RESUMO
Expression of progesterone receptor (PR) mRNA is indicative of a normal gene regulation mechanism mediated by functional estrogen receptor (ER). A simple assay which can reliably detect and quantitate PR mRNA levels in a small amount of tissue will be of value for studying functional status of ER. We have developed a quantitative nucleic acid hybridization assay for PR mRNA in breast carcinoma. The assay, which is based on the branched DNA (bDNA) technology, is simple, highly specific, and reproducible, requires 20 mg of tissue, and correlates reasonably well (r = 0.86) with an established methodology. The assay has a dynamic range of 3 x 10(3)-6 x 10(7) copies of PR mRNA per well. PR message as high as 3.9 x 10(5) copies per well could be detected in normal breast tissues. Thus a sensitivity of 3 x 10(3) PR copies per well was sufficient for testing clinical samples. In the present studies, accurate measurement of tissue weight enabled direct reporting of the PR mRNA values as the end point results. The bDNA assay provides a useful tool for the detection and quantitation of PR mRNA in research and routine clinical laboratories.
Assuntos
Neoplasias da Mama/química , Neoplasias Mamárias Animais/química , RNA Mensageiro/análise , RNA Neoplásico/análise , Receptores de Progesterona/análise , Animais , Neoplasias da Mama/genética , Cricetinae , Sondas de DNA , DNA de Neoplasias/análise , Feminino , Humanos , Neoplasias Mamárias Animais/genética , Receptores de Progesterona/genética , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The First International Workshop on Soluble HLA antigens focused on the comparison of immunoassay procedures for quantitation of soluble HLA (sHLA) class I antigens and the selection of a sHLA class I antigen international standard. Several sets of serum, plasma, and cell culture supernatant specimens were assayed blindly for levels of sHLA class I antigens by 15 participating laboratories using different immunoassay formats. The sandwich ELISA using (i) for antigen capture: an anti-HLA class I heavy chain monoclonal antibody (mAb) specific for a monomorphic epitope, and (ii) for antigen detection: an anti-beta 2 microglobulin antibody-enzyme conjugate, was the assay format of choice. There was a high inter-laboratory correlation among the majority of laboratories. All serum and plasma specimens from normal donors, and from a single transplant patient, had detectable levels of sHLA class I antigens. Paired serum and plasma specimens had similar levels of sHLA class I antigens, although plasma sHLA antigens seemed more stable than serum sHLA antigens. sHLA-A2 and sHLA-B7 antigens were detected in all specimens from HLA-A2 and HLA-B7 donors, respectively, using allele-specific ELISAs. No difference in reactivity was observed for quantitation of native sHLA class I antigens whether the capture mAb was TP25.99 (alpha 3 domain-specific) or W6/32 (alpha 2 + alpha 3-specific). However, a human-mouse chimeric sHLA class I antigen reacted weakly in assays which used TP25.99 mAb. The wide variation among laboratories in their reporting of micrograms/ml units pointed to the need for an inter-laboratory standardization based on a calibrated sHLA antigen preparation. T.sB7, an sHLA-B7 antigen derived from a cell line transfected within human beta 2 microglobulin and HLA-B7 genes, was accepted as the First sHLA class I Antigen International Standard at the workshop meeting.
