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

1988 ◽  
Vol 59 (01) ◽  
pp. 068-072 ◽  
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.

QUANTIFICATION OF ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) ANTIGEN BY AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

1987 ◽  
Author(s):  
I R MacGregor ◽  
N A Booth ◽  
N R Hunter ◽  
B Bennet
Keyword(s):  
Cell Line ◽  
Carcinoma Cell ◽  
Specific Activity ◽  
Carcinoma Cell Line ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Breast Carcinoma Cell Line ◽  
Standard Samples ◽  
Wide Range ◽  
Pai 1

An ELISA to measure PAI-1 antigen has been developed using PAI-1 purified from human endothelial cell conditioned medium and a monospecific antiserum raised against it in the rabbit. Test and standard samples diluted in assay buffer containing non-immune rabbit serum were incubated in microplate wells coated with anti-PAI-1 IgG. Then biotinylated anti-PAI-1 IgG was added to the wells followed by a streptavidin-biotinylated horseradish peroxidase complex. Tetramethylbenzidine was used as substrate and the optimised ELISA had a detection limit of 0.2 ng PAI-1 ml-1 sample, using purified PAI-1 of known concentration as a standard for the assay.PAI-1 antigen was readily detectable in human plasmas and higher concentrations were invariably detected in the corresponding whole blood sera.α2antiplasmin and antithrombin III which have been shown to have high degrees of sequence homology with PAI-J were undetectable in the ELISA at concentrations of 10 ug ml-1 . Sera from baboon and Rhesus and Cynomologus monkeys exhibited partial cross reactivity in the ELISA while no crossreactivities were observed with a wide range of non-primate sera that included dog, goat, cow, horse, pig and rat.A variety of human cell cultures were assayed for PAI-1 antigen. Endothelial cells from umbilical cord veins and arteries and from adult sapgenous veins secreted, typically,1-2 ug PAI-1 24-1 hr per 10 cells. This represented approximately 2-4% of the total secreted protein. Detectable but considerably lower levels of PAI-1 were secreted by keratinocytes and fibroblasts as well as a lung carcinoma cell line A549.No PAI-1 was detected in media conditioned by two melanoma cell lines, a breast carcinoma cell line MCF7 or a monocytic leukaemia JIII. The ELISA proved suitable for monitoring altered cellular PAI-1 metabolism, and in conjunction with functional assays, for investigating changes in PAI-1 specific activity.

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

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Stef J. Koppelman ◽  
Ashley L. Lardizabal ◽  
Lynn Niemann ◽  
Joe L. Baumert ◽  
Steve L. Taylor
Keyword(s):  
Sandwich Elisa ◽  
Food Product ◽  
Food Products ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Allergic Reactions ◽  
Arctica Islandica ◽  
Limit Of Quantification ◽  
Detection And Quantification

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.

scholarly journals Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1758-1764 ◽  
Author(s):  
J Kassis ◽  
J Hirsh ◽  
TJ Podor
Keyword(s):  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Postoperative fibrinolytic shutdown has been attributed to an increase in plasma levels of type I plasminogen activator inhibitor (PAI-1) activity and may contribute to postoperative venous thrombosis. The purpose of this study was to determine whether the postoperative increase in PAI-1 is contributed to by a plasma mediator(s) that stimulates PAI-1 synthesis and secretion by vascular endothelium. Plasma samples collected from patients (N = 11) before and after surgery for total hip replacement were (1) assayed for endogenous plasma PAI-1 antigen and activity, and (2) incubated with cultured human umbilical vein endothelial cells (HUVECs) and PAI-1 antigen and activity measured in the conditioned medium (CM). Eighteen hours after surgery, endogenous plasma levels of PAI-1 antigen and activity were increased by 225% (P = .003) and 190% (P = .04), respectively over the preoperative values. In addition, compared with preoperative plasma, postoperative plasma increased HUVEC secretion of PAI-1 antigen and activity by 99% (P = .001) and 66% (P = .002), respectively. This increase in HUVEC PAI-1 secretion reflects an increase in PAI-1 mRNA expression and protein biosynthesis as confirmed by metabolic radiolabeling, immunoprecipitation, and Northern blot analysis. Ultra- filtration experiments indicate that the postoperative plasma mediator(s) that stimulates HUVEC PAI-1 biosynthesis is in a molecular weight (MW) range of approximately 30 to 100 Kd. Heat treatment (56 degrees C; 30 minutes) of postoperative plasma abolished the induction of HUVEC PAI-1 production. Enzyme-linked immunosorbent assay and immunoneutralization experiments indicate that tumor necrosis factor- alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) do not contribute to the postoperative plasma effect on HUVEC PAI-1 synthesis. These observations demonstrate that postoperative patient plasma contains a factor(s) that may stimulate endothelial cell PAI-1 biosynthesis in vivo and thus mediate postoperative fibrinolytic shut- down.

scholarly journals A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen

1988 ◽  
Vol 256 (2) ◽  
pp. 433-440 ◽  
Author(s):  
R A Maciewicz ◽  
D J Etherington
Keyword(s):  
Cathepsin B ◽  
Specific Activity ◽  
Cathepsin L ◽  
Cross Reactivity ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Collagenolytic Activity ◽  
Cysteine Proteinases ◽  
Isoelectric Points ◽  
Multiple Charge

We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.

PURIFICATION AND CHARACTERIZATION OF PAI-1 FROM HUMAN ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
N A Booth ◽  
I R MacGregor ◽  
N R Hunter ◽  
B Bennett
Keyword(s):  
Endothelial Cells ◽  
Specific Activity ◽  
Umbilical Vein ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Protein Band ◽  
Single Chain ◽  
Step Procedure ◽  
Umbilical Vein Endothelial Cells ◽  
Pai 1

Platelet α-granules and endothelial cells contain an inhibitor of plasminogen activator, which inhibits both t-PA and u-PA. The inhibitor (PAI-1) is detectable after SDS-PAGE and zymography on fibrin/plasmin-ogen/u-PA detector gels. We have purified endothelial PAI-1 by a simple two-step procedure. Serum-free conditioned medium from human umbilical vein endothelial cells, grown in microcarrier culture, was fractionated on Sephadex CM-50, a cation exchanger, followed by gel filtration on Sephacryl S-200. Aprotinin was included throughout the procedure to maintain the activity of the inhibitor. The PAI-1 was purified 2000-fold with a recovery of about 7%. The purified protein had a specific activity of 8500 U/mg protein and the activity could be stimulated 14-fold by 4M guanidine. The purified PAI-1, of M 48000, was a single-chain glycoprotein.The ptoduct wasapparently homogeneous on a silver-stained SDS-polyacrylamide gel, the protein band co-migrating with PAI activity. Further, a rabbit antiserum raised against the purified PAI-1 revealed only a single band on immunoblots of material from each stage of the purification. The immunoglobulin fraction ofthe antiserum, incorporated into the detector gel for zymographic analysis, neutralised the inhibitor from plasma, platelets and endothelial cells, confirming their identity. Preincubation of PAI-1 from these sources with the immunoglobulin prevented formation of a complex with t-PA or u-PA. This purification procedure, in which no denaturants are employed, provides a homogeneous preparation of PAI-1 that is useful for studies on the stimulatory effects of denaturants. The antiserum raised has allowed the development of a sensitive ELISA, specific for PAI-1.

Comparison of Real-Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for Sensitive and Quantitative Detection of Hazelnuts in Nut Pastes

2018 ◽  
Vol 101 (6) ◽  
pp. 1864-1867 ◽  
Author(s):  
Ľubica Piknová ◽  
Veronika Janská ◽  
Tomáš Kuchta ◽  
Peter Siekel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sandwich Elisa ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Wide Range ◽  
Order Of Magnitude ◽  
Polymerase Chain

Abstract Background: Hazelnuts, being a frequent agent of allergenic reactions, need to be detected in food products. Thus, it is necessary to develop and further investigate appropriate methods for detection. Objective: The aim of the study was to compare the analysis of nut pastes (peanut paste spiked with different amounts of hazelnut paste) as a model of contamination of confectionery. Methods: Real-time PCR and sandwich ELISA (RidaScreen Hazelnut Fast Kit) were used. Results: For real-time PCR, LOQ of 2 mg/kg and a quantification range from 2 to 10 000 mg/kg were determined. For ELISA, LOQ of 1 mg/kg and a quantification range from 1 to 100 mg/kg were determined. Conclusions: The comparison shows that the methods had comparable sensitivity with LOQs in the same order of magnitude. Although ELISA was slightly more sensitive, it required dilution of samples at higher concentrations of the analyte because of its narrow quantification range. Results of this study suggest that real-time PCR and ELISA are both suitable methods for the analysis of nut pastes over a wide range of concentrations. Achieved results could be useful for control as well as for technological purposes. Highlights: Real-time PCR analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. Sandwich ELISA analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. The analytical parameters of real-time PCR and ELISA methods are compared.

scholarly journals Evidence for a Phytoreovirus Associated with Tobacco Exhibiting Leaf Curl Symptoms in South Africa

1999 ◽  
Vol 89 (4) ◽  
pp. 303-307 ◽  
Author(s):  
Marie Emma Christine Rey ◽  
Elvera D'Andrea ◽  
Jennifer Calvert-Evers ◽  
Maria Paximadis ◽  
Guido Boccardo
Keyword(s):  
South Africa ◽  
Inner Core ◽  
Sequence Similarity ◽  
First Record ◽  
Outer Core ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Class I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leaf Curl

Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 × 106 Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.

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

Foods ◽  
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.

scholarly journals Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1758-1764
Author(s):  
J Kassis ◽  
J Hirsh ◽  
TJ Podor
Keyword(s):  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Activator Inhibitor ◽  
Pai 1

Postoperative fibrinolytic shutdown has been attributed to an increase in plasma levels of type I plasminogen activator inhibitor (PAI-1) activity and may contribute to postoperative venous thrombosis. The purpose of this study was to determine whether the postoperative increase in PAI-1 is contributed to by a plasma mediator(s) that stimulates PAI-1 synthesis and secretion by vascular endothelium. Plasma samples collected from patients (N = 11) before and after surgery for total hip replacement were (1) assayed for endogenous plasma PAI-1 antigen and activity, and (2) incubated with cultured human umbilical vein endothelial cells (HUVECs) and PAI-1 antigen and activity measured in the conditioned medium (CM). Eighteen hours after surgery, endogenous plasma levels of PAI-1 antigen and activity were increased by 225% (P = .003) and 190% (P = .04), respectively over the preoperative values. In addition, compared with preoperative plasma, postoperative plasma increased HUVEC secretion of PAI-1 antigen and activity by 99% (P = .001) and 66% (P = .002), respectively. This increase in HUVEC PAI-1 secretion reflects an increase in PAI-1 mRNA expression and protein biosynthesis as confirmed by metabolic radiolabeling, immunoprecipitation, and Northern blot analysis. Ultra- filtration experiments indicate that the postoperative plasma mediator(s) that stimulates HUVEC PAI-1 biosynthesis is in a molecular weight (MW) range of approximately 30 to 100 Kd. Heat treatment (56 degrees C; 30 minutes) of postoperative plasma abolished the induction of HUVEC PAI-1 production. Enzyme-linked immunosorbent assay and immunoneutralization experiments indicate that tumor necrosis factor- alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) do not contribute to the postoperative plasma effect on HUVEC PAI-1 synthesis. These observations demonstrate that postoperative patient plasma contains a factor(s) that may stimulate endothelial cell PAI-1 biosynthesis in vivo and thus mediate postoperative fibrinolytic shut- down.

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

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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