[62]
ANTIPOLYSACCHARIDE ANTIBODIES
537
of avidin and biotin-linked enzyme to a tube containing PBS-Tween. Incubate for 10 min at 37 °, and add the complex to the wells. Incubate the plate for 30 min at 37°. Wash the plate 5 times with PBS-Tween. Add appropriate substrate. Incubate the plate at 37 ° or room temperature until the weakly positive control has visible color equivalent to an optical density of approximately 0.1 measured at the appropriate wavelength. Comments. For each sample calculate the virus-specific activity by subtracting the mean activity of the wells coated with the nonimmune IgG from that measured in wells coated with the antiviral IgG. To ensure accurate quantification, specimens giving readings of greater than 1.2 optical density units should be diluted 1 : 10 and retested. Calculate the mean and standard deviation of the virus activity of the negative controls. A specimen is considered positive if its mean activity is greater than 2 standard deviations above the mean activity of the negative controls. Alternatively, a specimen can be considered positive if its specific activity is greater than that of the weakly positive control. If qualitative visual determinations are used, a specimen is considered positive if the degree of color in the antiviral IgG wells is different than the amount of color generated in the nonimmune wells and in the wells containing the weakly positive control samples. Note that if the fl-lactamase starch-iodine system is utilized, a positive reaction will be manifested by a decrease in color, whereas in the case of most other enzymes, the binding of enzyme will result in an increase in color production. Acknowledgment This work was supported by contract N01-AI-52579from the National Institute of Allergies and Infectious Diseases.
[62]
Enzyme-Linked
Immunosorbent
Assay
By WILLIE F. VANN, ANN SUTTON, and RACHEL SCHNEERSON Capsular polysaccharides are important in invasive bacterial disease as virulence factors and as immunogens. Since polysaccharides and oligosaccharides often do not adhere to a plastic solid phase as well as proteins, the measurement of antibodies to polysaccharides by enzymeMETHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
538
APPLICATIONS
[62]
4).>-
A-,o,o
Q.,.,,.
FIG. 1. Schematic illustration of avidin-biotin-based antipolysaccharide EklSA.
linked immunosorbent assay (ELISA) has been difficult. ~ Avidin-biotin complex formation has been used to enhance ELISA sensitivity by the preparation of biotinylated antibodies or indicator enzyme conjugates, z,3 In the assay described here, the avidin-biotin complex is used to enhance the binding of the polysaccharide antigen to the solid phase. 4,5 The polysaccharide is derivatized with biotin via an amino or hydrazide functional group. 4 The biotin moieties are then used to complex the polysaccharide antigen to an avidin-coated solid phase as illustrated in Fig. 1. The resulting solid phase can then be used in a routine ELISA to measure polysaccharide-specific antibody. 2,5 This assay has also been used to measure the ability of carbohydrate inhibitors to bind antibody specific for immobilized polysaccharide antigen) 1 L. T. Callahan, A. F. Woodhour, J. B. Meeker, and M. R. Hilleman, J. Clin. Microbiol. 10, 459 (1979). 2 p. Tijssen, in "Laboratory Techniques in Biochemistry and Molecular Biology" (R. H. Burdon and P. H. van Knippenberg, eds.), Vol. 15, p. 329. Elsevier, Amsterdam, 1985. 3 J.-L. Guesdon, T, Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 4 E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). 5 A. Sutton, W. F. Vann, A. B. Karpas, K. E. Stein, and R. Schneerson, J. lmmunol. Methods 82, 215 (1980).
[62]
ANTIPOLYSACCHARIDE ANTIBODIES
539
TABLE I METHODS OF PREPARING POLYSACCHARIDEHYDRAZIDES
Polysaccharide
Available function
Method of attaching hydrazide
Haemophilus influenzae b Streptococcus pneumoniae 12F Staphylococcus aureus type 8 Streptococcus pneumoniae 6A
--OH --COOH --COOH --OH
CNBr RN~---C~----NR' RN=C~NR' CNBr
Preparation of Biotinylated Polysaccharides Polysaccharides are derivatized with biotin via adipic acid hydrazide groups. The method of attachment of the hydrazide or amino groups is determined by the structure of the polysaccharide (Table I). Haemophilus influenzae b Polysaccharide Hydrazide. Adipic acid hydrazide groups are attached to Haemophilus influenzae b (Hib) polysaccharide 6 by cyanogen bromide activation of hydroxyl groups. A 10-ml solution of polysaccharide (5-10 mg/ml) is adjusted to pH 10.5 with 0.1 N NaOH. Cyanogen bromide (0.4 mg/ml polysaccharide) is added (1 g/ml prepared by dissolving 2 g CNBr in 1 ml acetonitrile to yield 2 ml), and the reaction mixture is maintained at 4° and pH 10.5. After 6 min, 10 ml of 0.5 M adipic dihydrazide in 0.5 M Na2CO3 (pH 8.5) is added, and the solution is stirred overnight at 4 °. The reaction mixture is then desalted on Sephadex PD-10 and lyophilized.
Streptococcus pneumoniae Type 12F Polysaccharide Hydrazide. Polysaccharide carboxyl groups are derivatized with adipic acid dihydrazide by the carbodiimide method, v Polysaccharide (Streptococcus pneumoniae type 12F, 23 mg) 8 is dissolved in 10 ml of 0.25 M adipic acid dihydrazide and adjusted to pH 4.75 at 4 °. Ethyldimethylaminopropylcarbodiimide (0.5 mmol) is added, and the pH is maintained with 0.1 N HCI for 3 hr. The reaction mixture is dialyzed against water and lyophilized. Yield, 20 mg. The degree of substitution of polysaccharides by hydrazide using either method of activation is determined by the trinitrobenzenesulfonic 6 R. M. Crisel, R. S. Baker, and D. E. Dorman, J. Biol. Chem. 250, 4926 (1975). 7 D. G. Hoare and D. E. Koshland, J. Biol. Chem. 242, 2447 (1967). 8 K. Leontein, B. Lindberg, and J. Lonngren, Can. J. Chem. 59, 2081 (1981).
540
APPLICATIONS
[62]
acid reaction. 9 The number of groups incorporated is dependent on the polysaccharide and usually varies between 100 and 400 nmol/mgP Biotinylation of Polysaccharide Hydrazides. All polysaccharide hydrazides are biotinylated by the following procedure. Streptococcus pneumoniae type 12F hydrazide (10.8 rag), dissolved in 1 ml of phosphate-buffered saline (PBS), is added to 0.5 ml of biotin-N-hydroxysuccinimide ester (8-10 mg/ml) dissolved in anhydrous amine-free dimethylformamide at 4 °. The resulting solution is stirred overnight and desalted on a Sephadex PD-10 column. Polysaccharides are detected by capillary precipitation using the appropriate antisera. Yield, 5 mg.
ELISA Reagents Phosphate-buffered saline (PBS, pH 7.4) Phosphate-buffered saline (pH 7.4) containing 1% bovine serum albumin, 0.1% Triton X-100, 0.1% sodium azide (PBS-BSA diluent) Avidin, egg white (Sigma Chemical Co., St. Louis, MO), 4/zg/ml in PBS Biotinylated polysaccharide antigen (2/.~g/ml in PBS-BSA, prepared immediately before use, 10 ml/plate) 2% BSA, 0.2% sodium azide in PBS, sterile Antiimmunoglobulin, species- and class-specific, conjugated to alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) p-Nitrophenyl phosphate (Sigma 104 substrate), 1 mg/ml in 1 M TrisHCI, 0.3 mM MgC12 (pH 9.8) Antisera, stock dilutions of test sera prepared in 2% BSA (usually 1 : 100 to 1 : 10,000 depending on antibody activity); antibody activity varies considerably and may require dilution outside this range Procedure. Wells of U-well microtiter plates (Dynatech polyvinyl or Nunc Immunoplate II) are coated with 100/.d of avidin in PBS. Plates are sealed, incubated overnight at room temperature, and washed four times with PBS. Biotinylated polysaccharide is diluted to 2/xg/ml in PBS-BSA immediately before use, and 100/~1 is added to avidin-coated plates. After incubation for 30-120 rain at room temperature, the plates are washed with PBS. Test sera are serially diluted with PBS-BSA (2-fold) pipetting 100 ~l/well. Plates are sealed, incubated overnight, and washed. Diluted goat antiimmunoglobulin, conjugated to alkaline phosphatase (100/~1), is added, and the plates are incubated for 4 hr and washed. The 9 A. F. S. A. Habeeb, Anal. Biochem. 14, 328 (1966).
[63]
RAPID DETECTIONOF HSV
541
dilution of anti-Ig conjugate (usually 1 : 1000) must be determined for each lot such that the maximum absorbance of the lowest reference serum dilution is approximately 1.0 after color development. 2 The color is developed by addition of 100/zl p-nitrophenyl phosphate to the washed plate and incubation for 30-60 min. The optical density is read at 405 nm. Comments
Several polysaccharides have been successfully biotinylated and used in this ELISA including Escherichia coli K1, S. pneumoniae 6A and 12F, dextran, and Staphylococcus aureus types 5 and 8. Some polysaccharides, however, either are insoluble and unusable after derivatization by this procedure (meningococcal Group C) or develop precipitates after storage at 4 ° (S. aureus type 5). Inherent with any assay method involving chemical modification of polysaccharide antigens is the risk that antibody binding could be altered. We have not observed a noticeable change in the ability of S. aureus and S. pneumoniae polysaccharides to bind antibody; however, there is a noticeable difference in the ability of H. influenzae type b polysaccharide to inhibit antibody binding. 5 No noticeable background was observed in experiments measuring antibody levels in mice or rabbits. Nonspecific binding of human sera to avidin-coated plates became evident in low-titered sera. This background reactivity can be controlled by addition of sufficient antigen to coat most of the avidin sites.
[63] R a p i d D e t e c t i o n o f H e r p e s S i m p l e x Virus By LATA S. NERURKAR
Herpes simplex virus (HSV) causes diverse clinical illnesses associated with its primary infection. Generalized infection during immunosuppression, neonatal infection, and HSV encephalitis (infection of the brain) are very devastating and in many instances fatal if not properly treated. Antiviral therapy that responds to both types (HSV type 1 and HSV type 2) is now available. This makes it essential and important to identify the virus so that chemotherapy can be initiated to save the life of the patient. In neonatal herpes, the baby often acquires the infection during birth from the asymptomatic mother who is shedding virus from the cervix or vagina. In this case, a laboratory viral culture is recommended as close to METHODS IN ENZYMOLOGY. VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ANTIPOLYSACCHARIDE ANTIBODIES
537
of avidin and biotin-linked enzyme to a tube containing PBS-Tween. Incubate for 10 min at 37 °, and add the complex to the wells. Incubate the plate for 30 min at 37°. Wash the plate 5 times with PBS-Tween. Add appropriate substrate. Incubate the plate at 37 ° or room temperature until the weakly positive control has visible color equivalent to an optical density of approximately 0.1 measured at the appropriate wavelength. Comments. For each sample calculate the virus-specific activity by subtracting the mean activity of the wells coated with the nonimmune IgG from that measured in wells coated with the antiviral IgG. To ensure accurate quantification, specimens giving readings of greater than 1.2 optical density units should be diluted 1 : 10 and retested. Calculate the mean and standard deviation of the virus activity of the negative controls. A specimen is considered positive if its mean activity is greater than 2 standard deviations above the mean activity of the negative controls. Alternatively, a specimen can be considered positive if its specific activity is greater than that of the weakly positive control. If qualitative visual determinations are used, a specimen is considered positive if the degree of color in the antiviral IgG wells is different than the amount of color generated in the nonimmune wells and in the wells containing the weakly positive control samples. Note that if the fl-lactamase starch-iodine system is utilized, a positive reaction will be manifested by a decrease in color, whereas in the case of most other enzymes, the binding of enzyme will result in an increase in color production. Acknowledgment This work was supported by contract N01-AI-52579from the National Institute of Allergies and Infectious Diseases.
[62]
Enzyme-Linked
Immunosorbent
Assay
By WILLIE F. VANN, ANN SUTTON, and RACHEL SCHNEERSON Capsular polysaccharides are important in invasive bacterial disease as virulence factors and as immunogens. Since polysaccharides and oligosaccharides often do not adhere to a plastic solid phase as well as proteins, the measurement of antibodies to polysaccharides by enzymeMETHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
538
APPLICATIONS
[62]
4).>-
A-,o,o
Q.,.,,.
FIG. 1. Schematic illustration of avidin-biotin-based antipolysaccharide EklSA.
linked immunosorbent assay (ELISA) has been difficult. ~ Avidin-biotin complex formation has been used to enhance ELISA sensitivity by the preparation of biotinylated antibodies or indicator enzyme conjugates, z,3 In the assay described here, the avidin-biotin complex is used to enhance the binding of the polysaccharide antigen to the solid phase. 4,5 The polysaccharide is derivatized with biotin via an amino or hydrazide functional group. 4 The biotin moieties are then used to complex the polysaccharide antigen to an avidin-coated solid phase as illustrated in Fig. 1. The resulting solid phase can then be used in a routine ELISA to measure polysaccharide-specific antibody. 2,5 This assay has also been used to measure the ability of carbohydrate inhibitors to bind antibody specific for immobilized polysaccharide antigen) 1 L. T. Callahan, A. F. Woodhour, J. B. Meeker, and M. R. Hilleman, J. Clin. Microbiol. 10, 459 (1979). 2 p. Tijssen, in "Laboratory Techniques in Biochemistry and Molecular Biology" (R. H. Burdon and P. H. van Knippenberg, eds.), Vol. 15, p. 329. Elsevier, Amsterdam, 1985. 3 J.-L. Guesdon, T, Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 4 E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). 5 A. Sutton, W. F. Vann, A. B. Karpas, K. E. Stein, and R. Schneerson, J. lmmunol. Methods 82, 215 (1980).
[62]
ANTIPOLYSACCHARIDE ANTIBODIES
539
TABLE I METHODS OF PREPARING POLYSACCHARIDEHYDRAZIDES
Polysaccharide
Available function
Method of attaching hydrazide
Haemophilus influenzae b Streptococcus pneumoniae 12F Staphylococcus aureus type 8 Streptococcus pneumoniae 6A
--OH --COOH --COOH --OH
CNBr RN~---C~----NR' RN=C~NR' CNBr
Preparation of Biotinylated Polysaccharides Polysaccharides are derivatized with biotin via adipic acid hydrazide groups. The method of attachment of the hydrazide or amino groups is determined by the structure of the polysaccharide (Table I). Haemophilus influenzae b Polysaccharide Hydrazide. Adipic acid hydrazide groups are attached to Haemophilus influenzae b (Hib) polysaccharide 6 by cyanogen bromide activation of hydroxyl groups. A 10-ml solution of polysaccharide (5-10 mg/ml) is adjusted to pH 10.5 with 0.1 N NaOH. Cyanogen bromide (0.4 mg/ml polysaccharide) is added (1 g/ml prepared by dissolving 2 g CNBr in 1 ml acetonitrile to yield 2 ml), and the reaction mixture is maintained at 4° and pH 10.5. After 6 min, 10 ml of 0.5 M adipic dihydrazide in 0.5 M Na2CO3 (pH 8.5) is added, and the solution is stirred overnight at 4 °. The reaction mixture is then desalted on Sephadex PD-10 and lyophilized.
Streptococcus pneumoniae Type 12F Polysaccharide Hydrazide. Polysaccharide carboxyl groups are derivatized with adipic acid dihydrazide by the carbodiimide method, v Polysaccharide (Streptococcus pneumoniae type 12F, 23 mg) 8 is dissolved in 10 ml of 0.25 M adipic acid dihydrazide and adjusted to pH 4.75 at 4 °. Ethyldimethylaminopropylcarbodiimide (0.5 mmol) is added, and the pH is maintained with 0.1 N HCI for 3 hr. The reaction mixture is dialyzed against water and lyophilized. Yield, 20 mg. The degree of substitution of polysaccharides by hydrazide using either method of activation is determined by the trinitrobenzenesulfonic 6 R. M. Crisel, R. S. Baker, and D. E. Dorman, J. Biol. Chem. 250, 4926 (1975). 7 D. G. Hoare and D. E. Koshland, J. Biol. Chem. 242, 2447 (1967). 8 K. Leontein, B. Lindberg, and J. Lonngren, Can. J. Chem. 59, 2081 (1981).
540
APPLICATIONS
[62]
acid reaction. 9 The number of groups incorporated is dependent on the polysaccharide and usually varies between 100 and 400 nmol/mgP Biotinylation of Polysaccharide Hydrazides. All polysaccharide hydrazides are biotinylated by the following procedure. Streptococcus pneumoniae type 12F hydrazide (10.8 rag), dissolved in 1 ml of phosphate-buffered saline (PBS), is added to 0.5 ml of biotin-N-hydroxysuccinimide ester (8-10 mg/ml) dissolved in anhydrous amine-free dimethylformamide at 4 °. The resulting solution is stirred overnight and desalted on a Sephadex PD-10 column. Polysaccharides are detected by capillary precipitation using the appropriate antisera. Yield, 5 mg.
ELISA Reagents Phosphate-buffered saline (PBS, pH 7.4) Phosphate-buffered saline (pH 7.4) containing 1% bovine serum albumin, 0.1% Triton X-100, 0.1% sodium azide (PBS-BSA diluent) Avidin, egg white (Sigma Chemical Co., St. Louis, MO), 4/zg/ml in PBS Biotinylated polysaccharide antigen (2/.~g/ml in PBS-BSA, prepared immediately before use, 10 ml/plate) 2% BSA, 0.2% sodium azide in PBS, sterile Antiimmunoglobulin, species- and class-specific, conjugated to alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) p-Nitrophenyl phosphate (Sigma 104 substrate), 1 mg/ml in 1 M TrisHCI, 0.3 mM MgC12 (pH 9.8) Antisera, stock dilutions of test sera prepared in 2% BSA (usually 1 : 100 to 1 : 10,000 depending on antibody activity); antibody activity varies considerably and may require dilution outside this range Procedure. Wells of U-well microtiter plates (Dynatech polyvinyl or Nunc Immunoplate II) are coated with 100/.d of avidin in PBS. Plates are sealed, incubated overnight at room temperature, and washed four times with PBS. Biotinylated polysaccharide is diluted to 2/xg/ml in PBS-BSA immediately before use, and 100/~1 is added to avidin-coated plates. After incubation for 30-120 rain at room temperature, the plates are washed with PBS. Test sera are serially diluted with PBS-BSA (2-fold) pipetting 100 ~l/well. Plates are sealed, incubated overnight, and washed. Diluted goat antiimmunoglobulin, conjugated to alkaline phosphatase (100/~1), is added, and the plates are incubated for 4 hr and washed. The 9 A. F. S. A. Habeeb, Anal. Biochem. 14, 328 (1966).
[63]
RAPID DETECTIONOF HSV
541
dilution of anti-Ig conjugate (usually 1 : 1000) must be determined for each lot such that the maximum absorbance of the lowest reference serum dilution is approximately 1.0 after color development. 2 The color is developed by addition of 100/zl p-nitrophenyl phosphate to the washed plate and incubation for 30-60 min. The optical density is read at 405 nm. Comments
Several polysaccharides have been successfully biotinylated and used in this ELISA including Escherichia coli K1, S. pneumoniae 6A and 12F, dextran, and Staphylococcus aureus types 5 and 8. Some polysaccharides, however, either are insoluble and unusable after derivatization by this procedure (meningococcal Group C) or develop precipitates after storage at 4 ° (S. aureus type 5). Inherent with any assay method involving chemical modification of polysaccharide antigens is the risk that antibody binding could be altered. We have not observed a noticeable change in the ability of S. aureus and S. pneumoniae polysaccharides to bind antibody; however, there is a noticeable difference in the ability of H. influenzae type b polysaccharide to inhibit antibody binding. 5 No noticeable background was observed in experiments measuring antibody levels in mice or rabbits. Nonspecific binding of human sera to avidin-coated plates became evident in low-titered sera. This background reactivity can be controlled by addition of sufficient antigen to coat most of the avidin sites.
[63] R a p i d D e t e c t i o n o f H e r p e s S i m p l e x Virus By LATA S. NERURKAR
Herpes simplex virus (HSV) causes diverse clinical illnesses associated with its primary infection. Generalized infection during immunosuppression, neonatal infection, and HSV encephalitis (infection of the brain) are very devastating and in many instances fatal if not properly treated. Antiviral therapy that responds to both types (HSV type 1 and HSV type 2) is now available. This makes it essential and important to identify the virus so that chemotherapy can be initiated to save the life of the patient. In neonatal herpes, the baby often acquires the infection during birth from the asymptomatic mother who is shedding virus from the cervix or vagina. In this case, a laboratory viral culture is recommended as close to METHODS IN ENZYMOLOGY. VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
