Development of a second generation Inhibin B ELISA.

Abstract

Inhibins are heterodimeric protein hormones secreted by the granulosa cells of the ovary in the female and the Sertoli cells of the testis in the male. Published research studies have assessed Inhibin B levels in Sertoli cell function, ovarian reserve and granulosa cell tumors. A two-step sandwich-type enzymatic microplate assay to measure Inhibin B levels within 3.5h is reported, and sample pre-treatment is not required. The assay measures Inhibin B in 50 μL of serum or Li-Hep plasma sample against Inhibin B calibrators (10-1000 pg/mL). The highly characterized antibody pair used in the assay measures 100% Inhibin B and no response was detected above the sensitivity of the assay with Inhibin A, Activin A, Activin B, Activin AB, AMH, FSH, LH or Follistatin 315 at the concentrations tested. The second generation Inhibin B assay was compared against two commercially available assays using 60 male and 60 female samples, ranging in age from 20 to 50 years. The assay showed significant positive linear correlations to Oxford Brooks Innovation (OBI) and Diagnostics Systems Laboratories (DSL) assays (r=0.99; P<0.0001; and r=0.97; P<0.0001), respectively. Method comparison to OBI and DSL resulted in the following slope and intercept (Gen II=1.03 OBI-6.77 pg/mL and Gen II=1.57 DSL+11.29 pg/mL), respectively. Matched serum and Li-Hep plasma samples (n=120) showed a correlation coefficient of >0.99 and a slope of 0.97 with zero intercept. Total imprecision calculated on three samples and two controls over 40 runs, three replicates per run, using NCCLS EP5-A guidelines was 6.8% at 19.34 pg/mL, 4.4% at 76.03 pg/mL, 4.3% at 275.3 pg/mL, 5.4% at 99.88 pg/mL, and 5.7% at 363.9 pg/mL. The LoQ for the assay at 20% CV was 4.8 pg/mL. Dilution and spiking studies showed an average recovery of 90-110%. A highly specific, sensitive, simplified and reproducible microplate Inhibin B assay has been developed to measure Inhibin B in serum and Li-Hep plasma. The performance of the assay is ideal for investigation into the physiologic role of Inhibin B in both men and women.

• Authors: Bhanu Kalra, Ajay Kumar, Kinita Patel, Amita Patel, M J Khosravi
• Journal: Journal of immunological methods (J Immunol Methods) Vol. 362 Issue 1-2 Pg. 22-31 (Oct 31 2010) ISSN: 1872-7905 [Electronic] Netherlands
• PMID: 20732325 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2010 Elsevier B.V. All rights reserved.

Chemical References

• inhibin B
• Inhibins

Topics

• Adult
• Enzyme-Linked Immunosorbent Assay (methods, standards)
• Female
• Granulosa Cells (immunology, metabolism)
• Humans
• Inhibins (blood, immunology)
• Male
• Middle Aged
• Reference Standards
• Sensitivity and Specificity
• Sertoli Cells (immunology, metabolism)