Testosterone RIA test

Description

The testosterone [¹²⁵I] radioimmunoassay system provides the quantitative in vitro determination of testosterone in human serum. Testosterone can be assayed in the range 0-35 nmol/l, using 50 µl serum sample. Each kit contains materials sufficient for 100 assay tubes, permitting the construction of one standard curve and the assay of 41 unknowns and 1 control in duplicate.

Introduction

The blood level of testosterone is an important indicator of a wide variety of pathologic conditions.

Elevated concentration of testosterone
in males is characteristic of precocious puberty, congenital 21-hydroxylase deficiency, adrenal hyperplasia (Cushing’s syndrome), testicular tumours
in females: ovarium or endometrium tumors, Stein-Leventhal syndrome, Adrenal hyperplasia (Cushing’s syndrome), hirsutism, glycocorticoid therapy

Decreased concentration of testosterone
in males is associated with Klinefelter’s syndrome, agonadism, anorchism, chryptorchidism, Kallman’s syndrome, Leydig cell aplasia, defects of the pituitary functions and
in females at postmenopausa.

Principle of method

This assay is based on the competition between unlabelled testosterone and a fixed quantity of ¹²⁵I-labelled testosterone for a limited number of binding sites on testosterone specific antibody. Allowing to react a fixed amount of tracer and antibody with different amounts of unlabelled ligand the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand. Upon addition of magnetisable immunosorbent the antigen-antibody complex is bound on solid particles which are then separated by either magnetic sedimentation or centrifugation. Counting the radioactivity of solid phase enables a standard curve to be constructed and samples to be quantitated.

Contents of the kit

1 vial	TRACER, concentrated solution < 150 kBq ¹²⁵I-testosterone in 1 ml organic solution
6 vials	STANDARDS_(1-6), lyophilized 0.5 ml per vial, containing 0 (S1), 0.43 (S2), 1.73 (S3), 4.3 (S4), 17.3 (S5), 35 (S6) nmol/l in 0.5 ml serum with 0.1% NaN₃
1 vial	ANTISERUM, lyophilized Contains polyclonal anti-testosterone (rabbit) IgG in buffer.
1 vial	CONTROL SERUM, lyophilized 0.5 ml human serum, containing 0.1% NaN₃. The concentration of control serum is specified in the quality certificate enclosed.
1 bottle	MAGNETIC IMMUNOSORBENT (MIS), ready to use 55 ml, containing paramagnetic particles in buffer with 0.1% NaN₃.
1 bottle	TRACER BUFFER, ready to use 24 ml phospate buffer containing 0.1% NaN₃
1 pc	Quality certificate
1 pc	Pack leaflet

Materials and equipment required

Round bottom polystyrene or polypropylene assay tubes (about 12 x 75 mm)
Plastic film to cover tubes
Precision pipettes (50, 100, 200 and 500 µl disposable tips)
Vortex mixer
Magnetic separator (or alternatively centrifuge)
Decanting racks
Gamma counter
Absorbent tissue

Recommended tools and equipment

Orbital shaker
Repeating pipettes

Preparation of reagents

Add 2-4 ml tracer buffer into the tracer concentrate and transfer the content into the tracer buffer bottle.

Reconstitute antiserum with 10 ml distilled water.

Add exactly 500 µl distilled water to each standard vial. Add exactly 500 µl distilled water to the lyophilized control serum. Ensure that complete dissolution is achieved, and allow the solutions to equilibrate at room temperature for at least 20 minutes.

Specimen collection and storage

Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Sera can be stored at 2-8^oC for two days after collection. For later analysis they should be stored deep-frozen. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens. Samples with a testosterone concentration higher than that of the most concentrated standard should be diluted and reassayed. Use the zero standard as diluent.

Assay procedure

(For a quick guide refer to Table 1)

1	Equilibrate all reagents to room temperature.
2	Label duplicate tubes for total counts (T), zero standard (Standard 1 = B₀), standards (S2-6), control (C) and samples (Sx).
3	Mix all reagents and samples thorougly before use. Avoid excessive foaming.
4	Pipette 50 µl each of standards, control and samples into the properly labelled tubes.
5	Pipette 200 µl of tracer solution into all tubes.
6	Pipette 100 µl of antiserum into all tubes except T.
7	Thoroughly vortex mix all tubes except T for 2-5 seconds. When having an orbital shaker, leave all tubes in the rack holder, fix the holder onto the plate of the shaker, and shake it gently for a few seconds.
8	Cover tubes with a plastic foil, and allow them to incubate at room temperature for 3 hours.
9	Place T tubes on a separate tube rack. Gently shake and swirl the bottle containing magnetic immunosorbent until homogeneity. Add 500 µl to each tube except T. When using a single pipette, swirl the bottle of MIS after every 15-20 tubes. With the use of a repeating pipette (e.g. Eppendorf), there is no need for repeated homogenization of MIS reagent.
10	Thoroughly vortex mix all tubes and incubate them for 5 minutes at room temperature.
11	Separate the bound fraction by using one of the following procedures. Magnetic separation Attach the rack on to the magnetic separator base and ensure that every tube is in contact with the base plate. Let the MIS particles settle for 5 minutes. Do not remove the rack from the separator base after the separation of the solid and liquid phases. Pour off and discard the supernatant. Keeping the separator inverted, place the tubes on a pad of absorbent tissue and allow to drain for 2 minutes. Centrifugation Centrifuge all tubes for 15 minutes at 1500 g or greater. Aspirate the supernatant. Take care of not disturbing the precipitate.
12	Count the radioactivity of each tube for at least 60 seconds or longer in a gamma counter.
13	Calculate the concentrations as described under Calculation of results.

Table 1 Assay Protocol, Pipetting Guide (all volumes in microliters)

T = total count, S _1-6 = standards, Sx = sample, C = control, NSB = Non-specific binding

Tubes Reagent	T	NSB	S_1-6	Sx	C
Distilled water		100
Standard			50
Sample				50
Control					50
Tracer	200	200	200	200	200
Anti-serum			100	100	100
Vortex mix. Incubate for 3 hours at room temperature
Magnetic immunosorbent		500	500	500	500
Vortex mix. Incubate for 5 minutes at room temperature
Place the tubes on the magnetic separator for 5 minutes or centrifuge for 15 minutes at 1500xg
Remove the supernatant and blot the tubes
Count all tubes

Calculation of results

The calculation is illustrated using representative data. Data obtained should be similar to those shown in Table 2.

Calculate the average counts per minute (CPM) for each pair of assay tubes.

Calculate the percent B₀/T for zero standard (S1) by using the following equation:

	S₁ - NSB
B₀ / T % =	————	x 100
	T

Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

	(S_2-6 [C, Mx] - NSB)
B / B₀ % =	——————————	x 100
	(S₁ - NSB)

Using a semi-logarithmic graph paper plot B/B₀ % for each standard versus the corresponding concentration of testosterone. Figure 1 shows a typical standard curve.

Determine the testosterone concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range.

Table 2 Typical Assay Data

Tubes	Counts CPM1	Counts CPM2	Mean CPM	B/T %	B/B₀ %
T	43864	44478	44171
S1	19136	19852	19494	43.1	100.0
S2	17972	17454	17713	39.1	90.6
S3	14976	14290	14633	32.1	74.5
S4	10821	10622	10722	23.3	53.9
S5	6300	6415	6358	13.4	31.0
S6	4436	4758	4597	9.4	21.8
C	8361	7823	8092	17.3	40.1
NSB	486	416	451	1.02

Typical standard curve for testosterone concentration

Figure 1
A typical standard curve
(Do not use to calculate sample values)

Conversion of SI units can be performed according to the following formula:

1 nmol/l = 0.29 ng/ml

Characterization of the assay

Assay parameters

NSB/T	< 3%
B₀/T	45.0 ± 10%
ED-50	4.2 ± 1.1 nmol/l

Specificity

Cross reactivity was defined by weight at the 50% displacement level in percent.

Analyte	Cross reactivity %
Testosterone	100
5a-dihydrotestosterone	35
5b-dihydrotestosterone	0.8
17b-estradiol	0.01
Cortisol	0.01

Expected reference values

Male: 9 –38 nmol/l (2.6 – 11 ng/ml)
Female: 0.9 – 4.5 nmol/l (0.26 – 1.3 ng/ml)

It is recommended that each laboratory establish its own reference intervals. The results obtained should only be interpreted in the context of the overall clinical picture. None of in vitro diagnostic kits can be used as the one and only proof of any disease or disorder.

Additional information

Storage

Store the reagents between 2 and 8 ^oC. At this temperature each reagent is stable until expiry date. Pay special attention to preventing magnetic immunosorbent suspension from freezing.

Availability

From stock.

Shelf life

The minimum shelf life of kit reagents is usually 8 weeks from the date of manufacturing. The actual expiry date is given on the package label and in the quality certificate. To make the maximum benefit of long-term stability it is recommended to adjust the date of ordering to new-batch manufacturing calendar issued each year. Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precaution

Radioactivity

This kit contains radioactive material. Receipt, acquisition, possession, or use of radioactive materials are subject to regulations, and a licence of (inter)national authorizing bodies. It is the responsibility of the user to ensure that local regulations or codes of practice are satisfied.

Potentially infectious materials

Human blood products provided as components of this product have been obtained from donors tested individually and found negative for Human Immunodeficiency Virus antibody (HIV-Ab) as well as for Hepatitis B surface Antigen (HBsAg) using approved EIA methods.

Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV), or other infectious agents are absent, and all human blood samples should be considered potentially infectious.

Chemical and other hazard

Some components contain sodium azide (0.1% w/v) as an antimicrobial agent. Dispose the waste by flushing it with copious amounts of water to avoid build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 82.5 mg.

Use by	In vitro diagnostic medical device	Control
Batch code	Manufacturer	Standard
Caution, consult accompanying documents	Radioactive material	Magnetic immunosorbent
Biological risk	Temperature limitation Store between 2-8 °C	Tracer
Consult operating instructions	Catalogue number	Antiserum

Testosterone RIA test