Page 66 - GPD-2-3

Gene & Protein in Disease Testosterone as a biomarker of colorectal cancer

drawn into ethylene diamine tetraacetic acid-filled sterile to seal the microtiter plate in a bag. The unopened kit was
tubes and then delivered to the immunobiology laboratory. stored at 2 – 8℃.
Serum and plasma were subsequently separated from the
blood samples and assayed for biochemical parameters, 2.3.3. Assay procedure
specifically focusing on the levels of testosterone. The desired number of coated wells were placed in the
holder. Next, 10 μL each of standards, samples, and
2.3. The testosterone assay
controls were dispensed into separate wells. Subsequently,
2.3.1. Enzyme immunoassay (EIA) 100 μL of testosterone-HRP conjugate reagent and 50 μL
Testosterones were directly measured in the serum using of rabbit anti-testosterone reagent were added to each
radioimmunoassay kits (Catalog number: 07BC-1115, respective well. After thorough mixing for 30 s, the mixture
Bangalore Genei Private Limited) with reference values . was incubated for 90 min at 37°C. The incubation mixture
[20]
The detection method is based on the competitive binding was then removed from the wells by flicking, followed
of testosterone in the test specimen with a testosterone- by rinsing each well five times with deionized water.
horseradish peroxidase (HRP) conjugate for a consistent Subsequently, each well received 100 μL of TMB reagent,
amount of rabbit anti-testosterone. During the incubation, which was gently mixed for 10 s and then incubated for
testosterone standards, control samples, patient samples, 20 min at room temperature. The reaction was stopped by
testosterone-HRP conjugate reagent, and rabbit anti- gently adding 100 μL of the stop solution (1N HCl) to each
testosterone reagent were all incubated for 90 min within well for 30 seconds. The optical density must be read at
goat anti-rabbit IgG-coated wells. Subsequently, the wells 450 nm using a microtiter well reader within 15 min of the
were rinsed to remove unbound testosterone-peroxidase complete conversion of all blue colors to yellow. Figure 1
conjugate, and the addition of tetramethylbenzidine (TMB) illustrates each step of the process, from sample collection
solution induced the development of a blue color. TMB is through spectrophotometric analysis.
a chromogenic substrate used in immunohistochemical
staining processes and as a visualizing reagent in enzyme- 2.4. Statistical analysis
linked immunosorbent tests. To measure the absorbance Descriptive data analysis utilized frequency distribution
spectrophotometrically at 450 nm, the color development and mean with standard deviation (SD) statistics. To
was halted. The standard curve served to determine the assess significant differences between discrete and
testosterone concentration of the samples and controls in continuous data variables, Chi-square and t-tests were
relation to the standards. used in inferential statistics. ANOVA was used to assess
group mean differences. All statistical calculations were
2.3.2. Reagents composition performed using the International Business Machines-
Microtiter plates with 96 antibody-coated wells, with Statistical Package for the Social Sciences (IBM-SPSS) tool.
goat anti-rabbit IgG coating, were used. The reference
standard set (1 mL/vial) contains preservative-free human 3. Results
serum with testosterone concentrations of 0, 0.1, 0.5, 2.0, In the present study, a total of 130 participants were involved,
6.0, and 18.0 ng/mL. The pink-colored, 7mL rabbit anti- with 65 being diagnosed with CRC, while the remaining 65
testosterone reagent comprises anti-testosterone in a bovine served as healthy, cancer-free controls. These participants
serum albumin buffer with preservatives. The testosterone- were further categorized into two groups, namely Group I
HRP conjugate reagent (12 mL/vial, blue color), designed (comprising subjects under 50 years old) and Group II
for the quantitative determination of testosterone in (comprising subjects over 50 years old), based on “age”
human serum or plasma, comprises only HRP-conjugated criteria. The average age of patients in Group I was 42.62 ±
testosterone. Testosterone control sets one and two each 6.10 (n = 24), while in Group II, it was 6.40 ± 2.03 (n = 41),
include approximately 0.6 and 11 ng/mL of testosterone representing 36.92% and 63.08% of the total, respectively.
in human serum (0.5 mL/vial). The 11 mL container of The age range of CRC-diagnosed patients ranged from 37
TMB reagent contains 3,3’,5,5’-TMB stabilized in a buffer – 78 years, while that of the control subjects ranged from
solution. In addition, a bottle of stop solution containing
11 mL of diluted hydrochloric acid (1N HCl) is included. 36 – 76 years. There was a deviation of ±2 years in the age
Before use, all reagents should be equilibrated to room distribution between the CRC-diagnosed patient groups
and controls.
temperature (18 – 25℃) and gently inverted or swirled
to ensure proper mixing. Testosterone concentrations in Patients were also divided based on “disease stage”
samples were quantified through dilution with diluents. To criteria, including Dukes stage A, which encompassed 8
prevent exposure to humid air, a desiccant was included subjects (12.31%); Dukes stage B, comprising 14 subjects

Volume 2 Issue 3 (2023) 3 https://doi.org/10.36922/gpd.1082