.5, the expression of the lectin gene was induced by 0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG) and the cell culture was shaken at 30 C for 3 hours. Cells were harvested, resuspended in a buffer consisting of 20 mM Tris/HCl, 100 mM NaCl, and 100 mM CaCl2 (pH 7.5). Cells were disintegrated by sonication (Soniprep 150; Schoeller Instruments). The lectins were purified using affinity chromatography accordingly to previously used and described methods (Gilboa-Garber, 1972; Kostlanova et al., 2005). The proteins were dialyzed against water and lyophilized for long-term storage. For experiments, the lectins were dissolved in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) to the desired concentration. Preparing of Red Blood Cells (RBC) Anonymized human blood treated with natrium citrate (obtained from Transfusion and Tissue Department, The University Hospital Brno) was washed three times with PBS, diluted to 50% with PBS with 0.005% (w/v) natrium azide and stored at 4 C. The desired concentration of RBC suspension was obtained by further diluting the RBC with the PBS buffer. Microscopy Research and Technique
843
Hemagglutination on Microtiter Plate Each well in a microtiter plate was filled with 50 mL PBS. Total of 50 mL of a lectin (1 mg/mL) was pipetted into the first well and serially diluted except for the last well (control). Total of 50 ml of 2%, 10%, or 20% RBC was added to each well and mixed. The incubation lasted approximately 30 min at room temperature until the control had fully sedimented and the results were recorded. Hemagglutination on Microscope Glass Slide Three aliquots of RBC of different concentrations were prepared (2, 10, and 20%). Similarly, three lectin solutions were prepared (10 mg/mL, 100 mg/mL, 1 mg/ mL). For each combination of RBC and lectin concentration, the RBC sample was pipetted onto a glass microscopy slide and mixed in a 10 mL:10 mL ratio with the lectin solution. The mixture was incubated for 5 minutes and observed using a motorized inverted fluorescence microscope IX81 (Olympus). Images were captured with a DP72 microscope digital camera (Olympus). In the blank experiment, 10 mL of the PBS buffer was mixed with RBC instead of the lectin solution and the sample was processed in the same way. Hemagglutination Inhibition Test on the Microscope Glass Slide Total of 10 mL of the lectin solution (1 mg/mL) was mixed with 10 mL of the sugar (50 mM) specific for the lectin (L-Fuc for RSL, D-Gal for PA-IL) and incubated for 10 min at room temperature. Total of 10 mL of this mixture was mixed on a glass slide with 10 mL of 20% RBC and incubated as mentioned above. As a negative control, a saccharide that the lectin does not bind (DGal for RSL, D-Glc for PA-IL) was incubated with the lectin. As a blank experiment, the lectin was incubated with 10 mL of the PBS buffer. Cytosol-induced Hemagglutination Lectins were produced in E. coli as mentioned above. After sonication, the disrupted cells were centrifuged at 14,000 g for 1 h and the supernatant was directly used as a lectin solution for incubation with RBC. As a negative control, non-expressing E. coli XL1 cells were cultivated in LB medium to the mid-log phase (OD6005 0.5) and processed in the same way as the expressing cells. For the blank experiment, PBS was incubated with RBC instead of the lectin sample. Hemolysis on the Microscope Glass Slide Sample of 10% RBC_0 was pipetted onto a glass microscopy slide and mixed in a 10 mL:10 mL ratio with the PA-IL (500 mg/mL) solution. The mixture was incubated for 5, 10, and 30 minutes at room temperature and observed using a motorized inverted fluorescence microscope IX81 (Olympus). Images were captured with a DP72 microscope digital camera (Olympus). In the blank experiment, 10 mL of the PBS buffer was mixed with RBC instead of the lectin solution and the sample was processed in the same way. RESULTS Hemagglutination on Microtiter Plate Hemagglutination was performed with the lectins PA-IL (blood groups 0, B) and RSL (blood group A). In
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ET AL. L. ADAMOVA
Fig. 2. Hemagglutination assay on microplate performed with RBC_0 and RBC_B and PA-IL (1 mg/mL), and RBC_A and RSL (1 mg/mL). Lectin concentration decreases from left to right by a ratio of 0.5 between two neighboring wells. Three different concentrations of RBC (2, 10, and 20%) suspension were used for the experiment.
Agglutinated red blood cells form a diffuse mat, whereas nonagglutinated red blood cells sediment and form a clear dot in the bottom of the well. Last wells represent control experiments in absence of lectins. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
both cases, the best results were obtained with the 2% RBC suspension (Fig. 2). When using higher concentrations of RBC, agglutination was not even observed. The strongest hemagglutination was visible using RSL and 2% RBC. The protein is able to agglutinate RBC_A at a concentration of about 30 mg/mL and above. PA-IL gives better results with RBC_B (agglutination ability
