Antibody performance

For microarray work, PLL-PEG-biotin-sAV monolayers adsorbed onto underlying titanium dioxide surfaces (pillars) replaced the b-SAM gold-coated glass surface. While oriented capture agents out-performed their random counterparts, distinct differences in Fab and antibody performance between this new surface and the b-SAM surface were apparent. 

The determination of antibody affinity (in M 1) is useful for comparison with other reported antibodies. However, the important test of affinity is how well the antibody performs. If more than one antibody has been prepared, a direct comparison of antigen binding curves will indicate the most avid preparation. 

If you have to use secondary antibodies, perform all further steps including washing and centrifugation at 2°C, to prevent recycling of the receptor. 

Appropriate extraction protocols and sample preparation procedures must be well-documented by the developer and presented in a manner clearly understood by someone unfamiliar with the method. The effects of extraction and sample preparation on antibody performance must be determined. Specific areas such as pH, organic solvents, and ionic strength should also be addressed. 

The majority of keratin immunostaining is performed on FFPE tissues. The duration of formalin fixation is a key factor when trying to optimize the technical performance of keratin immunoperoxidase stains. The fixation time is closely related to the time required for enzymatic predigestion.212 Generally, tissue fixed in 10% formalin for more than 2 days requires greater antigen retrieval, with less time required for tissues fixed briefly (hours) in 10% formalin. Most, if not all, keratin antibodies require epitope retrieval (depending on antibody and fixation duration) for optimal keratin antibody performance. 

KS Joshi, JE Butler. The immunochemistry of sandwich ELISAs. V. The capture antibody performance of polyclonal antibody-enriched fractions prepared by various methods. Mol Immunol 29 971, 1992. 

Whole serum can be used in preliminary evaluation of antibody performance, but the final lateral flow assay should be developed with purified antibody. Affinity chromatography with protein A or G can purify total antibodies from the serum, but purification on immobilized antigen is preferred, since it isolates only analyte-specific antibodies (2). Using only antibodies specific to the target helps minimize background and increase assay sensitivity. 

In each collected fraction, measure protein concentration by absorbance at 280nm and antibody performance by microplate ELISA. If necessary, make serial dilutions of the fractions. 

Antibody performance should be tested at all stages of lateral flow assay development, starting with the animal bleeds. ELISA of unfractionated antisera can help eliminate poorly performing antibodies and save time in assay development. The main goal of antibody testing is to evaluate potential assay sensitivity and specificity. 

Depending on the secondary antibody, perform either of the following steps ... 

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