Homologous ELISA for detection of prednisolone in human serum

Figures & data

Figure 1. Comparison of UV spectra of PSL, PSL-21-HS, BSA and PSL-21-HS-BSA.

Figure 2. Composite dose–response curve of homologous ELISA of prednisolone using PSL-21-HS-BSA-antibody with PSL-21-HS-HRP-enzyme conjugate. Each value is a mean ± SD of eight assays (In duplicate). The coefficient of variation at each concentration is shown in parentheses.

Table 1. Slope (m), Intercept (c), Sensitivity, Affinity, ED₅₀ and R² of prednisolone assay, using PSL-21-HS-BSA-antibody with PSL-21-HS-HRP-enzyme conjugate.

Assay combination	Slope (m) and intercept (c)		Sensitivity of the ;?>assay (ng/mL)	Affinity of the ;?>assay (L/mol)	ED50 ;?>(ng/mL)	R²
	M	C
PSL-21-HS-BSA-antibody with ;?>PSL-21-HS-HRP-enzyme conjugate	−1.814	0.768	0.078	6.36 × 10^−8	2.64	1.0

Table 2. Cross-reactivity of steroid compounds with Prednisolone in homologous and bridge heterologous assays of Prednisolone using PSL-21-HS-BSA-antibody with PSL-21-HS-HRP-enzyme conjugates by ELISA.

Steroid measured	% Cross reactions of PSL-21-HS-BSA-antibody with PSL-21-HS-HRP
C-27 Steroid
Cholesterol	<0.025
C-22 Steroid
Danazol	<0.025
C-21 Steroid
Progesterone	1.76
5 α Dehydro Progesterone	<0.025
5 β Dehydro Progesterone	<0.025
P-17α 20β Dioh	<0.025
P-17α 20 α Dioh	<0.025
11α OH Progesterone	<0.025
16-dehydroprogesterone	<0.025
Medroxy Progesterone Acetate (MPA)	<0.025
Pregnenolone	<0.025
17α-OH progesterone	5.89
17α-OH pregnenolone	<0.025
5 Pregnene 3 β 20 α diOL	<0.025
5α Pregnane 3β, 20α diOL	<0.025
5α Dihydropregnanolone	<0.025
5β-pregnane-3,20-dione	<0.025
Betamethosone	<0.025
17α, 20-dioh, 1, 4, Pregnadiene, 3, 11, 20 trione	<0.025
Pregnanediol	<0.025
Cortisol	7.69
5 α Dihydro Cortisol	<0.025
5 β Dihydro Cortisol	<0.025
Prednisolone	100
Prednisone	1.13
Aldosterone	<0.025
Dexamethasone	<0.025
Flutamide	<0.025
Corticosterone	<0.025
5 α Dihydro Corticosterone	<0.025
5 β Dihydro Corticosterone	<0.025
Cortisone	<0.025
5 α Dihydro Cortisone	<0.025
5 β Dihydro Cortisone	<0.025
Deoxycorticosterone (DOC)	<0.025
C-19 Steroid
Testosterone	<0.025
6 Dehydro Testosterone	<0.025
17 α Methyl Testosterone	<0.025
11 Keto testosterone	<0.025
Dihydrotestosterone (DHT)	<0.025
Etiocholanolone	<0.025
Dehydroepiandrosterone (DHEA)	<0.025
Dehydroepiandrosterone Sulfate (DHEA -S)	<0.025
Dehydroisoandrosterone	<0.025
Androstenedione	<0.025
Androstanedione	<0.025
Androsten 3 β, 17 β -Diol	<0.025
Androsten 3, 17, Dione	<0.025
Androstenediol	<0.025
Mesterolone	<0.025
Nandrolone	<0.025
C-18 Steroid
Estrone	<0.025
Estrone 3-Glu (E1 3G)	<0.025
Estradiol	<0.025
Estriol	<0.025

Bold signifies cross reactivity.

Table 3. Recovery of prednisolone from exogenously spiked serum pools.

Human serum pools	Prednisolone added (ng/mL)	Prednisolone observed (ng/mL)	Prednisolone expected (ng/mL)	% Recovery
Pool A (Basal)	–	0.69	0.69	–
Pool B	0.62	1.28	1.31	97.70
Pool C	1.25	1.84	1.94	94.84
Pool D	5.0	5.70	5.69	100.17
Pool E	15.0	15.20	15.69	96.87
Pool F	25.0	26.65	25.69	99.85

Table 4. Inter- and intra-assay CV % for the measurement of prednisolone in serum pool.

Variation	Sample value (ng/mL) (mean ± S.D.)	Coefficient of variation (%)
Intra-assay n = 8	0.65 ± 0.04	6.15
	2.49 ± 0.10	4.01
	5.20 ± 0.18	3.46
	15.06 ± 0.21	1.39
	25.70 ± 0.44	1.71
Inter-assay N= 8	0.83 ± 0.06	7.22
	2.41 ± 0.09	3.73
	5.04 ± 0.08	1.58
	15.22 ± 0.54	3.54
	25.57 ± 0.68	2.65

Figure 3. Regression graph of correlation between the serums prednisolone concentrations as estimated by the developed ELISA and an established ELISA kit (plotted by Prism 6.0 software).

Table 5. Determination of PSL in patient receiving PSL medication for different patient by using developed ELISA.

Variable	Healthy		Asthma		Rheumatic		Allergic
Gender	Male	Female	Male	Female	Male	Female	Male	Female
N	38	22	17	16	18	14	22	13
Age	18–64		10–72		40–62		16–60
Prednisolone ;?>mean ± 2 SD (ng/mL)	0.004 ± 0.0004		22.20 ± 7.77		10.05 ± 3.57		2.09 ± 1.36
Reference range	0.002–0.0068		12.42–25.87		4.12–15.45		0.66–5.25