Radioimmunoassay for dynorphin A 1-13
Abstract
The antisera against dynorphin A 1-13 were raised and the radioimmunoassay (RIA) for dynorphin A 1-13 was developed in the present work.
The immunogen was prepared by coupling synthetic dynorphin A 1-13 to porcine thyroglobulin with the method of glutaraldehyde. Male New Zealand rabbits were immunized by multiple intradermal inoculations.
The antisera possessed high titre (1:70 000), high affinity (detection limit 6 pg, IC50 345 pg, Kaff=l×1010 L/mol) and high specificity (no cross-reactivity to dynorphin A 1-6, dynorphin A 1-7, dynorphin A 1-8, dynorphin A 1-10, dynorphin B and other 7 endogenous peptides).
By using this method, the contents of immunoreactive dynorphin A1-13 were measured in pituitary gland, spinal cord, and some brain areas of normal and urethane-anaesthetized rats. Our results showed that the values in urethane-anaesthetized rats were significantly reduced by 89, 74, 74, 73, 72, 62, 62, 35 and 33% in striatum, hippocampus, midbrain, pons/medulla oblongata, spinal cord, thalamus, cortex, pituitary and hypothalamus, respectively (p<0.01).
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The immunogen was prepared by coupling synthetic dynorphin A 1-13 to porcine thyroglobulin with the method of glutaraldehyde. Male New Zealand rabbits were immunized by multiple intradermal inoculations.
The antisera possessed high titre (1:70 000), high affinity (detection limit 6 pg, IC50 345 pg, Kaff=l×1010 L/mol) and high specificity (no cross-reactivity to dynorphin A 1-6, dynorphin A 1-7, dynorphin A 1-8, dynorphin A 1-10, dynorphin B and other 7 endogenous peptides).
By using this method, the contents of immunoreactive dynorphin A1-13 were measured in pituitary gland, spinal cord, and some brain areas of normal and urethane-anaesthetized rats. Our results showed that the values in urethane-anaesthetized rats were significantly reduced by 89, 74, 74, 73, 72, 62, 62, 35 and 33% in striatum, hippocampus, midbrain, pons/medulla oblongata, spinal cord, thalamus, cortex, pituitary and hypothalamus, respectively (p<0.01).