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Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE
| Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
| Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
| Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
| Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
All papers submitted to Gut and Liver are reviewed by the editorial team before being sent out for an external peer review to rule out papers that have low priority, insufficient originality, scientific flaws, or the absence of a message of importance to the readers of the Journal. A decision about these papers will usually be made within two or three weeks.
The remaining articles are usually sent to two reviewers. It would be very helpful if you could suggest a selection of reviewers and include their contact details. We may not always use the reviewers you recommend, but suggesting reviewers will make our reviewer database much richer; in the end, everyone will benefit. We reserve the right to return manuscripts in which no reviewers are suggested.
The final responsibility for the decision to accept or reject lies with the editors. In many cases, papers may be rejected despite favorable reviews because of editorial policy or a lack of space. The editor retains the right to determine publication priorities, the style of the paper, and to request, if necessary, that the material submitted be shortened for publication.
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Ji-Hyun Seo*, Jin-Su Jun*, Hee-Shang Youn**, Jung Sook Yeom*, Ji Sook Park*, Chan-Hoo Park*, Hyang-Ok Woo*, Woo-Kon Lee†, Myung-Je Cho†, and Kwang-Ho Rhee†
*Department of Pediatrics, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea.
†Department of Microbiology, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea.
Correspondence to: Hee-Shang Youn. Department of Pediatrics, Gyeongsang National University School of Medicine, 79 Gangnam-ro, Jinju 660-702, Korea. Tel: +82-55-750-8158, Fax: +82-55-752-9339, hsyoun@gnu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gut Liver 2013;7(4):437-442. https://doi.org/10.5009/gnl.2013.7.4.437
Published online June 11, 2013, Published date July 31, 2013
Copyright © Gut and Liver.
We aimed to develop a quantitative enzyme-linked immunosorbent assay (ELISA) using whole-cell lysates of
Data from 300 plates were obtained by two different operators. Standard sera were used to make a standard curve to analyze the quantity of anti-
The mean IgG-antibody titers in
Using the current ELISA based on whole-cell lysates of
Keywords:
Enzyme-linked immunosorbent assay (ELISA) has long been used to detect immunoglobulin G (IgG) for screening
Quantitative evaluation of anti-
The usefulness of most commercial ELISA kits is generally limited by the specificity and sensitivity of antigens used for the assays.7 For instance, ELISA for anti-
We reasoned that if quantitative ELISA measurements of anti-
To make a standard curve, 96-well flat-bottomed ELISA plates (EIA plate, Costar 3590; Costar, Cornig, NY, USA) were coated overnight with 50 µL per well of purified antigen diluted with coating buffer (10 µg/mL) containing 50 mM sodium carbonate buffer (pH9.6; Bioshop, Burlington, ON, Canada) at 4℃. To initiate the assay, plates were blocked with 150 µL/well of blocking buffer containing 3% bovine serum albumin in PBS with Tween (PBST) (BioShop) for 3 hours at 37℃. Independent triplicate dilutions of standard pooled serum (IgG 1:400, and IgA 1:100) were prepared in dilution buffer (PBST) and 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) in PBS. Diluted sera were added to antigen-coated wells (50 µL/well) and incubated for 1 hour at 37℃. After four washing with washing buffer (PBST), 50 µL of peroxide-conjugated goat antihuman IgG (Bethyl Laboratories Inc., Montgomery, TX, USA; diluted 1:30,000) and IgA (Bethyl Laboratories Inc.; diluted 1:5,000) were added to the wells and incubated at 37℃ for 1 hour. The plates were then washed five times with PBST and wells were added with 0.8 mg of o-nitrophenyl phosphate (Sigma-Aldrich). The reaction was stopped after 30 minutes at room temperature by adding 25 µL of stopping buffer which contained 2N H2SO4. Immediately after this addition, optical density (OD) was measured at 492 nm using a VERSA max microplate reader (Molecular Devices Co., Sunnyvale, CA, USA).
Sera from 300 patients selected for strong positivity in in-house immunoblot assay10 were used to prepare a positive reference standard (standard pooled sera). After making the appropriate pool, all standard sera were aliquoted (200 µL of each) and maintained at -80℃ until required. Antibody units were assigned to each standard serum following the methods indicated by Miura et al.11 First, one of the aliquots was diluted in 2-fold steps from 1:1,000 to 1:526,000, 1:2,500 to 1:2,560,000, 1:5,000 to 1:5,120,000, and 1:7,500 to 7,680,000 with dilution buffer. These serially diluted sera were applied on an ELISA plate as primary antibodies. Four wells per set of serially diluted standard did not receive any samples or buffer during this step (blank wells), but other ELISA steps (adding blocking buffer, incubating with secondary antibody, adding substrate, and stopping buffer) were performed as those for other wells. These blank wells were used as the negative control wells (reciprocal number of dilution was assigned as 0). The relationship between reciprocal number of the dilution and OD492 was approximated by a 4-parameter hyperbolic curve (SOFTmax PRO version 5; Molecular Devices Co., Sunnyvale, CA, USA). If the R2 of that curve fit was less than 0.997, another standard serum aliquot was thawed and the entire procedure was repeated. Based on the constants of the equation, antibody units were assigned to the standard as the reciprocal of the dilution giving an OD492=1. Once the antibody units of a standard serum were determined, the number was used invariably for all samples tested by ELISA against that standard. Thereafter a reference standard was used on each ELISA plate to make a standard curve. To do this, standard serum aliquots were freshly thawed and two independent serial dilutions (2-fold steps, 10 different dilutions) were prepared starting with a dilution of 20 antibody units. These were applied to the wells on the ELISA plate and four blank wells (assigned zero antibody units) were also designated. The OD values were then fitted to a 4 parameter standard curve (antibody units=C{[(A-OD492)/(OD492-D)]^(1/B)}) using SOFTmax PRO version 5 software; Molecular Devices, Sunnyvale, CA, USA). The antibody units in test sera were then calculated from their OD492 values using the parameters estimated from this standard curve.
To evaluate the validity of this ELISA, sera collected from
Pooled sera were serially diluted and analyzed by ELISA. Two numbers were generated for each test serum. Using a single OD value which was closest to 1 in each set of diluted sera, the antibody units were calculated referring to a standard curve. Using all OD values from serially diluted sera, the reciprocal dilution giving an OD492=1 calculated based on a 4-parameter fitted hyperbolic curve and the reciprocal dilution giving an OD492 of 1 calculated from full dilution curves were significantly correlated (Spearman rank correlation, 1.0; p<0.0001) (Fig. 1). Thus to obtain the antibody units, we reasoned that a single dilution was enough if serially diluted standard serum was tested in the same ELISA plate. The antibody units of IgG and IgA contained in the pooled sera were determined to be 1,200 and 200 IU/mL, respectively.
To determine the criteria for acceptable quality of an ELISA plate, data from 300 plates were analyzed by two different operators using sera from whole cell lysate of
The quality of data obtained with standard sera was examined using two data sets. Standard sera were serially diluted over 100 times and antibody units for each dilution of standard sera were calculated. The relative units were calculated by comparison with the antibody units of the sera that came from the dilution showing an OD492 closest to 1.0.
Based on the data above, we determined the criteria for acceptable quality for ELISA plates as the following:
1) R2 of the standard curve should be equal to or greater than 0.997.
2) The OD492 of blank wells should be less than 0.04.
If the R2 of standard curve and blank values did not match these criteria, all data were discarded.
Following the criteria listed above, antibody units of standard sera were analyzed by ELISA to evaluate the reproducibility of the data. Pooled standard sera were aliquoted and frozen at -80℃ before use, and these sera were tested at an interval of several months. The concordance between the two sets of the data is highly significant judged by the linear regression analysis (coefficient was 1.01; 95% confidence interval [CI], 0.97 to 1.06; p<0.001). The reproducibility of data obtained with 224 antisera by two different operators using the same standard was also high (coefficient was 0.84; 95% CI, 0.97 to 1.06; p<0.001, data not shown).
One hundred fourteen
The reliability of ELISA test is required with the standard curve for distinguishing positive and negative sera in infectious diseases, and for reproducibility. In this study, the pooled standard sera were used for making a standard curve. There are some advantages to using the pooled standard sera to make a standard curve in analyzing the quantity of antibody in
In a previous study, we showed that the Genedia IgG ELISA kit, which used
To evaluate the validity of our ELISA, we used it to compare antibody titers between
There are some limitations to the present study including the lack of comparison with other commercially available-ELISAs, the absence of negative standard sera, and a small study population for each age group to evaluate its validity.
In conclusion, we have developed a reliable ELISA method to detect anti-
Fig. 1.Comparison of enzyme-linked immunosorbent assay antibody units derived from single versus multiple serial dilutions. (A) Immunoglobulin G (IgG). (B) IgA. Antibody units were calculated from the OD492 reading of a single dilution referred to a standard curve and plotted on the X-axis. The standard sera were serially diluted to generate a complete dilution curve. The reciprocal of the dilution giving an OD492 reading of 1 was calculated (on the Y-axis) from the dilution curve.OD, optical density.
Fig. 2.Quality-control parameters for an enzyme-linked immunosorbent assay standard curve. The R2 of a standard curve (fitted to a 4-parameter hyperbolic curve) is plotted on the X-axis, and the arithmetic mean value of OD492 of 4 blank wells on the same plate is plotted on the Y-axis. (A) Immunoglobulin G (IgG). (B) IgA.OD, optical density.
Fig. 3.Comparison of immunoglobulin G (IgG) antibody titers in *p<0.0001.
Fig. 4.Comparison of immunoglobulin A (IgA) antibody titers in the *p<0.05.
IgG, immunoglobulin G; IgA, immunoglobulin A.
*p=0.021.
*p=0.021.
Gut Liver 2013; 7(4): 437-442
Published online July 31, 2013 https://doi.org/10.5009/gnl.2013.7.4.437
Copyright © Gut and Liver.
Ji-Hyun Seo*, Jin-Su Jun*, Hee-Shang Youn**, Jung Sook Yeom*, Ji Sook Park*, Chan-Hoo Park*, Hyang-Ok Woo*, Woo-Kon Lee†, Myung-Je Cho†, and Kwang-Ho Rhee†
*Department of Pediatrics, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea.
†Department of Microbiology, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea.
Correspondence to: Hee-Shang Youn. Department of Pediatrics, Gyeongsang National University School of Medicine, 79 Gangnam-ro, Jinju 660-702, Korea. Tel: +82-55-750-8158, Fax: +82-55-752-9339, hsyoun@gnu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
We aimed to develop a quantitative enzyme-linked immunosorbent assay (ELISA) using whole-cell lysates of
Data from 300 plates were obtained by two different operators. Standard sera were used to make a standard curve to analyze the quantity of anti-
The mean IgG-antibody titers in
Using the current ELISA based on whole-cell lysates of
Keywords:
Enzyme-linked immunosorbent assay (ELISA) has long been used to detect immunoglobulin G (IgG) for screening
Quantitative evaluation of anti-
The usefulness of most commercial ELISA kits is generally limited by the specificity and sensitivity of antigens used for the assays.7 For instance, ELISA for anti-
We reasoned that if quantitative ELISA measurements of anti-
To make a standard curve, 96-well flat-bottomed ELISA plates (EIA plate, Costar 3590; Costar, Cornig, NY, USA) were coated overnight with 50 µL per well of purified antigen diluted with coating buffer (10 µg/mL) containing 50 mM sodium carbonate buffer (pH9.6; Bioshop, Burlington, ON, Canada) at 4℃. To initiate the assay, plates were blocked with 150 µL/well of blocking buffer containing 3% bovine serum albumin in PBS with Tween (PBST) (BioShop) for 3 hours at 37℃. Independent triplicate dilutions of standard pooled serum (IgG 1:400, and IgA 1:100) were prepared in dilution buffer (PBST) and 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) in PBS. Diluted sera were added to antigen-coated wells (50 µL/well) and incubated for 1 hour at 37℃. After four washing with washing buffer (PBST), 50 µL of peroxide-conjugated goat antihuman IgG (Bethyl Laboratories Inc., Montgomery, TX, USA; diluted 1:30,000) and IgA (Bethyl Laboratories Inc.; diluted 1:5,000) were added to the wells and incubated at 37℃ for 1 hour. The plates were then washed five times with PBST and wells were added with 0.8 mg of o-nitrophenyl phosphate (Sigma-Aldrich). The reaction was stopped after 30 minutes at room temperature by adding 25 µL of stopping buffer which contained 2N H2SO4. Immediately after this addition, optical density (OD) was measured at 492 nm using a VERSA max microplate reader (Molecular Devices Co., Sunnyvale, CA, USA).
Sera from 300 patients selected for strong positivity in in-house immunoblot assay10 were used to prepare a positive reference standard (standard pooled sera). After making the appropriate pool, all standard sera were aliquoted (200 µL of each) and maintained at -80℃ until required. Antibody units were assigned to each standard serum following the methods indicated by Miura et al.11 First, one of the aliquots was diluted in 2-fold steps from 1:1,000 to 1:526,000, 1:2,500 to 1:2,560,000, 1:5,000 to 1:5,120,000, and 1:7,500 to 7,680,000 with dilution buffer. These serially diluted sera were applied on an ELISA plate as primary antibodies. Four wells per set of serially diluted standard did not receive any samples or buffer during this step (blank wells), but other ELISA steps (adding blocking buffer, incubating with secondary antibody, adding substrate, and stopping buffer) were performed as those for other wells. These blank wells were used as the negative control wells (reciprocal number of dilution was assigned as 0). The relationship between reciprocal number of the dilution and OD492 was approximated by a 4-parameter hyperbolic curve (SOFTmax PRO version 5; Molecular Devices Co., Sunnyvale, CA, USA). If the R2 of that curve fit was less than 0.997, another standard serum aliquot was thawed and the entire procedure was repeated. Based on the constants of the equation, antibody units were assigned to the standard as the reciprocal of the dilution giving an OD492=1. Once the antibody units of a standard serum were determined, the number was used invariably for all samples tested by ELISA against that standard. Thereafter a reference standard was used on each ELISA plate to make a standard curve. To do this, standard serum aliquots were freshly thawed and two independent serial dilutions (2-fold steps, 10 different dilutions) were prepared starting with a dilution of 20 antibody units. These were applied to the wells on the ELISA plate and four blank wells (assigned zero antibody units) were also designated. The OD values were then fitted to a 4 parameter standard curve (antibody units=C{[(A-OD492)/(OD492-D)]^(1/B)}) using SOFTmax PRO version 5 software; Molecular Devices, Sunnyvale, CA, USA). The antibody units in test sera were then calculated from their OD492 values using the parameters estimated from this standard curve.
To evaluate the validity of this ELISA, sera collected from
Pooled sera were serially diluted and analyzed by ELISA. Two numbers were generated for each test serum. Using a single OD value which was closest to 1 in each set of diluted sera, the antibody units were calculated referring to a standard curve. Using all OD values from serially diluted sera, the reciprocal dilution giving an OD492=1 calculated based on a 4-parameter fitted hyperbolic curve and the reciprocal dilution giving an OD492 of 1 calculated from full dilution curves were significantly correlated (Spearman rank correlation, 1.0; p<0.0001) (Fig. 1). Thus to obtain the antibody units, we reasoned that a single dilution was enough if serially diluted standard serum was tested in the same ELISA plate. The antibody units of IgG and IgA contained in the pooled sera were determined to be 1,200 and 200 IU/mL, respectively.
To determine the criteria for acceptable quality of an ELISA plate, data from 300 plates were analyzed by two different operators using sera from whole cell lysate of
The quality of data obtained with standard sera was examined using two data sets. Standard sera were serially diluted over 100 times and antibody units for each dilution of standard sera were calculated. The relative units were calculated by comparison with the antibody units of the sera that came from the dilution showing an OD492 closest to 1.0.
Based on the data above, we determined the criteria for acceptable quality for ELISA plates as the following:
1) R2 of the standard curve should be equal to or greater than 0.997.
2) The OD492 of blank wells should be less than 0.04.
If the R2 of standard curve and blank values did not match these criteria, all data were discarded.
Following the criteria listed above, antibody units of standard sera were analyzed by ELISA to evaluate the reproducibility of the data. Pooled standard sera were aliquoted and frozen at -80℃ before use, and these sera were tested at an interval of several months. The concordance between the two sets of the data is highly significant judged by the linear regression analysis (coefficient was 1.01; 95% confidence interval [CI], 0.97 to 1.06; p<0.001). The reproducibility of data obtained with 224 antisera by two different operators using the same standard was also high (coefficient was 0.84; 95% CI, 0.97 to 1.06; p<0.001, data not shown).
One hundred fourteen
The reliability of ELISA test is required with the standard curve for distinguishing positive and negative sera in infectious diseases, and for reproducibility. In this study, the pooled standard sera were used for making a standard curve. There are some advantages to using the pooled standard sera to make a standard curve in analyzing the quantity of antibody in
In a previous study, we showed that the Genedia IgG ELISA kit, which used
To evaluate the validity of our ELISA, we used it to compare antibody titers between
There are some limitations to the present study including the lack of comparison with other commercially available-ELISAs, the absence of negative standard sera, and a small study population for each age group to evaluate its validity.
In conclusion, we have developed a reliable ELISA method to detect anti-
OD, optical density.
OD, optical density.
*p<0.0001.
*p<0.05.
Table 1 Antibody Titers in
IgG, immunoglobulin G; IgA, immunoglobulin A.
*p=0.021.
*p=0.021.
pISSN 1976-2283
eISSN 2005-1212
OD, optical density.|@|~(^,^)~|@|Quality-control parameters for an enzyme-linked immunosorbent assay standard curve. The R2 of a standard curve (fitted to a 4-parameter hyperbolic curve) is plotted on the X-axis, and the arithmetic mean value of OD492 of 4 blank wells on the same plate is plotted on the Y-axis. (A) Immunoglobulin G (IgG). (B) IgA.
OD, optical density.|@|~(^,^)~|@|Comparison of immunoglobulin G (IgG) antibody titers in
*p<0.0001.|@|~(^,^)~|@|Comparison of immunoglobulin A (IgA) antibody titers in the
*p<0.05.
Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE
| Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
| Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
| Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
| Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
All papers submitted to Gut and Liver are reviewed by the editorial team before being sent out for an external peer review to rule out papers that have low priority, insufficient originality, scientific flaws, or the absence of a message of importance to the readers of the Journal. A decision about these papers will usually be made within two or three weeks.
The remaining articles are usually sent to two reviewers. It would be very helpful if you could suggest a selection of reviewers and include their contact details. We may not always use the reviewers you recommend, but suggesting reviewers will make our reviewer database much richer; in the end, everyone will benefit. We reserve the right to return manuscripts in which no reviewers are suggested.
The final responsibility for the decision to accept or reject lies with the editors. In many cases, papers may be rejected despite favorable reviews because of editorial policy or a lack of space. The editor retains the right to determine publication priorities, the style of the paper, and to request, if necessary, that the material submitted be shortened for publication.
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