Antibody concentrations

The concentration of antibody in tissue cultures of the hybridoma is low (10-60"gml- ) but the use of large culture vessels can obviate this. The hybridoma can also be propagated in mice where the antibody concentration in the serum and other body fluids can reach lOrngml. ... 

CV measurements showed that the reversible eleetrode reaetion of the [Fe(CN)6]" redox eouple was suppressed to some extent by the treatment with the DNA. The addition of the anti-DNA antibody further suppressed the redox reaetion thus decreasing the magnitudes of the CV peak currents. This is most likely caused by a steric hindrance of the bulky protein, which binds to the DNA double strands on the electrode surface, to mainly reduce the effective area of the electrode. The electrostatic repulsive effect may also contribute to the electrode response, since the isoelectric point of mouse IgM is commonly in the range of 4.5 to 7.0. Figure 11 shows the relationship between the decrease in the anodic peak current (A/p ) and the antibody concentration. As seen in this figure, the electrode system responded to the anti-DNA antibody in the concentration range of 1 — 100 nM. For the case of the mouse IgM, which does not interact with double-stranded DNA, the present system gave almost no response. The sensor did not respond to other serum proteins as well (data not shown). 

Figure 17.6 Dialyzed Chemostat Monoclonal antibody concentration (raw and smoothed measurements) during initial batch start-up and subsequent dialyzed continuous operation with a dialysis flow rate of 5 L/d. [reprinted from the Journal of Biotechnology Bioengineering with permission from J. Wiley],...

All steps should be performed at room temperature, optimally on a gently moving rotary shaker. If the cells are very delicate (e.g., neurons) and cannot withstand gentle shaking, incubations may be performed without shaking, but times or antibody concentrations may need to be increased. 

Figure 7.7 Peptide spot color intensity as a function of doubling dilutions of primary (PR) antibody. PR peptides and PR+ tissue sections were both placed on the same slides and stained with various dilutions of the PR mAb. Color intensity of the peptide spots (square symbols) or tumor cells (triangle symbols) was measured and plotted on the y-axis. The figure shows a linear decline in intensity with decreasing antibody concentrations for both the peptide spots and the tissue sections. Tissue color intensity is measured as optical density on a 0-2 scale. Peptide spot color is measured as mean pixel intensity on a 1-256 scale. Copied with permission from Sompuram et al.6...

Add TCEP to a final concentration of 20 mM. For partial reduction of antibody disulfides in the hinge region while maintaining a biospecific IgG molecule, add TCEP in a 2.75-fold molar excess over that of the antibody concentration. 

Dissolve the protein or antibody to be conjugated in 0.1 M sodium phosphate, 0.15M NaCl, pH 7.4. The antibody solution should be as concentrated as possible, given the amount of antibody available for modification. This general protocol will work for protein or antibody concentrations ranging from about 0.5mg/ml to lOmg/ml, but an increase in the molar excess of either SANH or SFB may have to be done at lower antibody concentrations to provide the same modification yields obtained at higher concentrations. 

Add a quantity of the crosslinker solution of choice (SANH or SFB) to the antibody solution to obtain the desired molar excess of reagent over the antibody. Typically, antibody modification procedures are done with 10- to 20-fold molar excess, but for dilute antibody concentrations, this may have to be doubled, depending on how many hydrazine or aldehyde groups are desired to be introduced on the modified antibody. 

Add 10—40 ul of the SATA stock solution per ml of lmg/ml antibody solution. This will result in a molar excess of approximately 12- to 50-fold of SATA over the antibody concentration (for an initial antibody concentration of lmg/ml). A 12-fold molar excess works well, but higher levels of SATA incorporation will potentially result in more maleimide-activated enzyme molecules able to couple to each thiolated antibody molecule. For higher concentrations of antibody in the reaction medium, proportionally increase the amount of SATA addition however do not exceed 10 percent DMF in the aqueous reaction medium. 

Pool the fractions containing protein. Adjust the antibody concentration to lOmg/ml for the conjugation step. The oxidized antibody should be used immediately. 

Supplementation of Gamborg s B5 medium with 0.1% KN03 (in addition to the 0.25% KN03 usually present in B5 medium) resulted in a significant increase in antibody levels in tobacco hairy root cultures [19]. The antibody concentration in the medium increased 2.8-fold and the total antibody accumulation increased by 90% relative to cultures without added nitrate. 

Biomolecules like antibodies attach to surfaces via a variety of mechanisms. This attachment phenomenon is controlled by the chemical properties of the surface, but can be influenced by factors such as pH and temperature. In the case of antibody coating to a solid support the use of so-called medium-binding plates is to be recommended. Coating conditions can be optimized by performing a checkerboard titration (in the following example the optimal coating antibody concentration is determined) ... 

A. The antigen is serially diluted down the plate (optimized with respect to antibody concentration as described in Sec. 1.3.1.), allowed to react with the immobilized antibody, and excess is washed away (PBS containing 0.1% Tween 20, PBST). 

Optimization of the immunoassay was performed with respect to tracer and antibody concentrations to obtain the required sensitivity. These conditions differed depending on the detection system used photographic detection required higher antibody and tracer concentrations than when the plate luminometer was used. A further complication arose from the very low affinity of the tracer for the antibody when using an antibody dilution of 1 3000 and a tracer dilution of 1 4000 less than 1% of the tracer was bound after a 2-h incubation. This means that the antibody, in the absence of clenbuterol, binds less than 10 pg of the... 

The optimal working concentration can be determined in a series of stains with the antibody in several dilutions of twofold increments (e.g., 1 25, 1 50, 1 100, 1 200, 1 400, 1 800, 1 1600). The resulting antibody concentration can be calculated according to the following formula. 

Antibody concentrations and affinities vary considerably. The optimal dilution for a given primary antibody must be determined empirically (although most companies will give an indicative range of dilutions). In general, early bleed serum or tissue culture supernatant are used at 1 100-1 1,000 dilution, and ascites fluid or serum from hyperimmunized animals at 1 1,000-1 100,000 dilution. Secondary antibodies are used at dilutions ranging from 1 2,000 to 1 10,000... 

The corrected peak area was plotted against antibody concentration for each component, and the results are shown in Figure 10. The % corrected peak areas for each component (HC, LC, and non-main) are linear over a wide protein concentration range, with coefficient of determination (R ) values greater than 0.99. 

Protein sample concentration can have a significant impact on cIEF peak resolution. Figure 18 shows the results of an experiment in which the antibody concentration was varied... 

Atopy is inborn and characterized by genetic markers and increased IgE-antibody concentration. The increased concentration must be accompanied by clinical symptoms of a disease. If only the level of IgE is exceeded, it is not a sign of a disease, however it may forecast developing of the allergy in the future. 

Since ammonium sulfate fractionation will also cause precipitation of other proteins, antibody concentrations obtained from absorbance measurements at 280 nm are only estimates. Alternatively, a sample of the dialyzed solution can be resolved on a SDS-polyacrylamide gel alongside a series of known concentrations of IgG. Staining the gel with Coomassie blue can then be used to estimate the amount of immunoglobulin obtained and can also give an estimate of purity. 

Antibody concentration should be determined by titration of the stock solution and testing on a known positive specimen. Usually, working concentrations are in the range of 10-20 pg/mL. However, depending on the source this concentration could vary significantly. For detailed instructions on titrating antibodies, see Chapter 24. 

Figure 17.11 Secondary response to an antigen challenge. Note the antibody concentration is a logarithm scale, so that the increase in concentration of antibody is considerably greater than would appear in the diagram.

---