Immunosensors antigens

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). 

Enzyme immunosensors are employed for the determination of Hepatitis B surface antigen, IgG, alpha-fetoprotein, estradiol, theophylline, insulin [9004-10-8] and alburnin (69,70). However, these immunosensors generally have slow response times and slow reversibiUty (57). 

AN AMPEROMETRIC ENZYME IMMUNOSENSOR BASED ON SCREEN-PRINTED ELECTRODE FOR THE DETERMINATION OF KLEBSIELLA PNEUMONIAE BACTERIAL ANTIGEN... 

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. 

The developed amperometric enzyme immunosensor was probed to determine the Klebsiella pneumoniae antigen in the human sera samples. The obtained results were juxtaposed with the data of the bacteriological analysis. 

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. 

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). 

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. 

In amperometry, the current produced by the oxidation or reduction of an electroactive analyte species at an electrode surface is monitored under controlled potential conditions. The magnitude of the current is then related to the quantity of analyte present. However, as both antibody and antigen are not intrinsically electroactive, a suitable label must be introduced to the immunocomplex to promote an electrochemical reaction at the immunosensors. In this respect, enzyme labels including the... 

Y.-Q. Miao and J.-G. Guan, Probing of antibody-antigen reactions at electropolymerized polyaniline immunosensors using impedance spectroscopy. Anal. Lett. 37, 1053—1062 (2004). 

Since immunosensors usually measure the signals resulting from the specific immu-noreactions between the analytes and the antibodies or antigens immobilized, it is clear that the immobilization procedures of the antibodies (antigens) on the surfaces of base transducers should play an important role in the construction of immunosensors. Numerous immobilization procedures have been employed for diverse immunosensors, such as electrostatic adsorption, entrapment, cross-linking, and covalent bonding procedures. They may be appropriately divided into two kinds of non-covalent interaction-based and covalent interaction-based immobilization procedures. 

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