I am trying to detect Avian leukosis virus from egg albumin by ELISA (Cat#CK422B, BioChek, the UK). I am getting OD of positive control - OD of negative control below 0.50, which should be more than 0.50 for test should be valid. What may be the causes? Thanks in advance.
Kais Khudhair al Hadrawi
Low sensitivity: ELISA assays may not be sensitive enough to detect low levels of ALV present in egg albumin, especially if the virus is present at low concentrations. Presence of inhibitors: Egg albumin contains various proteins and substances that can interfere with the ELISA assay, leading to false-positive or false-negative results. These inhibitors may affect the binding of antibodies to the target antigen. Cross-reactivity: There may be cross-reactivity with other proteins or substances present in egg albumin, leading to nonspecific binding and false-positive results. Sample preparation: Proper sample preparation is crucial for ELISA assays. Improper handling or preparation of egg albumin samples may lead to degradation of the virus or interference with the assay. Optimization of ELISA protocol: Optimization of the ELISA protocol, including antigen retrieval, antibody concentration, incubation times, and washing steps, can improve sensitivity and specificity. Use of specific antibodies: Utilizing highly specific antibodies against ALV can minimize cross-reactivity with other proteins present in egg albumin. Sample dilution or concentration: Adjusting the sample dilution or concentration can help minimize the effects of inhibitors and improve the detection sensitivity of the assay. Validation of results: Validation of ELISA results using alternative detection methods, such as PCR or virus isolation, can confirm the presence of ALV in egg albumin samples. Quality control: Implementing rigorous quality control measures, including positive and negative controls, can help ensure the reliability and reproducibility of ELISA results.
Detecting avian leukosis virus (ALV) from egg albumin using ELISA can be challenging due to several factors:
To overcome these challenges, several strategies can be employed:
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