Immunosensors antibodies

The carboxylic acid derivative of ferrocene N-(ferrocenylmethyl)caproic acid 9 after activation with the carbodiirriide DCC and NHS was also covalently coupled to GOx though its lysines. The resulting ferrocene-tethered enzyme was used as part of a model anti-DNP antibody immunosensor device based on the insulation of electrical contact between Fc-tethered GOx and the electrode by antigen-antibody association as depicted in Fig. 6.3 [21]. 

MPCs functionalized with large biomolecules including proteins, enzymes, and antibodies (immunosensors) are also abundant in the literature. A Ti02 nanotube array was decorated with antibody-labeled Au MPCs to allow for binding with a target analyte and amperometric detection. Typically, sensors must achieve detection limits of at least ng mL" to be a viable sensing platform for low-abundance proteins, and this sensor could lead to detection limits as low was 0.01 ng mL". Similar to the vast improvement in sensitivity for the chemiresistor that IDA electrodes made, miniaturization of electrodes into high aspect ratio arrays for this sensor led to enhanced electrochemical detection in comparison to flat electrode surfaces by 10-fold. ... 

Fig. 8. Basic components of a biosensor. In the case of an immunosensor, the antibody (or antigen) would be immobilized onto the transducer.

Fig. 9. Immunosensor approaches where A is the analyte, is the labeled analyte, and Y is the antibody, (a) Direct immunosensors where the actual antigen—antibody interaction is measured (b) indirect immunosensors 1 and 2 which utilize formats similar to competitive and displacement...

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

The aim of our investigation was the development of the amperometric enzyme immunosensor for the determination of Klebsiella pneumoniae bacterial antigen (Ag), causes the different inflammatory diseases. The biosensing pail of the sensors consisted of the enzyme (cholinesterase) and antibodies (Ab) immobilized on the working surface of the screen-printed electrode. Bovine seiaim albumin was used as a matrix component. 

The working conditions of the immunosensor (enzyme and antigen concentrations, dilutions of the antibodies, pH of the buffer solution) were found. The cholinesterase immobilized demonstrated the maximum catalytic activity in phosphate buffer solution with pH 8.0. The analytical chai acteristics of the sensor - the interval of the working concentrations and detection limit - have been obtained. The proposed approach of immunoassay made possible to detect 5T0 mg/ml of the bacterial antigen. 

A.M. Campbell, Monoclonal Antibody and Immunosensor Technology the Production and Application of Rodent and Human Monoclonal Antibodies, Elsevier, Amsterdam (1991). 

Figure 2 shows the most abundant class of antibodies found in blood serum and lymph - immunoglobulin G (IgG). IgG of molecular mass about 156 000, is most frequently used as a receptor in immunosensors. According to X-ray data6 8, IgG is a Y-shaped molecule consisting of two identical antigen binding Fab arms of dimensions 6.5 nm by 3.5 nm and an inactive Fc shank of dimensions 5 nm by 3.5 nm. 

Brynda E., Elouska M., Brandenburg A., Wikerstal A., Skvor J., The detection of human P2-microglobulin by grating coupler immunosensor with three-dimensional antibody networks, Biosens. Bioelectron. 1999 14 363-368. 

V.B. Kandimalla, N.S. Neeta, N.G. Karanth, M.S. Thakur, K.R. Roshini, B.E.A. Rani, A. Pasha, and N.G.K. Karanth, Regeneration of ethyl parathion antibodies for repeated use in immunosensor a study on dissociation of antigens from antibodies. Biosens. Bioelectron. 20, 903-906 (2004). 

M.A. Gonzalez-Martinez, S. Morais, R. Puchades, A. Maquieira, A. Abad, and A. Montoya, Monoclonal antibody-based flow-through immunosensor for analysis of carbaryl. Anal. Chem. 69, 2812—2818 (1997). 

K. Grennan, G. Strachan, A.J. Porter, A.J. Killard, and M.R. Smyth, Atrazine analysis using an amperometric immunosensor based on single-chain antibody fragments and regeneration-free multi-calibrant measurement. Anal Chim. Acta 500, 287-298 (2003). 

There is a continuing demand for fast and simple analytical methods for the determination of many clinical, biochemical and environmental analytes. In this respect, immunoassays and immunosensors that rely on antibody-antigen interactions provide a promising means of analysis owing to their specificity and sensitivity. High specificity... 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

There are many other examples of competitive electrochemical immunoassays and immunosensors for detecting clinically important analytes [12-14], Despite simplicity, a disadvantage of competitive immunoassays is that labeling the analyte may reduce, or totally remove, its binding affinity for antibody. This would occur if the analyte were labeled at a site that is closely associated with an epitope. 

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