ELISA validation

For lasalocid assay, polyclonal antibodies were raised in sheep (98). These antisera were applied in an ELISA validated for chicken serum, liver, and muscle. Bridge homology in the ELISA was overcome by absorbing unspecific antisera onto a conjugate between salinomycin and chicken serum albumin, which was immobilized onto Biosilon beads. The assay was highly specific for lasalocid and was capable of detecting it at concentrations less than 0.15 ppb. 

In September 2010, CAA annoimced the addition of ALLERGENEYE ELISA series of kits for egg, milk, wheat, buckwheat, and peanut as Japanese official methods based on their validation determined by the Japanese validation protocol. 

Since the MHLW designated shrimp/prawn and crab for mandatory labeling in June 2008 due to the almost unlimited use of crustacean in the processed foods in Japan and the status as a frequent cause of adverse food reactions in allergic patients, two ELISA methods for the determination of crustacean protein in processed foods have been developed (Seiki et al, 2007 Shibahara et al., 2007) EA test EIA-Crustacean [Nissui] produced by Nissui Pharmaceutical Co., Ltd. and Crustacean Kit [Maruha ] produced by Maruha Nichiro Eoods, Inc. Both kits have been validated according to the Japanese validation protocol (Sakai et al., 2008) and are commercially available. All the commercial ELISA kits are shown in Table 4.9. 

Other attempts have been made to detect BPA at a low concentration range. Thus Kodaira et al. [274] analyzed BPA in urine samples with an assay that showed a working range between 0.5 and 5 pg L The assay was validated by HPLC. DeMeulenaer et al. [275] developed an indirect competitive ELISA using PAbs obtained from chicken egg yolk, but the assay achieved an IC50 value of only 570 pg L-1. 

Validation of ELISAs for the detection and quantification of gums in foodstuffs. 

Finally, there are custom two-step quantitation methods such as chromatography or ELISA that require a capture step for isolating the protein and then a quantitation step based on a standard curve of the purified target protein. The preliminary capture step may also concentrate the protein for increased sensitivity. These techniques are typically not available in a commercial kit form and may require extensive method development. They are more labor intensive and complex than the colorimetric or absorbance-based assays. In addition, recovery of the protein from and reproducibility of the capture step complicate validation. Despite these disadvantages, the custom two-step quantitation methods are essential in situations requiring protein specificity. 

ELISAs can be used for identification and quantitation of a biopharmaceutical product or for quantitation of impurities or contaminants as discussed previously. They can be used throughout the manufacturing process as well as in quality control or the product release stage just as they are used in all the other stages of product development. To be used for quality control, GMP practices must be followed. All methods need to be validated so that the assay s performance is documented. ELISAs should have internal quality controls to monitor assay... 

ELISAs can be used for identification and quantitation of the product as well as impurities in the various purification steps (as discussed previously). They can be used to document the removal of known impurities and contaminants, and in process validation to demonstrate batch-to-batch consistency of manufacturing. 

In general, the strengths of the ELISA are its selectivity and specificity, whereas its weaknesses are related to precision of measurement. Each assay varies — depending on the antibody, enzyme, enzyme conjugate, and measurement, as well as on assay format. Each should be validated so that its unique performance characteristics are known. 

Because of antibody-based selectivity, ELISAs are capable of handling samples that are impure or only semipurihed. It is possible to perform ELISAs in a variety of matrices. This is in contrast to other methods such as HPLC that require relatively pure material. During the development and validation of the ELISA method, it needs to be demonstrated that the ELISA is not affected by interfering substances that could be in the test sample, such as buffers, salts, contaminating proteins, and excipients. It also needs to be demonstrated that the conjugated antibody does not bind nonspecihcally to the coated solid phase. 

Assays Validated methods of analysis (e.g., ELISA for MAb), QPCR for residual DNA, and potency assays for vaccines... 

Two important observahons were noted. First, no single microarray substrate satisfied all performance criteria for all antibodies. Second, in other work, certain antibodies worked well in conventional ELISA but not in microarray format. Thus, results from ELISA do not necessarily qualify an antibody for use on microarrays. If is besf to validate each antibody for use on the intended substrate in a microarray-based assay. 

Currently there are few methods for specific investigation of immunotoxic effects, which are regarded as sufficiently validated for routine use (EC 2003). The plaque forming assay or the equivalent using the ELISA method (Enzyme-linked Immunosorbent Assay) are recommended to identify altered T-cell-dependent humoral responses. Of particular value for hazard assessment are the so-called host resistance models, in which the clinical relevance of immunotoxicity can be evaluated. Other methods may also be of value to provide information on the mode of immunotoxic action, e.g., mitogen stimulation tests and leucocyte phenotyping. However, further work is needed on standardization and validation of these test methods. 

Sehr P, Zumbach K, Pawlita M. (2001) A generic capture ELISA for recombinant proteins fused to glutathione S-transferase validation for HPV serology. J Immunol Methods 253, 153-62. 

The other approach is based largely on informatics. In such an approach, tumors would be profiled in contrast to normal tissue. Tumor-specific markers would be identified via microarrays, as has been done in many publications already. From here, the list of tumor-specific markers would be analyzed to determine if any of these markers represented proteins which were likely to be secreted out of the cell and which may be detected in the peripheral blood stream. Preferably multiple markers would be identified that could be tested using multiplex ELISA assays (antibody arrays). Such work will take time, however, because once the potential markers are identified, antibodies must be generated, validated, and tested for effectiveness as an early diagnostic tool. Such work is being done, but little has been published so far. 

JA Itak, EG Olson, JR Flee-Ker, DP Herzog. Validation of a paramagnetic particle-based ELISA for the quantitative determination of carbaryl in water. Bull Environ Contain Toxicol 51 260-267, 1993. 

Guidance for ELISA, HPLC, GC, Validated assay Commercial... 

Pharmacotoxic and pharmacokinetic studies carried out for the new antitumor drug Aviscumine (rViscumin) were supported by a robust quantitative IPCR assay developed by Adler et al. [66, 85], The potency of this protein-based drug, derived from recombinant mistletoe lectine, required initial doses well below the quantification range accessible by conventional ELISA. An IPCR assay was adapted and validated for the quantification of rViscumin in standardized human serum [66, 85, 86] and subsequently... 

Meulenberg, E.P., L.G. de Vree, and J. Dogterom (1999). Investigation of indicative methods in the Netherlands Validation of several commercial ELISAs for pesticides. Anal. Chim.Acta, 399(1/2) 143-149. 

Development and Validation of an ELISA Method for an Antibody Drug... 

Drug X is an Ab against a target macromolecule Y . An ELISA method was developed and validated for the determination of X in human and monkey plasma. Since the purposes of these methods were to support preclinical and clinical PK studies, the method validation and sample assays were conducted under an in-house SOP, which is GLP-compliant with IA considerations according to De-Silva et al. [10]. 

Enfuvirtide (Fuzeon , T-20, Ro 29-9800, Hoffmann-La Roche) is a 36-amino acid synthetic peptide with a molecular weight of 4492 Da. It selectively inhibits human immunodeficiency virus (HIV) fusion to the host cell membranes [73]. The N-terminus of the molecule is acetylated and the C-terminus is amidated. A metabolite, M-20, is deamidated at the C-terminus. An ELISA method was initially used during drug development of this compound, but the decision was made to develop and validate an LC-ESI-MS/MS method for the simultaneous determination of enfuvirtide and M-20 for PK studies to support the NDA submission of this product [53]. Some of the issues of LC-ESI-MS/MS application for peptide bioanalysis are highlighted in the following. 

Cetuximab serum concentrations were measured using validated enzyme-linked immunosorbent assay (ELISA) methods or a validated surface plasmon resonance assay. These bioanalytical assays were crossvalidated to allow pooling of PK data across studies. 

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