The present invention relates to the method of detecting rupture of the amniotic membranes in pregnant mammals including humans using an immunoassay and reagents useful in such an assay. The method describes how to prepare a suitable protein antigen form amniotic fluid, gives criteria for the selection of this protein from the mixture of proteins present in amniotic fluid using the techniques of protein purification, gives criteria for assessing a sufficient degree of antigen purity for raising antibodies to the antigen and shows how the resultant antibodies can be used in immunoassays to detect the presence of amniotic fluid in the vagina and consequently to detect rupture of the amniotic membranes. The method also relates to the detection of amniotic fluid in other situations.