Preparation Of Immunoaffinity Column Research Articles

Preparation of an immunoaffinity column and its application in sample cleanup for methandrostenolone residues detection

Methandrostenolone (MA) is a steroid used as veterinary medicine on stockbreeding to promote animal growth. The use of MA has been strictly regulated because of its harmful effect on consumers. This paper describes the production of polyclonal antibody (pAb) against MA, the preparation of immunoaffinity column (IAC) and its potential application to the selective extraction of MA residues from animal tissue and feed samples. The produced pAb exhibited good sensitivity to MA with an IC 50 value of 5.6 ng/mL. The cross-reactivity values of the antibody with MA structurally related compounds of testosterone propionate (TP) and trenbolone (TR) were lower than 0.6%. By coupling the produced antibody with CNBr-activated Sepharose 4B, an IAC was prepared. 2% methanol and 80% methanol were selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was approximately 334 ng/mL gel. The average recovery of 20, 40 and 60 ng/mL MA standard solutions from IACs was 97.9% with the relative standard deviation (RSD) among columns of 6.7%. After 3 times of repeated usage, the column capacity and recovery rate still remained 82.0% and 92.6% respectively. The IACs were then challenged with MA-fortified animal tissue and feed samples, recoveries of MA were found to be in the range of 83.5–99.7%.

Preparation of immunoaffinity column of zearalenone

To prepare immunoaffinity column of zearalenone. The zearalenone immunoaffinity column (IAC) was prepared by coupling CNBr-activated Sepharose 4 Fast Flow (4FF) with the anti-zearalenone monoclonal antibody which was purified by caprylic acid-ammonium sulfate method. The coupling reaction was identified by UV-absorbance measurements, and the IAC prepared was evaluated by indirect-competition ELISA and HPLC. The column capacity was determined to be 0.40 microg when using 0.5 ml of CNBr activated Sepharose 4FF and 350 microg of purified anti-zearalenone monoclonal antibody. The mean true recoveries were in the range 76.33% - 90.10% and RSD was 6.68% - 10.93% at levels of 60 microg/kg - 300 microg/kg. 30 samples of wheat and maize were detected by the anti-ZEN IAC produced by the laboratory, 17 samples were observed to be contaminated in a comparable range from 31.33 microg/kg - 377.84 microg/kg. Detection limit based on a signal-to-noise ratio 3:1 was 10. 00 microg/kg for ZEN in wheat and maize. IAC, a simple separating method which is used in ZEN extraction from cereals, is able to purify and condense ZEN in one step. The cost of detection can be lowered down because the IAC developed is hopefully to substitute the imported IAC.

IgG purification.

AbstractSeveral immunological procedures can be successfully carried out using nonpurified antibodies, such as unfractionated antisera or ascitic fluid/culture supernatant containing monoclonal antibodies (MAbs). However, a much “cleaner” result can often be obtained if some form of enrichment or isolation of immunoglobulin (Ig) is employed. Some procedures, such as conjugation with isotopes, fluorochromes, or enzymes, and preparation of immunoaffinity columns cannot usually be efficiently performed with nonpurified immunoglobulin; other procedures may yield artifactual results if whole antiserum or ascitic fluid is used as a source of antibody. Purification of immunoglobulin is therefore at least useful and sometimes essential for a range of immunological methods. This process may consist of purification of total IgG or subpopulations (e.g., subclasses) of IgG from antisera/ascitic fluid/culture supernatant or the isolation of a particular antigen-binding fraction of Ig from such fluids. The former can be achieved by biochemical procedures, whereas the latter usually requires some type of affinity purification.KeywordsAffinity ChromatographyColumn VolumeAmmonium Sulfate PrecipitationFume HoodCaprylic AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.