Chapter 20 Quantification of Total Human Alpha-1 Antitrypsin by Sandwich ELISA Qiushi Tang, Alisha M. Gruntman, and Terence R. Flotte Abstract In this chapter we describe an enzyme-linked immunosorbent assay (ELISA) to quantitatively measure human alpha-1 antitrypsin (AAT) protein levels in serum, other body fluids or liquid media. This assay can be used to measure the expression of the human AAT (hAAT) gene in a variety of gene transfer or gene downregulation experiments. A hAAT-specific capture antibody and a HRP-conjugated anti-AAT detection antibody are used in this assay. The conjugated anti-AAT used in this protocol, instead of the typical sandwich which employs an unconjugated antibody followed by a specifically conjugated IgG, makes the assay simpler and decreases variability. This provides a useful tool to evaluate the AAT levels in clinical and research samples and can allow fairly rapid testing of a large number of samples. Key words Alpha-1 antitrypsin, AAT, Enzyme-linked immunosorbent assay, Sandwich ELISA
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Introduction The following protocol details the performance of an enzymelinked immunosorbent assay (ELISA) to quantitate levels of human alpha-1 antitrypsin in both research and clinical samples. The assay is species-specific for human AAT in the background of mouse serum, but will not differentiate between hAAT from endogenous serum AAT in nonhuman primate species [1]. The assay is not allele-specific. Therefore, it is not generally useful for measuring expression of wild-type (PiM) hAAT protein in the serum of patients with AAT deficiency due to the common missense E342K (PiZ) mutation, but could be used to measure total (wildtype þ mutant) hAAT levels in that setting. The human alpha-1 antitrypsin (AAT) “sandwich” ELISA assay relies on an AAT-specific capture antibody which will bind the AAT protein to the surface of the plate, after which any unbound antigen is removed by washes. A skim milk blocking buffer is then used to inhibit any nonspecific binding to the well surface. Samples to be
Florie Borel and Christian Mueller (eds.), Alpha-1 Antitrypsin Deficiency: Methods and Protocols, Methods in Molecular Biology, vol. 1639, DOI 10.1007/978-1-4939-7163-3_20, © Springer Science+Business Media LLC 2017
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Fig. 1 Assay sensitivity: The assay was performed as described in the protocol, with increasing amounts of AAT in the protein standard. OD values for the standard are: 1.582, 1.367, 1.076, 0.734, 0.434, 0.222, 0.115, and 0.062. As shown here, the linear range of the standard curve is between 1.582 and 0.222, which represents the dynamic range for this assay
assayed are added and human AAT is bound by the capture antibody. Then the detection antibody, which is conjugated to horseradish peroxidase (HRP) (an enzyme leading to a signal readout) is added. 3,30 ,5,50 -tetramethylbenzidine (TMB) microwell peroxidase substrate system is used to develop a deep blue color in the presence of peroxidase-labeled conjugates. The density of yellow coloration after addition of the stop solution is directly proportional to the amount of alpha-1 antitrypsin bound in the well. The ELISA is sensitive and has a wide dynamic range (the standard curve linear range is from 1 to 100 ng/ml) (Fig. 1). Since AAT is a secreted protein, this ELISA can detect AAT in a wide range of samples, including human serum, plasma, cell culture supernatant, and milk as well as in AAT transgenic mouse serum (mice expressing human ZAAT). We have tested the ELISA using serum from multiple species, including several nonhuman primates, and proven that the native AAT produced in those species does not cross-react with the human antibody. Tested species include: rhesus macaques, cynomolgus macaques, African green monkeys, marmosets, ferrets, and wild-type C57/B6 mice. However, the ELISA as described does not differentiate between normal AAT and mutant human AAT.
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Materials The reagents should be prepared as described below and stored at room temperature unless otherwise noted. Any material or sample waste should be disposed of according to your institution’s waste
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handling regulations. Millipore water should be used for all reagents unless the protocol specifies otherwise. 1. Clear flat-bottom nonsterile 96-well plate 2. Microplate sealing films, nonsterile. 3. Voller’s buffer: Add the follow chemicals: 2.76 g Na2CO3 (106.0 g/mol), 1.92 g NaHCO3 (84.01 g/mol), 0.2 g NaN3 (65.01 g/mol), bring total volume to 1 l using water. The final pH should be adjusted to 9.6 with NaOH. 4. Wash buffer: PBS–Tween: 500 μl Tween 20 added to 1 l of 1 PBS 5. Blocking buffer: 1% skim milk in wash buffer (above). Add 1.0 g of skim milk powder to 100 ml of wash buffer. 6. Coating antibody: Cappel goat antiserum to human alpha-1 antitrypsin (Cat # 55111, Cappel Laboratories, MP Biomedicals, Santa Ana, CA). Make 5 mg/ml stock by reconstituting with 2 ml of dH2O. Make 23 μl aliquots and store at 80 C. Working concentration: add 22 μl of aliquoted antibody to 11 ml of coating buffer (1:500 dilution). 7. Human AAT standard: Human alpha-1 antitrypsin (Cat# 1616-011609, Athens Research and Technology, Athens, GA) make the stock solution 1 mg/ml by reconstituted in 1 ml PBS. Make 20 μl aliquots and store at 80 C. The dilution scheme for the standard is demonstrated in the table (Table 1) below. All dilutions are performed using blocking buffer (see above). Add 100 μl of standard from steps 1–9 to individual wells of the assay plate. It is recommended to run the standard in duplicate (see Note 1). Table 1 Standards dilution chart
Step
Final hAAT concentration (ng/ml)
hAAT Standard
Sample diluent w/blocking buffer
0
1000
10 μl of 1 mg/ml stock
10 ml
1
100
100 μl from step 0
900 μl
2
50
500 μl from step 1
500 μl
3
25
500 μl from step 2
500 μl
4
12.5
500 μl from step 3
500 μl
5
6.25
500 μl from step 4
500 μl
6
3.125
500 μl from step 5
500 μl
7
1.5625
500 μl from step 6
500 μl
8
0.78125
500 μl from step 7
500 μl
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8. Unknown samples: Optimal sample dilutions are dependent on the condition and should be determined by the user. Samples are diluted to desired concentrations in the standard range with blocking buffer. Duplicate wells are recommended (see Notes 1 and 2). 9. Positive control: Human serum (Cat#14-102E, BioWhittaker™ Media, Lonza Walkersville, Inc., Walkersville, MD). 20 μl aliquots stored at 80 C. Working dilution is 1:200,000 diluted in blocking buffer. Duplicate wells (2/plate) are recommended. 10. Secondary antibody: Anti-alpha-1 antitrypsin antibody (HRP conjugated) (Cat#7635, Abcam, Cambridge, UK) should be diluted 1:10 in 1 PBS. Store 23 μl aliquots at 80 C. Working concentration: 22 μl of aliquoted antibody diluted in 11 ml of blocking buffer (1:500 dilution). 11. TMB peroxidase substrate (TMB microwell peroxidase substrate system, SeraCare Life Sciences) stored at 4 C: prepare freshly by mixing 6 ml of TMB solution A with 6 ml of TMB solution B per 96-well plate being run. 12. Stop solution: 10 ml 95–98% of sulfuric acid, 90 ml of water. 13. Plate washer (optional) (MultiWash III Microplate Washer, TriContinent, Grass Valley, CA). 14. Microplate reader with 450 nm filter, such as VersaMax (Molecular Devices, Sunnyvale, CA). 15. Software (optional), such as SoftMax Pro (Molecular Devices).
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Methods 1. Add 100 μl of diluted coating antibody (see above) to each well of a 96-well plate. 2. Cover plate with pressure-sensitive film and incubate at room temperature for 1 h or at 4 C overnight. 3. Aspirate each well and wash with at least 200 μl of wash buffer, repeat two times (three total washes) (see Note 3). 4. Add 100 μl of blocking solution to each well, cover plate with pressure-sensitive film and incubate at room temperature for 1 h (see Note 4). 5. Repeat washes as in step 3. 6. Add 100 μl each of hAAT standard (dilutions 1–9), samples, and positive controls to their respective wells (Fig. 2). Load one well with blocking buffer to serve as a “blank”. Cover the plate
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Fig. 2 ELISA plate layout
with pressure-sensitive film and incubate at room temperature for 1 h (see Note 5). 7. Repeat washes as in step 3. 8. Add 100 μl of diluted secondary antibody to each well, cover the plate with pressure-sensitive film and incubate at room temperature for 1 h. Wash the plate six times with 200 μl of washing buffer per well. 9. Add 100 μl of TMB peroxidase substrate 1:1 dilution solution (see above) to each well. Develop the plate in the dark at room temperature for 30 min (see Notes 6 and 7). 10. Stop reaction by adding 50 μl of stop solution to each well (see Note 8). 11. Measure absorbance on a plate reader at 450 nm. 12. Calculate the concentration of unknown samples. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the samples [2]. (a) Calculate the mean absorbance for each set of duplicate standards, controls, and samples. Subtract the mean of blank control standard absorbance from each. (b) Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. (c) To determine the concentration of the unknowns, find the unknowns’ mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
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Notes 1. It is very important to vortex all standard vials well between each dilution step and to vortex again as each standard is loaded to the well. Be cautious not to contaminate any sample wells with the standard samples while loading the plate. 2. Keep the samples on ice to thaw. 3. Do not let the plate dry out after coating antibody added. The plate can be blocked for a longer time until the standard and sample preparation is finished. Always leave the washing buffer in the well until the solution for the next step is ready. 4. Incubation at room temperature is about 22–25 C. 5. Save at least one well for a blank control (load with blocking buffer). This will be used to determine the baseline background of the absorbance. 6. Bring the TMB peroxidase substrate to room temperature before use; this can be done by setting Solutions A and B on the bench an hour before it is needed (when the secondary antibody is added). Solution A and B must be protected from light. 7. Do not cover the plate with pressure-sensitive film on TMB peroxidase substrate development step. The solution must be kept in the dark for this step; we often accomplish this by placing the plate on a paper towel in a drawer during the incubation. 8. Read the plate within 30 min of the stop solution being added.
References 1. Song S, Scott-Jorgensen M, Wang J, Poirier A, Crawford J, Campbell-Thompson M, Flotte TR (2002) Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects. Mol Ther 6(3):329–335
2. Sono Biological Inc (online) ELISA protocolcalculation of results. ELISA encyclopedia http://www.elisa-antibody.com/generalelisa-protocol
1
Introduction The following protocol details the performance of an enzymelinked immunosorbent assay (ELISA) to quantitate levels of human alpha-1 antitrypsin in both research and clinical samples. The assay is species-specific for human AAT in the background of mouse serum, but will not differentiate between hAAT from endogenous serum AAT in nonhuman primate species [1]. The assay is not allele-specific. Therefore, it is not generally useful for measuring expression of wild-type (PiM) hAAT protein in the serum of patients with AAT deficiency due to the common missense E342K (PiZ) mutation, but could be used to measure total (wildtype þ mutant) hAAT levels in that setting. The human alpha-1 antitrypsin (AAT) “sandwich” ELISA assay relies on an AAT-specific capture antibody which will bind the AAT protein to the surface of the plate, after which any unbound antigen is removed by washes. A skim milk blocking buffer is then used to inhibit any nonspecific binding to the well surface. Samples to be
Florie Borel and Christian Mueller (eds.), Alpha-1 Antitrypsin Deficiency: Methods and Protocols, Methods in Molecular Biology, vol. 1639, DOI 10.1007/978-1-4939-7163-3_20, © Springer Science+Business Media LLC 2017
211
212
Qiushi Tang et al.
Fig. 1 Assay sensitivity: The assay was performed as described in the protocol, with increasing amounts of AAT in the protein standard. OD values for the standard are: 1.582, 1.367, 1.076, 0.734, 0.434, 0.222, 0.115, and 0.062. As shown here, the linear range of the standard curve is between 1.582 and 0.222, which represents the dynamic range for this assay
assayed are added and human AAT is bound by the capture antibody. Then the detection antibody, which is conjugated to horseradish peroxidase (HRP) (an enzyme leading to a signal readout) is added. 3,30 ,5,50 -tetramethylbenzidine (TMB) microwell peroxidase substrate system is used to develop a deep blue color in the presence of peroxidase-labeled conjugates. The density of yellow coloration after addition of the stop solution is directly proportional to the amount of alpha-1 antitrypsin bound in the well. The ELISA is sensitive and has a wide dynamic range (the standard curve linear range is from 1 to 100 ng/ml) (Fig. 1). Since AAT is a secreted protein, this ELISA can detect AAT in a wide range of samples, including human serum, plasma, cell culture supernatant, and milk as well as in AAT transgenic mouse serum (mice expressing human ZAAT). We have tested the ELISA using serum from multiple species, including several nonhuman primates, and proven that the native AAT produced in those species does not cross-react with the human antibody. Tested species include: rhesus macaques, cynomolgus macaques, African green monkeys, marmosets, ferrets, and wild-type C57/B6 mice. However, the ELISA as described does not differentiate between normal AAT and mutant human AAT.
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Materials The reagents should be prepared as described below and stored at room temperature unless otherwise noted. Any material or sample waste should be disposed of according to your institution’s waste
Quantification of Total Human Alpha-1 Antitrypsin by Sandwich ELISA
213
handling regulations. Millipore water should be used for all reagents unless the protocol specifies otherwise. 1. Clear flat-bottom nonsterile 96-well plate 2. Microplate sealing films, nonsterile. 3. Voller’s buffer: Add the follow chemicals: 2.76 g Na2CO3 (106.0 g/mol), 1.92 g NaHCO3 (84.01 g/mol), 0.2 g NaN3 (65.01 g/mol), bring total volume to 1 l using water. The final pH should be adjusted to 9.6 with NaOH. 4. Wash buffer: PBS–Tween: 500 μl Tween 20 added to 1 l of 1 PBS 5. Blocking buffer: 1% skim milk in wash buffer (above). Add 1.0 g of skim milk powder to 100 ml of wash buffer. 6. Coating antibody: Cappel goat antiserum to human alpha-1 antitrypsin (Cat # 55111, Cappel Laboratories, MP Biomedicals, Santa Ana, CA). Make 5 mg/ml stock by reconstituting with 2 ml of dH2O. Make 23 μl aliquots and store at 80 C. Working concentration: add 22 μl of aliquoted antibody to 11 ml of coating buffer (1:500 dilution). 7. Human AAT standard: Human alpha-1 antitrypsin (Cat# 1616-011609, Athens Research and Technology, Athens, GA) make the stock solution 1 mg/ml by reconstituted in 1 ml PBS. Make 20 μl aliquots and store at 80 C. The dilution scheme for the standard is demonstrated in the table (Table 1) below. All dilutions are performed using blocking buffer (see above). Add 100 μl of standard from steps 1–9 to individual wells of the assay plate. It is recommended to run the standard in duplicate (see Note 1). Table 1 Standards dilution chart
Step
Final hAAT concentration (ng/ml)
hAAT Standard
Sample diluent w/blocking buffer
0
1000
10 μl of 1 mg/ml stock
10 ml
1
100
100 μl from step 0
900 μl
2
50
500 μl from step 1
500 μl
3
25
500 μl from step 2
500 μl
4
12.5
500 μl from step 3
500 μl
5
6.25
500 μl from step 4
500 μl
6
3.125
500 μl from step 5
500 μl
7
1.5625
500 μl from step 6
500 μl
8
0.78125
500 μl from step 7
500 μl
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8. Unknown samples: Optimal sample dilutions are dependent on the condition and should be determined by the user. Samples are diluted to desired concentrations in the standard range with blocking buffer. Duplicate wells are recommended (see Notes 1 and 2). 9. Positive control: Human serum (Cat#14-102E, BioWhittaker™ Media, Lonza Walkersville, Inc., Walkersville, MD). 20 μl aliquots stored at 80 C. Working dilution is 1:200,000 diluted in blocking buffer. Duplicate wells (2/plate) are recommended. 10. Secondary antibody: Anti-alpha-1 antitrypsin antibody (HRP conjugated) (Cat#7635, Abcam, Cambridge, UK) should be diluted 1:10 in 1 PBS. Store 23 μl aliquots at 80 C. Working concentration: 22 μl of aliquoted antibody diluted in 11 ml of blocking buffer (1:500 dilution). 11. TMB peroxidase substrate (TMB microwell peroxidase substrate system, SeraCare Life Sciences) stored at 4 C: prepare freshly by mixing 6 ml of TMB solution A with 6 ml of TMB solution B per 96-well plate being run. 12. Stop solution: 10 ml 95–98% of sulfuric acid, 90 ml of water. 13. Plate washer (optional) (MultiWash III Microplate Washer, TriContinent, Grass Valley, CA). 14. Microplate reader with 450 nm filter, such as VersaMax (Molecular Devices, Sunnyvale, CA). 15. Software (optional), such as SoftMax Pro (Molecular Devices).
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Methods 1. Add 100 μl of diluted coating antibody (see above) to each well of a 96-well plate. 2. Cover plate with pressure-sensitive film and incubate at room temperature for 1 h or at 4 C overnight. 3. Aspirate each well and wash with at least 200 μl of wash buffer, repeat two times (three total washes) (see Note 3). 4. Add 100 μl of blocking solution to each well, cover plate with pressure-sensitive film and incubate at room temperature for 1 h (see Note 4). 5. Repeat washes as in step 3. 6. Add 100 μl each of hAAT standard (dilutions 1–9), samples, and positive controls to their respective wells (Fig. 2). Load one well with blocking buffer to serve as a “blank”. Cover the plate
Quantification of Total Human Alpha-1 Antitrypsin by Sandwich ELISA
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Fig. 2 ELISA plate layout
with pressure-sensitive film and incubate at room temperature for 1 h (see Note 5). 7. Repeat washes as in step 3. 8. Add 100 μl of diluted secondary antibody to each well, cover the plate with pressure-sensitive film and incubate at room temperature for 1 h. Wash the plate six times with 200 μl of washing buffer per well. 9. Add 100 μl of TMB peroxidase substrate 1:1 dilution solution (see above) to each well. Develop the plate in the dark at room temperature for 30 min (see Notes 6 and 7). 10. Stop reaction by adding 50 μl of stop solution to each well (see Note 8). 11. Measure absorbance on a plate reader at 450 nm. 12. Calculate the concentration of unknown samples. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the samples [2]. (a) Calculate the mean absorbance for each set of duplicate standards, controls, and samples. Subtract the mean of blank control standard absorbance from each. (b) Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. (c) To determine the concentration of the unknowns, find the unknowns’ mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
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Notes 1. It is very important to vortex all standard vials well between each dilution step and to vortex again as each standard is loaded to the well. Be cautious not to contaminate any sample wells with the standard samples while loading the plate. 2. Keep the samples on ice to thaw. 3. Do not let the plate dry out after coating antibody added. The plate can be blocked for a longer time until the standard and sample preparation is finished. Always leave the washing buffer in the well until the solution for the next step is ready. 4. Incubation at room temperature is about 22–25 C. 5. Save at least one well for a blank control (load with blocking buffer). This will be used to determine the baseline background of the absorbance. 6. Bring the TMB peroxidase substrate to room temperature before use; this can be done by setting Solutions A and B on the bench an hour before it is needed (when the secondary antibody is added). Solution A and B must be protected from light. 7. Do not cover the plate with pressure-sensitive film on TMB peroxidase substrate development step. The solution must be kept in the dark for this step; we often accomplish this by placing the plate on a paper towel in a drawer during the incubation. 8. Read the plate within 30 min of the stop solution being added.
References 1. Song S, Scott-Jorgensen M, Wang J, Poirier A, Crawford J, Campbell-Thompson M, Flotte TR (2002) Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects. Mol Ther 6(3):329–335
2. Sono Biological Inc (online) ELISA protocolcalculation of results. ELISA encyclopedia http://www.elisa-antibody.com/generalelisa-protocol
