Animal cell antibodies / Neurosteroids/Neurobiology

Method description for use of Agrisera anti-allopregnanolone antibodies
Procedure for radioimmunoassay for allopregnanolone after extraction and chromatography

Extraction:
Allopregnanolone is measured with radioimmunoassay (RIA) after pre-assay diethylether extraction and HPLC or celite chromatography purification. The 0.2 ml serum sample was extracted using 3.0 ml diethylether (Merck, Pro Analysi) by shaking. Thereafter the ether phase is evaporated under nitrogen.

High Performance Liquid Chromatography HPLC:
For purification of sample before quantification of allopregnanolone one may use preparative High Performance Liquid Chromatography followed by Radioimmunoassay (HPLC-RIA). Plasma samples are analyzed in duplicates. Evaporated samples were resolved in 1 ml ethanol: water 1:1 (V/V) prior to analysis Our HPLC system consisted of a Waters 1515 Isocratic Pump, delivering the mobile phase (methanol : water, 60:40, V/V) at a flow rate of 1.0 ml/min. A Waters 717 plus Auto-sampler was used for injection of samples (200 µL) into a Symmetry C18 3,5 µm 4,6 x 75 mm separation column (Waters), heated to 45°C in a Waters 1500 Column Heater. Detection of retention times of standards and cross-reacting steroids was at 206 nm using a Waters 2487 Dual λ Absorbance Detector. The detector output was recorded on a PC-based Waters Breeze Chromatography Software (version 3.20). In the preparative HPLC 5 ml fractions were symmetrically collected around the retention time for allopregnanolone, retention was found from injection of a standard sample before the start of analysis. A Waters Fraction Collector II was used for collection of samples, for further analysis with RIA. No cross-reacting steroids had retention times close to the collected fraction, as analyzed by injection of 20 nmol of standard samples (Table 1).

Celite chromatography:
The evaporated sample is dissolved in 1.0 ml isooctane (Merck, Pro-analysi) saturated with ethyleneglycol (J.T. Baker, for analysis), before application to the column. The celite column chromatography was performed as follows. Glass columns (50 mm x 5 mm i.d.) were tightly packed with a mixture of celite (Mansville, Denver CO, USA, heated to 600ºC over night), and propylene glycol (Merck, Pro-analysi), weight : volume = 1 : 1. Isooctane (10 ml) was percolated through the columns before sample applications. The elution pattern was: First sample was applied. Thereafter 1.0 ml isooctane wash, followed by 1.5 ml isooctane to obtain 5 alpha- and 5β-DHP, additional 4.0 ml isooctane to obtain progesterone, additional 3.0 ml isooctane to obtain allopregnanolone and in the next 4.0 ml 4-pregnen-3alpha-ol-20-one was eluted but 5 alpha-pregnan-20ß-ol-3-one is not eluted with isooctane. Thereafter a mixture (60:40) isooctane : toluene (Fisher chemicals, for analysis) was percolated. Pregnanolone was eluted and with the first 4 ml of the mixture and 5 ß-pregnan-3alpha,21-diol-20-one and 5alpha-pregnan-3alpha,21-diol-20-one were eluted with further 5.0 ml isooctane : toluene (60:40). The allopregnanolone containing fraction is evaporated under nitrogen. Recovery is determined for each assay using 300-500 cpm of tritium-labeled allopregnanolone (New England Nuclear, Boston, USA) added to a plasma sample before extraction and measuring the amount recovered after chromatography. The recovery of allopregnanolone was 78%.

RIA.
The allopregnanolone antiserum, was raised against 3alpha-hydroxy-20-oxo-5 alpha-pregnan-11-yl carboxymethyl ether coupled with bovine serum albumin. The antigen was made by Dr. Robert H. Purdy, Department of Psychiatry, College of Medicine, University of California, San Diego, CA, USA in the same way as earlier published (Purdy et al., 1990). Immunization is made in Hen and IgY antibodies are made (Agrisera AB, Vannas, Sweden). The cross-reactivity of the antibody is shown in table 2. The standard curve was established by preparing duplicate tubes containing eight concentrations of unlabeled allopregnanolone to give a range from 0 to 5,000 pg. Antibody solution was prepared using tritium labeled steroid, 3 million cpm/30 ml ready made solution, 65 mM boric acid (Merck) buffer, pH=8.0, bovine serum albumin solution 100 mg/ml (Sigma, St Louise, USA), human gamma globulin solution 20 mg/ml (Octapharma, Sweden) and antiserum in ratio 30:1:1:0.0009. The solution was allowed to equilibrate overnight at 8ºC. Antibody solution (200 µl) was added to all standard and sample tubes and the mixture allowed to stand overnight at 8ºC. After the addition of 200 µl saturated ammonium-sulfate each tube was again mixed and centrifuged at 20 000 RPM for 20 minutes. Thereafter the supernatant was aliquoted into a counting vial and diluted with 3.0 ml Optiphase scintillation medium (Wallac, Finland). The samples were counted in a RackBeta (Wallac, Finland) scintillation counter. The sensitivity of the assays was 25 pg, with an intraassay coefficient of variation for allopregnanolone is 6.5% and interassay coefficient of variation is 8.5%.

Table 1. HPLC retention times of Cross-reacting steroids to anti allopregnanolone antibody using a Symmetry C18 3,5 µm 4,6 x 75 mm separation column (Waters), heated to 45°C and a mobile phase (methanol: water, 60:40, V/V) at a flow rate of 1.0 ml/min.

Steroid                                       Retention time, min.

Table 2: Crossreactivity test of Agrisera’s IgY antibody from HEN1102-batch1219-collected 20030919, titer 1/1000. As comparison is shown the crossreactivity pattern from Purdys antiserum using the same antigen 3-hydroxy-20-oxo-5-pregnan-11-yl carboxymethyl ether coupled with bovine serum albumin (Purdy et al 1990).

Reference
Purdy R.H., Moore Jr P.H., Rao P.N., Hagino N., Yamaguchi T., Schmidt P., Rubinow D.R., Morrow A.L. and Paul S.M. (1990) Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma. Steroids 55, 290-296.