Development of a Sandwich Enzyme-Linked Immunosorbent Assay for Detection and Quantification of Clam Residues in Food Products

Seafood is a frequent cause of allergic reactions to food globally. The presence of undeclared trace amounts of clam can cause allergic reactions in sensitive individuals. Limited tools are available to test food products for the presence of traces of clam. We report on the development of a sandwich ELISA that can detect and quantify clam protein in food. Antisera against a mix of two commercially important clam species, Atlantic Surf (Spisula solidissima) and ocean quahog (Arctica islandica), were raised in rabbit and sheep. A sandwich ELISA was constructed with this antisera, and sensitivity and specificity were evaluated. Also, model food products spiked with clam protein were analyzed to assess the performance of the ELISA. Comparison was made with a commercially available ELISA for crustacea. The lower limit of quantification of the sandwich ELISA is 2.5 ppm clam protein in food samples, allowing the detection of low amounts of clam that may trigger a reaction in clam allergic patients. The sandwich ELISA was highly specific with cross-reactivity only noted for other molluscan shellfish (mussel and scallop). Clam protein in tomato juice and potato cream soup was detected well with recoveries ranging from 65 to 74% and from 74 to 113%, respectively. However when potato cream soup was retorted, the recover fell to 20%, imposing the risk of underestimating the clam content of a food product. A commercially available crustacean ELISA test was not suitable to detect clam protein. The sandwich ELISA described here is suitable for detection and quantification of clam protein in food products. Care should be taken with food products that have been retorted as the results may be underestimated.

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Protein Quantification, Sandwich ELISA, and Real-Time PCR Used to Monitor Industrial Cleaning Procedures for Contamination with Peanut and Celery Allergens

Journal of AOAC International ◽

10.1093/jaoac/87.6.1448 ◽

2004 ◽

Vol 87 (6) ◽

pp. 1448-1457 ◽

Cited By ~ 34

Author(s):

Oliver Stephan ◽

Nancy Weisz ◽

Stefan Vieths ◽

Tanja Weiser ◽

Burghard Rabe ◽

...

Keyword(s):

Real Time ◽

Sandwich Elisa ◽

The United States ◽

Protein Quantification ◽

Enzyme Linked Immunosorbent Assay ◽

Allergic Reactions ◽

Cleaning Process ◽

Cleaning Procedures ◽

Washing Water

Abstract In the United States, peanut is one of the main sources of food allergens. Similarly, celery is a common allergenic food in Western Europe. Severe allergic reactions to both foods are common. Unexpected allergic reactions can occur after the consumption of celery-and peanut-free foods as a result of inadvertent cross-contaminations during manufacturing. Therefore, in cooperation with a flavor manufacturer, we monitored the cleaning process of slurry preparation equipment with regard to contaminations of follow-up products with celery and peanut compounds. Washing water samples taken after different cleaning steps and follow-up products were analyzed for the presence of celery and peanut traces with a celery-specific real-time polymerase chain reaction (PCR) and a peanut-specific sandwich enzyme-linked immunosorbent assay (ELISA). PCR and ELISA were compared with a nonspecific protein assay to evaluate whether the detection of protein traces can be a fast and cost-effective method for monitoring the effectiveness of wet cleaning procedures. Additionally, the allergenic potential of the celery and peanut mush, which were used as source material, were measured by a mediator release assay using a rat basophilic leukemia (RBL) cell line. In conclusion, the quantification of total protein in washing water was suitable for monitoring the cleaning process. Our study also revealed evidence that, in cases where wet cleaning is applicable, allergenic traces can be removed with high efficiency.

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New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus

Viruses ◽

10.3390/v13091680 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1680

Author(s):

Joan Miquel Bernabé-Orts ◽

Covadonga Torre ◽

Eduardo Méndez-López ◽

Yolanda Hernando ◽

Miguel A. Aranda

Keyword(s):

Internal Control ◽

Plant Protection ◽

Genetic Resistance ◽

Diagnostic Techniques ◽

Plant Viruses ◽

Elisa Test ◽

Cross Reactivity ◽

Sensitive Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Effective Prevention

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

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An Enzyme-Linked Immunosorbent Assay (ELISA) Used to Study the Cellular Secretion of Endothelial Plasminogen Activator Inhibitor (PAI-1)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646771 ◽

1988 ◽

Vol 59 (01) ◽

pp. 068-072 ◽

Cited By ~ 23

Author(s):

I R MacGregor ◽

N A Booth

Keyword(s):

Specific Activity ◽

Umbilical Vein ◽

Sandwich Elisa ◽

Secretory Protein ◽

Cross Reactivity ◽

Polyclonal Antiserum ◽

Enzyme Linked Immunosorbent Assay ◽

Wide Range ◽

Pai 1 ◽

Cellular Secretion

SummaryA two-site sandwich ELISA was developed to measure PAI-1 antigen and utilised a polyclonal antiserum produced against PAI-1 purified from human endothelial cell secretory products. The assay was calibrated against a preparation of pure PAI-1 whose protein concentration had been determined by amino acid analysis and the detection limit was 30 pg PAI-1 ml“3 sample. PAI-1 was detected in primate sera but not in a wide range of nonprimate sera and no cross-reactivity with α2-antiplasmin or antithrombin III was observed.The ELISA was used to study cellular secretion of PAI-1 which was confirmed as a major secretory protein in human umbilical vein endothelial cells (HUVEC). PAI-1 antigen accumulated in the medium in a linear fashion with time and accounted for ≃10% of total secreted protein. Specific activity of intracellular PAI1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t½ for inactivation of secreted PAI-1 of 0.53 h was calculated. Purified endotoxin stimulated the secretion of PAI-1 antigen and raised the intracellular levels in HUVEC cultures showing that the anti-fibrinolytic actions of endotoxin are effected by increasing the rate of synthesis and secretion of PAI-1.

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Development, Optimization and Validation of a Sandwich ELISA for Detection of Recombinant Human Factor VII

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.5(1).p26-31 ◽

2015 ◽

Vol 5 (1) ◽

pp. 26-31

Author(s):

Sheila Sarial ◽

Rozhan Sonboli ◽

Shayan Maleknia ◽

Fatemeh Ashori ◽

Sara Rezaeiravesh ◽

...

Keyword(s):

Low Dose ◽

Human Factor ◽

Sandwich Elisa ◽

Factor Vii ◽

Enzyme Linked Immunosorbent Assay ◽

P Value ◽

Limit Of Quantification ◽

Intermediate Precision ◽

Downstream Processes

Recombinant human factor VII (rh-FVII) is produced by engineered BHK cells at low dose. Accordingly, establishment of a precise method is crucial to reliably measuring expression level of this protein during manufacturing pro-cesses. We developed and established a reproducible sandwich enzyme-linked immunosorbent assay (ELISA) method for measuring amount of FVII during biopharmaceutical in upstream and downstream processes of Aryo-Seven. A sandwich ELISA was designed using two different high affinity mon-oclonal antibodies (mAb1 and mAb2) against h-FVII. The bounded FVII to the first antibody was revealed by the use of a second mouse anti-FVII monoclo-nal antibody (1F1-B11), labeled with HRP that binds to another antigenic determinant of the FVII. Then, validation was done by determination of spec-ificity, linearity, accuracy, precision, reproducibility, detection and quantifica-tion limit and robustness according to ICH Q2 (R1) guideline. In developed ELISA, no interference was found between FVII and proteins derived from BHK which commonly exist in the supernatant. Linear range of detection was from 25-1.56 ng/mL with P-Value <0.001. In accuracy, spiked samples showed 109±2% recovery. Intra and intermediate precision assays showed %RSD not more than20. Detection limit of this assay was 0.99 ng /mL and limit of quantification was 2.99 ng/ml. The sandwich ELISA was found to be useful tool for measuring FVII/ FVIIa. The ELISA approach was precise, repro-ducible, and accurate. The ELISA therefore, is offered as an assured kit for detection of recombinant human factor.

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Enzyme-Linked Immunosorbent Assay Detection of Melamine in Infant Formula and Wheat Food Products

Journal of Food Protection ◽

10.4315/0362-028x-73.4.701 ◽

2010 ◽

Vol 73 (4) ◽

pp. 701-707 ◽

Cited By ~ 15

Author(s):

ERIC A. E. GARBER ◽

VICKERY A. BREWER

Keyword(s):

Sample Preparation ◽

Infant Formula ◽

Immunosorbent Assay ◽

Cyanuric Acid ◽

Food Products ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Limits Of Detection ◽

Wheat Products ◽

Assay Detection

The adulteration of food products with melamine to inflate the nitrogen content necessitates the establishment of analytical methods that can distinguish between proteinaceous ingredients and such adulterants. The specificity and ability to detect melamine by two commercial enzyme-linked immunosorbent assay (ELISA) kits were evaluated along with three protocols for sample preparation. Both ELISAs displayed cross-reactivity with ammeline, but neither was able to detect ammelide or cyanuric acid, indicating either a requirement for the 4,6-diamino-1,3,5-triazine structure or inability to bind 1,3,5-triazine-4,6-diones. The limits of detection for melamine in powder infant formula ranged from 0.2 to 3 μg/g depending on the ELISA kit and the method used to prepare the sample. The limits of detection for melamine in liquid infant formula and wheat products were <1 μg/ml and <2.5 μg/g, respectively. The ELISA kits provide an effective alternative for the analysis of samples suspected of containing melamine without relying on extensive sample preparation or expensive instrumentation.

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Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR

Foods ◽

10.3390/foods10020440 ◽

2021 ◽

Vol 10 (2) ◽

pp. 440

Author(s):

Raquel Madrid ◽

Aina García-García ◽

Pablo Cabrera ◽

Isabel González ◽

Rosario Martín ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Polyclonal Antibodies ◽

Juglans Regia ◽

Sandwich Elisa ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa Kit ◽

Specificity And Sensitivity ◽

Direct Elisa

Labeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.

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A pre- and during Pandemic Survey of Sars-Cov-2 Infection in Stray Colony and Shelter Cats from a High Endemic Area of Northern Italy

Viruses ◽

10.3390/v13040618 ◽

2021 ◽

Vol 13 (4) ◽

pp. 618

Author(s):

Eva Spada ◽

Fabrizio Vitale ◽

Federica Bruno ◽

Germano Castelli ◽

Stefano Reale ◽

...

Keyword(s):

Rectal Swab ◽

Elisa Test ◽

Cross Reactivity ◽

Northern Italy ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Endemic Region ◽

Lombardy Region ◽

Stray Cats ◽

Stray Cat

Cats are susceptible to infection with severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Whilst a number of studies have been performed worldwide on owned cats, limited data are available on stray, colony or shelter cats. We investigated SARS-CoV-2 infection in a stray cat population before and during human outbreaks of SARS-CoV-2 in cities in the Lombardy region in northern Italy, a high endemic region for SARS-CoV-2, using serological and molecular methods. A cohort of different samples were collected from 241 cats, including frozen archived serum samples from 136 cats collected before the 2019 coronavirus disease (COVID-19) pandemic and serum, pharyngeal and rectal swab samples from 105 cats collected during the SARS-CoV-2 outbreak. All pre-pandemic samples tested seronegative for antibodies against the nucleocapsid of SARS-CoV-2 using indirect enzyme linked immunosorbent assay (ELISA) test, while one serum sample collected during the pandemic was seropositive. No serological cross-reactivity was detected between SARS-CoV-2 antibodies and antibodies against feline enteric (FECV) and infectious peritonitis coronavirus (FIPC), Feline Immunodeficiency Virus (FIV), Feline Calicivirus (FCV), Feline Herpesvirus-1 (FHV-1), Feline Parvovirus (FPV), Leishmania infantum, Anaplasma phagocytophilum, Rickettsia spp., Toxoplasma gondii or Chlamydophila felis. No pharyngeal or rectal swab tested positive for SARS-CoV-2 RNA on real time reverse transcription-polymerase chain reaction (rRT-PCR). Our data show that SARS-CoV-2 did infect stray cats in Lombardy during the COVID-19 pandemic, but with lower prevalence than found in owned cats. This should alleviate public concerns about stray cats acting as SARS-CoV-2 carriers.

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A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1

Frontiers in Immunology ◽

10.3389/fimmu.2021.670626 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xin Zhang ◽

Haipeng Zhu ◽

Xu Zheng ◽

Yunjie Jiao ◽

Lulu Ning ◽

...

Keyword(s):

Limit Of Detection ◽

Sandwich Elisa ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Detection ◽

Serum Samples ◽

Double Antibody ◽

Programmed Death ◽

Prokaryotic Expression Vector ◽

Das Elisa

Fibrinogen-like protein 1 (FGL1), a member of the fibrinogen family, is a specific hepatocyte mitogen. Recently, it has been reported that FGL1 is the main inhibitory ligand of lymphocyte activating gene 3 (LAG3). Furthermore, the FGL1-LAG3 pathway has a synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway and is regarded as a promising immunotherapeutic target. However, swine FGL1 (sFGL1) has not been characterized and its detection method is lacking. In the study, the sFGL1 gene was amplified from the liver tissue of swine and then inserted into a prokaryotic expression vector, pQE-30. The recombinant plasmid pQE30-sFGL1 was transformed into JM109 competent cells. The recombinant sFGL1 was induced expression by isopropyl-β-d-thiogalactoside (IPTG) and the purified sFGL1 was used as an antigen to produce mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb). After identification, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for sensitive and specific detection of sFGL1 was developed. Swine FGL1 in samples was captured by anti‐sFGL1 mAb followed by detection with anti‐sFGL1 rabbit pAb and HRP-conjugated goat anti-rabbit IgG. The limit of detection of the developed sFLG1-DAS-ELISA is 35 pg/ml with recombinant sFLG1. Besides, it does not show cross‐reactivity with the control protein. Then serum samples of PRRSV-negative and -positive pigs were tested with the established DAS-ELISA and calculated according to the equation of y=0.0735x+0.0737. The results showed that PRRSV infection enhanced the serum FGL1 levels significantly. Our research provides a platform for the research on the functional roles of swine FGL1.

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Indirect Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Plasma Apolipoprotein E

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456329603300204 ◽

1996 ◽

Vol 33 (2) ◽

pp. 119-126 ◽

Cited By ~ 3

Author(s):

Patrick Kee ◽

Renze Bais ◽

Stan K Sobecki ◽

Susan Branford ◽

Kerry-Anne Rye ◽

...

Keyword(s):

Apolipoprotein E ◽

Low Density Lipoprotein ◽

Sandwich Elisa ◽

Density Lipoprotein ◽

Gel Permeation Chromatography ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Lipoprotein Subfractions ◽

Apo E ◽

Plasma Apolipoprotein

We have developed an indirect sandwich ELISA for measuring plasma apolipoprotein E (apo-E), using commercially available antibodies. A monoclonal anti-apo-E was used as the capture antibody and the captured apo-E detected with polyclonal anti-apo-E antiserum (goat). The detecting antibody was quantitated using horseradish peroxidase-conjugated rabbit immunoglobulin to goat immunoglobulins. There was no detectable cross-reactivity between the three antisera. Interference with the assay by apolipoprotein A-1, bilirubin and haemoglobin was not significant up to 1·7 g/L, 1250 μmol/L and 13·0 g/dL, respectively. The ELISA method showed high correlation with an established immunonephelometric method (ELISA apo-E=104 Immunonephelometric apo-E+16; r2 = 0·954, P < 0·0001, n = 39). The assay has a measuring range between 5 and 560 mg/L. The coefficient of duplicates was 20%, within-run coefficients of variation (CV) ranged from 3·7 to 6·0% and between-run CV from 6·1 to 15·1%. The reference range determined for 168 normotriglyceridaemic subjects was 20 to 130 mg/L. In an analysis of the lipoprotein subfractions isolated by ultracentrifugation as the fraction of density less than 1·25 g/mL and separated by gel permeation chromatography, apo-E was found to be associated with very low-density lipoprotein and large high-density lipoprotein.

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Nano-Biosensing Platforms for Detection of Cow’s Milk Allergens: An Overview

Sensors ◽

10.3390/s20010032 ◽

2019 ◽

Vol 20 (1) ◽

pp. 32 ◽

Cited By ~ 4

Author(s):

Monika Nehra ◽

Mariagrazia Lettieri ◽

Neeraj Dilbaghi ◽

Sandeep Kumar ◽

Giovanna Marrazza

Keyword(s):

Life Quality ◽

Milk Proteins ◽

Analytical Techniques ◽

Food Products ◽

Cross Reactivity ◽

Cow Milk ◽

Food Allergies ◽

Enzyme Linked Immunosorbent Assay ◽

Labeling Regulations ◽

Significant Research

Among prevalent food allergies, cow milk allergy (CMA) is most common and may persist throughout the life. The allergic individuals are exposed to a constant threat due to milk proteins’ presence in uncounted food products like yogurt, cheese, and bakery items. The problem can be more severe due to cross-reactivity of the milk allergens in the food products due to homologous milk proteins of diverse species. This problem can be overcome by proper and reliable food labeling in order to ensure the life quality of allergic persons. Therefore, highly sensitive and accurate analytical techniques should be developed to detect the food allergens. Here, significant research advances in biosensors (specifically immunosensors and aptasensors) are reviewed for detection of the milk allergens. Different allergic proteins of cow milk are described here along with the analytical standard methods for their detection. Additionally, the commercial status of biosensors is also discussed in comparison to conventional techniques like enzyme-linked immunosorbent assay (ELISA). The development of novel biosensing mechanisms/kits for milk allergens detection is imperative from the perspective of enforcement of labeling regulations and directives keeping in view the sensitive individuals.

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