- Email: [email protected]
Comparison of different ELISA protocols for the detection of IgA against influenza nucleoproteins in trachea of vaccinated chickens
Comparison of different ELISA protocols for the detection of IgA against influenza nucleoproteins in trachea of vaccinated chickens V. A. Kuttappan,∗ L. R. Bielke,∗ 1 A. D. Wolfenden,∗ L. R. Berghman,† G. Tellez,∗ B. M. Hargis,∗ and O. B. Faulkner∗ ∗
Department of Poultry Science, University of Arkansas, Fayetteville, AR; and † Department of Poultry Science, Texas A&M University, College Station, TX
Key words: Avian influenza, mucosal immunity, nucleoprotein, ELISA, trachea 2015 Poultry Science 94:181–184 http://dx.doi.org/10.3382/ps/peu054
INTRODUCTION
munity can reduce the chances of disease and mortality, only the mucosal portion of this adaptive immune response is capable of protecting animals from infection. The key to inducing both an adaptive systemic and mucosal response has traditionally been through the use of the mucosa as a portal of entry for attenuated vaccines, with oral and respiratory routes being the preferred and most common routes of administration. A major challenge in measuring the efficacy of mucosal vaccination is the quantification of secretory IgA, especially in poultry, because techniques and reagents are not as readily available and plentiful as other species of animal commonly used in research, such as rodents. Agar gel immunodiffusion, antigen neutralization, and hemagglutination inhibition are examples of commonly used serological techniques used for the identification and quantification of antibodies (Barrett, 1978; Hawkes et al., 1983; Stanley, 2002), but these techniques often require large amounts of both antigens and antibodies. Enzyme-linked immunosorbent assay (ELISA) is an effective and comparatively inexpensive serological method that is widely used to detect antibodies both in serum and mucosal secretions. Various studies
Many infectious diseases affecting poultry have a mucosal route of entry, making mucosal protection by vaccination an important method of disease prevention. Primarily killed vaccines, which generally must be administered parenterally, have been applied to protect against systemic infections, and although they have been shown to reduce colonization and shedding, the protection provided by these vaccines has limited ability to prevent initial mucosal infection. They predominantly stimulate both humoral (circulating IgM and IgG) and cell-mediated responses but are quite ineffective at generating mucosal immunity because secretory IgA antibody stimulation is very low through this type of vaccination. This is important because, whereas both systemic (humoral and cell-mediated) and mucosal im C 2015 Poultry Science Association Inc. Received August 8, 2014. Accepted September 16, 2014. 1 Corresponding author: L. R. Bielke. POSC 0–114, University of Arkansas, Fayetteville, AR 72701. Tel: +479-575-8495; Fax: +479-5758490; Email: [email protected]
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was performed on NP1- or NP2-positive trachea samples, negative trachea samples, and blank wells with different levels of NP1 and NP2 coating peptides (5 or 10 μg/mL) using two different secondary antibodies (Gene Tex, GT, or Thermo Scientific, TS), with or without an acetate wash, and using maximum, medium, or low binding ELISA plates. The TS antibody resulted in a higher background signal compared to GT. Furthermore, coating plate wells with NP2 resulted in very high background compared to NP1. An acetate buffer wash resulted in the muffling of signals, and medium and low binding plates used in the study resulted in better results than maximum binding plates. These results suggest that the selection of appropriate secondary antibodies, binding plates, and ELISA reagent protocols all play important roles in determining NP1- or NP2specific IgA levels in trachea samples.
ABSTRACT Vaccines targeting mucosal immunity are important for the control of infection by pathogens with mucosal portals of entry, such as avian influenza. However, reliable and effective methods for determining levels of mucosal IgA stimulated by vaccination are not well developed in poultry and are necessary for determining efficacy. The objective of the present study was to compare different ELISA protocols to evaluate levels of mucosal IgA against two different sequences of nucleoprotein (NP), a highly conserved internal protein in avian influenza virus, in trachea. Positive control tracheas were obtained through hyperimmunization of birds with adjuvated NP1 and NP2 peptide conjugated with keyhole limpet hemocyanin administered both orally and parenterally; negative birds received no antigen. Trachea samples were homogenized, and supernatant fluid was collected to separate IgA. ELISA
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MATERIALS AND METHODS Hyperimmunization of birds All procedures involving live birds in this study were approved by the University of Arkansas Institutional Animal Care and Use Committee. For positive control immune samples, broiler chickens were hyperimmunized with NP1 and NP2 peptides conjugated with keyhole limpet hemocyanin (GenScript Corp., Piscataway Township, NJ) diluted in sterile water to 1mg/mL. The birds received parenteral (a subcutaneous injection of 0.17mg/bird) administration of conjugated peptide at 4, 20, 37, and 56 d of age, along with oral administration (0.33mg/bird with modified chitosan adjuvant) at 37 and 56 d. Three birds per peptide were hyperimmunized, but serum or tissue sample from a single bird was tested in the ELISA. Negative control birds were given no immunization. All birds were euthanized on day 66 of age by CO2 inhalation. Trachea samples were collected immediately after dissection of the birds and kept frozen at −20◦ C until further processing.
Tissue processing Trachea samples were collected, minced, weighed, and transferred to 2 mL tubes and diluted 2fold (w/v) in a phosphate buffered saline solution (PBS)-protease inhibitor (Complete, Roche Diagnostics GmbH, Mannheim, Germany) mixture of 1 tablet per 100 mL of PBS. The tubes were incubated overnight at room temperature, after which trachea samples were homogenized (Bio-Gen Pro200, PRO Scientific Inc., Oxford, CT) for 5 s each, after which samples were centrifuged (Z 233 M-2, HERMLE Labortechnik GmbH, Wehingen, Germany) at 17,500 rcf for 5 min at 4◦ C, and supernatant was collected. The supernatant was kept frozen at −20◦ C until further analysis.
Enzyme-linked immunosorbent assay An indirect ELISA was performed to quantify IgA present in supernatant obtained from processed tracheas. Two 96-well plates (Nunc MaxiSorp, Thermo Fisher Scientific, Rochester, NY) were coated with either 5 or 10 μg/mL of BSA-conjugated NP1 or NP2 peptide diluted in 0.05M sodium carbonate each. The plates were covered with a lid and allowed to incubate with the peptide for 2 h at room temperature or overnight at 4◦ C. Then the contents of the plates were emptied, tapped on a dry paper towel, and rinsed with 265 μL/well PBS + 2% Tween (Sigma Life Science, St. Louis, MO; PBS-T) 5 times. Individual wells were then blocked (265 μL/well) with 20% Superblock (Pierce Inc., Rockford, IL) in PBS for 45 min at room temperature. The plates were again emptied and rinsed as described above. Supernatant obtained from processed tracheas (both positive and negative control birds) were thawed to room temperature and diluted in Superblock (1:50), and 100 μL was added to the respective wells, while blank wells received only Superblock. Plates were then incubated for 1 h at room temperature and rinsed with PBS-T. Two types of affinity-purified HRP-conjugated goat anti-chicken IgA polyclonal antibodies – Gene Tex (GT, Irvine, CA) or Thermo Scientific (TS, Rockford, IL) – were diluted at 1:5,000 or 1:10,000 and added to individual wells (100 uL/well), incubated for 1 h at room temperature, and again rinsed with PBS-T. After washing, 100 uL of the tetramethylbenzidine substrate (TMB, BD Biosciences, San Diego, CA) was added to each well and incubated for 30 min at room temperature. The reaction was stopped with 25 uL of 1N sulfuric acid solution, and absorbance was measured at 450nm using an ELISA plate reader (Synergy HT, multi-mode microplate reader, BioTek Instruments, Inc., Winooski, VT, USA). For the NP1 peptide, ELISA was performed with plates having different adsorption characteristics using the procedure explained above. The 96 different well plates included Nunc MaxiSorp (Thermo Scientific, Rockford, IL) with high hydrophilic (++) binding capacity, Nunc Medisorp medium
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have reported the use of ELISA for the detection of IgA in bile (Mockett and Rose, 1986), intestinal (Cawthraw et al., 1994), and tracheal mucus (Yagahshi and Tajima, 1986; Hawkes et al., 1983). Typically, ELISAs require fewer antibodies, antigens, and reagents than the aforementioned methodologies, and the assay can be manipulated for direct or indirect (sandwich assay) detection of antibodies, depending on the desired level of specificity and detection signal. Yagahshi and Tajima (1986) reported that ELISA detection of antibodies against Mycoplasma gallisepticum from infected chickens was 100 times more sensitive than hemagglutination inhibition and tube agglutination assays, suggesting that ELISA is a viable option for the detection of antibodies in low concentrations or from small sample sizes. Specific methodologies and variations of ELISAs are largely dependent on sample type and quantity, available reagents, and what is being detected. For example, to measure total IgA, total antibody could be captured in a nondiscriminatory manner, and detection antibodies labeled anti-IgA would report only IgA within each well. Or, if antigen-specific IgA were desired, a well could be coated with antigen to capture IgA, and detection antibodies labeled anti-IgA would report only those IgA present that are specific for the antigen. Low concentrations of antibody or signal can be amplified by including secondary and detection antibodies. Additionally, assays can be affected by blocking reagents, the affinity of detection antibodies, and substrates used for detection. The present study aims to compare the use of different ELISA protocols in an attempt to optimize the detection of secretory IgA in tracheas of chickens in response to the administration of a recombinant Bacillus-vectored vaccine targeted to 2 different NP sequences from avian influenza virus.
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RESEARCH NOTE Table 1. OD of positive, negative, and blank from ELISAs with NP1 coating peptides with Thermo Scientific (TS) or Gene Tex (GT) secondary antibodies (1:5000 or 1:10000) with or without acetate and using plates with different binding properties. NP1 (10ug/mL)
NP1 (5ug/mL)
TS Positive∗ Negative∗ Blank ∗
GT
TS (1:10,000)
GT (1:10,000)
1:5,000
1:10,000
1:5,000
1:10,000
No acetate
acetate
No acetate
acetate
0.439 0.156 0.218
0.262 0.100 0.139
0.314 0.124 0.124
0.192 0.087 0.071
0.405 0.144 0.117
0.063 0.045 0.043
0.273 0.114 0.084
0.058 0.044 0.042
Samples from the same two birds (positive or negative) were used for all variables across the table.
Table 2. OD of positive, negative, and blank from ELISAs with NP2 coating peptides with Thermo Scientific (TS) or Gene Tex (GT) secondary antibodies (1:5,000 or 1:10,000) with or without acetate. NP2 (10ug/mL)
Positive∗ Negative∗ Blank ∗
NP2 (5ug/mL) GT
TS (1:10,000)
GT (1:10,000)
1:5,000
1:10,000
1:5,000
1:10,000
No acetate
Acetate
No acetate
Acetate
0.353 0.187 0.922
0.216 0.116 0.492
0.292 0.157 0.383
0.179 0.105 0.189
0.336 0.183 0.578
0.065 0.051 0.141
0.261 0.134 0.386
0.058 0.050 0.113
Samples from the same two birds (positive or negative) were used for all variables across the table.
binding plate with moderate hydrophilic (+) binding capacity, and Nunc low binding plate flat bottom Polystrene untreated plate (Thermo Fisher Scientific, Rochester, NY). However, a combination of TS and GT secondary antibodies (1:5,000 each) was used. Both positive and negative samples for NP1 and NP2, as well as the respective blanks, were plated in duplicate and the results reported as mean values. No statistical analysis was conducted because each data point is a mean of two samples.
RESULTS AND DISCUSSION The results from the present study (Table 1) showed that a higher NP1 peptide concentration of 10 μg/mL to coat wells resulted in a higher background signal with TS secondary antibodies, both at 1:5,000 and 1:10,000 secondary antibody dilutions, compared to GT secondary antibodies. This could be due to increased nonspecific binding by secondary antibodies to coated peptides or, perhaps, incomplete blocking. However, the absence of such a higher signal in the negative wells indicates that the addition of tracheal extract could have blocked some of the nonspecific binding sites. Moreover, the lack of higher background at a lower NP1 peptide concentration, as well as a higher background signal for higher concentration of secondary antibodies, confirms the possibility of nonspecific binding between TS secondary antibodies and NP1– BSA-conjugated peptide. On the other hand, TS secondary antibodies always produced numerically better separation between the positive and negative controls compared to GT, regardless of peptide or secondary antibody concentration. This implies that TS secondary antibodies have better sensitivity but lack specificity. The use of acetate
buffer as a wash between the addition of secondary antibody and substrate is usually employed to produce an acidic environment, which could result in better enzyme–substrate reactions. However, in the present study, the use of acetate buffer resulted in the muffling of signals in the case of both TS and GT secondary antibodies (Table 1). When absorbent plates with different binding characteristics (with TS and GT secondary antibodies) were considered, medium (positive 0.507; negative 0.221; blank 0.121) and low (positive 0.403; negative 0.175; blank 0.098) binding plates showed numerically higher signals and better separation between positive and negative samples when compared to the Maxisorp (positive 0.232; negative 0.107; blank 0.073) plates. However, Maxisorp plates showed the numerically lowest blank signal, suggesting that, perhaps, the blocking agent did not completely coat wells in the medium and low binding plates. Additionally, this decreased binding capacity and resultant low blockage may have increased nonspecific binding of samples to the wells, which may have caused high negative control signals. With NP2-coated wells, background signals were very high in almost all cases, which could be due to a higher affinity of chicken IgA-specific secondary antibodies to NP2 peptides (Table 2). Wu et al. (2007) and LaMere et al. (2011) demonstrated that a NP–ELISA could be an effective method for screening influenza antibodies present in serum samples. However, the present study showed that the protocol of IgA extraction from trachea samples and NP– ELISA in mucosal samples needs to be better standardized before being used effectively. Even though the data presented in these studies were completed with few samples, the trend seen in the data may be helpful for designing future experiments.
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TS
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REFERENCES Barrett, J. T. 1978. Textbook of immunology: An introduction to immunochemistry and immunobiology. Third edition. The C. V. Mosby Company, St. Louis, MO. Cawthraw, S., R. Ayling, P. Nuijten, T. Wassenaar, and D. G. Newell. 1994. Isotype, specificity, and kinetics of systemic and mucosal antibodies to Campylobacter jejuni antigens, including flagellin, during experimental oral infections of chickens. Avian Dis. 38: 341–349. Epstein, S. L., W. P. Kong, J. A. Misplon, C. Y. Lo, T. M. Tumpey, L. Xu, and G. J. Nabel. 2005. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine 23:5404–5410. Fiers, W., M. De Filette, A. Birkett, S. Neirynck, and W. M. Jou. 2004. A “universal” human inffiuenza A vaccine. Virus Res. 103:173–176. Hawkes, R. A., J. H. Darbyshire, R. W. Peters, A. P. A. Mockett, and D. Cavanagh. 1983. Presence of viral antigens and antibody in the trachea of chickens infected with avian infectious bronchitis virus. Avian Pathol. 12:331–340.
Layton, S. L., D. R. Kapczynski, S. Higgins, J. Higgins, A. D. Wolfenden, K. A. Liljebjelke, W. G. Bottje, D. Swayne, L. R. Berghman, Y. M. Kwon, B. M. Hargis, and K. Cole. 2009. Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge. Poult. Sci. 88:2244–2252. LaMere, M. W., A. Moquin, F. E. Lee, R. S. Misra, P. J. Blair, L. Haynes, T. D. Randall, F. E. Lund, and D. A. Kaminski. 2011. Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance. J. Virol. 85:5027– 5035. Mockett, A. P., and M. E. Rose. 1986. Immune responses to eimeria: Quantification of antibody isotypes to Eimeria tenella in chicken serum and bile by means of the ELISA. Parasite Immunol. 8:481– 489. OIE. 2013. http://www.oie.int/?id=1682 accessed on August 20. Patel, A., K. Tran, M. Gray, Y. Li, Z. Ao, X. Yao, D. Kobasa, and G. P. Kobinger. 2009. Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1viruses. Vaccine 27:3083–3089. Rao, S. S., W. P. Kong, C. J. Wei, N. V. Hoeven, J. P. Gorres, M. Nason, H. Andersen, T. M. Tumpey, and G. J. Nabel. 2010. Comparative efficacy of hemagglutinin, nucleoprotein, and matrix 2 protein gene-based vaccination against H5N1 influenza in mouse and ferret. PLoS 5:e9812 1–11. Stanley, J. 2002. Essentials of immunology and serology. Thomson Delmar Learning. Albany, NY. Suarez, D. L. 2008. Influenza A virus. Pages 3–22 in Avian influenza. 1st ed. D. E. Swayne Blackwell Publishing, Ames, IA. Swayne, D. E., and D. A. Halvorson. 2008. Influenza. Pages 153–184 in Diseases of Poultry. 12th ed. Y. M. Saif, A. M. Fadley, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Blackwell Publishing Professional, Ames, IA. Swayne, D. E., and D. R. Kapczynski. 2008. Pages 407–452 in Avian influenza. 1st ed. D. E. Swayne Blackwell Publishing, Ames, IA. Tompkins, S.M., Z. S. Zhao, C. Y. Lo, J.A. Misplon, T. Liu, Z. Ye, R. J. Hogan, Z. Wu, K. A. Benton, T. M. Tumpey, and S. L. Epstein. 2007. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg. Infect. Dis. 13:426–435. Tong, S., Y. Li, P. Rivailler, C. Conrardy, D. A. Alvarez Castillo, L. M. Chen, S. Recuenco, J. A. Ellison, C. T. Davis, I. A. York, A. S. Turmelle, D. Moran, S. Rogers, M. Shi, Y. Tao, M. R. Weil, K. Tang, L. A. Rowe, S. Sammons, X. Xu, M. Frace, K. A. Lindblade, N. J. Cox, L. J. Anderson, C. E. Rupprecht, and R.O. Donis. 2012. A distinct lineage of influenza A virus from bats. PNAS 109:4269–4274. Wu, R., S. Hu, Y. Xiao, Z. Li, D. Shi, and D. Bi. 2007. Development of indirect enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection and quantification of antibodies against avian influenza virus. Vet. Res. Commun. 31:631– 641. Yagihashi, T., and M. Tajima. 1986. Antibody responses in sera and respiratory secretions from chickens infected with Mycoplasma gallisepticum. Avian Dis. 30:543–550.
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In conclusion, results from the present study showed that detection of IgA in trachea samples can be highly dependent on the binding peptide and specificity of the secondary antibodies. Furthermore, the low, medium, and maximum binding properties of the plates used may affect the results. In fact, this research note is a description of our experiences, which may help readers to optimize IgA detection in birds with the understanding that each assay appears to be dependent on multiple variables beyond just sample type and antigen. Presently, these results (and other unpublished data) suggest a high degree of variability from experiment to experiment among different tissues, antigens, and reagents for chicken IgA detection. Also, this study suggests that anti-chicken IgA detection antibodies are variable and highly dependent on a number of factors that differ from experiment to experiment. A major issue with the trachea extracts could be the occurrence of higher amounts of impurities with lower concentrations of IgA extracted from the tissue, which might have influenced the ELISA assay. Thus, future studies are required for the quantification of IgA using an ELISA protocol with different blocking agents, a different IgA extraction method from the trachea, or other tissue samples like GI scrapings.
Department of Poultry Science, University of Arkansas, Fayetteville, AR; and † Department of Poultry Science, Texas A&M University, College Station, TX
Key words: Avian influenza, mucosal immunity, nucleoprotein, ELISA, trachea 2015 Poultry Science 94:181–184 http://dx.doi.org/10.3382/ps/peu054
INTRODUCTION
munity can reduce the chances of disease and mortality, only the mucosal portion of this adaptive immune response is capable of protecting animals from infection. The key to inducing both an adaptive systemic and mucosal response has traditionally been through the use of the mucosa as a portal of entry for attenuated vaccines, with oral and respiratory routes being the preferred and most common routes of administration. A major challenge in measuring the efficacy of mucosal vaccination is the quantification of secretory IgA, especially in poultry, because techniques and reagents are not as readily available and plentiful as other species of animal commonly used in research, such as rodents. Agar gel immunodiffusion, antigen neutralization, and hemagglutination inhibition are examples of commonly used serological techniques used for the identification and quantification of antibodies (Barrett, 1978; Hawkes et al., 1983; Stanley, 2002), but these techniques often require large amounts of both antigens and antibodies. Enzyme-linked immunosorbent assay (ELISA) is an effective and comparatively inexpensive serological method that is widely used to detect antibodies both in serum and mucosal secretions. Various studies
Many infectious diseases affecting poultry have a mucosal route of entry, making mucosal protection by vaccination an important method of disease prevention. Primarily killed vaccines, which generally must be administered parenterally, have been applied to protect against systemic infections, and although they have been shown to reduce colonization and shedding, the protection provided by these vaccines has limited ability to prevent initial mucosal infection. They predominantly stimulate both humoral (circulating IgM and IgG) and cell-mediated responses but are quite ineffective at generating mucosal immunity because secretory IgA antibody stimulation is very low through this type of vaccination. This is important because, whereas both systemic (humoral and cell-mediated) and mucosal im C 2015 Poultry Science Association Inc. Received August 8, 2014. Accepted September 16, 2014. 1 Corresponding author: L. R. Bielke. POSC 0–114, University of Arkansas, Fayetteville, AR 72701. Tel: +479-575-8495; Fax: +479-5758490; Email: [email protected]
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was performed on NP1- or NP2-positive trachea samples, negative trachea samples, and blank wells with different levels of NP1 and NP2 coating peptides (5 or 10 μg/mL) using two different secondary antibodies (Gene Tex, GT, or Thermo Scientific, TS), with or without an acetate wash, and using maximum, medium, or low binding ELISA plates. The TS antibody resulted in a higher background signal compared to GT. Furthermore, coating plate wells with NP2 resulted in very high background compared to NP1. An acetate buffer wash resulted in the muffling of signals, and medium and low binding plates used in the study resulted in better results than maximum binding plates. These results suggest that the selection of appropriate secondary antibodies, binding plates, and ELISA reagent protocols all play important roles in determining NP1- or NP2specific IgA levels in trachea samples.
ABSTRACT Vaccines targeting mucosal immunity are important for the control of infection by pathogens with mucosal portals of entry, such as avian influenza. However, reliable and effective methods for determining levels of mucosal IgA stimulated by vaccination are not well developed in poultry and are necessary for determining efficacy. The objective of the present study was to compare different ELISA protocols to evaluate levels of mucosal IgA against two different sequences of nucleoprotein (NP), a highly conserved internal protein in avian influenza virus, in trachea. Positive control tracheas were obtained through hyperimmunization of birds with adjuvated NP1 and NP2 peptide conjugated with keyhole limpet hemocyanin administered both orally and parenterally; negative birds received no antigen. Trachea samples were homogenized, and supernatant fluid was collected to separate IgA. ELISA
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MATERIALS AND METHODS Hyperimmunization of birds All procedures involving live birds in this study were approved by the University of Arkansas Institutional Animal Care and Use Committee. For positive control immune samples, broiler chickens were hyperimmunized with NP1 and NP2 peptides conjugated with keyhole limpet hemocyanin (GenScript Corp., Piscataway Township, NJ) diluted in sterile water to 1mg/mL. The birds received parenteral (a subcutaneous injection of 0.17mg/bird) administration of conjugated peptide at 4, 20, 37, and 56 d of age, along with oral administration (0.33mg/bird with modified chitosan adjuvant) at 37 and 56 d. Three birds per peptide were hyperimmunized, but serum or tissue sample from a single bird was tested in the ELISA. Negative control birds were given no immunization. All birds were euthanized on day 66 of age by CO2 inhalation. Trachea samples were collected immediately after dissection of the birds and kept frozen at −20◦ C until further processing.
Tissue processing Trachea samples were collected, minced, weighed, and transferred to 2 mL tubes and diluted 2fold (w/v) in a phosphate buffered saline solution (PBS)-protease inhibitor (Complete, Roche Diagnostics GmbH, Mannheim, Germany) mixture of 1 tablet per 100 mL of PBS. The tubes were incubated overnight at room temperature, after which trachea samples were homogenized (Bio-Gen Pro200, PRO Scientific Inc., Oxford, CT) for 5 s each, after which samples were centrifuged (Z 233 M-2, HERMLE Labortechnik GmbH, Wehingen, Germany) at 17,500 rcf for 5 min at 4◦ C, and supernatant was collected. The supernatant was kept frozen at −20◦ C until further analysis.
Enzyme-linked immunosorbent assay An indirect ELISA was performed to quantify IgA present in supernatant obtained from processed tracheas. Two 96-well plates (Nunc MaxiSorp, Thermo Fisher Scientific, Rochester, NY) were coated with either 5 or 10 μg/mL of BSA-conjugated NP1 or NP2 peptide diluted in 0.05M sodium carbonate each. The plates were covered with a lid and allowed to incubate with the peptide for 2 h at room temperature or overnight at 4◦ C. Then the contents of the plates were emptied, tapped on a dry paper towel, and rinsed with 265 μL/well PBS + 2% Tween (Sigma Life Science, St. Louis, MO; PBS-T) 5 times. Individual wells were then blocked (265 μL/well) with 20% Superblock (Pierce Inc., Rockford, IL) in PBS for 45 min at room temperature. The plates were again emptied and rinsed as described above. Supernatant obtained from processed tracheas (both positive and negative control birds) were thawed to room temperature and diluted in Superblock (1:50), and 100 μL was added to the respective wells, while blank wells received only Superblock. Plates were then incubated for 1 h at room temperature and rinsed with PBS-T. Two types of affinity-purified HRP-conjugated goat anti-chicken IgA polyclonal antibodies – Gene Tex (GT, Irvine, CA) or Thermo Scientific (TS, Rockford, IL) – were diluted at 1:5,000 or 1:10,000 and added to individual wells (100 uL/well), incubated for 1 h at room temperature, and again rinsed with PBS-T. After washing, 100 uL of the tetramethylbenzidine substrate (TMB, BD Biosciences, San Diego, CA) was added to each well and incubated for 30 min at room temperature. The reaction was stopped with 25 uL of 1N sulfuric acid solution, and absorbance was measured at 450nm using an ELISA plate reader (Synergy HT, multi-mode microplate reader, BioTek Instruments, Inc., Winooski, VT, USA). For the NP1 peptide, ELISA was performed with plates having different adsorption characteristics using the procedure explained above. The 96 different well plates included Nunc MaxiSorp (Thermo Scientific, Rockford, IL) with high hydrophilic (++) binding capacity, Nunc Medisorp medium
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney on March 15, 2015
have reported the use of ELISA for the detection of IgA in bile (Mockett and Rose, 1986), intestinal (Cawthraw et al., 1994), and tracheal mucus (Yagahshi and Tajima, 1986; Hawkes et al., 1983). Typically, ELISAs require fewer antibodies, antigens, and reagents than the aforementioned methodologies, and the assay can be manipulated for direct or indirect (sandwich assay) detection of antibodies, depending on the desired level of specificity and detection signal. Yagahshi and Tajima (1986) reported that ELISA detection of antibodies against Mycoplasma gallisepticum from infected chickens was 100 times more sensitive than hemagglutination inhibition and tube agglutination assays, suggesting that ELISA is a viable option for the detection of antibodies in low concentrations or from small sample sizes. Specific methodologies and variations of ELISAs are largely dependent on sample type and quantity, available reagents, and what is being detected. For example, to measure total IgA, total antibody could be captured in a nondiscriminatory manner, and detection antibodies labeled anti-IgA would report only IgA within each well. Or, if antigen-specific IgA were desired, a well could be coated with antigen to capture IgA, and detection antibodies labeled anti-IgA would report only those IgA present that are specific for the antigen. Low concentrations of antibody or signal can be amplified by including secondary and detection antibodies. Additionally, assays can be affected by blocking reagents, the affinity of detection antibodies, and substrates used for detection. The present study aims to compare the use of different ELISA protocols in an attempt to optimize the detection of secretory IgA in tracheas of chickens in response to the administration of a recombinant Bacillus-vectored vaccine targeted to 2 different NP sequences from avian influenza virus.
183
RESEARCH NOTE Table 1. OD of positive, negative, and blank from ELISAs with NP1 coating peptides with Thermo Scientific (TS) or Gene Tex (GT) secondary antibodies (1:5000 or 1:10000) with or without acetate and using plates with different binding properties. NP1 (10ug/mL)
NP1 (5ug/mL)
TS Positive∗ Negative∗ Blank ∗
GT
TS (1:10,000)
GT (1:10,000)
1:5,000
1:10,000
1:5,000
1:10,000
No acetate
acetate
No acetate
acetate
0.439 0.156 0.218
0.262 0.100 0.139
0.314 0.124 0.124
0.192 0.087 0.071
0.405 0.144 0.117
0.063 0.045 0.043
0.273 0.114 0.084
0.058 0.044 0.042
Samples from the same two birds (positive or negative) were used for all variables across the table.
Table 2. OD of positive, negative, and blank from ELISAs with NP2 coating peptides with Thermo Scientific (TS) or Gene Tex (GT) secondary antibodies (1:5,000 or 1:10,000) with or without acetate. NP2 (10ug/mL)
Positive∗ Negative∗ Blank ∗
NP2 (5ug/mL) GT
TS (1:10,000)
GT (1:10,000)
1:5,000
1:10,000
1:5,000
1:10,000
No acetate
Acetate
No acetate
Acetate
0.353 0.187 0.922
0.216 0.116 0.492
0.292 0.157 0.383
0.179 0.105 0.189
0.336 0.183 0.578
0.065 0.051 0.141
0.261 0.134 0.386
0.058 0.050 0.113
Samples from the same two birds (positive or negative) were used for all variables across the table.
binding plate with moderate hydrophilic (+) binding capacity, and Nunc low binding plate flat bottom Polystrene untreated plate (Thermo Fisher Scientific, Rochester, NY). However, a combination of TS and GT secondary antibodies (1:5,000 each) was used. Both positive and negative samples for NP1 and NP2, as well as the respective blanks, were plated in duplicate and the results reported as mean values. No statistical analysis was conducted because each data point is a mean of two samples.
RESULTS AND DISCUSSION The results from the present study (Table 1) showed that a higher NP1 peptide concentration of 10 μg/mL to coat wells resulted in a higher background signal with TS secondary antibodies, both at 1:5,000 and 1:10,000 secondary antibody dilutions, compared to GT secondary antibodies. This could be due to increased nonspecific binding by secondary antibodies to coated peptides or, perhaps, incomplete blocking. However, the absence of such a higher signal in the negative wells indicates that the addition of tracheal extract could have blocked some of the nonspecific binding sites. Moreover, the lack of higher background at a lower NP1 peptide concentration, as well as a higher background signal for higher concentration of secondary antibodies, confirms the possibility of nonspecific binding between TS secondary antibodies and NP1– BSA-conjugated peptide. On the other hand, TS secondary antibodies always produced numerically better separation between the positive and negative controls compared to GT, regardless of peptide or secondary antibody concentration. This implies that TS secondary antibodies have better sensitivity but lack specificity. The use of acetate
buffer as a wash between the addition of secondary antibody and substrate is usually employed to produce an acidic environment, which could result in better enzyme–substrate reactions. However, in the present study, the use of acetate buffer resulted in the muffling of signals in the case of both TS and GT secondary antibodies (Table 1). When absorbent plates with different binding characteristics (with TS and GT secondary antibodies) were considered, medium (positive 0.507; negative 0.221; blank 0.121) and low (positive 0.403; negative 0.175; blank 0.098) binding plates showed numerically higher signals and better separation between positive and negative samples when compared to the Maxisorp (positive 0.232; negative 0.107; blank 0.073) plates. However, Maxisorp plates showed the numerically lowest blank signal, suggesting that, perhaps, the blocking agent did not completely coat wells in the medium and low binding plates. Additionally, this decreased binding capacity and resultant low blockage may have increased nonspecific binding of samples to the wells, which may have caused high negative control signals. With NP2-coated wells, background signals were very high in almost all cases, which could be due to a higher affinity of chicken IgA-specific secondary antibodies to NP2 peptides (Table 2). Wu et al. (2007) and LaMere et al. (2011) demonstrated that a NP–ELISA could be an effective method for screening influenza antibodies present in serum samples. However, the present study showed that the protocol of IgA extraction from trachea samples and NP– ELISA in mucosal samples needs to be better standardized before being used effectively. Even though the data presented in these studies were completed with few samples, the trend seen in the data may be helpful for designing future experiments.
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In conclusion, results from the present study showed that detection of IgA in trachea samples can be highly dependent on the binding peptide and specificity of the secondary antibodies. Furthermore, the low, medium, and maximum binding properties of the plates used may affect the results. In fact, this research note is a description of our experiences, which may help readers to optimize IgA detection in birds with the understanding that each assay appears to be dependent on multiple variables beyond just sample type and antigen. Presently, these results (and other unpublished data) suggest a high degree of variability from experiment to experiment among different tissues, antigens, and reagents for chicken IgA detection. Also, this study suggests that anti-chicken IgA detection antibodies are variable and highly dependent on a number of factors that differ from experiment to experiment. A major issue with the trachea extracts could be the occurrence of higher amounts of impurities with lower concentrations of IgA extracted from the tissue, which might have influenced the ELISA assay. Thus, future studies are required for the quantification of IgA using an ELISA protocol with different blocking agents, a different IgA extraction method from the trachea, or other tissue samples like GI scrapings.