Antibody titration

A clear demonstration that aldolase is a mandatory catalyst in liver PPP involved the immunochemical evidence of Bleakely et al. (43) who showed the total cessation of hexose 6-P formation when liver aldolase antibody titrated the removal of aldolase from the system where Rib 5-P was reacted with the same rat liver enzyme preparation that established the Fig. 2 scheme. Irrespective of the other contrary data, this evidence alone showed another reaction mechanism involved Aid in liver PPP. The claim that aldolase is an essential enzyme in the PPP was also supported by data of (60) in which, using an in vitro construction of a PPP preparation for the complete oxidation of Glc, noted the formation of Oct-P and the need to include aldolase and sedoheptulose 1,7-bisphosphatase for the construction system to work. 

Antibody too dilute Check antibody titration increase concentration lengthen incubation time increase temperature of reaction check amount of rinsing buffer left on slide... 

Antibody titration records are required for new antibody lots or when new antibodies are introduced into the laboratory. For newly introduced antibodies, this is accomplished by running the assay at various antibody dilutions using a known positive and negative control to determine the appropriate concentration for maximum sensitivity and specificity. Since incubation times, buffers, specimen processing, pretreatment conditions, fixation, and other processing reagents will affect the dilution used, optimal dilutions must be determined by each laboratory under its own special conditions. Parallel testing for new lots of established antibodies to verify optimum antibody titer and controls is usually sufficient. 

Check antibody titrations and dilutions to insure they were done properly. 

Note that the same pretitrated system can be used for both antigen and antibody titration. The respective analytical sensitivities of the systems as adapted... 

Capture antibody titrated is in columns 1X11, and constant antigen A12 to H12, conjugate diluted is in rows A XG. Readings are out of accurate range for reader. 

Figure 38 is a graph relating the antibody titration curves to the IgG concentrations on the wells. From these data we can do the following ... 

Dilute primary antibody in 1% BSA in 0.1% Triton X-IOO/PBS. Rat anti-CXCR4 (1 100) in combination with mouse anti-EEAl (1 1000) or anti-LAMPl (1 1000). EEAl is used as a marker for early endosomes and LAMP2 is used as a marker for late endosomes/lysosomes. Primary antibody titrations should be performed to identify optimal antibody dilutions to use for staining. 

Dilute secondary antibody in 1% BSA in 0.1% Triton X-IOO/PBS. Secondary antibody titration experiments should be performed to identify optimal secondary antibody dilutions to use for immunostaining. 

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... 

Fig. 8.10 Titers of antibodies at day 50 induced by plant-derived CTB-2L21 recombinant protein. Balb/c mice were intraperitoneally immunized with leaf extract from CTB-2L21 transgenic plants. Animals were boosted at days 21 and 35. Each mouse received 20 pg of CTB-2L21 recombinant protein. Individual samples of mouse serum were titrated against 2L21 synthetic peptide,VP2 protein and a control peptide (amino acids 122-135 of hepatitis B virus surface antigen). Titers were expressed as the highest serum dilution to yield twice the absorbance mean of preimmune sera. M1-M6 mice 1 to 6 2L21 epitope from the VP2 protein of the canine parvovirus CTB cholera toxin B VP2 protein of the canine parvovirus that includes the 2L21 epitope.

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) ... 

Livingstone, J.R. 1996. Antibody characterization by isothermal titration calorimetry. Nature 384 491 192. 

Storing and titrating antibodies correctly are often easy solutions when no staining is observed. Some antibodies are extremely sensitive to repeat freeze-thaw cycles and others have a limited shelf life, as evidenced by a short expiry dates, ft is important to check the compatibility of the primary and secondary antibodies before starting IHC, as using the wrong secondary IgG is a common but easily corrected problem. As mentioned, antigen retrieval can be a problem when tissue has been overfixed, so special attention should be paid to fixation time and, once optimized, should be held constant for all subsequent runs. 

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. 

The working concentration of antibody mnst be determined empirically by serial dilation of the stock solution in PBSG with 10% bovine serum albumin. Usual concentrations are in the range of 10-20 pg/mL. Depending on the individual reagent, this could vary considerable. See Chapter 24 for additional instructions on performing titrations. 

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