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Published on: December 12, 2011
A supersensitive immunofluorometric assay for rat luteinizing hormone
A M Haavisto1, K Pettersson, M Bergendahl
1Department of Physiology, University of Turku, Finland.
Insights
A new immunofluorometric assay (IFMA) offers a faster and more sensitive method for measuring rat luteinizing hormone (LH) compared to traditional RIA. This enhanced assay accurately detects suppressed LH levels, crucial for understanding reproductive and endocrine functions.
Area of Science:
- Endocrinology
- Assay Development
- Reproductive Biology
Background:
- Accurate quantification of rat luteinizing hormone (LH) is essential for reproductive and endocrine research.
- Existing radioimmunoassay (RIA) methods are time-consuming and lack the sensitivity required for detecting low LH concentrations.
Purpose of the Study:
- To develop and validate a highly sensitive and rapid immunofluorometric assay (IFMA) for quantifying rat LH.
- To compare the performance of the new IFMA with the standard NIDDK rat LH RIA and a bioassay.
Main Methods:
- Development of an IFMA utilizing monoclonal antibodies against rat LH and time-resolved fluorescence detection with a europium label.
- Validation of the assay's sensitivity, specificity, and cross-reactivity against other rat pituitary hormones.
- Comparison of IFMA results with RIA and a bioassay using rat serum and pituitary homogenates, including studies on suppressed LH levels.
Main Results:
- The IFMA demonstrated significantly higher sensitivity (0.75 pg/tube) and speed (4 hours) compared to RIA (3 days).
- The assay showed minimal cross-reactivity with other rat hormones and high correlation (0.93) with a bioassay.
- IFMA accurately measured suppressed LH levels in testosterone-treated and fasted rats, where RIA failed to show significant changes.
Conclusions:
- The developed IFMA is a sensitive, specific, and rapid assay for rat LH measurement.
- This assay provides more accurate and meaningful data, especially for suppressed LH levels, compared to RIA.
- The IFMA is a valuable tool for endocrine and reproductive research in rats.
Abstract:
We have developed an immunofluorometric assay (IFMA) for rat (r) LH, which is based on two monoclonal antibodies, one to bovine and the other to human LH. Signal detection occurs by time-resolved fluorescence evoked by a europium label (Delfia, Wallac). The method is fast in comparison to the standard RIA with the NIDDK reagents (4 h vs. 3 days). The sensitivity of the IFMA assay (0.75 pg/tube; NIDDK rLH RP-2) is over 30-fold higher than that of the NIDDK RIA (usual detection limit, 20-30 pg/tube). Using 25-microliters serum samples, the sensitivity of IFMA is 0.03 micrograms/liter; with 100-microliters samples, it is 0.0075 micrograms/liter. The cross-reactivity of the IFMA assay is 0.3% with rFSH, 3% with rTSH, and less than 0.05% with rGH, rPRL, and the rat alpha-subunit. A linear correlation between IFMA and RIA values is seen at serum levels above 0.4 micrograms/liter. Below this level, only IFMA is able to detect concentration differences between samples. In practice, this means that only IFMA is able to provide meaningful measurements of suppressed levels of serum LH. The correlation coefficient between IFMA and the mouse interstitial cell in vitro bioassay for LH in randomly selected rat pituitary homogenates was 0.93 (n = 47). The serum concentration of LH determined by IFMA is 0.57 +/- 0.10 micrograms/liter in intact adult male rats (mean +/- SEM; n = 12) and 0.41 +/- 0.10 micrograms/liter (n = 10) in randomly cycling females. The level in hypophysectomized rat serum is 0.035 +/- 0.0033 micrograms/liter (n = 8), if the limit of sensitivity (0.03 microgram/liter) is assigned to unmeasurable levels. One-week treatment of male rats with 2-cm Silastic implants containing testosterone suppressed serum LH, measured by IFMA, from 0.56 +/- 0.057 to 0.086 +/- 0.057 micrograms/liter (P < 0.01). The suppression of LH measured in the same samples by RIA was lower, from 0.73 +/- 0.057 to 0.44 +/- 0.048 micrograms/liter (P < 0.01). A 5-day starvation of intact male rats suppressed serum LH from 0.57 +/- 0.10 to 0.30 +/- 0.05 microgram/liter by IFMA (P < 0.01), whereas the decrease determined by RIA was not significant (0.80 +/- 0.07 vs. 0.66 +/- 0.13 micrograms/liter).(ABSTRACT TRUNCATED AT 400 WORDS)
