Efficient hybridoma screening technique using capture antibody based microarrays.

The hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs), particularly if the target of the antibody is a low-molecular weight analyte. This work presents an advanced screening method that makes use of antibody microarrays generated by contact printing of the hybridoma cell supernatant samples on glass chips initially coated with capture antibodies. The noncompetitive immunoassay is based on the specific binding of an analyte-horseradish peroxidase conjugate and is performed in an automated fashion using a chemiluminescence readout system. Compared to the standard ELISA screening, the work load is reduced due to a higher degree of automation. The quality of the generated data is comparable to data generated by a previously optimized microplate-based immunoassay method. Among a reference set of 373 hybridoma cell supernatant samples, three out of four high-affinity MAbs were identified as true positive, whereas none of the samples were detected as a false positive.

Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods.

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.

Comparison of hybridoma screening methods for the efficient detection of high-affinity hapten-specific monoclonal antibodies.

This study compares diverse microplate-based hybridoma screening methods for the generation of hapten-(aflatoxin-) specific monoclonal antibodies (MAbs). Standard indirect enzyme-linked immunosorbent assay (ELISA) screenings (with immobilization of hapten-protein conjugate and use of enzyme-labeled anti-mouse IgG as tracer) were compared with direct ELISAs (with antibody immobilization and use of a hapten-enzyme conjugate as tracer). Although direct ELISA is rarely used for routine hybridoma screenings, it showed considerable advantages compared to the indirect assays. Standard indirect ELISA screening can lead to a considerable number of false positives (up to about 50% false positives of all 373 supernatants tested) if the antibody concentrations in the supernatants are too high. Direct ELISAs gave useful screening results for the different supernatant dilutions chosen. At most 3 false positives were detected out of 373 supernatants. However, the sensitivity of the direct ELISA screening is generally lower compared to indirect ELISA, and individual high-affinity MAbs might be classified as false negative. Therefore, a modified indirect ELISA screening was also developed. It includes pre-incubation of the supernatants in anti-mouse IgG-coated microplates which are then transferred into the (indirect) hapten conjugate-coated microplates. This screening method leads to excellent results with good overall selectivity and sensitivity. It can also be conveniently combined with the direct ELISA screening. Using these improved screening methods, aflatoxin-specific MAbs could be generated with IC50 values down to 3 ng/l (aflatoxin concentration).

Novel aflatoxin derivatives and protein conjugates.

Aflatoxins, a group of structurally related mycotoxins, are well known for their toxic and carcinogenic effects in humans and animals. Aflatoxin derivatives and protein conjugates are needed for diverse analytical applications. This work describes a reliable and fast synthesis of novel aflatoxin derivatives, purification by preparative HPLC and characterisation by ESI-MS and one- and two-dimensional NMR. Novel aflatoxin bovine serum albumin conjugates were prepared and characterised by UV absorption and MALDI-MS. These aflatoxin protein conjugates are potentially interesting as immunogens for the generation of aflatoxin selective antibodies with novel specificities.