Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays

OBJECTIVEDiabetes is associated with various vascular complications and is suggested to induce a prothrombotic state. In the current study, we characterized antiplasmin incorporation into fibrin in relation to other fibrinolytic compounds in patients with type 1 diabetes. RESEARCH DESIGN AND METHODSA total of 236 patients with type 1 diabetes and 78 control subjects were investigated. The incorporation of antiplasmin into the fibrin network and the plasma levels of plasminogen activator inhibitor type 1 (PAI-1) activity, tissue plasminogen activator (tPA) activity, tPA/PAI-1 complex, plasmin-antiplasmin complex, antiplasmin, factor XIII, and D-dimer were measured. In addition, we used global assays to study fibrinolysis. RESULTSThe incorporation of antiplasmin into the fibrin network was significantly higher in patients with type 1 diabetes than in control subjects without diabetes (1.65 6 0.25 vs. 1.35 6 0.18 mg/L, respectively; P < 0.0001). The patients also had lower PAI-1 activity (2.19 units/mL [interquartile range 0.96-5.42] vs. 4.25 units/mL [1.95-9.0]; P = 0.0012) and antiplasmin level in plasma (78.5 6 13.3 vs. 83.2 6 15.4 mg/L; P < 0.05), resulting in a higher fibrinolytic capacity (shorter clot lysis time; P = 0.0090). We did not find any important sex differences regarding fibrinolysis in the patients or in the control subjects. CONCLUSIONSPatients with type 1 diabetes incorporate more antiplasmin into the fibrin network than control subjects without diabetes do and have a reduced PAI-1 activity and a shorter clot lysis time. These results suggest that patients with type 1 diabetes produce a fibrin clot that is more resistant to fibrinolysis, which, however, may be counteracted by an increased fibrinolytic potential in plasma.Diabetes is linked to microvascular disturbances causing neuropathy, retinopathy, and nephropathy, as well as macrovascular complications such as stroke, coronary heart disease, and nonhealing foot ulcers (1). Hypercoagulability and impaired fibrinolysis are pathophysiologically important processes of vascular complications (2). Diabetes has been considered a prothrombotic state (3), partly due to impaired fibrinolysis (4), but has also been associated with increased risk of bleeding when

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“…For detection, the antibodies were conjugated with horseradish peroxidase (16). The CV of the method was ,5% both within and between series.…”

Section: Antiplasminmentioning

confidence: 99%

Increased Incorporation of Antiplasmin Into the Fibrin Network in Patients With Type 1 Diabetes

et al. 2014

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“…The recorded current will then be proportional to the glucose concentration in the sample. Previously it has been clearly shown that using polymeric bound mediators and especially polymeric mediators covalently bound with the enzyme is beneficial to obtain stable biosensors [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60]. However, in the case stipulated in this work when coimmobilising GOD and HRP it has been possible to achieve long term stable sensors.…”

Section: Resultsmentioning

confidence: 98%

“…These can be further reacted with the amine functions of GOD forming Schiff bases hereby linking the two enzymes together increasing the trapping efficiency of the produced H 2 O 2 by GOD [56][57][58][59]. The quantities given below are for paste preparations based on 400 mg of graphite powder as starting point.…”

Section: The Basic Preparation Procedures For the Modified Carbon Pastementioning

confidence: 99%

The synergetIc effect of redox mediators and peroxidase in a bienzymatic biosensor for glucose assays in FIA

Rondeau

Larsson²,

Boujtita

et al. 1999

Analusis

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Amperometric biosensors based on enzymes are interesting due to their high sensitivity, excellent selectivity, simplicity, low cost and rapid response. The most frequently used enzymatic methods for glucose determination employ glucose oxidase (GOD), due to its high selectivity towards ß-D-glucose. In the presence of GOD, the oxidation of glucose occurs, producing H 2 O 2 . Most commonly, the electrochemical detection is performed through the anodic oxidation of H 2 O 2 [1]. A drawback with this detection technique is that high overvoltages are required, and an applied potential in the range of 0.6-1.1 V vs. Ag/AgCl is necessary. Working at such high potentials increases the risk of interference from easily oxidisable compounds such as ascorbate, ureate, acetaminophen, paracetamol, phenols, etc. which are common compounds in biological samples.A possible way of improving the selectivity of such a biosensor is to couple a second enzyme (peroxidase) to the oxidase, creating a bienzymatic sensor. Peroxidase catalyses the electrochemical reduction of H 2 O 2 produced by GOD through a direct electron transfer reaction at a much lower applied potential compared with electrochemical oxidation of H 2 O 2 and maximum catalytic current can be obtained at carbon electrodes in a potential range of -0.1 and 0 V [2-14]. However, the kinetics of the direct electron transfer from an electrode to the peroxidase is a sluggish process [15][16][17] hence the advantage of using a mediator.The introduction of a mediator in the bienzymatic system (GOD/HRP) leads to an acceleration of the electron transfer, a decrease in the applied working potential (the reduction of the mediator is detected at the electrode) and an increase in sensitivity. The use of mediators is a well-known technique although the way of adding the mediator to the bienzymatic system (oxidase/peroxidase) may vary. A number of different mediators have been used as additions into sample solutions such as, potassium hexacyanoferrate (II) [18][19][20][21][22][23][24] [44,45], and Nafion-Nmethyl phenazinium [46]. The advantages of using the redox polymers are several, but mainly they result in more stable biosensors since leaking of the mediator from the electrode is minimised and higher and faster responses are observed due to the proximity between the enzyme and the mediator. Although, choosing an appropriate bulk matrix and mediator concentration these properties can be achieved with nonpolymeric mediators and recently a ferrocene mediated bienzymatic graphite-Teflon electrode has been developed for batch and flow-injection measurements [47]. On the other hand several biosensors based on monoenzymatic electrodes 649The synergetIc effect of redox mediators and peroxidase in a bienzymatic biosensor for glucose assays in FIA Abstract. A bienzymatic biosensor incorporating a mediator has been developed in order to achieve a fast and selective detection of glucose in a flow injection system. The working electrode is based on a carbon paste matrix bulk modified with glucose ...

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“…The reactivity of anti-T. gondii IgG pAb against SAG1 antigen was measured by indirect ELISA with some modifications from the original method of Engvall and Perlman (15). Labeling of anti-SAG1 IgG pAb with horseradish peroxidase with the periodate method was performed as previously described (22).…”

Section: Participantsmentioning

confidence: 99%

Novel IMB-ELISA Assay for Rapid Diagnosis of Human Toxoplasmosis Using SAG1 Antigen

et al. 2015

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SUMMARY: Nanotechnology is important for medical diagnosis. Various nanoparticles have presented tremendous potential for diagnosing disease markers, pre-cancerous cells, fragment of viruses, specific proteins, antibodies, and other disease indicators. In general, nanoparticles are smaller than 1,000 nm and produced from different materials in different shapes such as spheres, rods, wires, and tubes. Our study aimed to develop a novel antigen-capture immunoassay based on IgG polyclonal antibody-coated magnetic microbead nanoparticles for the rapid detection of circulating surface antigen 1 of Toxoplasma gondii in human serum samples. Sandwich ELISA elicited a sensitivity of 92z, a specificity of 92.7z, a positive predictive value (PPV) of 92z, and a negative predictive value (NPV) of 92.7z. Immunomagnetic bead-ELISA showed sensitivity (98z), specificity (96.4z), PPV (96z), and NPV (98.1z) higher than that of sandwich ELISA. It is obvious that the use of magnetic microbead nanoparticles offers the potential advantage of improving the diagnostic testing of toxoplasmosis.

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Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays

Cited by 226 publications

References 13 publications

Increased Incorporation of Antiplasmin Into the Fibrin Network in Patients With Type 1 Diabetes

Increased Incorporation of Antiplasmin Into the Fibrin Network in Patients With Type 1 Diabetes

The synergetIc effect of redox mediators and peroxidase in a bienzymatic biosensor for glucose assays in FIA

Novel IMB-ELISA Assay for Rapid Diagnosis of Human Toxoplasmosis Using SAG1 Antigen

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