Antibodies anti-analyte

Competitive inhibition. A constant amount of anti-analyte antibody (primary antibody) and a series of solutions containing increasing amounts of analyte are added to the prepared microtiter plate wells. During incubation, the free analyte and bound... 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

Competitive immunoassays can be based on different formats [3, 22, 23] (1) an anti-analyte antibody is immobilized on a solid support and the analyte in the sample competes with an added labeled analyte for a limited number of antibody binding sites (2) a solid phase is coated with an antibody that binds to the antianalyte antibody and the assay is carried out with the analyte and the labeled derivative mixture (3) the immobilized antigen approach consists of the competition of the analyte in the sample with the immobilized antigen for binding to labeled antibody molecules. 

For epitope elucidation of the polyclonal anti-c APP antibody, the analytical scheme summarised in Figure 3 was employed (Macht et al. 1996 Tian et al. 2005). The antibody was first immobilised on Sepharose embedded in a microcolumn (s. Materials and Methods). The synthetic peptide cAPP(724-770) was used as antigen and initially cleaved by trypsin and Glu-C protease, yielding the proteolytic fragments summarised in Table 1. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

Fig. 4. Sandwich Immunoassay (A) In the absence of analyte, antibody-conjugated liposomes are captured by a species-specific secondary antibody at the control zone, thus proving that the assay was run correctly. (B) In the presence of analyte, the anti-analyte tagged liposomes form a sandwich complex at the capture zone. Unbound liposomes are captured by a species-specific secondary antibody at the control zone.

Sandwich immunoassays, such as those described above, often use two antibodies that recognize different epitopes on the analyte being quantified. The first antibody (capture antibody) is immobilized in the wells of an assay plate and binds the analyte of interest. A second anti-analyte antibody (detection molecule) binds to the captured... 

Analyses were performed on serum samples containing a mixture of goat [gC, mouse IgG, human IgG. and chicken IgY antibodies (0-125 ng/mL). In this assay, the target analytes were also antibodies (IgG and gY). and so the capture and detection antibodies were antibodies toward antibodies (anti-IgG aqd anti-lgY). Analyte solutions were added to the sensor well, followed by a mixture of detection antibodies. To perform the electrochemical measurements, the biosensor was connected to a multichannel computer-controlled po-tentiostai via the edge contacts of the array, and current measurements were made on all eight working electrodes. 

In the sandwich immunoassay, the analyte antibody (anti-biotin IgG in the example shown in Figure 25-27) is brought into contact with the working electrode where it binds specifically with the biotin on the surface of the dendrimer. Alkaline phosphatase-conjugated IgG is then added, and it binds specifically with the analyte. The working electrode is then immersed in a buffer containing 1 mM p-aminophenylphosphate (p-APP) as shown in the figure. This species is converted by alkaline phosphatase to p-aminophenol (p-AP), which then diffuses to the surface of the dendrimer. Ferrocene (Fc) on the surface of the dendrimer... 

Immunosorbents have also found applicability in on-line SPE analysis. An antibody is immobilized on to a silica support and used as an affinity ligand to retain targeted analytes. Components not recognized by the antibody are not retained and some degree of selectivity is attained. Recoveries of 87-103% were obtained for atrazine, simazine, DEA, propazine, and terbuthylazine at the 0.2 xgL concentration level when using immunosorbent SPE (80 mg silica and 2 mg anti-atrazine and anti-chlortoluron antibodies) on-line with LC/APcI-MS however, this method is not applicable to DIA (0% recovery). This compound may be better retained when using an... 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Figure 1 Schematic of an enzyme immunoassay. (1, 2) The test solution and enzyme conjugate are added to a tube or well pre-coated with anti-anal) e antibodies. (3) After the inhibition step, the solid phase is washed, and only antibody-bound material is retained. (4A-C) Colorless substrate is added and is converted to a visible color in inverse proportion to the amount of analyte in the sample...

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. 

---