Thyroxine (T.sub.4) radioimmunoassay
First Claim
1. A composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T4) in a measured quantity of human serum, the reagent comprising in the absence of non-radioactive T4 :
- a blocking agent in sufficient quantity to displace essentially all of the T4 in the measured quantity of serum bound to thyroxine-binding globulin (TBG);
an antibody in sufficient quantity to bind a significant quantity of the T4 in the measured quantity of serum;
polyethylene glycol (PEG) in sufficient quantity to precipitate essentially all the antibody; and
a buffering agent in sufficient quantity to inhibit binding the T4 in the measured quantity of serum to thyroxine-binding prealbumin.
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Accused Products
Abstract
It has been discovered polyethylene glycol can be added as a separator to the other reagents of a T4 radioimmunoassay of unextracted serum prior to introduction of the serum, without impairing the sensitivity of the measurement. A single, highly stable composite reagent is prepared to measure the concentration of T4 in a measured quantity of human serum; the composite reagent comprises, in freeze dried form, a blocking agent in sufficient quantity to displace essentially all the T4 in the measured quantity of serum bound to TBG, an antibody in sufficient quantity to bind a significant quantity of the T4 in the measured quantity of serum, PEG in sufficient quantity to precipitate essentially all the antibody, and a buffering agent in sufficient quantity to inhibit binding the T4 in the measured quantity of serum to TBP. To run a radioimmunoassay, the human serum being assayed and quantities of standard serums containing known concentrations of T4 are added to a mixture of the composite reagent reconstituted with water and radioactive T4 in sufficient quantity to give a measurable counting rate. After an incubation period, the antibody precipitated by the PEG, including the antibody bound T4, is removed by centrifuging and aspirating the supernatants. The radio activity of the precipitates is then counted to correlate the T4 in the serum being assayed with the standard serums.
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Citations
24 Claims
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1. A composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T4) in a measured quantity of human serum, the reagent comprising in the absence of non-radioactive T4 :
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a blocking agent in sufficient quantity to displace essentially all of the T4 in the measured quantity of serum bound to thyroxine-binding globulin (TBG); an antibody in sufficient quantity to bind a significant quantity of the T4 in the measured quantity of serum; polyethylene glycol (PEG) in sufficient quantity to precipitate essentially all the antibody; and a buffering agent in sufficient quantity to inhibit binding the T4 in the measured quantity of serum to thyroxine-binding prealbumin. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. The method of preparing a composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T4) in a measured quantity of serum, the method comprising the following steps in the order recited:
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dissolving in an aqueous buffer solution having a pH of about 8.6 sufficient gamma globulin to provide a visible precipitate after centrifusion and supernatant aspiration and sufficient 8-anilino-1-naphthalene-sulfonic acid (ANS) to displace all the T4 bound to thyroxine binding globulin in the measured quantity of serum; slowly adding sufficient polyethylene glycol (PEG) to precipitate substantially all the gamma globulin, thereby producing a thick slurry; adding to the slurry an antibody capable of binding a significant quantity of T4 ; and thoroughly mixing the recited ingredients. - View Dependent Claims (12, 13, 24)
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14. A method of measuring the concentration of thyroxine (T4) in a measured quantity of human serum, the method comprising the steps of:
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combining the following to form a mixture; a blocking agent in a quantity sufficient to displace essentially all the T4 in the measured amount of human serum from thyroxine binding globulin (TBG); an antibody in sufficient quantity to bind a significant quantity of the T4 in the measured quantity of serum; polyethylene glycol (PEG) in sufficient quantity to precipitate essentially all the antibody; and a buffering agent in sufficient quantity to inhibit binding the T4 in the measured quantity of serum to thyroxine-binding prealbumin; adding the mixture to the measured quantity of human serum and radioactive T4 in sufficient quantity to give a measurable counting rate; incubating the serum and radioactive T4 with the mixture added to allow reaction of the T4 in the serum and the radioactive T4 with the antibody to proceed substantially to equilibrium, thereby producing antibody bound T4, a percentage of which is radioactive; removing the antibody precipitated by the PEG including the antibody bound T4 from the supernatant; and measuring the quantity of radioactive T4 in the removed precipitate or the supernatant. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23)
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Specification