Antibody Dilution Matrix

The Antibody Dilution Matrix (after Hoffman et al 2008) is needed to determine the correct dilution of antibodies. For a different detection method, the matrix is the same, but the dilutions of the H antibody and other reagents may be a different antibody Dilution Matrix has different concentration ranges for different detection systems. To test the antibody Dilution Matrix with indirect fluorescent immunocy-tochemistry, run a preliminary experiment that uses eight samples. The results of this preliminary experiment will show the dilutions for both the D antibody and the 2° antibody. This one experiment will save so much time that its importance cannot overemphasized. 

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Table 10.2 Antibody dilution matrix Antibody dilution matrix...

F) Incubate 1° antibody - In this example experiment, the anti-Ag A 1° antibody has not been used in the lab and therefore its dilutions had not been determined. Using the Antibody Dilution Matrix described in this chapter, the conditions for this 1° antibody were found to be mouse anti-Ag A, the dilution is 1 500 and... 

Antibody Dilution Matrix - a method to determine the optimal dilution for both the 1° antibody and the 2° antibody... 

In the second category, 1° antibody, antigens are listed in separate columns so that all of the reagents associated with the E antibody can be seen. In this example, the 1° antibody to antigen Ag A is made in mouse. The dilutions of the 1° antibody for anti-Ag-A is not known, as this antibody has not be used previously, so the dilution will be determined by the Dilution Matrix when the procedure is developed in Chapter 10 (Single Antibody Procedure). 

In planning experiments, it is important to look at the entire experiment and know that all of the reagents and conditions have been considered. Table 10.1 is the Experimental Design Chart for a single antibody experiment. The most common use for single antibody experiments is to test a new 1° antibody and to perform the Dilution Matrix described later. Before planning the details needed to perform each of the steps in the experiment, this chart must be completed. 

Plan to add controls that confirm successful blocking steps between two sets of antibodies (Table 12.2). Because of the sequential addition of antibodies, the controls are different from other experiments with the indirect method of immunocyto-chemistry. The first 1° antibody is not eliminated because there are no competing antibodies for the first 1° antibody. Also, the no 1° antibody control for the first 1° antibody was done previously when the Dilution Matrix showed it was bound specifically by the 2° antibody. The controls here test the potential binding of the second 1° antibody and second 2° antibody to the first set of antibodies. 

The 2° antibody as found in the Dilution Matrix is goat anti-mouse 488 fluorophore dilution is 1 1000... 

Dilution of 1° and 2° antibody incorrect - The dilutions of the 1° and 2° antibodies had not been tested rather dilutions were selected from those recommended. To investigate the antibody dilutions, a Dilution Matrix was done, and the results are shown in Fig. 14.2. These results show that at the dilutions... 

Fig. 14.2 Case No. 1 solution. A dilution matrix experiment with the rabbit anti-ribosomal protein antibody, (a) With 1° antibody 1 100 and 2° antibody at 1 100, specific labeling was seen with high background, (b) With 1° antibody 1 1000 and 2° antibody at 1 100, no specific labeling was found, (c) With no 1° antibody and 2° antibody at 1 100, no specific labeling was found, (d) With 1° antibody 1 100 and 2° antibody at 1 1000, slight specific labeling was seen, (e) With 1° antibody 1 1000 and 2° antibody at 1 1000, no specific labeling was seen, (f) With no 1° antibody and 2° antibody at 1 1000, no specific labeling was seen...

Dilution of 1° and 2° antibody - A Dilution Matrix was performed previously and so the dilutions of the 1° and 2° antibodies were correct. 

Dilution of the D antibodies is incorrect - The Dilution Matrix had been run and the dilutions of the D and 2° antibodies were correct. 

A monoclonal antibody-based ELISA has been utilized to determine ceftiofur levels in milk. The authors noted that matrix interference occurred, but a 1 100 dilution lowered the interference, and a 1 1000 dilution eliminated the matrix interference. Because of the high dilution, samples could not be measured below l.Opgkg The assay measured ceftiofur, its major metabolite desfuroylceftiofur, and ceftiofur protein conjugates and has been utilized to measure residues in milk from cows treated with therapeutic doses of the drug. The results from the incurred residue correlated well with a previous study using radiolabeled ceftiofur, confirming the detection of a metabolite that was not detected by HPLC. 

Strong crossreactivity to chlortetracycline has been also observed when a commercialized kit (70) was applied to analyze tetracycline, chlortetracycline, and oxytetracycline residues in honey (71). The detection limit for both tetracycline and chlortetracycline was at 15 ppb, but for oxytetracycline at 250 ppb due to the low crossreactivity of the used antibodies to this analyte. Experiments using honey free of tetracyclines showed that dilution of honey with buffer at a ratio of a 1 50 was sufficient to eliminate matrix interferences. 

Halofuginone can be also analyzed in chicken serum by a competitive ELISA developed on the basis of monoclonal antibodies (99). In this study, a serum matrix effect that afforded a higher sensitivity for the detection of halofuginone in chicken serum than in assay buffer or in highly diluted serum was observed. The sensitivity of the ELISA improved when used in more concentrated serum. 

The diligent analyst would develop a robust method with rigorous matrix effect tests on multiple lots, including hemolyzed and lipidemic samples. An initial test would be a spike-recovery evaluation on at least six individual lots. Samples should be spiked at or near the LLOQ, and at a high level near the ULOQ. If matrix interference were indicated by unacceptable relative error (RE) percentage in certain lots, the spiked sample of the unacceptable lots should be diluted with the standard calibrator matrix to estimate the minimum dilution requirement (MDR) at and above which the spike-recovery is acceptable. The spike-recovery test should then be repeated with the test samples diluted at the MDR. Note that this approach will increase the LLOQ for a less sensitive assay. If sensitivity is an issue, then other venues will be required to address the matrix effect problem. For example, the method can be modified to include sample clean-up, antibodies and/or assay conditions may be changed, or the study purpose may be tolerable to acknowledge that the method may not be selective for a few patients (whose data may require special interpretation). 

Matrix effects lead to a decrease in binding of analyte to antibody resulting in reduced sensitivity and falsely increased or decreased concentration depending on the assay principle. The matrix effects can be reduced or eliminated by diluting the sample. 

Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) Mass spectra of native and denatured antibodies were obtained with a PerSeptive Biosystems (Farmingham, MA.) Voyager Elite mass spectrometer operated in the linear mode with a Laser Sciences Inc., 337 nm nitrogen laser. hAB-1 was denatured by boiling the sample in 1.0 M guanidine-HCl, 50 mM Tris pH 7.5 buffer. Native and denatured samples were diluted with 20 mM Tris, 10 mM octylglucoside (Tris/OG) pH 6.8 buffer prior to MALDl-MS analysis. Proteins were spotted on the sample plate as a sandwich between two layers of the matrix. The bottom layer consisted of 100 mM sinapinic acid in acetonitrile and the top layer consisted of 50 mM sinapinic acid in 30% acetonitrile / 70% H2O / 0.07% TFA. The m/z scale of the instrument was calibrated using a Hewlett-Packard protein standard mixture. 

Qualitative conclusions of the presence or absence of cross-reactants in the sample matrix can easily be drawn by measuring the analytical response at different dilutions of the sample non-parallel curves for the standard and sample demonstrate a lack in structural identity [1]. This simple result is based on the principle that differences in molecular structme are likely to lead to differences in reaction thermodynamics and kinetics, even when identical epitopes are involved in the antibody-antigen reaction. Such differences are generally manifested as differences in the apparent affinity constants governing the reactions, which can be revealed as alterations of assay characteristics [1,16]. 

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