BIOASSAY OF RELAXIN USING A REFERENCE STANDARD: A SIMPLE AND RELIABLE METHOD UTILIZING DIRECT MEASUREMENT OF INTERPUBIC LIGAMENT FORMATION IN MICE

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Abstract
The estrogen-primed mouse responds to single injections of relaxin with dose-proportional increases in the length of the interpubic ligament. This effect is specific for relaxin and has served as the basis for published bioassay procedures. A simple and accurate method for direct measurement of interpubic ligament length was previously developed, employing transillumination and magnification of the exposed pubic symphysis. A quantitative bioassay procedure was devised in which relaxin-containing preparations were evaluated in terms of a reference standard. The present report is an extension of this earlier work. Detailed evidence is presented to justify each step of the assay procedure. The data demonstrate that precision and reproducibility of the assay depends primarily on control of the following variables: selection of mice, adequacy of estrogen priming, selection of injection vehicle, rate of ligament formation, and seasonal shifts in responsiveness of mice. Thus, mice which fail to gain weight during the estrogen priming period are not reliable for assay. Uterine enlargement is correlated with the effectiveness of the estrogen priming, and is used as a criterion of assay eligibility. Rate of interpubic ligament formation is dependent on the vehicle used for relaxin injection. Several commercial strains of mice were used for assay weekly over a period of 2 years. Significant seasonal shifts in response to the relaxin reference standard were observed. Thus, unknowns must be evaluated in terms of a concomitant standard. Response units are unreliable. When these precautions are used, limits of error of potency estimates generally fall within -27 to + 35% at p =0.95, and [lambda] is 0.2-0.4.

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