Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Abstract

An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10−19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.

Memory block: A consequence of conflict resolution

“…Thus, the occurrence of an insulin spike correlates with the acquisition and retention of associative learning. In the near future, we will determine the trace amount of insulin concentration in the Lymnaea CNS (Watabe et al, 2014).…”

An increase in insulin is important for the acquisition conditioned taste aversion in Lymnaea

“…Thus, an insulin spike may also occur in the Lymnaea CNS that is caused only by CS stimulation of the appropriate receptors. However, in order to confirm this, we will need to develop the appropriate antibodies for MIPs and develop a system with the necessary sensitivity to measure the trace amounts of MIPs that are thought to be released (Watabe et al, 2014). However, the data showing that the antibody directed against the mammalian insulin receptor blocks the memory formation in Day 5 snails fed sucrose seems to be consistent with a molluscan cephalic phase insulin release into the CNS.…”

Function of insulin in snail brain in associative learning

“…Thus, it is conceivable that trace amounts of molluscan insulin-related peptides could be measured in the CNS of snails, maybe even in single neurons. We are optimistic that this will occur, because the Ito group has succeeded in developing an ultrasensitive ELISA method for human insulin in the blood at the level of 10 −19 mol/ assay (Watabe et al 2014). Thus, if we are successful in being able to measure such low levels of molluscan insulinrelated peptides in the CNS of Lymnaea exhibiting CTA-LTM, our insulin scenario will be completed.…”