Anti-rabbit IgG

Figure 2. Binding of [ IJpalytoxin to anti-palytoxin (Rabbit 633) before ( ) and after primary (o), first boost (A), second boost (A), and third boost ( , Rabbit 633D-24) of immunization and after absorption of IgG in Rabbit 633D-24 with goat anti-rabbit IgG ( ).

Incubate with goat anti-rabbit IgG/10 nm colloidal gold without dilution for 5 min. 

An example of an evanescent wave fiber optic immunoassay and the associated optics has been described in detail for measurement of anti-rabbit IgG.(130) Rabbit antibody is immobilized on the distal face of an optical fiber. Unlabeled anti-rabbit IgG competes with fluorescein-labeled anti-rabbit IgG for rabbit antibody binding sites... 

L/mole). It cross-reacts at >90% with saxitoxin but at <1% with neosaxitoxin. This antibody, when used in an anti-rabbit IgG "second antibody" radioimmunoassay format, can detect pmole quantities of saxitoxin. This assay has been shown to be a simple and efficient method for the analysis of saxitoxin in clam extracts. The lack of antibody cross-reactivity to the neosaxitoxin sub-group of the paralytic shellfish poisons limits the general utility of the assay to neurophysiology studies and to certain clam species which preferentially accumulate saxitoxin. However, the radioimmunoassay serves as a good precursor in the development of an enzyme immunoassay for the paralytic shellfish poisons. 

Step 4. Add anti-STXOL serum (1 ul) and incubate for 30 minutes. Step 5. Add 60-120 ul of second antibody (anti-rabbit IgG Sigma Chemical Co.). The exact quantity must be optimized. Incubate for 60 minutes. 

Dilute the respective antiserum, e.g., goat anti-(rabbit IgG) antiserum, to about 10 mg/ml with Soln. A and centrifuge or filter through a 0.45-pm membrane filter. 

After being washed with TBST for 5 min four times, the membrane was further incubated with 1 5,000 horseradish-peroxidase-conjugated anti-rabbit IgG antibody (Promega) in TBST containing 1% skim milk for 60 min. 

Fig. 5. (a) Layout of a slide that was spotted and immobilized using the anti goat IgG rabbit antibody and the anti rabbit IgG goat-antibody, (b) Fluorescent image after incubation of a Cy5-labeled goat IgG. (c) Fluorescent image after incubation of a Cy5-labeled rabbit IgG. 

In 1982, the first enzyme immunoassay of clenbuterol was described (134). It was used to determine clenbuterol levels in plasma of human patients treated by oral route with this drug. It was a highly sensitive double-antibody and heterologous immunoassay based on a competition for binding to a clenbuterol-specific antibody between a diazotized clenbuterol analogue labeled with -galactosidase and unlabeled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. The assay could detect levels as low as 0.5 pg clenbuterol per tube. 

Anti-rabbit IgG (whole molecule)-biotin conjugate (Sigma-Aldrich, B-7389). 

Remove the supernatant and resuspend the cells in 10 pL PBS containing antimouse or anti-rabbit IgG biotin conjugate as required (1 10 dilution). Leave on ice for 10 min and wash the cells by addition of 180 pL PBS and centrifugation. 

Fig. 13.1 Cytoprotective action of adenosine in a quantitative model of mouse hindlimb ischemia and reperfusion (I/R) injury. Adult wild type mice were injected with (a) sterile vehicle (0.1% DMSO in phosphate-buffered saline, pH 7.4) or various adenosine receptor agonists (b, c) or antagonist (d, f). (a) Following ischemia and reperfusion, skeletal muscle showed a significant uptake of Evans Blue dye (EBD) in vehicle-treated mice (first part of (a), representative of 7 mice). The contra-lateral leg not subjected to ischemia-reperfusion showed virtually no EBD uptake. In the second part of (a), the same section was stained with rabbit polyclonal anti-skeletal muscle actin antibodies followed by staining with goat anti-rabbit IgG conjugated with FITC. (b) The nonselective adenosine agonist R-PIA caused a large reduction in the EBD-stained area (see both first and second parts to this figure, representative of six mice)...

Cholera toxin B subunit-biotin labeled (lyophilized powder, biotin content 0.9mol/mol protein), peroxidase-labeled IgG anti-rabbit antibody (HRP-Ab, from goat, protein content 0.8mg/ml, affinity isolated antibody), anti-cholera toxin (from rabbit, protein content 48mg/ml, purified toxin from Vibrio cholerae), biotin monoclonal anti-rabbit IgG -y-chain specific (from mouse, protein content 4.2mg/ml), glucose oxidase-biotinamidocaproyl labeled (GOX-B, from Aspergillus niger, lyophilized powder containing 40-70% protein, 137 U/mg), polyoxyeth-ylene-sorbitan monolaurate (Tween 20), bovine serum albumin (fraction... 

Incubation of biotin labeled monoclonal anti-rabbit IgG (0.5mg/ml) suspended in buffer (1% (w/v) BSA/PBST) for 20 min. 

A competitive ELISA was developed to detect stem peptide generated from the coupled enzyme reaction as well as to measure the cross-reactivity of the antibody with small peptides. The ELISA format is pictured in Figure 4. BSA-pentapeptide (BSA-PP) was immobilized onto the wall of a microtiter plate. Samples or standards containing pentapeptide were then added, followed by rabbit anti-pentapeptide antibody. The amount of anti-pentapeptide antibody bound to immobilized BSA-PP was visualized by adding a second anti-rabbit IgG antibody... 

FIGURE 5.18 Illustration of the flow confinement concept. In microchannels, a sample flow is joined with a confinement flow (e.g., water or sample medium) in a perpendicular orientation. Under laminar flow conditions, no mixing occurs. The sample flow is confined into a thin layer of higher velocity. For immunoassay application, rabbit IgG is immobilized on a planar waveguide and Cy5-labeled anti-rabbit IgG is introduced as analyte in the sample flow [694]. Reprinted with permission from the American Chemical Society. 

In the on-chip heterogeneous immunoassay for human carcinoembry-onic antigen (CEA) in serum, three antibodies (mouse anti-human CEA, rabbit anti-human CEA, and anti-rabbit IgG-colloidal gold) were used, (a) What is the purpose of colloidal gold (b) Why isn t the colloidal gold directly attached to rabbit anti-human CEA to save a step [1021] (3 marks)... 

With the application of protein microarrays in mind, Spencer and coworkers immobilized poly(lysine) with grafted PEG side chains on various metal or semiconductor oxide surfaces via electrostatic adsorption [200], Part of the PEG side chain was functionalized with biotin at the distal end. Streptavidin was bound to the surface-tethered biotin in a subsequent step, and the remaining unoccupied binding pockets of streptavidin were then used to immobilize biotinylated capture antibodies. As an example of an immunoassay, biotinylated goat anti-rabbit IgG was immobilized, which then specifically bound rabbit IgG. 

In immunoautoradiographs of rat brain sections exposed to these antibodies and then to [35S]IgG anti-rabbit IgG, the strongest immunolabeling was in the... 

An even more indirect method which does not require the use of a fluorescence microscope is to use enzyme linked antibodies, e.g. the peroxidase anti-peroxidase (PAP) method of Stemberger et al. (1970). Here, after the antigen has been reacted with a rabbit antibody preparation this is conjugated to sheep anti-rabbit IgG which in turn is conjugated to a complex of rabbit anti-horse-radish peroxidase and horseradish peroxidase. This then reacts with diaminobenzidine and hydrogen peroxide when a brown colour indicates the presence of the antigen. 

Snitkoff et al. reported a specific immunoassay for monitoring the levels of ciprofloxacin in human samples [63], Serum was incubated at 37°C with shaking for 2 h, with rabbit anti-ciprofloxacin keyhole limpet haemocyanin primary antibody in microtiter wells coated with cipro-floxacin-BSA conjugate. Further incubation with horseradish peroxidase-labeled goat anti-rabbit IgG was carried out. The samples were shaken in 0.1 M citric buffer (pH 4.5), plates were incubated for 10 min, and then read at 450 nm. Calibration graphs were linear for 10 pg/mL to 10 ng/mL of ciprofloxacin, and the within-day RSD was less than 10%. 

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