Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial growth factor and epidermal growth factor-like domain 7.

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.

Review of Implant Gonadotrophin-Releasing Hormone Agonist Use: Experience in a Single Academic Center.

BACKGROUND: Gonadotrophin-releasing hormone agonists (GnRHas) are used for puberty suppression in central precocious puberty (CPP) and gender dysphoria (GD). Guidelines on biochemical monitoring are not defined. OBJECTIVES: The aim of this study was to evaluate the utility of biochemical monitoring of GnRHa therapy in patients with CPP or GD. METHODS: This is a retrospective chart review of patients 18 years or younger who received GnRHa therapy from January 1, 2018, to March 20, 2021. RESULTS: A total of 103 patients were evaluated, 43 with CPP and 60 with GD. Using thresholds of basal luteinizing hormone (LH) <2 IU/L and stimulated LH <4 IU/L, biochemical pubertal suppression occurred in all but 2 patients. Basal LH frequently remained above prepubertal range. CONCLUSIONS: Laboratory assessment for puberty suppression on GnRHa therapy may be unnecessary in CPP and GD patients monitored with physical exams.