Transfer of offal detection assay to an ELISA platform

Transfer of offal detection assay to an ELISA platform

FINAL REPORT FA0106 August 2012

Reviewed by Authenticity Methods Working Group: August 2012

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Project Code FA0106

Title Transfer of offal detection assay to an ELISA platform

Contractor Professor E. Ellen Billett Nottingham Trent University

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CONTENTS

1. Executive summary 6 2. Abbreviations 7 3. Introduction 8

3.1 Background 8 3.2 Outline of project 9

4. Materials and Methods 11

4.1 Materials 11 4.2 Methods 11

4.2.1 Preparation of crude protein extracts from meat and offal 11 4.2.2 Bio-Rad protein assay 11 4.2.3 ELISA detection of marker proteins 12 4.2.4 1-D SDS PAGE of protein extracts 12 4.2.5 Western blot detection of marker proteins 12

5. Results and Discussion 13 5.1 Review of commercially available antibodies for the detection and quantification of

heart and lung marker protein 13 5.2 Review of facilities available in Public Analysts laboratories for performing ELISAs 15 5.3 Specificity of heart and lung marker protein antibodies 17 5.3.1 Overview 17 5.3.2 Reactivity of offals and skeletal muscle with heart marker protein antibodies 17 5.3.2.1 Reactivity of offals and skeletal muscle with anti-cTnT antibodies 17 5.3.2.2 Reactivity of offals and skeletal muscle with anti-cTnI antibodies 18 5.3.3 Reactivity of offals and skeletal muscle with lung marker protein antibodies 21 5.3.3.1 Reactivity of offals and skeletal muscle with anti-SP-A antibodies 21 5.3.3.2 Reactivity of offals and skeletal muscle with anti-PRDX6

antibodies 23 5.3.3.3 Reactivity of offals and skeletal muscle with alternative anti-SP-A antibodies 25 5.3.3.4 Reactivity of heart and lung marker protein antibodies with food additives/ingredients by Western blot analysis 35 5.3.4 Summary of marker protein antibody specificities by ELISA and Western blot analysis 39 5.4 Development of an ELISA for the detection of heart 40 5.4.1 Sensitivity of heart ELISA for detection of cTnT and cTnI 40 5.4.2 Optimisation of cTnI ELISA for detection of heart 41 5.4.3 Reactivity of anti-cTnI [4C2] antibodies with food additives/ingredients by ELISA 47 5.4.4 Summary of heart ELISA optimisation 48 5.5 Validation of heart quantification using standards and blind samples prepared

in-house 49 5.5.1 Quantification of heart in blind samples prepared in-house by ELISA and

Western blot analysis 49 5.5.2 Summary of analysis of blind samples 51 5.6 Determination of heart in processed commercial meat products 52 5.6.1 Introduction 52 5.6.2 Quantification of heart in commercial products by ELISA and Western blot

analysis 52 5.6.3 Summary of the analysis of processed commercial products 54 6. General Conclusions 55

6.1 Future work 55 7. Acknowledgements 55 8. References 55 9. Appendices 57

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List of Figures Figure 1 – Reactivity of skeletal muscle and offal extracts with anti-cTnT [1F11] antibody by ELISA. 18 Figure 2 – Reactivity of skeletal muscle and offal extracts with anti-cTnT [1F11] antibody by Western blotting. 19 Figure 3 – Reactivity of skeletal muscle and offal extracts with anti-cTnI [4C2] antibody by ELISA. 20 Figure 4 – Reactivity of skeletal muscle and offal extracts with anti-cTnI [4C2] antibody by Western blotting. 21 Figure 5 – Reactivity of skeletal muscle and offal extracts with anti-bovine SP-A antibody (Abcam ab78173) by ELISA. 22 Figure 6 – Reactivity of skeletal muscle and offal extracts with anti-bovine SP-A antibody (Abcam ab78173) by Western blotting. 23 Figure 7 – Reactivity of skeletal muscle and offal extracts with anti-PRDX6 antibody (Abcam ab59543) by ELISA. 24 Figure 8 – Reactivity of skeletal muscle and offal extracts with anti-PRDX6 antibody (Abcam ab59543) by Western blotting. 25 Figure 9 – Reactivity of skeletal muscle and offal extracts with anti-SPA antibody (Abcam ab103789) by ELISA. 26 Figure 10 – Reactivity of skeletal muscle and offal extracts with anti-SP-A antibody (Abcam ab103789) by Western blotting. 27 Figure 11 - Reactivity of skeletal muscle and offal extracts with anti-SP-A antibody (Abcam ab87674) by ELISA. 28 Figure 12 – Reactivity of skeletal muscle and offal extracts with anti-SP-A antibody (Abcam ab87674) by Western blotting. 29 Figure 13 – Reactivity of skeletal muscle and offal extracts with anti-SPA [PE10] antibody by ELISA. 30 Figure 14 – Reactivity of skeletal muscle and offal extracts with anti-SP-A [PE10] antibody by Western blotting. 31 Figure 15 – Reactivity of skeletal muscle and offal extracts with anti-SPA [32E12] antibody by ELISA. 32 Figure 16 – Reactivity of skeletal muscle and offal extracts with anti-SPA [1G8] antibody by ELISA. 33 Figure 17 – Reactivity of skeletal muscle and offal extracts with anti-SP-A [32E12] antibody by Western blotting. 34 Figure 18 – Reactivity of skeletal muscle and offal extracts with anti-SP-A [1G8] antibody by Western blotting. 35 Figure 19 – Reactivity of anti-cTnT [1F11] antibody with meat containing food additives/ ingredients by Western blot analysis. 36 Figure 20 – Reactivity of anti-cTnI [4C2] antibody with meat containing food additives/ingredients by Western blot analysis. 37 Figure 21 – Reactivity of anti-bovine SP-A antibody (Abcam ab78173) with meat containing food additives/ingredients by Western blot analysis. 38 Figure 22 – Reactivity of the ant-PRDX6 antibody Abcam ab59543) with meat containing food additives/ingredients by Western blot analysis. 39 Figure 23 – Reactivity of meat and meat mixtures containing heart with anti-cTnT [1F11] and anti-cTnI [4C2] antibodies by ELISA. 40 Figure 24 – Comparison of different TMB developing reagents on the detection of heart by ELISA. 41 Figure 25 – Comparison of different antigen coating conditions on the detection of heart by ELISA. 42 Figure 26 – Titration of anti-cTnI [4C2] antibody at different antigen coating concentrations. 44 Figure 27 – Comparison of different primary antibody incubation conditions on detection of heart by ELISA. 45 Figure 28 – Effect of ELISA development time on detection of beef heart. 46 Figure 29 – Reactivity of anti-cTnI [4C2] antibody with meat containing food additives/ingredients by ELISA. 48 Figure 30 – Standard calibration curve for quantification of added beef heart by ELISA. 49

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List of Tables Table 1 – Summary of antibodies for the detection of marker proteins available from major suppliers / distributors 14 Table 2 – Public Analyst Laboratories who responded to the questionnaire 15 Table 3 - Summary of responses to Public Analyst’s facilities survey 16 Table 4 – Indirect ELISA using anti-cTnT [4C2] antibodies for the detection of beef heart in meat. 47 Table 5 - Quantification of beef heart in blind samples by detection of cTnI by ELISA. 50 Table 6 - Quantification of beef heart in blind samples by detection of cTnI by Western blot analysis 51 Table 7 – Offal content of commercial products used for detection of heart marker protein 52 Table 8 - Quantification of heart in commercial meat products by detection of cTnI by ELISA 53 Table 9 - Quantification of heart in commercial meat products by detection of cTnI by Western blot analysis 54 List of Appendices Appendix 1 – Summary of commercially available anti-cTnI antibodies that have reactivity against bovine and/or porcine cTnI 57 Appendix 2 – Summary of commercially available anti-cTnT antibodies that have reactivity against bovine and/or porcine cTnT 59 Appendix 3 - Summary of commercially available anti-SP-A antibodies 62 Appendix 4 - Summary of commercially available anti-PRDX6 antibodies 64 Appendix 5 – Questionnaire circulated to PA laboratories by Defra 67

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1. EXECUTIVE SUMMARY From the point of view of labelling of products containing meat as an ingredient, ‘meat’ is defined as skeletal muscle with naturally included or adherent fat and connective tissues (The Food Labelling (Amendment), (England) Regulations, 2003, and parallel legislation in Scotland, Wales and Northern Ireland). These regulations state that other types of muscle, such as heart, tongue etc., are excluded from this definition. The regulations also state that certain parts of the carcass, such as liver, kidney and lung have to be explicitly labelled as such; the generic term ‘offal’ is not permitted. It is believed that offal is used to increase the apparent meat content of some fresh and processed products. Consequently there is a need for methods for the detection and quantification of various offals in meat and meat products. Previously our laboratory at Nottingham Trent University (NTU) developed proteomic methods to identify offals (heart, liver, kidney and lung) from beef, pork and lamb in meat products (Food Authenticity Programme Project [Q01105]). Tissue-specific protein biomarkers were identified for each offal and procedures established to quantify low levels of the offal biomarkers in raw and oven cooked minced meat samples (by Western blotting) and establish the species of origin of the offal (by mass spectrometry). It was subsequently recognised that there was a need for a simpler test for the detection and semi-quantification of offals in meat and meat products that is suitable for enforcement purposes. Attempts were therefore made to develop enzyme linked immunosorbent assays (ELISAs) for the detection of heart and lung using biomarker proteins and antibodies identified during project Q01105. An ELISA has been developed which routinely detects 1% beef heart in meat using cTnI as the target antigen. Anti-cTnI antibodies did not react with any of the fourteen food additives/ingredients of relevance to burgers, and other minced meat products when screened by ELISA. Both the ELISA and Western blot assays gave good estimates for the amount of heart in blind samples prepared in-house, but overall the intra- and inter-assay repeatability was better for the ELISA than the Western blot assay. The ELISA and Western blot assays also confirmed the presence of heart in 4 meat products that were found to contain undeclared heart during an independent survey of retail meat products and also 2 commercial products with heart declared as an ingredient None of the 6 anti-SP-A antibodies or the anti-PRDX6 antibody had the required specificity or sensitivity to detect lung tissue by ELISA. However, an alternative anti-SP-A antibody that can detect beef, sheep and pork SP-A by Western blotting was found which could be used for detection of lung from all species by Western blotting. Further work is needed to establish the limit of detection of lung in meat using this antibody by Western blotting. Although the fourteen ingredients/additives screened in this project did not interfere with the heart detection ELISA, the limit of detection of the assay may be affected by the presence of these additives/ingredients or combinations of these; this could be form part of a future work plan.

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2. ABBREVIATIONS 1-D One dimensional AIDA Advanced Image Data Analyser BSA Bovine serum albumin cTnI Cardiac specific troponin I cTnT Cardiac specific troponin T CV Coefficient of variation Da Daltons DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid ECL Enhanced Chemiluminescence HRP Horseradish peroxidase PA Public Analyst PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PRDX6 Peroxiredoxin 6 rpm Revolutions per minute SD Standard deviation SDS Sodium dodecyl sulphate SP-A Surfactant protein A TEMED N,N,N’N’-tetramethylethylenediamine

TMB 3,3’,5,5’-tetramethylbenzidine

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3. INTRODUCTION 3.1 Background Food fraud comes in many guises and includes the adulteration of meat and meat products. Preventing adulteration is important for economic, health, food safety and religious reasons and the accurate labelling of meat products is mandatory. From the point of view of labelling of products containing meat as an ingredient, ‘meat’ is defined as skeletal muscle with naturally included or adherent fat and connective tissues (The Food Labelling (Amendment), (England) Regulations, 2003, and parallel legislation in Scotland, Wales and Northern Ireland). These regulations state that other types of muscle, such as heart, tongue etc., are excluded from this definition. The regulations also state that certain parts of the carcass, such as liver, kidney and lung have to be explicitly labelled as such; the generic term ‘offal’ is not permitted. It is believed that offal is used to increase the apparent meat content of some fresh and processed products. Consequently there is a need for methods for the detection and quantification of various offals in meat and meat products. Our laboratory at Nottingham Trent University (NTU) was commissioned by the Food Standards Agency to develop proteomic methods to identify offals (heart, liver, kidney and lung) from beef, pork and lamb in meat products (Food Authenticity Programme Project [Q01105]). Two tissue-specific protein biomarkers were identified for each offal and the utility of the biomarkers was tested using both immunoassays (Western blotting) and mass spectrometry. The procedures can quantify low levels of offals in raw and oven cooked minced meat samples and they can also establish the species of origin of the offal (by mass spectrometry). Our procedures can also detect and quantify offals in processed meat products such as burgers, haggis and dog food. It was subsequently recognised that there was a need for a simpler test for the detection and semi-quantification of offals in meat and meat products that is suitable for enforcement purposes. Attempts were therefore made to develop enzyme linked immunosorbent assays (ELISAs) using marker proteins and antibodies identified during project Q01105. The aim was to have an assay which was as simple and robust as possible in order to facilitate successful transfer to Public Analysts. In a limited independent survey of commercially available meat/meat products, conducted (using Western blotting) by our group at NTU, undeclared offals were detected; offals present at substantial levels were heart and lung. These offals were detected in minced meat and burgers from retailers and catering suppliers, with beef heart being the most common adulterant. Therefore the aim was to design ELISAs to monitor heart and lung in meat and meat products. Two marker proteins for heart and two for lung tissue were established during the Food Authenticity Programme Project (Q01105). Antibodies capable of detecting the marker proteins on Western blots were identified.

The marker proteins established for beef, pork and sheep heart were cardiac specific troponin I (cTnI) and cardiac specific troponin T (cTnT). Both proteins are involved in the regulation of heart muscle contraction and are specific to cardiac myocytes. cTnT and cTnI form a regulatory complex of myofibrillar proteins with cTnC, a complex which binds to the thin actin myofilament via tropomyosin to regulate muscle contraction (Gomes et al., 2002).

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Owing to the high degree of sequence homology between species, antibodies raised against respectively human cTnI and cTnT detected cTnI and cTnT extracted from cooked beef, pork and sheep heart. Our Western blot assays confirmed that both proteins are detectable in heart, but not in skeletal muscle, liver, kidney or lung, in cows, pigs and sheep. Indeed, using the Western blot assay the antibodies can detect heart in meat mixtures containing < 1% cooked beef, pork and sheep heart. Despite the high homology, proteolytic digestion produces peptide fragments of different masses for both cTnI and cTnT in the three species; which can be separated by time-of-flight (Tof) mass spectrometry, thus allowing the species to be distinguished.

The marker proteins established for beef, pork and sheep lung during project Q01105 were surfactant protein A (SP-A) and peroxiredoxin 6 (PRDX6). Our Western blot assays confirmed that both proteins are detectable in lung (but not in skeletal muscle, liver, kidney or heart), in cows, pigs and sheep.

SP-A belongs to a family of collagen containing, C-type lectins, called collectins. It is produced by alveolar type II cells and Clara cells and binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung (Hermans and Bernard, 1999). Expression of SP-A mRNA has been reported in non-pulmonary tissues such as the pancreas and prostate gland, but SP-A protein has not been reported outside the lung (Madsen et al., 2003).

Owing to the high sequence homology between beef and sheep SP-A, a commercial antibody raised against bovine SP-A detected beef and sheep SP-A extracted from cooked meat mixtures containing 1% beef lung and 2% sheep lung. This antibody did not detect pork SP-A, and to date no antibody against pork SP-A has been sourced (although a suitable antibody may be found following a more extensive search). It is possible to distinguish between species by mass spectrometric analysis of peptides from SP-A.

PRDX6 is a unique member of the peroxiredoxin family of antioxidant enzymes. The protein has glutathione peroxidase and phospholipase A2 activities and can also reduce phospholipid hydroperoxides. In rats, high levels of mRNA and protein have been detected in lung, mRNA was detected in other tissues at lower levels, but immunoreactive protein was not detected outside the lung (Kim et al., 1998). However, Seo et al., (2000) reported high levels of PRDX6 protein in rat lung and brain and much lower levels in other tissues. In beef tissues, quantification of PRDX6 by RT-PCR revealed high levels in the lungs and spinal cord (Leyens et al., 2003). Using Western blotting we detected beef PRDX6 protein in the lung only. 3.2 Outline of Project The overall aim was to provide Public Analysts with much required simple assays for the detection of specific offals (heart and lung) from beef, lamb and pork in meat and meat products. Offal is not dangerous to the consumer, but it is illegal to have products not correctly labelled. Meat products hold a substantial share of the food market and, as noted earlier a small in-house survey at NTU has revealed that offals are being added to meat products without being declared. The specific objectives were defined as follows:

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1. Desk based review of (a) commercially available antibodies for the detection and quantification of the offal marker proteins and (b) facilities commonly available in Public Analyst laboratories.

2. Analysis of the specificity of commercially available antibodies for heart and lung marker proteins.

3. Establishing semi-quantitative ELISA(s) suitable for the detection of offal by Public Analysts with a sensitivity of 2.5% offal in meat.

4. Testing the ELISA(s) for analysis of a small number of commercial products.

5. Production of SOP(s) and final report.

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4. MATERIALS AND METHODS Brief details only are given in these sections; the development of the methods are outlined in the Results and Discussion section, and the final method is outlined in the SOP. 4.1 Materials Matched sets of beef, sheep and pork skeletal muscle, heart, liver, lung and kidney samples were obtained from an independent slaughterhouse and butcher 1-day after

slaughter. All tissues were stored at 4C until 2-days post mortem and then processed (see section 4.2.1) or stored at -80°C. Commercial meat products and marvel milk powder were purchased from local supermarkets. Ammonium persulphate, bovine serum albumin (BSA), bromophenol blue, copper phthalocyanine 3,4’,4’’,4’’’ tetrasulphonic acid (tetrasodium salt), sodium hydroxide, TEMED and Tween 20 were from Sigma-Aldrich (Poole, UK). Di-sodium hydrogen orthophosphate, sulphuric acid and 1-StepTM Ultra TMB-ELISA substrate were from Thermo Fisher Scientific (Loughborough, UK). Sodium chloride, sodium dodecyl sulphate (SDS), urea, glycine and Tris base were from Melford (Ipswich, UK). Potassium chloride and potassium dihydrogen orthophosphate were from Merck (Nottingham, UK). ProtoGel® Resolving and Stacking Buffers (Tris/SDS) and AccuGelTM 29:1 were from National Diagnostics (Hessle, UK). iBlot® nitrocellulose transfer stacks were from Invitrogen. Goat anti-mouse and goat anti-rabbit immunoglobulins labelled with horseradish peroxidase (HRP) were from DakoCytomation (Ely, UK). EZ-ECL Chemiluminescence detection kit for HRP was from Biological Industries (Beit Haemek, Israel). Bio-Rad Protein assay kit was from Bio-Rad (Hemel Hempsted, UK). NUNC Maxisorp clear flat bottom ELISA plates were from Scientific Laboratory Supplies (Nottingham, UK). SiLi Type ZY beads (1.4 – 1.6 mm) were from Sigmund Lindner UK (Oldham, UK). 4.2 Methods 4.2.1 Preparation of crude protein extracts from meat and offal Matched sets of beef, sheep and pork skeletal muscle and offals (heart, liver, kidney and lung) were stored at 4°C until 2-days post-mortem. Fifty gram samples were individually wrapped in foil and cooked to a minimum core temperature of 80°C for 15 minutes. Samples were removed from the oven and allowed to cool at room temperature for 1 hour before use. Five gram representative samples of cooked meat, offal or meat mixtures, or commercial meat products were extracted in 30 ml of buffer using a MP FastPrep® 24 homogeniser fitted with a 50 ml rotor and tubes with 15 g of SiLi beads Type ZY beads (1.4 – 1.6 mm). The homogenate was centrifuged at 1,500xg for 15 min and the supernatant was collected and stored in aliquots at -20°C. 4.2.2 Bio-Rad protein assay The Bio-Rad protein assay was performed according to the manufacturer’s instructions, using BSA as a standard. 4.2.3 ELISA detection of marker proteins

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ELISA plate wells were coated with diluted antigen (50µl/well) at 37°C for 1 hour. Wells were washed twice with PBS-Tween (PBS containing 0.05% [v/v] Tween 20) and twice with distilled water and then blocked for 2 hours at room temperature with PBS containing 3% (w/v) Marvel milk powder. After washing twice with PBS-Tween and distilled water, wells were incubated overnight at 4°C with appropriately diluted primary antibody in blocking solution (50µl/well). After washing, each well was incubated for 90 min with 1:1,000 HRP-conjugated anti-mouse or rabbit secondary antibodies in blocking solution. Wells were washed twice with PBS-Tween and twice with distilled water and then 100 µl of 1-StepTM Ultra TMB-ELISA substrate was added to each well. When the blue colour was dark enough, the reaction was stopped with 50 µl /well 2.5 M H2SO4 and the absorbance values read at 450 nm. 4.2.4 1-D SDS PAGE of protein extracts Proteins were solubilised with 5x gel loading buffer (250 mM Tris-HCl, 50% glycerol, 5% SDS, 0.5 M DTT and 0.05% [w/v] bromophenol blue, pH 6.8) and heated at 100°C for 5 minutes. Samples were electrophoresed using SDS-PAGE in a polyacrylamide gel (resolving gel concentration of 10 to 15% [w/v] depending on the separation required) at 200-V constant voltage. Following SDS PAGE proteins were transferred to nitrocellulose for immunodetection of marker proteins (section 4.2.5). 4.2.5 Western blot detection of marker proteins After 1-D SDS-PAGE proteins were transferred to a nitrocellulose membrane using an Invitrogen iBlot® transfer device as described by the manufacturer. Electroblotted membranes were blocked for 1 hour at room temperature in PBS containing 3% (w/v) Marvel milk powder. After blocking, membranes were incubated overnight at 4°C with appropriately diluted primary antibody in blocking solution. After 3 washes in PBS-Tween (PBS containing 0.1% [v/v] Tween 20) for 10 minutes each, the membrane was incubated for 90 min with 1:2,000 HRP-conjugated anti-mouse or rabbit secondary antibodies in blocking solution. Membranes were washed three times with PBS-Tween for 10 min each and once with PBS for 5 min. Antibody binding was detected by enhanced chemiluminescence using a Fujifilm LAS 3100 CCD camera (Raytek Scientific Ltd). Band intensities were measured using AIDA software according to the manufacturer’s instructions, only bands that were non-saturated were used for analysis.

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5. RESULTS AND DISCUSSION

5.1 Review of commercially available antibodies for the detection and quantification of heart and lung marker proteins

Two marker proteins for heart and two for lung tissue were established during the Food Authenticity Programme Project (Q01105) and antibodies capable of detecting the marker proteins on Western blots were identified. A review of commercially available antibodies for the four marker proteins was conducted and Table 1 provides an overview of the companies supplying antibodies to the marker proteins. This review was conducted in order to identify all potential antibodies for the development of ELISAs.

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Table 1 – Summary of antibodies for the detection of marker proteins available from major suppliers / distributors The first number is the total number of antibodies the company sells and the number in brackets is the number of antibodies that are reported to react with the beef and/or pork marker protein.

Company

Marker protein

cTnI

cTnT SP-A PRDX6

HyTest Ltd

30 (8) 12 (9) 0 0

Abcam plc. 38 (6 HyTest + 2 others)

33 (8 HyTest + 2 others)

7 (3) 11 (2)

AbD Serotec 9 (5 HyTest + 1 other)


0 0

Abnova

51 (8 HyTest) 18 (9 HyTest) 6 (0) 9 (0)

Acris Antibodies GmbH



4 (0) 12 (0)

Cell Signalling Technology, Inc.

1 (0) 1 (0) 0 0

Enzo Life Science, Inc.

1 (0) 0 0 0

GeneTex, Inc. 20 (7 HyTest) 15 (10 HyTest + 1 other)

0 3

GenWay Biotech, Inc.

19 (7 HyTest) 13 (9 HyTest +1 other)

1 (0) 7 (0)

Immuno-Biological Laboratories Co., Ltd

0 0 1 (0) 0

Invitrogen Ltd 0 0 0 0

LifeSpan Biosciences, Inc. (LS Bio)

88 (8 HyTest + 1 other)


8 (1) 0

Millipore

3 (2 HyTest) 1 (0) 7 (1) 2 (0)

Novus Biologicals 58 (4 HyTest + 2 others)


1 (0) 10 (0)

Sigma-Aldrich Co. 5 (0) 3 (0) 0 6 (0)

Santa Cruz Biotechnology, Inc. (SC)



5 (2) 8 (4)

Thermo Scientific 20 (7 HyTest + 1 other)

4 (2 HyTest +2 others)

2 2

HyTest Ltd manufacture high-quality immunological reagents for cardiac biomarkers, including cTnT and cTnI. They supply anti-human cTnT and cTnI antibodies, some of which react with the pork and/or beef proteins. Many antibody distributors sell these HyTest antibodies. Many of the other antibodies are sold by several of the suppliers. Appendices 1 and 2 summarise the available antibodies for the detection of beef and/or pork cTnI and cTnT respectively. Appendices 3 and 4 summarise all available antibodies for the detection of SP-A and PRDX6, respectively. Following consultation with the Food Authenticity and Standards Team in the Food Policy Unit at Defra (Tanya Gurung, Theresa Ekong and Sandy Primrose) it was decided that

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the antibodies against cTnI, cTnT, SP-A and PRDX6 used during project Q01105 would be tested / screened first. If these antibodies did not provide the required specificity and sensitivity the most suitable alternatives would then be selected from those listed in Appendices 1 – 4. It was agreed that alternative antibodies for the pork lung marker protein, SP-A, would be selected, as the antibody used during project Q01105 didn’t recognise pork SP-A by Western blot analysis. 5.2 Review of facilities available in Public Analysts laboratories for performing ELISAs To ensure successful transfer of assays to Public Analysts (PAs) a review of commonly available equipment in PA Laboratories for conducting ELISAs was carried out with assistance of Tanya Gurung from the Food Authenticity and Standards Team in the Food Policy Unit at Defra. The responding laboratories are listed in Table 2 and a summary of the responses are detailed in Table 3. A copy of the questionnaire can be found in Appendix 5. Table 2 – Public Analyst Laboratories who responded to the questionnaire

Country Laboratory

England Bristol City Council Scientific Services

Kent Scientific Services

Leicestershire County Council Scientific Services

London – Eurofins Laboratories Ltd

Lancashire County Laboratory

Hampshire Scientific Services

Somerset Scientific Services

West Yorkshire Analytical Services

Wolverhampton - Eurofins Laboratories Ltd

Scotland Dundee City Council Scientific Services

Wales Cardiff – Minton, Treharne & Davies Ltd

The Eurofins Laboratory in London does not perform ELISAs so their responses have not been included when compiling Table 3.

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Table 3 - Summary of responses to Public Analyst’s facilities survey

Question Responses positive responses (/10)

(1) Cooking facilities

Oven

8

Microwave oven

10

Water bath to 100°C

9

Water bath to 80°C

10

(2) Centrifuges

Tube size (up to 50 ml) speed up to 10,000 rpm

2

Tube size (up to 2 5ml) speed up to 3,000 rpm

10

Micro-centrifuge Tube size (up to 1.5 ml) speed up to 13,000 rpm

6 (4 did not specify)

(3) Plate washers

None

6

Biotek ELX50

1

Thermo electron corp. Wellwash 54 mk 2

1

Labtech LT-3000

1

Nunc-Immuno 8 channel washer 470174

1

(4) Plate reader wavelengths

405 nm

9

450 nm

10

490/492 nm

7

570 nm (562 nm BCA protein assay, used successfully 540 – 590 nm)

1

620/630 nm (595 nm - Bradford protein assay, used successfully 575 – 615 nm)

9

(5) Pipettes Multi-channel

7

Repeater

6

Methods were developed that incorporate procedures that can be carried out at the majority of Public Analyst Laboratories. It was agreed that a plate washer should be used and oven cooking was acceptable.

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5.3 Specificity of heart and lung marker protein antibodies 5.3.1 Overview As we have previously demonstrated that cooking allows enrichment of the marker proteins of interest, and since we have wide experience of the analysis of cooked samples, we decided to design the assays for cooked sample. It is likely that assays for cooked samples will be more sensitive than for raw samples.

Samples of skeletal muscle and offals (heart, liver, kidney and lung) from cows, pigs and sheep were minced and cooked in an oven to a minimum core temperature of 80°C for 15 minutes. Samples were homogenised in both PBS and PBS 8 M urea and soluble proteins collected by centrifugation at 1,500x g for 15 minutes. The specificity of the selected commercial antibodies for beef, pork and sheep offals was assessed using an indirect ELISA. Protein extracts (containing the marker proteins/antigens) were coated onto high protein-binding microtiter plates and the reactivity of primary antibodies against the bound antigen detected using an enzyme-labelled secondary antibody. Comparisons with antibody specificity using Western blot analysis were made in order to check that the antibodies were binding to a protein of the predicted molecular mass. It is realised that the buffer used for extraction may affect antigen binding to the plate and/or antigen presentation to the antibody. 5.3.2 Reactivity of offals and skeletal muscle with heart marker protein antibodies 5.3.2.1 Reactivity of offals and skeletal muscle with anti-cTnT antibody A mouse monoclonal antibody against cTnT [1F11] was screened for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and the results are presented in Figures 1 – 2.

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The anti-cTnT antibody detected cTnT in cooked beef, lamb and pork heart extracts, a much stronger signal was observed in the PBS 8 M urea extracts compared to the PBS extracts. The antibody had little or no reactivity with PBS 8 M urea extracts of skeletal muscle and the other offals. Therefore PBS 8 M urea needs to be used to extract cTnT from cooked heart tissue.

Figure 1 – Reactivity of skeletal muscle and offal extracts with anti-cTnT [1F11] antibody by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-cTnT [1F11] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)

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Key B – Beef Mu – Skeletal muscle L – Lamb He – Heart P – Pork Li – Liver Ki – Kidney Lu – Lung

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The anti-cTnT antibody detected cTnT in the cooked beef, lamb and pork heart extracts; the antibody did not react with proteins in the skeletal muscle and other offal extracts. 5.3.2.2 Reactivity of offals and skeletal muscle with anti-cTnI antibody A mouse monoclonal antibody against cTnI [4C2] was screened for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and the results are presented in Figures 3 – 4.

BEEF LAMB PORK Mu He Li Ki Lu Mu He Li Ki Lu Mu He Li Ki Lu

Figure 2 – Reactivity of skeletal muscle and offal extracts with anti-cTnT [1F11] antibody by Western blotting. PBS 8 M urea extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-cTnT [1F11] antibody. Arrow signifies position of cTnT.

Key Mu – Muscle He – Heart Li – Liver Ki – Kidney Lu – Lung

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The anti-cTnI antibody detected cTnI in PBS 8 M urea extracts of cooked beef, lamb and pork heart; only lamb and pork heart were detected in PBS extracts. The antibody had little or no reactivity with PBS 8 M urea extracts of skeletal muscle and the other offals. Therefore PBS 8 M urea needs to be used to extract cTnI from cooked heart tissue.

Figure 3 – Reactivity of skeletal muscle and offal extracts with anti-cTnI [4C2] antibody by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)

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The anti-cTnI antibody detected cTnI in the cooked beef, lamb and pork heart extracts; the antibody did not react with proteins in the skeletal muscle and other offal extracts. 5.3.3 Reactivity of offals and skeletal muscle with lung marker protein antibodies 5.3.3.1 Reactivity of offals and skeletal muscle with anti-SP-A antibody The rabbit polyclonal antibody used to detect beef and sheep SP-A during project Q01105 was used to screen for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and the results are presented in Figures 5 – 6.


Key Mu- Skeletal muscle He – Heart Li – Liver Ki – Kidney Lu – Lung

Figure 4 – Reactivity of skeletal muscle and offal extracts with anti-cTnI [4C2] antibody by Western blotting. PBS 8 M urea extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-cTnI [4C2] antibody. Arrow signifies position of cTnI.

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The anti-bovine SP-A antibody (Abcam ab78173) detected SP-A in PBS and PBS 8 M urea extracts of cooked beef, lamb and pork lung, but the signals were very low even though these extracts contained 100% lung.

Figure 5 – Reactivity of skeletal muscle and offal extracts with anti-bovine SP-A antibody (Abcam ab78173) by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-bovine SP-A antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)

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The anti-bovine SP-A antibody (Abcam ab78173) detected protein in the cooked beef and sheep lung extracts (Figure 6 (A) and (B) Lu); the antibody did not react with proteins in the skeletal muscle or other offal extracts. Only a faint band was detected in the pork lung extract (Figure 6 (A); Lu). Higher levels of SP-A were detected in the PBS extracts compared to the PBS 8 M urea extracts. 5.3.3.2 Reactivity of offals and skeletal muscle with anti-PRDX6 antibody The rabbit polyclonal antibody used to detect beef, sheep and pork PRDX6 during project Q01105 was used to screen for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and results are presented in Figures 7 – 8.



Figure 6 – Reactivity of skeletal muscle and offal extracts with anti-bovine SP-A antibody (Abcam ab78173) by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-bovine SP-A antibody. (A) PBS extracts and (B) PBS 8 M urea extracts. Arrow signifies position of PRDX6. (A) (B)


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The anti-PRDX6 antibody (Abcam ab59543) displayed poor sensitivity and specificity for lung tissue by ELISA.

Figure 7 – Reactivity of skeletal muscle and offal extracts with anti-PRDX6 antibody (Abcam ab59543) by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-PRDX6 antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) (B)

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The anti-PRDX6 antibody (Abcam ab59543) reacted with a protein of the expected molecular mass in PBS extracts from all three species; however it also reacted with a band of similar mass in pork liver and, additionally, reacted with higher mass proteins in skeletal muscle and heart. The specificity of the anti-PRDX6 antibody was very poor with PBS 8 M urea extracts. 5.3.3.3 Reactivity of offals and skeletal muscle with alternative anti-SP-A antibodies As neither of the lung marker protein antibodies used in project Q01105 displayed the required specificity or sensitivity in an ELISA, alternative antibodies were selected from those identified during the literature search (summarised in Appendix 3). The first to be tested was an anti-SP-A rabbit polyclonal antibody (Abcam ab103789) raised to a synthetic peptide corresponding to amino acids 221-236 (KEQCVEMYTDGQWNDR) of human SP-A. It was predicted to react with dog and pig SP-A due to sequence homology. The peptide immunogen shares 93.8% identity with pork and 81.3% identity with beef and sheep SP-A. The rabbit polyclonal antibody was used to screen for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and the results are presented in Figures 9 – 10.



Figure 8 – Reactivity of skeletal muscle and offal extracts with anti-PRDX6 antibody (Abcam ab59543) by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-PRDX6 antibody. (A) PBS extracts and (B) PBS 8 M urea extracts. Arrow signifies position of PRDX6. (A) (B)


26

The Abcam ab103789 anti-SP-A antibody did not detect SP-A in lung extracts by ELISA.

Figure 9 – Reactivity of skeletal muscle and offal extracts with anti-SPA antibody (Abcam ab103789) by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-SP-A antibody (Abcam ab103789) was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)


27

The abcam ab103789 anti-SP-A antibody did not detect SP-A in any of the lung extracts by Western blotting. Abcam ab87674 raised to a synthetic peptide of human SP-A and predicted to react with pig SP-A was used to screen for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and the results are presented in Figures 11 – 12.

Figure 10 – Reactivity of skeletal muscle and offal extracts with anti-SP-A antibody (Abcam ab103789) by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti- SP-A antibody (Abcam ab103789). (A) PBS extracts and (B) PBS 8 M urea extracts. Arrow signifies position of SP-A. BEEF LAMB PORK

Mu He Li Ki Lu Mu He Li Ki Lu Mu He Li Ki Lu



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The Abcam ab87674 anti-SP-A antibody did not detect SP-A in lung extracts by ELISA.

Figure 11 - Reactivity of skeletal muscle and offal extracts with anti-SP-A antibody (Abcam ab87674) by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-SP-A antibody (Abcam ab87674) was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)

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The Abcam ab87674 anti-SP-A antibody detected protein in the PBS extracts of cooked beef, sheep and pork lung extracts (Figure 12 (A) Lu); the antibody did not react with proteins in the skeletal muscle or other offal extracts. Higher signals were obtained with the PBS extracts compared to the PBS 8 M urea extracts, no SP-A was detected in the PBS 8 M urea extract of pork lung. Although this antibody detected SP-A from all species by Western blotting, it failed to work in an ELISA and therefore another antibody was selected for screening. Monoclonal anti-human SP-A [PE10] was selected, the epitope mapped to the C-terminus of human SP-A (Hiraike et al., 1995) and the antibody has been used to detect pork SP-A by immunohistochemistry (Paananen et al., 1999), no data were available on its use in ELISAs. The monoclonal antibody was used to screen for reactivity against beef, sheep and pork skeletal muscle and offal extracts by ELISA and Western blotting and the results are presented in Figures 13 – 14.

BEEF LAMB PORK

Mu He Li Ki Lu Mu He Li Ki Lu Mu He Li Ki Lu


Figure 12 – Reactivity of skeletal muscle and offal extracts with anti-SP-A antibody (Abcam ab87674) by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-human SP-A antibody (Abcam ab87674). (A) PBS extracts and (B) PBS 8 M urea extracts. Arrow signifies position of SP-A. (A) (B)

Key Mu – Skeletal muscle He – Heart Li – Liver Ki – Kidney Lu – Lung

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The monoclonal anti-human SP-A [PE10] antibody did not detect SP-A in lung extracts by ELISA.

Figure 13 – Reactivity of skeletal muscle and offal extracts with anti-SPA [PE10] antibody by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-SP-A [PE10] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)


31

The monoclonal anti-human SP-A [PE10] antibody did not detect SP-A in lung extracts by Western blotting. Two other monoclonal antibodies were also tested for reactivity with beef, sheep and pork SP-A by Western blotting and ELISA. Anti-SP-A [32E12] (Abcam ab49566) raised to a fragment of human SP-A (aa 104 – 246) and anti-SP-A [1G8] (Abnova H00653509-M03) raised to a different fragment of human SP-A (aa 133-248). ELISA and Western blot results are presented in Figures 15-18.




Figure 14 – Reactivity of skeletal muscle and offal extracts with anti-SP-A [PE10] antibody by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti- SP-A [PE10] antibody. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)

32

The monoclonal anti-human SP-A [32E12] antibody did not detect SP-A in lung extracts by ELISA.

Figure 15 – Reactivity of skeletal muscle and offal extracts with anti-SPA [32E12] antibody by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-SP-A [32E12] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)


33

The monoclonal anti-human SP-A [1G8] antibody had very low reaction with lung in PBS extracts, and reacted non-specifically with urea extracts (some reaction with pork heart). It was not deemed suitable for use in an ELISA for detection lung.

Figure 16 – Reactivity of skeletal muscle and offal extracts with anti-SPA [1G8] antibody by ELISA. Wells were coated with cooked protein extracts (40 µg/ml) and reactivity of bound antigen with anti-SP-A [1G8] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) PBS extracts and (B) PBS 8 M urea extracts. (A) (B)


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The monoclonal anti-human SP-A [32E12] antibody did not detect SP-A in lung extracts by Western blotting; but reacted with a high molecular weight protein in the PBS extracts of heart, kidney and lung.



Figure 17 – Reactivity of skeletal muscle and offal extracts with anti-SP-A [32E12] antibody by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti- SP-A [32E12] antibody. (A) PBS extracts and (B) PBS 8 M urea extracts. Arrow signifies position of SP-A. (A) (B)

Key Mu – Skeletal muscle He – Heart Li – Liver Ki – Kidney Lu – Lung

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The monoclonal anti-human SP-A [1G8] antibody did not specifically detect SP-A in lung extracts by Western blotting. 5.3.3.4 Reactivity of heart and lung marker protein antibodies with food additives/ingredients by Western blot analysis The reactivity of the commercial antibodies with potential food additives and ingredients of relevance to minced meat, burgers, and other minced meat products was assessed initially by Western blotting. Potential food additives and ingredients (e.g. soya, rice flour, wheat flour, corn flour, yeast extract and gelatine) were added to minced meat; cooked samples were homogenised in both PBS and PBS 8 M urea and soluble proteins collected by centrifugation at 1,500xg for 15 minutes. Western blots of extracts probed with the antibodies are presented in Figures 19 - 22. Any non-specific reactivity with potential food additives will render an antibody un-suitable for use in an ELISA.



Figure 18 – Reactivity of skeletal muscle and offal extracts with anti-SP-A [1G8] antibody by Western blotting. Proteins were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-SP-A [1G8] antibody. (A) PBS extracts and (B) PBS 8 M urea extracts. Arrow signifies position of SP-A. (A) (B)

Key Mu- Skeletal muscle He – Heart Li – Liver Ki – Kidney Lu - Lung

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The anti-cTnT antibody [1F11] did not react with any protein bands in the meat mixtures containing these food additives / ingredients. This antibody is therefore unlikely to produce non-specific signals when these ingredients are present in samples screened by ELISA.

Figure 19 – Reactivity of anti-cTnT [1F11] antibody with meat containing food additives/ingredients by Western blot analysis. PBS 8 M urea extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti- cTnT [1F11] antibody.

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The anti-cTnI antibody [4C2] did not react with any protein bands in the meat mixtures containing these food additives / ingredients. This antibody is therefore unlikely to produce non-specific signals when these ingredients are present in samples screened by ELISA.

Figure 20 – Reactivity of anti-cTnI antibody [4C2] with meat containing food additives/ingredients by Western blot analysis. PBS 8 M urea extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti- cTnI [4C2] antibody.

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The anti-bovine SP-A antibody (Abcam ab78173) reacted non-specifically with protein bands in a number of the meat mixtures containing food additives / ingredients. There was also a high background in the lanes containing 10% pork gelatine and 10% beef gelatine, this antibody is therefore likely to produce high non-specific signals when these ingredients are present in samples screened by ELISA.

Figure 21 – Reactivity of anti-bovine SP-A antibody (Abcam ab78173) with meat containing food additives/ingredients by Western blot analysis. PBS 8 M urea extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti-bovine SP-A antibodies.

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The anti-PRDX6 antibody (Abcam ab59543) reacted non-specifically with protein bands in all extracts, including the control which contained no additives. The antibody also reacted strongly with many protein bands in the lanes containing 10% pork gelatine and 10% beef gelatine, this antibody is therefore likely to produce high non-specific signals when these ingredients are present in samples screened by ELISA. 5.3.3.5 Summary of marker protein antibody specificities by ELISA and Western blot analysis (1) Anti-cTnT [1F11] and anti-cTnI [4C2] antibodies are specific for beef/sheep/pork heart by ELISA and Western blot analysis. (2) These anti-cTnT [1F11] and anti-cTnI [4C2] antibodies do not react with any of the food additives/ingredients when screened by Western blotting. (3) None of the 6 anti-SP-A antibodies tested have the required specificity or sensitivity to detect lung tissue by ELISA. (4) The anti-PRDX6 antibody (Abcam ab59543) did not have the required specificity and sensitivity to be used in an ELISA. (5) An alternative anti-SP-A antibody (Abcam ab87674) capable of detecting beef, sheep and pork SP-A by Western blotting was found. However, this was not suitable for use in an ELISA.

Figure 22 – Reactivity of the ant-PRDX6 antibody (Abcam ab59543) with meat containing food additives/ingredients by Western blot analysis. PBS 8 M urea extracts were separated by 1-D SDS PAGE, transferred to nitrocellulose and the Western blot probed with anti- PRDX6 antibody.

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5.4 Development of an ELISA for the detection of heart Development of ELISAs for offal detection has focused on heart as no suitable antibodies for the detection of lung marker proteins by ELISA are currently available. 5.4.1 Sensitivity of heart ELISA for detection of cTnT and cTnI The specificity and sensitivity of the anti-cTnT [1F11] and anti-cTnI [4C2] antibodies will dictate which target antigen will be utilised for the detection of heart by ELISA. An indirect ELISA was tested using cooked standards produced in-house (minced meat [beef, pork and lamb] containing 0 - 10% heart [beef, pork and lamb]) and the results are presented in Figure 23.

cTnT was detected in extracts containing 5% beef and lamb heart and 10% pork heart. cTnI was detected in extracts containing 5% beef, lamb and pork heart. In all cases the signal strength was very low and 2% heart could not be detected. As detection of pork heart using anti-cTnT antibodies was poor and cTnI was detected in 5% heart extracts from all species it was selected as the target antigen for developing an ELISA for the detection of heart. 5.4.2 Optimisation of cTnI ELISA for detection of heart

Figure 23 – Reactivity of meat and meat containing heart with anti-cTnT [1F11] and anti-cTnI [4C2] antibodies by ELISA. Wells were coated with cooked extracts of meat/heart mixtures (40 µg/ml) and reactivity of bound antigen with (A) anti-cTnT [1F11] and (B) anti-cTnI antibodies was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. (A) (B)

Key B – Beef L – Lamb P – Pork He – Heart

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Using cTnI as the target antigen for ELISA development, attempts were made to optimise the assay and provide the required sensitivity of 2.5% heart in meat. Firstly two commercially available TMB developing reagents were compared to the one being prepared in-house and the results are presented in Figure 24.

Commercial TMB based reagents gave an approximately 2-fold increase in signal for heart detection using the anti-cTnI antibody. Further ELISA development was performed using developer 3 (Thermo Scientific 1-StepTM Ultra TMB-ELISA) as this produced the strongest signal across the 3 species. The indirect ELISA was further optimised to try and achieve the desired sensitivity using cooked standards produced in-house (minced meat [beef, pork and lamb] containing 0 - 10% heart [beef, pork and lamb]). Different parameters were adjusted, including, antigen coating buffer, antigen coating concentration, antigen coating temperature, blocking agent, time and concentration; primary antibody dilution/incubation time and temperature, secondary antibody dilution/incubation time and temperature, development time. Some of the optimisation results are presented in Figures 25 – 29. Figure 25 shows the effects of antigen coating buffer, antigen coating concentration and antigen coating temperature on the detection of 0 – 10% beef heart in beef meat.

Figure 24 – Comparison of different TMB developing reagents on the detection of heart by ELISA. Wells were coated with cooked extracts of meat/heart mixtures (40 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells.

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B – Beef Developer 1 – Homemade TMB developer L – Lamb Developer 2 – Thermo Scientific TMB Substrate kit P – Pork Developer 3 – Thermo Scientific 1-StepTM Ultra TMB-ELISA He – Heart

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When carbonate pH 9.6 coating buffer was used the signal for heart detection increased as the antigen concentration increased, even at 1.6 mg/ml (80 µg/well) the signal was still increasing (Figure 25 (A) and (B)). With PBS coating buffer the optimum antigen coating concentration was 400 µg/ml (Figure 25 (C) and (D)), higher concentrations resulted in a decrease in signal. These coating concentrations are much higher than the binding capacity of the plates suggesting that binding of cTnI could be preferential.

Figure 25 – Comparison of different antigen coating conditions on the detection of beef heart by ELISA. Wells were coated with cooked extracts of beef meat/heart mixtures and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. Antigen coating using (A) carbonate binding buffer at 4°C for 16 hours (B) carbonate binding buffer at 37°C for 2 hours, (C) PBS binding buffer at 4°C for 16 hours and (D) PBS binding buffer at 37°C for 2 hours.

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When using carbonate coating buffer, the assay signal was higher when coating was performed at 4°C; whilst when PBS coating buffer was used the assay signal was higher when coating was performed at 37°C. Further optimisation was performed using PBS coating buffer. Antigen coating at 37°C for 1 hour was found to be preferential to 2 hours (data not shown). Primary antibody titer was explored using a range of antigen coating concentrations and the results are presented in Figure 26.

Figure 26 – Titration of anti-cTnI [4C2] antibody at different antigen coating concentrations. Wells were coated with cooked extracts of 2% or 10% beef heart in beef meat and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Data points represent the corrected mean A450nm ± SD of three replicate wells. (A) 2% beef heart in beef meat and (B) 10% beef heart in beef meat.

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Primary antibody saturation was observed at a dilution of 1/1000 (2 µg/ml). There was little difference in the signal when the antigen coating concentration was varied between 200 µg/ml and 600 µg/ml. An antigen coating concentration of 300 µg/ml and a primary antibody concentration of 2 µg/ml were used for further assay optimisation. Figure 27 demonstrates the effects of primary antibody incubation time and temperature on the assay signal.

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Figure 27 – Comparison of different primary antibody incubation conditions on detection of heart by ELISA. Wells were coated with cooked extracts of meat/heart mixtures (300 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells. Primary antibody incubation performed at (A) 37°C for 1 hour, (B) 25°C for 1 hour, (C) 37°C for 2 hours, (D) 25°C for 2 hours and (E) 4°C for 16 hours.

Key B – Beef L – Lamb P – Pork

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For beef, lamb and pork heart standards the strongest signal was observed when the primary antibody incubation was performed at 4°C for 16 hours (Figure 27 (E)). The optimal concentration of goat anti-mouse HRP conjugated secondary antibody was 1/1000, with an incubation time of 2 hours at 20°C or 1 hour at 25°C (results not shown). Assay development time was also monitored to ensure the reaction was stopped when product formation was linear, the results are presented in Figure 28.

Product formation was linear up to approximately 15 minutes for the 2% and 10% beef heart standards. Typically the assay is developed for 7 – 12 minutes. Table 4 shows typical absorbance values for standards and controls for the optimised heart ELISA.

Figure 28 – Effect of ELISA development time on detection of beef heart. Wells were coated with cooked extracts of beef meat/heart mixtures (300 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Data points represent the corrected mean A450nm ± SD of three replicate wells. (A) 2% beef heart in meat and (B) 10% beef heart in meat. (A) (B)

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Table 4 – Indirect ELISA using anti-cTnI [4C2] antibody for the detection of beef heart in beef meat. Wells were coated with cooked extracts of beef meat/heart mixtures (300 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Values represent the average A450nm ± SD of three replicate wells.

Absorbance at 450nm

Average A450nm

± SD

Corrected

A450nm ± SD well 1

well 2 well 3

No 1° ab/ No 2° ab

0.053

0.05 0.048 0.050 ± 0.003

No 1° ab 0.043

0.04 0.047 0.043 ± 0.004

No 2° ab 0.045

0.043 0.04 0.043 ± 0.003

Buffer blank 0.051

0.041 0.042 0.045 ± 0.006

0% B He 0.050

0.048 0.056 0.051 ± 0.004 0.000 ± 0.004

1% B He 0.125

0.118 0.120 0.121 ± 0.004 0.070 ± 0.004

2% B He 0.193

0.186 0.195 0.191 ± 0.005 0.140 ± 0.005

5% B He 0.625

0.643 0.678 0.649 ± 0.027 0.597 ± 0.027

10% B He 1.182

1.281 1.303 1.255 ± 0.064 1.204 ± 0.064

Antibody controls were performed using the 10% beef heart standard and were consistently below 0.05 Absorbance units. The assay routinely detects 1% beef heart in meat. 5.4.3 Reactivity of anti-cTnI [4C2] antibody with food additives/ingredients by ELISA The reactivity of the anti-cTnI [4C2] antibody with potential food additives and ingredients of relevance to minced meat, burgers, and other minced meat products was assessed using the optimised heart detection ELISA. Potential food additives and ingredients (e.g. soya, rice flour, wheat flour, corn flour, yeast extract and gelatine) were added to minced meat; cooked samples were homogenised in PBS 8 M urea and soluble proteins collected by centrifugation at 1,500xg for 15 minutes. Extracts were assayed alongside extracts of meat containing 0 – 10% beef, lamb and pork heart and results are presented in Figure 29.

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The anti-cTnI [4C2] antibody did not react with any meat mixtures containing these food additives / ingredients. 5.4.4 Summary of heart ELISA optimisation (1) An ELISA has been developed which routinely detects 1% beef heart in meat (2) Anti-cTnI antibodies did not react with the tested food additives / ingredients when screened by ELISA

Figure 29 – Reactivity of anti-cTnI [4C2] antibody with meat containing food additives/ingredients by ELISA. Wells were coated with cooked extracts of meat mixtures (300 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Bars represent the corrected mean A450nm ± SD of three replicate wells.

Key B – Beef L – Lamb P – Pork He – Heart

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5.5 Heart ELISA validation using standards and blind samples prepared in-house 5.5.1 Quantification of heart in blind samples prepared in-house by ELISA and Western blot analysis To validate the ELISA, beef heart standards were run alongside blind beef heart/ meat mixtures prepared in-house. Standard curves were produced and used to estimate the heart content of the blind samples, a typical standard curve is presented in Figure 30. Standards and blind samples were run in triplicate on each ELISA plate and the ELISA was repeated 9 times over several days to monitor intra- and inter-assay variation. Estimates of the amount of heart in the blind samples are summarised in Table 5.

Figure 30 – Standard calibration curve for quantification of added beef heart by ELISA. Wells were coated with cooked extracts of beef heart/meat mixtures (300 µg/ml) and reactivity of bound antigen with anti-cTnI [4C2] antibody was detected using an HRP-labelled secondary antibody. Data points represent the corrected mean A450nm ± SD of three replicate wells.

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Table 5 – Summary of the quantification of beef heart in blind samples by detection of cTnI by ELISA.

blind A

blind B blind C

% Added beef Heart ± SD (% CV)

Actual

3.0

7.5

12.0

ELISA 1

2.95 ± 0.15 (5.20)

8.34 ± 0.20 (2.37)

14.38 ± 0.11 (0.79)

ELISA 2

2.90 ± 0.10 (3.35)

8.52 ± 0.53 (6.20)

14.77 ± 0.42 (2.83)

ELISA 3

3.04 ± 0.12 (3.92)

7.91 ± 0.45 (5.68)

15.07 ± 0.64 (4.27)

ELISA 4

2.90 ± 0.13 (4.45)

8.42 ± 0.08 (0.98)

14.66 ± 0.30 (2.01)

ELISA 5

3.41 ± 0.34 (10.09)

7.76 ± 0.06 (0.80)

14.33 ± 0.66 (4.60)

ELISA 6

2.93 ± 0.20 (6.83)

7.92 ± 0.13 (1.62)

14.08 ± 0.35 (2.51)

ELISA 7

3.23 ± 0.25 (7.87)

8.42 ± 0.28 (3.31)

14.61 ± 0.39 (2.67)

ELISA 8

3.11 ± 0.56 (18.05)

8.52 ± 0.21 (2.52)

14.63 ± 0.02 (0.17)

ELISA 9

3.19 ± 0.12 (3.69)

8.19 ± 0.75 (9.21)

14.42 ± 1.11 (7.72)

AVERAGE ± SD

(% CV)

3.07 ± 0.18

(5.75)

8.22 ± 0.29

(3.53)

14.55 ± 0.29

(1.97)

The estimates obtained for the blind samples were close to the expected values for sample A and B, the heart estimate for blind sample C was higher than the actual amount but the estimate was consistent across the 9 assays. Intra-assay repeatability was good with low coefficients of variation (CV), the CV was only greater than 10% for 2 out of 27 estimates. Inter-assay repeatability was also good with the overall CV below 5% for all three blind samples. To compare the ELISA with the Western blot method developed in project Q01105, estimates for the heart content of the blind samples were also obtained by probing Western blots for cTnI. Densitometry analysis using AIDA image analysis software was used to produce standard curves and estimate the heart content of the blind samples. Blind samples were run in triplicate on each gel and the gel was repeated on 3 separate occasions to monitor reproducibility; estimates of the amount of heart in the blind samples are presented in Table 6.

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Table 6 - Quantification of beef heart in blind samples by detection of cTnI by Western blot analysis

blind A

blind B blind C

% Added beef Heart (% CV)

Actual

3.0

7.5

12.0

Gel 1

2.02 ± 0.12 (5.94)

7.29 ± 0.69 (9.47)

11.26 ± 0.71 (6.31)

Gel 2

1.94 ± 0.14 (7.22)

6.76 ± 0.29 (4.29)

10.22 ± 0.90 (8.81)

Gel 3

3.89 ± 0.23 (5.94)

5.67 ± 0.63 (11.14)

10.52 ± 1.86 (17.68)

Average ± SD

(% CV)

2.62 ± 1.10

(42.17)

6.57 ± 0.83

(12.57)

10.67 ± 0.54

(5.02)

The average estimates obtained for the blind samples were close to the expected values but lower than the actual amount for all blind samples. Intra-assay repeatability was good with low values for the CV, the CV was greater than 10% for 2 out of 9 estimates. Inter-assay repeatability was good for sample B and C but poor for sample A which suggests quantification of lower levels of heart is less accurate when using Western blotting. 5.5.2 Summary of analysis of blind samples (1) Both the ELISA and Western blot assays gave good estimates for the amount of heart in the blind samples (2) Overall the intra- and inter-assay repeatability was better for the ELISA than the Western blot analysis.

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5.6 Determination of heart in processed commercial meat products 5.6.1 Introduction It was important to determine whether our assays were capable of detecting the heart marker protein in processed meat products that contain heart. Two processed meat products were chosen that were labelled as containing heart and a further 4 products that were found to contain heart during an independent survey of retail meat products; in this case using Western blotting. Table 7 summarises the declared offal content of the products. Table 7 – Offal content of commercial products used for detection of heart marker protein

Product

Offal content

Haggis

33% pork lung 11% pork liver 1% pork heart

Dog Food

Min. 4% heart

Survey sample 10 Beef product with no declared offal. Heart detected using Western blotting during survey (Nov 2010)

Survey sample 47 Beef product with no declared offal. Heart detected using Western blotting during survey (Jan 2011)



5.6.2 Quantification of heart in commercial products by ELISA and Western blot analysis For quantification of heart in the commercial samples, cTnI was detected in PBS 8 M urea extracts of cooked in-house prepared heart standards and cooked commercial products by ELISA and Western blotting. Beef heart standards were assayed alongside the commercial products and standard curves were produced and used to estimate the heart content of the commercial products. Standards and commercial products were run in triplicate on each ELISA plate and the ELISA was repeated on 3 separate occasions to monitor intra- and inter-assay variation. Estimates of the amount of heart in the commercial products are presented in Table 8.

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For Western blots, densitometry analysis using AIDA image analysis software was used to produce standard curves and estimate the heart content of the commercial products. The commercial products were run in triplicate on each gel and the gel performed on 3 separate occasions to monitor intra- and inter-assay variation. Table 8 - Quantification of heart in commercial meat products by detection of cTnI by ELISA

Product

% Heart declared

% Heart detected (% CV)

ELISA 1

ELISA 2 ELISA 3 Average

Haggis

1 1.36 ± 0.22 (16.42)

1.60 ± 0.11 (6.75)

1.54 ± 0.05 (2.96)

1.49 ± 0.12 (8.14)

Dog Food

Min. 4 7.95 ± 0.11 (1.37)

6.89 ± 0.52 (7.54)

8.41 ± 0.02 (0.26)

7.75 ± 0.78 (10.06)

Survey sample 10

None 10.93 ± 0.29 (2.68)

9.32 ± 0.09 (0.96)

9.45 ± 0.01 (0.10)

9.90 ± 0.89 (9.03)

Survey sample 47

None 6.31 ± 0.35 (5.56)

5.68 ± 0.18 (3.17)

6.11 ± 0.38 (6.22)

6.03 ± 0.32 (5.34)

Survey sample 43

None 11.77 ± 0.34 (2.90)

10.02 ± 0.36 (3.62)

10.72 ± 0.25 (2.38)

10.84 ± 0.88 (8.13)

Survey sample 44

None 16.49 ± 0.18 (1.08)

12.61 ± 1.21 (9.60)

13.99 ± 0.76 (5.42)

14.36 ± 1.97 (13.69)

ND – not detected

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Table 9 - Quantification of heart in commercial meat products by detection of cTnI by Western blot analysis

Product

% Heart declared

% Heart detected (% CV)

Gel 1

Gel 2 Gel 3 Average

Haggis

1 1.06 ± 0.16 (14.98)

0.83 ± 0.13 (15.73)

0.63 ± 0.04 (5.85)

0.84 ± 0.22 (25.62)

Dog Food

Min. 4 4.23 ± 0.40 (9.50)

5.85 ± 0.72 (12.34)

5.66 ± 0.25 (4.48)

5.25 ± 0.89 (16.88)

Survey sample 10

none 13.01 ± 1.13 (8.69)

6.58 ± 1.00 (15.20)

10.41 ± 0.60 (5.74)

10.00 ± 3.23 (32.35)

Survey sample 47

None 5.31 ± 0.74 (13.94)

4.13 ± 0.41 (9.93)

7.34 ± 0.63 (8.58)

5.29 ± 1.62 (29.03)

Survey sample 43

None 13.42 ± 0.42 (3.13)

8.70 ± 1.45 (16.63)

10.19 ± 1.53 (15.00)

10.77 ± 2.41 (22.40)

Survey sample 44

None 18.25 ± 0.59 (3.23)

out of range of standards

out of range of standards

ND – not detected Heart was detected in the six products tested using the ELISA and Western blot assay. Intra-assay repeatability was good for the ELISA with CV <10% for all estimates except one. Inter-assay repeatability for the ELISA was also good with the overall CV < 15% for all estimates. Intra and inter-assay repeatability was higher for the ELISA than the Western assay. The ELISA and Western blot assay gave similar average values for the heart estimates for the haggis, dog food and 3 of the survey samples (10, 47 and 43). Survey sample 44 had a much higher heart content estimate by the Western blot assay compared to the ELISA, in 2 out of 3 of the gels the level was well above the range of the standards. As no heart was declared on the label we can’t be sure which assay has better accuracy. 5.6.3 Summary of the analysis of processed commercial products (1) The ELISA and Western blot assays detected heart in the processed commercial products labelled as containing heart (2) Both the ELISA and Western blot assays detected heart in all 4 of the survey samples previously found to contain heart (2) Overall the intra- and inter-assay repeatability was better for the ELISA than the Western blot assay

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6. GENERAL CONCLUSIONS An ELISA has been developed which routinely detects 1% beef heart in meat using cTnI as the target antigen. Anti-cTnI antibodies did not react with the tested food additives / ingredients when screened by ELISA. Both the ELISA and Western blot assays gave good estimates for the amount of heart in blind samples prepared in-house, but overall the intra- and inter-assay repeatability was better for the ELISA than the Western blot analysis. None of the 6 anti-SP-A antibodies or the anti-PRDX6 antibody had the required specificity or sensitivity to detect lung tissue by ELISA. However, an alternative anti-SP-A antibody that can detect beef, sheep and pork SP-A by Western blotting was found. Although this antibody didn’t have the required sensitivity by ELISA it can be used for detection of lung from all species by Western blotting. Further work is needed to establish the limit of detection of lung in meat using this antibody by Western blotting. 6.1 Future work To further validate the heart ELISA inter-procedure repeatability needs to be established by comparing the estimates for blind samples extracted and assayed on separate occasions. The limit of detection for pork heart and lamb heart by ELISA also needs to be established. Although the fourteen ingredients/additives screened in this project did not interfere with the heart detection ELISA, the limit of detection of the assay may be affected by the presence of these additives/ingredients or combinations of these, this could be investigated in the future. Additionally the interference of combinations of ingredients/additives was not looked at and this could also form part of a future work plan. The indirect ELISA developed routinely detects 1% beef heart in meat spiked with beef heart. If required, assay sensitivity could potentially be increased if a sandwich ELISA could be developed using a pair of monoclonal antibodies directed at distinct antigenic sites of the marker protein. It may also be possible to develop a sandwich ELISA that could distinguish between heart from different species. 7. ACKNOWLEDGEMENTS We acknowledge with gratitude the funding from Defra. 8. REFERENCES Gomes AV, Potter JD and Szczesna-Cordary D (2002) Life 54, 323-333 Hermans, C and Bernard A (1999) Am. J. Respir. Crit. Care Med. 159, 656-678 Hiraike N, Sohma H, Kuroki Y & Akino T (1995) Biochemica et Biophysica Acta 1257, 214 – 222 Kim T-S, Dodia C, Chen X, Hennigan BB, Jain M, Feinstein SI and Fisher AB (1998) Am. J. Physiol 274, L750-L761

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Leyens G, Donnay I and Knoops B (2003) Comp. Biochem. Phys. B 136, 943-955 Madsen J, Tornoe I, Nielsen O, Koch C, Steinhilber W and Holmskov U (2003) Am. J. Respir. Cell Mol. Biol. 29, 591-597 Paananen R, Glumoff V & Hallman M (1999) FEBS Letters 452, 141 – 144 Seo MS, Kang SW, Kim K, Baines I, Lee TH and Rhee SG (2000) J. Biol. Chem. 275, 20346-20354

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Transfer of offal detection assay to an ELISA platform