ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

doi:10.21203/rs.3.rs-2748047/v1

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ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

https://doi.org/10.21203/rs.3.rs-2748047/v1

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The introduction of spacers in coating steroid protein complex and / or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced different homobifunctional spacers between enzyme and prednisolone (PSL) moiety having atomic length 3 to 10 and studied their influence on functional parameters such as sensitivity and specificity of PSL enzyme immunoassays. In this study, four enzyme conjugates of PSL-21-HS and HRP were prepared by N-Hydroxy Succinimide mediated carbodiimide reaction, these were PSL-21-HS-adipic acid dihydrazide (ADH)-HRP, PSL-21-HS- carbohydrazide (CH)-HRP, PSL-21-HS-ehylenediamine (EDA)-HRP and PSL-21-HS- urea (U)-HRP. The assays were developed using these enzymes conjugate with antibody raised against PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of bridge in enzyme conjugate were 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL and 0.018 ng/mL with ADH, CH, EDA and urea as spacer respectively. Among all four combinations, PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity and showed cross-reaction with less number of steroids. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32-102.50 %. The intra and inter assay CV % was < 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r²= 0.98). The present study, suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.

Biological sciences/Biological techniques/Immunological techniques/Antibody generation

Health sciences/Medical research/Translational research

Heterologous ELISA

Bridge/Spacers

Prednisolone

Antibody

Horseradish Peroxidase

The spacer length is not significant as the spacer structure; it plays a remarkable role in improving of sensitivity of ELISA.
The physicochemical nature of the bridge and steroid is determinant in defining the assay parameters.
The in-house developed assay is simple, sensitive, direct, and convenient to use for prednisolone in serum.
The procedure adopted for coating primary antibody does not require affinity purified antibody.

The PSL (1, 4-Pregnadiene-11β, 17α, 21-Triol-3, 20-Dione / 1-Dehydrocortisol) is a synthetic analogue of endogenous cortisol, having potent glucocorticoid and low mineralocorticoid activity, making it useful for the treatment and management of a broad range of diseases of autoimmune diseases (such as lupus, arthritis, nephritis, etc.). It is also widely used in veterinary medicine particularly to treat inflammatory diseases, shock, stress, circulatory collapse, and acetonemia, to lower fever and reduce pain.

There is evidence that long-term exposure to low concentrations of glucocorticoid may thrive negative toxic effects on public health such as diabetes, metabolic disorders etc. Whereas prolonged use of therapy doses may cause depression, hypertension, weight loss, muscle atrophy, bone pain, increased susceptibility to infection, sleep disturbances, delayed wound healing, reduce sex hormone production, and decrease growth rates in children. Therefore, there is a growing demand for more sensitive, specific, rapid and cost-effective methods for the determination of the low concentration of PSL residues in the samples.

Compared with physico-chemical methods (HPLC, LC-MS/MS, GC-MS etc.), Enzyme-linked immunosorbent assay (ELISA) is rapid, simple, effective, and needs less or no sample preparation. An ELISAs are presently the most used and successful technique for immunologically-based detection of a wide variety of antigens. Its popularity has been increased rapidly due to its high-throughput capabilities, where it is capable of analyzing many samples in a relatively short period of time. It is not always easy to develop specific and sensitive ELISA for small molecule like prednisolone, a synthetic steroid which is not naturally present in the body. One of the most important factors that determine the sensitivity and specificity is the combination of antibody and enzyme conjugate used in the assay ^1–4.

ELISA for steroids (hapten) developed so far are either in a heterologous or homologous combination. The heterologous or homologous combinations of antibody and enzyme conjugate in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition that affects sensitivity of the assay. In heterologous assays, the haptens used as antigen to produce the antibody are different from the haptens used for labeling the enzyme. In homologous immunoassays, the haptens are similar in both cases. It’s often found that in a heterologous assay ^5–11certain differences, such as that of site, bridge, or antigen (steroid), exist between the steroid derivatives used for preparation of immunogen and enzyme conjugate, while the assay is more sensitive due to improved fitting of steroid into antibody binding pocket and reduced bridge recognition ^5–11. On the other hand, homologous combination does not provide satisfactory sensitivity, because the binding affinity of the labeled antigen to antibody is higher than that of the antigen to be measured^12,13. But few sensitive homologous assays for steroids have been developed ^1–4,12,13.

A remarkable improvement in the sensitivity of ELISA has been observed by various researchers using spacers between antigens and coupling protein when used as coating antigen in antigen immobilized format ^14–17 or between antigen and enzyme when used as enzyme conjugate in antibody immobilized format ^5,13. It was observed that in the antigen-immobilized format, varying lengths of spacer arm of the coating antigen had significant effect on the sensitivity of ELISA ^14–17. A 19-atom linker, an oligoethylene glycol (OEG) was conjugated between progesterone and ovalbumin (OVA) and used as coating antigen in the surface plasmon resonance (SPR) flow-through biosensor format with end-point detection using a BIAcore instruments. Its hydrophilic nature allows good projection of the antigen (progesterone) into the aqueous mobile phase ¹⁸.

In the antibody-immobilized format, spacers have been coupled between steroid derivative and label protein to minimize the conventional bridge recognition effects and to overcome steric constraints between the two high molecular weight proteins, i.e., antibody and label ^{5–11,14,19,20}. Shrivastav et al. studied differential behavior of spacers like ADH, EDA, CH, urea, gamma amino butyric acid-ADH (GABA-ADH) and 6-amino caproic acid-ADH (6ACA-ADH) between steroid-HRP enzyme conjugate in direct ELISA format. The study revealed that the use of a spacer between the steroid and enzyme in enzyme-conjugate increases the sensitivity and specificity of the assay. It has also been reported that the spacer length does not bear any correlation with the assay sensitivity in ELISA of steroids^{1,5–11,14,19,20}. Further, the nature of the spacer (hydrophilic, hydrophobic, flexible, or rigid) is related to assay sensitivity and not to spacer length ^5–11,14. This differential behavior of spacers towards the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates ⁹. Sathe et al. coupled spacers (urea, ADH and oxy diamine (ODA), bis-oxy diamine (BODA)) between organo phosphorous pesticide and HRP and studied the effect of spacer on sensitivity of the assay. The use of deliquescent spacers has helped to increased antibody binding signal and improved sensitivity of the assay ²⁰.

To the best of our knowledge, there are no any reported study exist on the development of ELISA for PSL using bridge heterology in enzyme conjugates. Also, the PSL derivatives of different bridge lengths are not available commercially to produce enzyme conjugate, as it is not easy to synthesize novel hapten derivatives with different bridge lengths. Because it requires knowledge on synthesis, isolation and purification of hapten derivative. To overcome this, we have first introduced spacer in enzyme and purified spacer incorporated enzyme by simple dialysis or column chromatography. To this spacer incorporated enzyme carboxyl derivative of PSL was coupled. We have incorporated four homobifunctional molecules, ADH (10 atomic length spacer), CH (5 atomic length spacer), EDA (4 atomic length spacer) and U (3 atomic length spacer) as spacer between PSL-21-HS and –NH₂ blocked HRP for preparation of the enzyme conjugates. The influences of these spacers on assay parameters like sensitivity, ED₅₀ and specificity have been studied with respect to their length and physiochemical nature.

2.1. Determination of hapten density by MALDI-TOF spectrometry

2.1.1. In immunogen

The molecular weights of PSL-21-HS, BSA and PSL-21-HS-BSA were determined by MALDI-TOF and found to be 483.737 Kelo Dalton (KDa), 69144.654 KDa and 66528.848 KDa respectively as shown in Fig. 1. The quantity of hapten (PSL) density was calculated by the formula given under section 2.6 and found to be 5.40 per molecule of BSA as shown in Table 1.

Table 1

Estimation of hapten density in immunogen.
Sample	Analyte MW (KDa)	Conjugate MW (KDa)	Change in mass (Conjugate MW – BSA MW)	Hapten density (Change in mass / hapten M.W.)
Prednisolone (PSL)-21-HS	483.737		-	-
Bovine Serum Albumin (BSA)	66528.848		-	-
PSL-21-HS-BSA	-	69144.654	2615.806	5.40

2.1.2. In enzyme conjugates.

The molecular weights of HRP, PSL-21-HS, PSL-21-HS-ADH-HRP, PSL-21-HS-CH-HRP, PSL-21-HS-EDA-HRPand PSL-21-HS-U-HRP were determined by MALDI-TOF and found to be 43348.001 KDa, 483.737 KDa, 50231.004 KDa, 49524.341 KDa, 46221.734 KDa, and 48962.172 KDa, respectively as shown in Fig. 2. The quantity of hapten (PSL) density was calculated by the formula given under section 2.6 and found to be 10.46, 10.76, 5.28 and 10.32 per molecule of PSL-21-HS-ADH-HRP, PSL-21-HS-CH-HRP, PSL-21-HS-EDA-HRP and PSL-21-HS-U-HRP enzyme conjugates respectively. The observed mass, changes in mass and the hapten density of enzyme conjugates are shown in Table 2.

Table 2

Estimation of hapten density in enzyme conjugate.
Sample	Analyte MW	Conjugate MW(KDa)	Change in mass (conjugate MW – HRP MW)	Hapten density (change in mass / hapten + spacer M.W.)
Horseradish peroxidase (HRP)	43348.001 KDa
Prednisolone-21-hemisuccinate (PSL-21-HS)	483.737 KDa
Adipic acid dihydrazide (ADH)	174.2 g/mol
PSL-21-HS-ADH-HRP	--	50231.004	6883.00	10.46
Carbohydrazide (CH)	90.09 g/mol
PSL-21-HS-CH-HRP	--	49524.341	6176.34	10.76
Ethylenediamine (EDA)	60.10 g/mol
PSL-21-HS-EDA-HRP	--	46221.734	2873.733	5.28
Urea (U)	60.06 g/mol
PSL-21-HS-U-HRP	--	48962.172	5614.171	10.32

2.2. Dose-Response study

The dose-response studies of the four enzyme conjugates PSL-21-HS-ADH-HRP, PSL-21-HS-CH-HRP, PSL-21-HS-EDA-HRP and PSL-21-HS-U-HRP were carried out with the antibody raised against PSL-21-HS-BSA. Figure 3 depicts the composite standard curves of ELISA for prednisolone using PSL-21-HS-BSA antibody with PSL-21-HS-HRP (with different length spacer) enzyme conjugates, where concentrations of PSL are plotted on the X axis and bound fraction (A/A0%) on the Y axis. The CVs for the A/A0 ratio of each standard ranged from 1.38 to 9.93% for the enzyme immunosorbent assays. Each value is a mean ± SD of eight assays (in duplicate). Thus, the standard curves obtained over several assays remained stable and precise. The slope and intercept of the curves were calculated by logit–log transformation of standard curve data, the affinity constant of the PSL antibody for the PSL was estimated by a Scatchard plot and the goodness of fit as the estimated non-linear squared correlation coefficient (R²) of the standard data and is shown in Table 3.

2.3. Sensitivity

The lower detection limit of the PSL assay after insertion of spacers in enzyme conjugate were 1.22 ng/mL (ADH spacer), 0.59 ng/mL (CH spacer), 0.48 ng/mL (EDA spacer) and 0.018 ng/mL (urea spacer) and shown in Table 3. Among all four combinations, PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity.

Table 3

Slope (m), Intercept (c), Sensitivity, Affinity, ED₅₀, and R² of prednisolone assay, using PSL-21-HS-BSA-antibody and PSL-21-HS-HRP (with spacers) enzyme conjugates.
Assay combination of PSL-21-HS-BSA- antibody with	Slope (m) and intercept (c)		Sensitivity of the assay (ng/mL)	Affinity of the assay (mole/L)	ED₅₀ (ng/mL)	R²
Assay combination of PSL-21-HS-BSA- antibody with	m	c	Sensitivity of the assay (ng/mL)	Affinity of the assay (mole/L)	ED₅₀ (ng/mL)	R²
PSL-21-HS-ADH-HRP Enzyme conjugate	-2.47	2.06	1.22	1.18 x 10⁻⁷	6.79	0.9
PSL-21-HS-CH-HRP Enzyme conjugate	-1.80	1.87	0.59	5.07 x 10⁻⁸	4.03	1.0
PSL-21-HS-EDA-HRP Enzyme conjugate	-2.15	1.61	0.48	4.71 x 10⁻⁸	3.60	1.0
PSL-21-HS-U-HRP Enzyme conjugate	-1.84	1.03	0.018	5.09 x 10⁻⁸	1.89	1.0

2.4. Specificity

The specificity of the anti- PSL-21-HS-BSA-antiserum with PSL-21-HS-U-HRP, PSL-21-HS-EDA-HRP, PSL-21-HS-CH-HRP, and PSL-21-HS-ADH-HRP was estimated as the percentage of cross-reaction with commercially available fifty-three analogous steroids, only 4 to 5 steroids showed cross-reaction and is shown in Table 4. Among all four combinations, PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better specificity. The percent cross-reaction was calculated from the following formula:

$$\text{c}\text{r}\text{o}\text{s}\text{s} \text{r}\text{e}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n} = \frac{\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{p}\text{r}\text{e}\text{d}\text{n}\text{i}\text{s}\text{o}\text{l}\text{o}\text{n}\text{e}, \text{r}\text{e}\text{q}\text{u}\text{i}\text{r}\text{e}\text{d} \text{f}\text{o}\text{r} 50\text{%} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}}{\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{r}\text{e}\text{l}\text{a}\text{t}\text{e}\text{d} \text{s}\text{t}\text{e}\text{r}\text{o}\text{i}\text{d}, \text{r}\text{e}\text{q}\text{u}\text{i}\text{r}\text{e}\text{d} \text{f}\text{o}\text{r} 50\text{%} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} }\text{X} 100$$

Table 4

Cross-reactivity of analogous steroids in bridge heterologous ELISA of PSL using PSL-21-HS-BSA antibody and PSL-21-HS-HRP (with spacers) enzymes conjugate.
Steroid Measured	% Cross reactions of with PSL-21-HS-EDA-HRP	% Cross reactions of with PSL − 21-HS-Urea-HRP	% Cross reactions of with PSL-21-HS-CH-HRP	% Cross reactions of with PSL-21-HS-ADH-HRP
C-27 Steroid
Cholesterol	< 0.025	< 0.025	< 0.025	< 0.025
C-22 Steroid
Danazol	< 0.025	< 0.025	< 0.025	< 0.025
C-21 Steroid
Progesterone	4.7	1.65	2.21	9.46
5 α Dehydro Progesterone	< 0.025	< 0.025	< 0.025	< 0.025
5 β Dehydro Progesterone	< 0.025	< 0.025	< 0.025	< 0.025
17α 20β Dioh Progesterone	< 0.025	< 0.025	< 0.025	< 0.025
17α 20 α Dioh Progesterone	< 0.025	< 0.025	< 0.025	< 0.025
11α OH Progesterone	< 0.025	< 0.025	< 0.025	< 0.025
16-dehydroprogesterone	< 0.025	< 0.025	< 0.025	< 0.025
Medroxy Progesterone Acetate (MPA)	< 0.025	< 0.025	< 0.025	< 0.025
Pregnenolone	< 0.025	< 0.025	< 0.025	< 0.025
17α-OH progesterone	21.25	6	12.5	14
17α-OH pregnenolone	< 0.025	< 0.025	< 0.025	< 0.025
5 Pregnene 3 β 20 α diOL	< 0.025	< 0.025	< 0.025	< 0.025
5α Pregnane 3β, 20α diOL	< 0.025	< 0.025	< 0.025	< 0.025
5α Dihydropregnanolone	< 0.025	< 0.025	< 0.025	< 0.025
5β-pregnane-3,20-dione	< 0.025	< 0.025	< 0.025	< 0.025
Betamethosone	< 0.025	< 0.025	< 0.025	< 0.025
Pregnanediol	< 0.025	< 0.025	< 0.025	< 0.025
Cortisol	11.6	4.5	12.5	10.4
5 α Dihydro Cortisol	< 0.025	< 0.025	< 0.025	< 0.025
5 β Dihydro Cortisol	< 0.025	< 0.025	< 0.025	< 0.025
Prednisolone	100	100	100	100
Prednisone	42	8	27	24
Aldosterone	< 0.025	< 0.025	< 0.025	< 0.025
Dexamethasone	< 0.025	< 0.025	< 0.025	< 0.025
Flutamide	< 0.025	< 0.025	< 0.025	< 0.025
Corticosterone	8.6	< 0.025	7.48	4.44
5 α Dihydro Corticosterone	< 0.025	< 0.025	< 0.025	< 0.025
5 β Dihydro Corticosterone	< 0.025	< 0.025	< 0.025	< 0.025
Cortisone	< 0.025	< 0.025	< 0.025	< 0.025
5 α Dihydro Cortisone	< 0.025	< 0.025	< 0.025	< 0.025
5 β Dihydro Cortisone	< 0.025	< 0.025	< 0.025	< 0.025
Deoxycorticosterone (DOC)	< 0.025	< 0.025	< 0.025	< 0.025
C-19 Steroid
Testosterone	< 0.025	< 0.025	< 0.025	< 0.025
6 Dehydro Testosterone	< 0.025	< 0.025	< 0.025	< 0.025
17 α Methyl Testosterone	< 0.025	< 0.025	< 0.025	< 0.025
11 Keto testosterone	< 0.025	< 0.025	< 0.025	< 0.025
Dihydrotestosterone (DHT)	< 0.025	< 0.025	< 0.025	< 0.025
Etiocholanolone	< 0.025	< 0.025	< 0.025	< 0.025
Dehydroepiandrosterone (DHEA)	< 0.025	< 0.025	< 0.025	< 0.025
Dehydroepiandrosterone Sulfate (DHEAS)	< 0.025	< 0.025	< 0.025	< 0.025
Dehydroisoandrosterone	< 0.025	< 0.025	< 0.025	< 0.025
Androstenedione	< 0.025	< 0.025	< 0.025	< 0.025
Androstanedione	< 0.025	< 0.025	< 0.025	< 0.025
Androsten 3 β, 17 β -Diol	< 0.025	< 0.025	< 0.025	< 0.025
Androstenediol	< 0.025	< 0.025	< 0.025	< 0.025
Mesterolone	< 0.025	< 0.025	< 0.025	< 0.025
Nandrolone	< 0.025	< 0.025	< 0.025	< 0.025
C-18 Steroid
Estrone	< 0.025	< 0.025	< 0.025	< 0.025
Estrone 3-Glu (E₁ 3G)	< 0.025	< 0.025	< 0.025	< 0.025
Estradiol	< 0.025	< 0.025	< 0.025	< 0.025
Estriol	< 0.025	< 0.025	< 0.025	< 0.025
2 Methoxy Estradiol	< 0.025	< 0.025	< 0.025	< 0.025

2.5. Selection of best combination of antibody and enzyme conjugate

Since, among all four combinations, anti-PSL-21-HS-BSA and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity, ED₅₀, affinity and specificity thus, this combination was further studied for analytical variables such as recovery, precision and correlation coefficient.

2.6. Recovery

The ability of an assay to accurately quantify PSL in serum was tested. Table 5 represents the percent recoveries of known amounts of PSL added to five aliquots of serum pools. After spiking, the concentration of PSL was measured and recovery was calculated for each pool. The recovery ranged from 88.32–102.50%.

The percentage of recovery was calculated by using the formula: (C - B)/A * 100. Where A = a known amount of analyte, B = a base (B) and C = measuring the concentration.

Table 5

Recovery of prednisolone from exogenously spiked human serum pools using PSL-21-HS-BSA-antibody and PSL-21-HS-Urea-HRP-enzyme conjugate.
Human serum pools	Prednisolone added (ng/mL)	Prednisolone observed (ng/mL)	Prednisolone expected (ng/mL)	% Recovery
Pool A (Basal)	–	0.75	--	–
Pool B	0.62	1.21	1.37	88.32
Pool C	1.25	2.05	2.00	102.50
Pool D	5.0	5.55	5.75	98.26
Pool E	15.0	`15.50	15.75	98.41
Pool F	25.0	25.80	25.75	100.19

2.7. Precision

The levels of precision are estimated by studying the intra-assay and inter assay variations. Serum pools of very low, low, medium, high-medium, and high PSL concentrations were utilized for determining the level of imprecision in assay by estimating PSL in each pool for eight times in assay and in eight different assays. Table 6 depicts the intra and inter-assay coefficient of variations. The coefficient of variation (CVs) of five serum pools for intra and inter-assay variation (n = 8 and N = 8, replicate of each pool) was < 8.46%. The % coefficient of variation is calculated as % CV = (SD/X) * 100 at a particular analyte level. Where, X = mean, SD = standard deviation and CV = coefficient of variation.

The intra-assay coefficients of variation range from 2.50–7.09% whereas, the inter-assay coefficients of variation range from 1.47– 8.46%.

Table 6

Inter and intra-assay coefficient of variation for the measurement of prednisolone in human serum pools using PSL-21-HS-BSA-antibody and PSL-21-HS-U-HRP-enzyme conjugate.
Variation	Sample value (ng/mL) (mean ± S.D.)	Coefficient of variation (%)
Intra-assay n = 8	0.80 ± 0.02 1.41 ± 0.10 5.45 ± 0.21 15.72 ± 0.84 25.85 ± 0.94	2.50 7.09 3.85 5.34 3.63
Inter-assay N = 8	0.77 ± 0. 05 1.30 ± 0.11 14.92 ± 0.22 15.72 ± 0.44 26.02 ± 1.04	6.49 8.46 1.47 2.79 3.99

2.8. Correlation coefficient

The newly developed technique must yield results analogous to those of the already available conventional method. The correlation coefficient for values of PSL in serum samples (n = 103) measured by developed ELISA and ELISA kit of prednisolone purchased from Sincere Biotech Co. Ltd., Beijing, China was found to be r² = 0.98. The regression analysis has been performed in which both X and Y was subject for measurement error. Figure 4 shows that method fit a straight line to a two dimensional data where both the variables, X and Y, are measured with errors that can accommodate differences in measurement error between the test and the reference method. The linear regression curve of the correlated data (plotted by Graph Pad Prism version 6.0 for MS windows).

2.9. Estimation of prednisolone in patient treated for different diseases

We determined the prednisolone concentration in human serum using samples from healthy volunteers (17 male, 11 female) and in human patient taking treatment with prednisolone drug through oral, local or injection form in diseases of chronic asthma (20 male & 05 female), rheumatoid arthritis (16 male & 11 female), allergic (11 male & 12 female) age group between 10 to 72 years. Samples value as determined by the developed ELISA are described in Table 7 and also presented in the form of Box-and-Whisker diagram (plotted by Graph Pad Prism version 6.0 for MS windows) shown in Fig. 5.

Table 7

Estimation of prednisolone by developed ELISA in human serum of healthy volunteers and patient taking treatment.
Variable	Healthy		Asthma		Rheumatic		Allergic
Gender	Male	Female	Male	Female	Male	Female	Male	Female
No. of samples	17	11	20	5	16	11	11	12
Age	18–64		10–72		40–62		16–60
Prednisolone (mean ± SD) (ng/mL)	(0.00 ± 0.00)		(24.06 ± 8.09)		(8.17 ± 3.15)		(1.80 ± 1.24)

In the present study, we described the, use of different spacers in enzyme conjugates for development of a sensitive and specific ELISA for prednisolone. For that we have incorporated four homo-bifunctional spacers namely; ADH, CH, EDA and U between PSL-21-HS carboxyl derivative of prednisolone and HRP. Spacer molecules containing an amine at both ends are often used to create a primary amine on a support for further modification or for the coupling of carboxylate containing ligands. These bifunctional spacers introduce few atoms chain between steroid moiety and carrier protein or enzyme or label. The terminal functional groups of these spacers do not spontaneously react with functional group of steroid/carrier protein/enzyme, and therefore, require some bioconjugate reagent as an intermediate to carry out the effective coupling of these spacers [10]. Carbodiimide along with N-hydroxysuccinimide has been the reagent of choice for coupling steroid and proteins through spacers, as they do not introduce any additional atoms in the conjugate.

In the present study, four enzyme conjugates were prepared using PSL-21-HS as a carboxylic derivative of prednisolone and horseradish peroxidase (HRP) as an enzyme label. These were PSL-21-HS -U-HRP (urea as 3 atomic length spacer), PSL-21-HS-EDA-HRP (ethylenediamine as 4 atomic length spacer), PSL-21-HS-CH-HRP (carbohydrazide as 5 atomic length spacer), and PSL-21-HS-ADH-HRP (adipic acid dihydrazide-aminocaproic acid as 10 atomic length spacer). The influence of different length spacers on the functional parameters of bridge heterologous prednisolone ELISA was studied.

The data of the present investigation reveal that in the lower detection limit of the PSL assay after insertion of spacers in enzyme conjugate were 1.22ng/mL (ADH spacer), 0.59ng/mL (CH spacer), 0.48ng/mL (EDA spacer) 0.018ng/mL (urea spacer). The ED₅₀ of the assay was 6.79 ng/mL, 4.03 ng/mL and 3.60 ng/mL and 1.89 ng/mL with ADH, CH, EDA and U respectively. Among all four combinations, PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity and ED₅₀.

Urea is a having three atomic length molecule and non-aliphatic, hydrophilic, rigid in nature due to the presence of double bond and lacks the flexibility in bending towards enzyme. This rigid nature in urea might have resulted in distance between the prednisolone moiety (PSL-21-HS) and enzyme and might have reduced the steric hindrance caused by enzyme due to its bulkiness and rendered easy access to unlabeled prednisolone to bind antibody. The present finding also suggests that the nature of the spacer (hydrophilic, hydrophobic, flexible, or rigid) is related to assay sensitivity and not to spacer length. Hence, introduction of urea between steroid derivative and enzyme helped in reduction of the local environment effect and steric hindrance between the two high molecular weight proteins, i.e. antibody and label. It has also been previously reported that the spacer length does not bear any correlation with the assay sensitivity in ELISA^{1–5,11,19,20} as it does have an effect in some other immunoassay and auxiliary binding systems ^9–11,20. The finding of present study also support the studies carried out by Nara et al. (2008), Shrivastav et al. (2012, 2013) and Sathe et al. (2016); in which insertion of hydrophilic spacer resulted in the enhancement of sensitivity and specificity of assay. Possibly the use of a hydrophilic and rigid spacer helped in projecting the hapten away from enzyme, leading to enhanced antibody binding signal and improved sensitivity of the assay ^9–11,20.

As the specificity of the antibody mostly depends on the site of the steroid molecule used to conjugate with the carrier protein to prepare the immunogen ¹⁴, it is not always possible to develop a specific assay using specific heterologous assay. The % cross-reaction of analogous steroids in the assay after insertion of spacers in enzyme conjugate were marginally different. The cross reaction data of present study indicated that enzyme conjugate having urea spacer showed less cross reaction as compared to other spacers (ADH, CH and EDA). This might be due to rigid nature of urea which might have projected PSL away from enzyme this might have kept bound antibody away thereby reduction in overall attraction between enzyme and antibody and allowed specific steroid and bound to antibody.

Since the assay with PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity, ED₅₀, affinity and specificity in comparison with other spacer containg enzyme conjugates, therefore; this combination was further studied for analytical variables such as recovery, precision, correlation coefficient and estimated prednisolone in clinical samples. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32 to 102.50%. The intra assay and inter assay CV% was < 8.46%. The correlation coefficient for values of prednisolone in serum samples (n = 103) measured by developed ELISA and commercially available assay was found to be r² = 0.98.

The results of the present study revealed that the developed bridge heterologous assay for detection of PSL is simpler than the commercial used method. The major difference of commercial ELISA and newly developed ELISA are: 1) In commercial kit standards were prepared freshly by serially diluting stock by standard diluent on the other hand newly developed assay do not required any such preparation. 2) The immunoassay procedure of commercial kit is two step whereas, in-house develop assay is a single step. 3) There are two washing steps in immunoassay procedure of commercial kit whereas, in-house developed immunoassay procedure has only one washing step. 4) The intra and inter assay % CV of immunoassay procedure of commercial kit was ≤ 15% whereas, intra assay and inter assay CV % of in-house developed immunoassay procedure was < 8.46%. 5). Recovery percent from exogenously spiked PSL and % cross-reaction with analogous steroids were not provided in the commercial kit used.

To the best of our knowledge, there is no any reported literature exists on the comparison of the bridge heterologous in enzyme conjugates on analytical variables of ELISA for the development of prednisolone. All the reagents such as buffers, immunogen, antigen-enzyme conjugate and antibody generation were indigenously developed in house.

In the present study, the increase in sensitivity and improvement in specificity of the developed assay was might be due to rigid structure of urea in comparison to other spacers. This rigidity in urea may be due to the resonance or mesomeric effect between carbonyl carbon (-C = O) and –NH₂ groups along the entire molecule. Due to this rigid nature of urea it might have projected PSL moiety away from enzyme that might have helped in keeping bound antibody away from enzyme thereby reduction in overall attraction between enzyme and antibody, simultaneously, resulting in reducing steric hindrance and rendering easy access to unlabelled PSL to bind to antibody.

The experimental protocols were approved by the institutional animal ethics committee (IAEC) of the National Institute of Health and Family Welfare (NIHFW), New Delhi, India. All animal experiments were performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

5.1 Materials

All solvents, chemicals, and salts used in the present study were of analytical grade and were used without prior purification. All steroids used for the conjugation and cross-reactivity were obtained from Steraloids, Inc. (Newport, RI, USA). Bovine serum albumin (BSA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC.HCl), Freund’s adjuvant complete (FAC), Dioxan, dimethyl formamide (DMF), Tetramethylbenzidine (TMB), Hydrogen peroxide urea (H₂O₂.U)/carbamide peroxide, ADH, EDA, CH, urea and thimerosal were purchased from Sigma Chemical Company (St. Louis, MO, USA). Horseradish peroxidase was purchased from Bangalore Genei, Bangalore, India. ELISA kit of prednisolone purchased from Sincere Biotech Co. Ltd., Beijing, China. Microtitre plates were procured from Corning Life sciences (Tewksbury, MA 01876, USA).

5.2. Instrumentation

Tecan Spectra micro-plate reader from Tecan Austria GmbH (5082 Grödig, Austria). Haryson lyophilizer from Haryson (New Delhi, India) and Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometer from Bruker daltonic, Germany.

5.3. Buffers compositions

Buffer ‘A’

Coating buffer pH 7.2 (10 mM PBS) was prepared using sodium phosphate dibasic (Na₂HPO₄.2H₂O) 1.1 g/L, sodium phosphate monobasic dihydrate (NaH₂PO₄.2H₂O) 0.52 g/L, sodium chloride (NaCl) 9 g/L and sodium azide (NaN₃) 1 g/L.

Buffer ‘B’

Enzyme conjugate dilution buffer was prepared by using Tris 2.42 g/L, NaCl 17.9 g/L, BSA 1.0 g/L, tween-80 1 mL/L, dextran 3 g/L, striped serum 50 mL/L, thimerosal, 0.5 g/L and glycerol 5 mL/L, pH adjusted to 8.0 with 1M HCl.

Buffer ‘C’

Antibody dilution buffer was prepared by using Na₂HPO₄.2H₂O 1.1 g/L, NaH₂PO₄.2H₂O 0.52 g/L, sucrose (C₁₂, H₂₂, O₁₁) 90 g/L, ammonium sulfate (NH₄)₂SO₄ 100 g/L, BSA 2 g/L and sodium azide (NaN₃) 0.5 g/L.

Buffer ‘D’

Blocking buffer was prepared using Tween Tris buffered saline (TTBS; 0.1% Tween 20, 50 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4) sucrose 92.0 g/L, bovine serum albumin (BSA) 2 g/L, glycerol 5 mL/L and thimerosal 0.5 g/L.

Buffer ‘E’

Buffer for washing ELISA plates (10 mM PBS containing 0.05% Tween-20). Prepared by adding, 500 µL Tween-20 to 1 litre of PBS.

Buffer ‘F’

Citrate-phosphate buffer, pH 5.0. Citric acid 22.2 mM and Na₂HPO₄ 51.3 mM.

HRP Substrate

Add 200 µL of 41 mM TMB solution in dimethyl sulfoxide (DMSO) having 25 mM sodium tartrate as stabilizer, 3 µL 30% H₂O₂ with 200 ppm of acetanilide as stabilizer to 9.8 ml citrate phosphate buffer, pH 5.0.

Stop solution

1N HCL.

5.4. Preparation of PSL-21-HS-BSA Immunogen

PSL-21-HS was coupled to BSA by using N-hydroxysuccinimide mediated carbodiimide reaction ²¹. The plan of chemical reaction is shown in Scheme 1. In brief, to 5 mg of PSL-21-HS each of 200 µL of DMF, dioxan, and 100 µL of distilled water containing 10 mg of NHS and 20 mg of EDAC.HCl were added into it. The reaction mixture was mixed and kept for activation at 4°C for overnight. Following the overnight activation, aqueous BSA solution (1 mg/0.5 mL) was added to the activated steroid reaction mixture and vortex-mixed and kept at 4 ºC for overnight for the formation of amide bond. The PSL-21-HS-BSA conjugate was dialyzed against distilled water for 3–4 changes at 4 ºC. The dialysate was frozen at -20 ºC, lyophilized and stored at 4 ºC in aliquots of 1 mg for immunization.

5.5. Preparation of Enzyme Conjugate

All the conjugation reactions were carried out as previously described by an active ester method with modification ¹⁰.

5.5.1. Preparation of enzyme conjugates with spacer (PSL-21-HS-ADH-HRP, PSL-21-HS-CH-HRP, PSL-21-HS-EDA-HRP and PSL-21-HS-U-HRP)

The enzyme conjugate with spacers were prepared as shown in the scheme 2 and 3 by active ester method ⁷.

5.5.1.1. Coupling of spacers to HRP

As per scheme 2; Five milligram of NHS-acetic acid was added to 10 mg of HRP per 1mL of milli q water. The reaction mixture was vortex-mixed and kept overnight at 4°C to form amide bond with –NH₂ group of HRP that lead to the formation of acetylated HRP (blocking of the –NH₂ group). Thereafter, the reaction mixture was extensively dialyzed against water. Carboxyl moieties of HRP were activated by addition of 10 mg of NHS plus 20 mg of EDAC.HCl. The activated HRP was then divided into four equal portions; 10 mg each of ADH, CH, EDA and urea was added to the respective activated HRP portion and kept overnight. After overnight incubation, the reaction mixture was extensively dialyzed against water.

5.5.1.2. Coupling of prednisolone to spacer containg HRP

As per scheme 3; to 10 mg of PSL-21-HS, 200 µL each of dioxan and DMF and 100 µL of distilled water containing 10 mg NHS and 20 mg EDAC were added; the reaction mixture was kept for activation for 24 hours at 4°C. Activated PSL-21-HS solution was divided into four equal parts, and each part was added to HRP-U, HRP-EDA, HRP-CH, and ADH-HRP, respectively, and kept for 24 hours at 4°C. Thereafter, each reaction mixture was passed separately through a G-25 column, previously equilibrated with 10 mM PBS containing 0.01% thimerosal. The brown colored fractions containing enzyme activity were pooled, and to them 1% of sucrose, ammonium sulfate, BSA, and an equal volume of ethylene glycol were added. The solution was kept at -30°C for future use ¹⁰.

5.6. Characterization of immunogen and enzyme conjugate by Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) spectrometer.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is effectively utilized for the molecular weight determination of various proteins with a claimed limit of 500 000 Da. The practical limit for routine analysis of proteins is approximately 300 000 Da with a sensitivity in the low pmol range and an accuracy of up to ± 0.1%. Protein-hapten conjugates were characterized by employing MALDI-TOF. The mass of PSL, BSA, HRP, PSL-BSA, PSL-HRP, PSL-ADH-HRP, PSL-CH-HRP, PSL-EDA-HRP and PSL-U-HRP were determined using MALDI-TOF spectrometer. The 1 mg of sample/mL of distilled water was mixed with 1 mL of matrix (3, 5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid) was prepared in 33% acetonitrile) in 1:1 ratio (v/v) thereafter 0.5 µL of 0.1% trifluoroacetic acid (TFA) was added. The samples were analyzed in linear mode with a 337 nm laser light at 30 kV and spectra for each sample was recorded at a threshold laser irradiance for 50 shots and data obtained was analyzed using the software provided with the system ^22,23. Each spectrum contained at least two peaks larger and shorter and are in singly (M + H) ⁺ and doubly (M + 2H)²⁺ charge states. The molecular weight of each sample was calculated from the singly centroid (M + H) ⁺ peak. Numbers of hapten attached to per molecule of protein were determined using the formula:

$$\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{h}\text{a}\text{p}\text{t}\text{e}\text{n} = \frac{\left[\text{c}\text{o}\text{n}\text{j}\text{u}\text{g}\text{a}\text{t}\text{e} \text{m}\text{o}\text{l}\text{e}\text{c}\text{u}\text{l}\text{a}\text{r} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \left(\text{M}\text{W}\right)\right]-\left[\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n} \right(\text{B}\text{S}\text{A}/\text{H}\text{R}\text{P}\left) \text{M}\text{W}\right]}{(\text{h}\text{a}\text{p}\text{t}\text{e}\text{n} \left(\text{P}\text{S}\text{L}\right)+\text{s}\text{p}\text{a}\text{c}\text{e}\text{r} \text{M}\text{W})}$$

5.7. Immunization of rabbits and collection of antiserum

The polyclonal antibody was raised in New Zealand white rabbits against PSL-21-HS-BSA as per procedure described elsewhere ²⁰. Briefly, PSL-21-HS-BSA (1mg) was dissolved in 0.9 %saline (0.5 mL) and emulsified with (0.5 mL) Freund’s (complete) adjuvant. The prepared emulsion (250 µL) was injected intramuscularly in each limb of the rabbits. Primarily, the first five booster injections were prepared with Freund’s adjuvant complete and given per week that was further followed by monthly booster injection prepared using Freund’s adjuvant incomplete. Further, the rabbits were bled after 9–14 days after the booster dose injections. The blood was collected from rabbit and after clotting, antiserum was separated by centrifugation at 1000 g for 15 min. The separated antiserum was collected and stored at -30 ºC.

5.8 Collection of normal rabbit serum (NRS) and generation of anti-rabbit anti-serum in goat (ARASG)

For the collection of normal rabbit serum, blood was withdrawn from the non-immunized New Zealand white rabbits. After clotting the blood was centrifuged at 2000 rpm for 10 min. The separated serum was collected and stored as NRS at -30 ºC until use. The anti-rabbit anti-serum was generated in goat (ARASG) by immunizing immunoglobulin (IgG) of rabbit ²¹. Following, the booster injection blood was collected from the goat and centrifuged after clotting. The obtained antiserum was collected and stored at -30 ºC for further use.

5.9. Checkerboard Assay

A checkerboard assay was performed to obtain the optimal dilutions of antibody and enzyme conjugate for the development of assay.

5.9.1. Coating of antibody to microtitre plates

The 96 well microtitre plates were coated using the immunobridge technique for primary antibody immobilization [11]. Briefly, 250 µL of normal rabbit serum (NRS) diluted (1: 250) in water was dispensed into each well and incubated overnight at 37 ºC. Following incubation, the plate was washed 2–3 times using washing solution (buffer E). To the NRS coated wells, 250 µL of 1:1000 diluted anti-rabbit anti-serums in goat (ARASG) was added and incubated for 5 hours at 37 ºC or overnight at 4 ºC. Thereafter the plate was washed thoroughly using washing solution (buffer E). The raised antiserum against immunogen (PSL-21-HS-BSA) was serially diluted in antibody dilution buffer to 1:500, 1:1000, 1:2000, 1:4000, and 150 µL was added per ARASG coated wells (single dilution per 8 well strip); for non-specific binding (NSB), 150 µL of antibody dilution buffer was added in a separate ARASG coated eight-well strip and incubated for 2 hours at 37 ºC. Unabsorbed antibody was then decanted and microtitre plate was washed with buffer E. Thereafter, 250 µL of buffer ‘D’ was added and kept at room temperature (RT) for 1 hour to block the unoccupied sites of the plate. The contents of the plate were decanted and the plate was dried at RT; packed and stored at 4 ºC in zip-lock bags for further use.

5.9.2. Determination of optimal dilution of primary antibody and enzyme conjugates

To determine the amount of immobilized primary antibody and enzyme conjugates required to develop the assay, 100 µL of serially diluted (1:500, 1:1000, 1:2000, and 1:4000) enzyme conjugate in buffer B (PSL-21-HS-ADH-HRP, or PSL-21-HS-CH-HRP, or PSL-21-HS-EDA-HRP, or PSL-21-HS-U-HRP) was added in the above coated plates for respective assays (one dilution per two wells in vertical fashion). The plates were incubated at RT for 1 hour. Unbound contents were decanted and plate was washed with buffer E. The bound enzyme activity was assessed by adding 100 µL of TMB/H₂O₂ substrate to each well followed by incubation at RT for 15 min. The reaction was stopped by adding 100 µL of 1 N HCl, and the color intensity was measured at 450 nm in a Tecan-spectra ELISA plate reader. The dilutions of antiserum and enzyme conjugate showed maximum zero binding (optical density more than 2.5) and least nonspecific binding (optical density less than 0.1) were selected for each combination for assay development.

5.9.3. Preparation of prednisolone standard

Eight PSL working standards (0.0, 0.62, 1.25, 2.5, 5.0, 10.0, 20.0 and 40.0 ng/mL) were prepared in stripped pooled serum, stripping of steroids from the serum was carried out by adding activated charcoal at a concentration of 50 mg/mL to pooled serum and stirred for two hours at 45 ºC and centrifuged at 3000 x g to remove the charcoal followed by filtration through 0.45 µM membrane filter. Thimerosal, 0.01% was added as a preservative.

5.9.4. Preparation of recovery pools

Six recovery pools were prepared in serum by spiking the serum with different known concentrations of prednisolone, viz., 0.0, 0.62, 2.5, 5.0, 15.0 and 25.0 ng/mL.

5.9.5. Standard displacement assay (assay procedure)

To the antibody-coated wells (PSL-21-HS-BSA-antibody), 100 µL of different concentrations of standard were added in duplicate. 100 µL of the working dilution of the enzyme conjugates (PSL-21-HS-ADH-HRP, or PSL-21-HS-CH-HRP, or PSL-21-HS-EDA-HRP, or PSL-21-HS-U-HRP) was added to all of the wells, the incubation, washing and measurement of bound enzyme activity was as described in section 2.9.2.

5.9.7. Measurement of serum prednisolone by commercial ELISA kit

Five working standards (0, 15, 30, 60, 120 and 240 pg/mL) were freshly prepared by serial diluting stock standard with diluent. To pre-coated wells 10 µL of different concentrations of standards or samples were added followed by 40 µL of sample diluent and incubated for 30 min at 37 ℃. Thereafter the plate was washed thoroughly using washing solution and 50 µL of HRP-conjugate was added to all the wells except the blank well and incubated at 37℃ for 30 min. The wells were again washed with washing solution, and the bound enzyme activity was developed by adding 100 µL of TMB/H₂O₂ substrate to each well and incubating at 37℃ for 15 min. The enzymatic reaction was stopped by adding 100 µL of stop solution, and the color intensity was measured at 450 nm in a Tecan-spectra ELISA plate reader (TECAN, Austria).

5.9.8. Sample collection and storage

Fasting blood collected during 8:00 AM to 9:00 AM from healthy volunteers and patients receiving PSL medication. After clotting, the blood was centrifuged at 2000 rpm for 10 min. The separated serum was collected and stored at -30 ºC until analysis.

6.1. Preparation of standard curve, determination of affinity constant, and sensitivity

Standard curve was plotted by software GraphPad Prism 6.0 and Microsoft Excel program. The concentration was plotted on the X-axis (log scale) and the A/A0x100 on the Y-axis. Values of the unknown samples were calculated by an in-house–developed personal computer program written in QBASIC language using the logit–log linear regression method according to the method described elsewhere ⁵. The affinity constant of the PSL antibody for PSL was estimated by a Scatchard plot according to method described elsewhere ¹⁰. The sensitivity of the developed method was determined according to the method described elsewhere ²¹.

6.2. Goodness of fit

Goodness of fit was expressed as the estimated non-linear squared correlation coefficient (R²) of the standard data. An R² value that approaches 1.0 is indicative of a precise fit for the data to the standard curve.

Prednisolone (PSL), hemisuccinate (HS), bovine serum albumin (BSA), horseradish peroxidase (HRP), adipic acid dihydrazide (ADH), carbohydrazide (CH), ethylenediamine (EDA), urea (U), gamma amino butyric acid (GABA), 6-amino caproic acid (6ACA), oxy diamine (ODA), bis-oxy diamine (BODA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC.HCl), Freund’s adjuvant complete (FAC), dimethyl formamide (DMF), tetramethylbenzidine (TMB), hydrogen peroxide urea (H₂O₂.U), Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and anti-rabbit anti-serum in goat (ARASG).

Authors' contributions

DK, TGT, HS, SCS Conceived and designed the experiments, DK, SCS, HSO, MK, PLB Carried out the experiments DK, HS, TGT, SCS & PLB Analyzed the data DS, HS, BCK, SCS & PLB Wrote and finalized the manuscript. All authors read and approved the final manuscript.

Acknowledgement:

We acknowledge the volunteers for providing their blood for prednisolone estimation, also thankful to Mr. Subodh Jain from Indian Institute of Technology Delhi (IITD) for MALDI-TOF study. Multidisciplinary Research Unit, Maulana Azad Medical College and Associated Hospital, New Delhi acknowledged for providing required facilities for running experiments.

Funding

This study was supported by the National Institute of Health and Family Welfare (NIHFW), New Delhi, India.

Competing interests

The authors declare that they have no competing interests.

Shrivastav, T. G. et al. Influence of different homologous and heterologous combinations of antibodies and enzyme conjugates of dehydroepiandrostosterone on the sensitivity and specificity of DHEA ELISA. J Immunoassay Immunochem 32, 114–127 (2011).
Shrivastav, T. G. et al. Development of heterologous enzyme linked immunosorbent assay for dehydroepiandrosterone in serum. J Immunoassay Immunochem 31, 266–278 (2010).
Shrivastav, T. G. et al. Enzyme linked immunosorbent assay for milk progesterone. J Immunoassay Immunochem 31, 301–313 (2010).
Shrivastav, T. G., Chaube, S. K., Kariya, K. P., Kumar, D. & Singh, R. Antigen heterologous enzyme linked immunosorbent assay for the measurement of DHEA in serum. J Immunoassay Immunochem 32, 326–341 (2011).
Shrivastav, T. G. et al. Influence of different length linker containing DHEA-7-CMO-enzyme conjugates on sensitivity and specificity of DHEA-17-CMO-antibody. J Immunoassay Immunochem 32, 269–283 (2011).
Basu, A. et al. The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten. Clin Chim Acta 366, 287–292 (2006).
Hosoda, H., Kobayashi, N., Ishii, N. & Nambara, T. Bridging phenomena in steroid immunoassays. The effect of bridge length on sensitivity in enzyme immunoassay. Chem Pharm Bull (Tokyo) 34, 2105–2111 (1986).
Shrivastav, T. G., Chaube, S. K., Prasad, P. K. V., Kariya, K. P. & Kumar, D. Heterologous enzyme linked immunosorbent assay for measurement of testosterone in serum. J Immunoassay Immunochem 33, 252–268 (2012).
Nara, S. et al. Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay. Anal Biochem 373, 18–25 (2008).
Shrivastav, T. G., Chaube, S. K., Kariya, K. P., Prasad, P. K. V. & Kumar, D. Influence of different length spacers containing enzyme conjugate on functional parameters of progesterone ELISA. J Immunoassay Immunochem 34, 94–108 (2013).
Shrivastav, T. G. et al. Influence of different length linker containing DHEA-7-cmo-enzyme conjugates on sensitivity and specificity of DHEA-3-hs-antibody. J Immunoassay Immunochem 33, 1–17 (2012).
Hallaj, S. et al. A Heterologous Enzyme Linked Immunosorbant Assay of Morphine Using Penicillinase as Label. Iran J Biotechnol 1, 239–246 (2003).
Tripathi, V. et al. Development of rapid and sensitive one-step direct enzyme linked immunosorbent assay for 17-alpha-OH-progesterone in serum. J Immunoassay Immunochem 29, 117–127 (2008).
Shrivastav, T. G. Incorporation of different bridge length linkers in enzyme and its use in the preparation of enzyme conjugates for immunoassay. J Immunoassay Immunochem 25, 215–225 (2004).
Yang, J. Y. et al. Development of fluorescence polarisation immunoassay for carbofuran in food and environmental water samples. Food Agric Immunol 26, 340–355 (2015).
Li, M. et al. Fluorescence polarization immunoassay for highly efficient detection of clothianidin in agricultural samples. Analytical Methods 6, 6541–6547 (2014).
Zhang, B. et al. Improving detection sensitivity by oriented bioconjugation of antibodies to quantum dots with a flexible spacer arm for immunoassay. RSC Adv 6, 50119–50127 (2016).
Zhang, Q. et al. Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion. Anal Chim Acta 596, 303–311 (2007).
Mitchell, J. S., Wu, Y., Mitchell, J. S. & Wu, Y. Surface Plasmon Resonance Biosensors for Highly Sensitive Detection of Small Biomolecules. Biosensors (Basel) (2010) doi:10.5772/7208.
Sathe, M., Srivastava, S., Agrawal, S. K. & Ghorpade, R. Effect of Spacer and the Enzyme-Linked Immunosorbent Assay. Def Sci J 66, 471–478 (2016).
Kumar, D., Singh, H. & Shrivastava, T. G. Homologous ELISA for detection of prednisolone in human serum. https://doi.org/10.1080/09540105.2017.1376184 29, 369–385 (2017).
Rajesh, K., Rana, K. V. & Suri, C. R. Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring. Bionanoscience 3, 137–144 (2013).
Adamczyk, M., Gebler, J. C. & Mattingly, P. G. Characterization of protein-hapten conjugates. 2. Electrospray mass spectrometry of bovine serum albumin-hapten conjugates. Bioconjug Chem 7, 475–481 (1996).

Schemes 1 and 2 are available in the Supplementary Files section

There is NO Competing Interest.

floatimage1.png
Graphical Abstract
Scheme1.png
Scheme 1. Conjugation of PSL-21-HS (a carboxylic derivative of prednisolone) to BSA.
Scheme2.png
Scheme 2. Blocking of amino group and coupling of spacers (ADH, CH, EDA & U) to HRP

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ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

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Abstract

Figures

Highlights

1. Introduction

2. Results

2.1. Determination of hapten density by MALDI-TOF spectrometry

2.1.1. In immunogen

2.1.2. In enzyme conjugates.

2.2. Dose-Response study

2.3. Sensitivity

2.4. Specificity

2.5. Selection of best combination of antibody and enzyme conjugate

2.6. Recovery

2.7. Precision

2.8. Correlation coefficient

2.9. Estimation of prednisolone in patient treated for different diseases

3. Discussion

4. Conclusions

5. Methods

5.1 Materials

5.2. Instrumentation

5.3. Buffers compositions

5.4. Preparation of PSL-21-HS-BSA Immunogen

5.5. Preparation of Enzyme Conjugate

5.5.1. Preparation of enzyme conjugates with spacer (PSL-21-HS-ADH-HRP, PSL-21-HS-CH-HRP, PSL-21-HS-EDA-HRP and PSL-21-HS-U-HRP)

5.5.1.1. Coupling of spacers to HRP

5.5.1.2. Coupling of prednisolone to spacer containg HRP

5.7. Immunization of rabbits and collection of antiserum

5.8 Collection of normal rabbit serum (NRS) and generation of anti-rabbit anti-serum in goat (ARASG)

5.9. Checkerboard Assay

5.9.1. Coating of antibody to microtitre plates

5.9.2. Determination of optimal dilution of primary antibody and enzyme conjugates

5.9.3. Preparation of prednisolone standard

5.9.4. Preparation of recovery pools

5.9.5. Standard displacement assay (assay procedure)

5.9.7. Measurement of serum prednisolone by commercial ELISA kit

5.9.8. Sample collection and storage

6. Data Analysis

6.1. Preparation of standard curve, determination of affinity constant, and sensitivity

6.2. Goodness of fit

Abbreviations

Declarations

References

Schemes

Additional Declarations

Supplementary Files

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