Aflatoxin: small molecule that needs low level detection and high sensitivity mAbs
Aflatoxins are a worldwide health problem as many as 5 billion persons may be exposed. Children are particularly affected by aflatoxin exposure, which leads to stunted growth, delayed development, liver damage and liver cancer. Adults have a higher tolerance but are also at risk. Aflatoxins have a negative economic impact on agriculture through reduced marketing options for crops and adverse health effects on livestock. Losses due to aflatoxins annually cost $ 900 million in Indonesia, the Philippines and Thailand alone.
Rat-LOU species to raise unique monoclonals against small molecules
Experiments conducted by SYnAbs conclude than the rat-LOU is better than the mouse to obtain high affinity mAbs against small molecules. In fact, at several occasions SYnAbs successfully obtained excellent rat mAbs against various haptens such as phosphorylated or methylated peptides, dinitrophenol, antibiotics, epigenetic modifications, steroids and carbohydrates. These multiple observations suggest to systematically use both rat-LOU and mouse to increase the probability to obtain high affinity mAbs to small molecules.
To reach sensitive detection in food stuff, SynAbs has developed antibodies in mouse and rat hosts. A Balb/C mouse and a LOU/C rat were immunized in the same manner with Aflatoxin B1
conjugated with KLH. Both were intraperitonally immunized at day 0, 15 and 30. The spleen was collected at day 33 and the fusion was performed. The mouse fusion was done with the SP2O fusion cell line and the rat fusion done with the IR983F fusion cell line.
The best monoclonal antibody (mAb) in both species has been produced in CeLLines and protein G or A purified. The mouse antibody, MA-afla, and the rat antibody, LOU-afla have been compared for their relative affinity against alfatoxin B1.
The titration assay using decreasing dilution of purified antibodies shows that the rat mAb displays a better relative affinity than the mouse one. Actually, 50% of the ELISA signal is lost at a 60 ng/ml dilution for the MA-afla, whereas for the LOU-afla mAb,
the signal loss occurs at 15 ng/ml (Fig. 1a & 1b).
A competition experiment was performed with increasing concentration of free aflatoxin B1 on an ELISA plate on which BSA-aflotoxin B1 was coated at 5 µg/ml and determine concentration of MA-afla or LOU-afla.
Using the mouse mAb, a concentration of 30 ng/ml of free aflatoxin B1 is necessary to detect a drop of the ELISA signal, whereas 4 ng/ml of free aflatoxin is enough to observe the
same effect on the LOU-afla mAb (Fig. 2a & 2b).
This competitive assay shows that the rat mAb has a better sensitivity than the mouse mAb for free aflatoxin. It means that the rat mAb can detect a concentration of about 4 ng/ml aflatoxin.
Currently, this value is above the admitted level which is 0,5 ng/ml in USA. However, an optimisation work could be done to increase the performance of the system.