Analytical Application of Flow Immunosensor in Detection of Thyroxine and Triiodothyronine in Serum

Abstract

In this study, an immunosensor based on kinetic exclusion analysis (KinExA) was used for thyroxine (T4) and triiodothyronine (T3) estimation. A KinExA™ 3200 instrument was used for this analysis, which is an automated flow fluorimeter designed to separate free unbound antibody binding sites in reaction mixtures of antibody, antigen, and antibody–antigen complex. A T3-BSA- and T4-BSA-coated polymethyl methacrylate (PMMA) bead microcolumn is generated inside the flow cell of the instrument. A sample mixture containing T3 and T4 with their respective monoclonal antibodies and their complexes are drawn past the microbead column. The unbound T3 or T4 monoclonal antibody binding sites are captured by their respective T3 and T4 antigens coated on the PMMA beads as bovine serum albumin conjugates. Fluorescently labeled secondary antibodies bind to the T3 or T4 antigen–antibody complex to generate fluorescence intensity for analysis. The limit of detection for the T3 and T4 assays was found to be 0.06 and 1.9 ng mL⁻¹ with acceptable precision values. The convenience of the automated KinExA format may be valuable in medical diagnostic laboratories.

Introduction

The normal amount of production of thyroid hormones is altered with thyroid diseases, and it is estimated that an average of 12%, which equals to almost 20 million of American population, have some kind of altered thyroid function as per the American Thyroid Association (ATA).¹ It has also been established that women are more susceptible to thyroid diseases than men and approximately 1 in 8 women develop a thyroid disorder during her life.^1,2 The thyroid gland produces hormones essential for normal body metabolism.³ TSH stimulates the production and release of primarily triiodothyronine (T3) and thyroxine (T4) from the thyroid gland.⁴ T3 and T4 comprise the major fraction of circulating thyroid hormones. These hormones control the rate at which the body uses energy and are regulated by a feedback system.

Most of the T3 and T4 circulates in the blood bound to thyroxin binding globulin (TBG), thyroxin binding prealbumin (TBPA), and albumin,⁵ whereas only a minute fraction of the circulating total T3 (0.5%) and T4 (0.03%) is unbound or “free.”^6,7 Blood estimations are performed to check the adequacy of the levels of thyroid hormones that can be normal, underactive, or overactive. Blood tests can measure total T3, free T3 or total T4, free T4.⁸ Therefore, it is being suggested to quantify free thyroid hormones for more accurate depiction of thyroid hormone function.⁹

Several new diagnostic tools are available now for the estimation of free thyroid hormone concentrations in serum.^10
–12 The introduction of radioimmune assay kits for free thyroid hormone estimation has attracted much attention than all other thyroid function tests, but has a drawback of handling radioactive antigen.¹³ Another accurate method of assessing thyroid function is to measure the concentration of free hormones using an equilibrium dialysis technique.^14,15 The equilibrium dialysis technique is time-consuming, expensive, and demands greater technical expertise. These drawbacks have led to the requirement for new, alternate, and competent diagnostic tools for the estimation of T3 and T4.

Immunosensors represent the most promising and outstanding technological progress in the field of clinical and biochemical estimations.^16

–19 These devices analyze the formation of antigen–antibody complex in real time, which is converted into a signal through a signal transducer and transmitted to a detector. The concentration of the analyte present is calculated as it is directly proportional to the signal generated. This study describes the development and validation of a new immunosensor-based assay for the estimation of total T3 and T4 in serum, employing the kinetic exclusion analysis (KinExA)-automated instrument. This instrument presents high sensitivity to quantify unchanged molecules in solution and hence provides an excellent platform to measure the interaction of binding partners (antibody/antigen).^20

–23 This assay is based on the measurement of signal generated from unbound T3 and T4 molecules in solution, instead of immobilized antigen or antibody on solid phase as is done in conventional enzyme-linked immunosorbent assays (ELISA) (Fig. 1).

Fig. 1.

Schematic representation of the immunosensor for estimation of T3 and T4. (A) The antigen and its antibody mixture are allowed to reach the equilibrium. (B) The equilibrated mixture consisting of free antigen, its specific antibody, and the antigen–antibody complex is rapidly drawn past the microbead column coated with antigen. Free antibodies in the equilibrated mixture are captured on the beads, whereas, an antigen–antibody complex and free antigen are washed away. (C) For estimation of the bound antibody as secondary antibody, fluorescently labeled anti-mouse IgG antibody is used. KinExA Pro 20.0.1.26 software is used to capture the data generated. (D) Curves generated (Curves 1–6) in the experiment corresponds represent the concentrations of antigen between zero and saturation. The change in the curve at position (arrow 1) indicates the introduction of fluorescently labeled secondary antibody; and the change in the curve at arrow 2 indicates the end of labeled secondary antibody and commencement of a buffer wash. T3, triiodothyronine; T4, thyroxine.

Materials and Methods

Instrument and Materials

KinExA™ 3200, KinExA Pro 20.0.1.26 software, and polymethyl methacrylate (PMMA) beads (140–170 mesh, 98 μm) were acquired from Sapidyne Instruments, Inc. (Boise, ID). Triiodothyronine-BSA conjugate, thyroxine-BSA conjugate, and their mouse monoclonal antibodies were obtained from MyBioSource (San Diego, CA), T3 and T4 hormones were purchased from Sigma Chemical Co., (St Louis, MO), and IgG secondary antibody (DyLight™ 649-conjugated Affini Pure goat anti-mouse secondary antibody) from Jackson Immuno Research Laboratories Inc. (West Grove, PA). Serum from healthy human volunteers was obtained from King Khalid University (Riyadh, Saudi Arabia). Ethical guidelines for the care and use of human samples in experiments were strictly followed. All the chemicals used, including BSA, were purchased from Sigma Chemical Co.

Preparation of Solutions

Blocking solution: 100 mg of BSA was weighed and dissolved in 10 mL of PBS to obtain a concentration of 10 mg mL⁻¹.

Standard KinExA sample buffer: 1 mg mL⁻¹ BSA and 0.02% sodium azide in PBS (pH 7.4).

The secondary antibody: Freshly prepared DyLight 649-conjugated goat anti-mouse IgG (1.5 mg mL⁻¹) was diluted with a sample buffer to get a final concentration of (150 ng mL⁻¹) for use in the analysis for both T3 and T4.

T3 and T4 Coating on PMMA Beads

T3-BSA conjugate and T4-BSA conjugate were coated (Table 1) separately on 200 mg of PMMA beads present in bead vials. One microliter of PBS containing 23 μg of T3-BSA conjugate and 35 μg of T4-BSA conjugate was added to separate PMMA bead vials and kept on rocking/tumbling apparatus (nutating mixer) for 2 h at room temperature. The vials were removed from the rocking/tumbling apparatus and allowed to settle down. Once the beads were settled at the base of vial, the supernatant was removed and replaced with blocking solution. The bead vials were again loaded over the rocking/tumbling apparatus for another 1 h. The vials were removed from the apparatus and stored at 2°C–8°C, and were stable for 1 week in this condition.

Table 1.

Experimental Protocol Table for T3 and T4

Step	Parameter	Value	Description
A. Bead coating (1–4)
1	PMMA beads 200 mg	23 μg	T3-BSA conjugate in 1 mL PBS
		35 μg	T4-BSA conjugate in 1 mL PBS
2	Nutating mixer	2 h	Room temperature
3	Supernatant removed and replaced	10 mg mL⁻¹	Replace with blocking solution
4	Nutating mixer	1 h	Room temperature and store
B. Sample preparation (6–8)
6	Antibody solution	45 ng mL⁻¹	Anti-T3 monoclonal antibody
		30 ng mL⁻¹	Anti-T4 monoclonal antibody
7	Calibration standards range	0.01 to 30 ng mL⁻¹	T3 Antigen
		0.5 to 1,000 ng mL-1	T4 Antigen
8	Equilibration time	1.5 h	T3 immune complex
		2.0 h	T4 Immune complex
C. Instrumental conditions (9–16)
Microbead column preparation
9	Flow rate	1.5 mL min⁻¹	T3-coated PMMA beads
		1.0 mL min⁻¹	T4-coated PMMA beads
10	Time of flow	35 s	T3-coated PMMA beads
		45 s	T4-coated PMMA beads
Sample run
11	Sample inlet	13	T3- or T4-equilibrated samples
12	Sample volume	700	T3-equilibrated complex
		500	T4-equilibrated complex
13	Flow rate	0.35 mL min⁻¹	T3-equilibrated complex
		0.25 mL min⁻¹	T4-equilibrated complex
14	Buffer wash	50 s	PBS
		30 s	PBS
15	Secondary antibody	833 μL	DyLight™ 649-conjugated goat anti-mouse IgG
		500 μL	DyLight 649-conjugated goat anti-mouse IgG
16	Buffer wash	2.0 mL	PBS
		1.5 mL	PBS

Step Notes

1–4. Coated PMMA beads obtained were stored at 2°C–8°C and were stable for 1 week.

6. Antibody solution was prepared in fresh standard KinExA buffer consisting of 1 mg mL⁻¹ BSA, 0.02% sodium azide in PBS (pH 7.4).

9. Bead column height was adjusted with help of flow cell camera, the beads were charged twice, and the first run was discarded to achieve proper bead column height.

11. Samples drawn from the inlets were passed over the bead column.

T3, triiodothyronine; T4, thyroxine; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PMMA, polymethyl methacrylate.

Preparation of Samples

Calibration standards for T3 and T4 were prepared by spiking blank human serum with T3 and T4 (Table 1). The calibration standards for T3 ranged from 0.01 to 30 ng mL⁻¹ and T4 ranged from 0.5 to 1,000 ng mL⁻¹. Anti-T3 monoclonal antibody (45 ng mL⁻¹) and anti–T4 monoclonal antibody (30 ng mL⁻¹) were prepared in standard KinExA buffer. The prepared anti-T3 and anti-T4 antibody solutions were distributed (constant volume) in separate tubes. The T3- and T4-spiked serum samples of similar volume and definite concentrations were added to their respective anti-T3 or anti-T4 monoclonal antibody antibodies. The resulting samples were filtered with syringe filters of 5 μm pore size to remove any solid matter that can choke the instrument lines. The filtered sample tubes were loaded on the sample rack with the sample inlet tubes inside these samples. The samples were placed in the order that inlet-1 contained the sample of highest concentration and inlet-12 the lowest. The inlet-13 contained the blank sample with only the anti-T3 monoclonal antibody or anti-T4 monoclonal antibody, without their respective antigens T3 or T4 (zero concentration). The sample lines were charged with these solutions and equilibrated for a period or 1.5 h in case of T3 and 2 h in case of T4 before the actual run.

KinExA Instrument Working and Analysis

The schematic working of KinExA instrument is provided in Figure 1 and experimental protocol in Table 1. The secondary antibody (fluorescently labeled goat anti-mouse IgG antibody) was infused through a separate inlet. The T3- and T4-coated bead vials were transferred to the bead bottle reservoir and 30 mL of PBS was added to this reservoir. The bottle reservoir was attached to the bead inlet of the instrument. The bead inlet of KinExA 3200 contains a stirrer, which operates at the start of each bead charge, ensuring the beads remain in suspension and not settle down at the bottom of the bottle reservoir while being charged into the microbead column. To ensure the proper bead height during the run of experiment, the beads were charged initially twice and the first charge was discarded. The instrument is provided with a camera in the flow cell to monitor the bead height inside the microcolumn. The desired column height is achieved by optimizing the rate of flow and volume of the microbeads from the reservoir. Under optimized conditions, identical and reproducible bead columns were produced for T3 at a flow rate of 1.5 mL min⁻¹ for 35 s, gave a flow volume 875 μL, and for T4 analysis at a flow rate of 1 mL min⁻¹ for 45 s, gave a flow volume 700 μL.

Once the sample equilibration is complete, the instrument starts to run the samples in the order from inlet 1 to inlet 13. As soon as the first run is over, a duplicate run for all the 13 samples starts and the results are defined as the mean of the two runs. For the analysis of T3 samples, 700 μL of each sample was injected at a flow rate of 0.35 mL min⁻¹ and passed over the T3-BSA-coated microbead column, whereas in case of T4 samples, 500 μL of each sample was injected at a flow rate of 0.25 mL min⁻¹ and passed over the T4-BSA-coated microbead column. After completion of each T3 or T4 injection, a buffer wash followed to remove unbound primary antibody. The buffer wash consisted of 208 μL of PBS for 50 s for T3 and 125 μL of PBS for 30 s for T4 at a flow rate of 0.25 mL min⁻¹. Injection of secondary antibody solution, 833 μL for 200 s in case of T3 and 500 μL for 120 s for T4 at a flow rate of 0.25 mL min⁻¹ that passed over the microbead column in a similar way as that of T3 and T4 samples, followed the washing step. This step was followed by another buffer wash of 2.0 mL for 120 s at a flow rate of 1 mL min⁻¹ for T3 and 1.5 mL for 90 s at a flow rate of 1 mL min⁻¹ for T4 to remove excess of labeled secondary antibody, which had remained unbound to the microbead column.

Kinetic Exclusion Assay instrument KinExA 3200 was run with the help of KinExA Pro 20.0.1.26 software (Sapidyne Instruments, Inc.). The analysis of the experimental results was performed using Graph Pad Prism 6 (Graph Pad Software, Inc., La Jolla, CA). The calibration curves were generated with the help of the 4 Parameter Logistic or 4PL nonlinear regression model represented by \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{F}} = { \rm{F}}0 - \{ \left( {{ \rm{F}}0 - { \rm{F}}1} \right) \ \left[ {{ \rm{T}}3} \right] / \left( {{ \rm{I}}{{ \rm{C}}_{50}} + \left[ {{ \rm{T}}3} \right] } \right) \} \qquad\qquad {{Equation \ 1}} \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{ F}} = { \rm{F}}0 - \{ \left( {{ \rm{F}}0 - { \rm{F}}1} \right) \ \left[ {{ \rm{T}}4} \right] / \left( {{ \rm{I}}{{ \rm{C}}_{50}} + \left[ {{ \rm{T}}4} \right] } \right) \} \qquad\qquad {{Equation \ 2}} \end{align*} \end{document}

In both equations 1 and 2, F represents fluorescence intensity at a certain known concentration of T3 or T4, respectively, F0 is the fluorescence intensity of the blank sample (zero concentration) of either T3 or T4, and F1 represents that fluorescence intensity of the saturating concentration of T3 or T4. IC₅₀ (50% inhibition of the total signal) in the equations represent the respective concentrations of T3 or T4. The unknown concentrations of T3 or T4 were determined by the interpolation of the standard curve.

Results and Discussion

The calibration curve consisted of equilibrated solution of T3 or T4 from zero to saturation (Fig. 2A, B). The calibration curves generated for T3 and T4 were in the range of (0.015–30 ng mL⁻¹) and (0.49–1,000 ng mL⁻¹) with correlation coefficients of (r = 0.991) and (r = 0.994), respectively, on four-parameter curve fit. The concentration of either T3 or T4, which caused 10% inhibition of the maximum signal, was considered the limit of detection. The limit of detection (LOD) for T3 and T4 was found to be 0.06 and 1.9 ng mL⁻¹, respectively. Each calibration curve was run in duplicates and two calibration curve runs were performed for T3 and T4. The normal reference range for estimation of T3 and T4 in clinical samples is 80–230 ng dL⁻¹ and 4.6–12 μg dL⁻¹, respectively.

Fig. 2.

Calibration curve (●) and precision profile (▲) of the proposed KinExA-based sensor for (A) T3 and (B) T4.

The most commonly employed technique for estimation of T3 and T4 are enzyme immunoassays and the commercially available kits can measure T3 and T4 in the concentration range of 0.5–10 ng mL⁻¹ and 20–250 ng mL⁻¹.^24
–26 The high sensitivity of the KinExA methods enables the estimation of T3 and T4 in diluted serum samples, thus avoiding the mass transport limitations and mobility effects encountered during conventional ELISA analysis. The high sensitivity of the KinExA is attributed to the microbeads used in the KinExA format, which are approximately 10,000 per column and provide a higher surface area (260 mm²) for capturing free antibody compared to microwell in the ELISA format (64 mm²).²⁷

The results of the assay precision obtained from the calibration standard samples are represented in Figure 2A and B. The precision results (%RSD) of calibration curves did not exceed 9.03% in case of T3 and 11.87% in case of T4. The evaluation of the intra- and interassay precision was carried out using three concentrations of T3 and T4 that were within the calibration range of T3 and T4, respectively. To analyze the intra-assay precision, three replicates of same concentration were assayed in a single run and the interassay precision was tested using three different concentrations in three separate runs. The results for interassay and intra-assay precision for assay of T3 and T4 are presented in Table 2. The %RSD values for intra-assay precision for T3 and T4 were in the range of 3.15%–8.98% and 4.92%–8.75%, respectively, whereas, interassay precision for T3 and T4 were in the range of 4.11%–7.06% and 4.16%–7.37%, respectively.

Table 2.

Precision of the Proposed Antibody-Based Biosensor for Measurement of T3 and T4

	Intra-assay Mean ± RSD^a (ng mL⁻¹)	Interassay Mean ± RSD (ng mL⁻¹)
T3 (ng mL⁻¹)
0.2	0.19 ± 8.98	0.19 ± 7.06
4	4.20 ± 5.17	4.24 ± 4.98
15	15.08 ± 3.15	15.28 ± 4.11
T4 (ng mL⁻¹)
50	49.36 ± 8.75	48.48 ± 7.37
100	99.49 ± 6.22	103.00 ± 5.71
500	498.63 ± 4.92	501.15 ± 4.16

The accuracy of the developed sensors for T3 and T4 and their applicability were determined with the help of recovery studies. The serum samples spiked with different concentrations of T3 within the calibration range of 0.015–30 ng mL⁻¹ or T4 within the calibration range of 0.49–1,000 ng mL⁻¹ were assayed for the T3 or T4 content as described in the experimental section. The recovery from these spiked samples was calculated as the ratio of recovered concentration to that of actual spiked concentration. The analytical recovery for T3 was in the range of 94.1%–109.7% with RSD ranging from 3.71% to10.77% and for T4 in the range of 95.4%–105.0% with RSD ranging from 3.63% to 9.63% (Table 3). The results infer that the developed methods were selective and had no interference from any endogenous substances present in the serum.²⁸

Table 3.

Analytical Recovery of T3 and T4 Samples Spiked into Three Different Batches of Serum

	Recovery^a (% ± RSD)
	Batch A	Batch B	Batch C
Spiked T3 (ng mL⁻¹)
0.2	96.6 ± 9.05	99.1 ± 10.77	94.1 ± 10.30
0.4	107.9 ± 9.42	100.4 ± 7.61	101.6 ± 7.23
0.8	104.3 ± 8.11	103.7 ± 7.58	103.3 ± 7.78
1.6	97.18 ± 5.96	105.4 ± 6.57	95.1 ± 5.40
3.2	106.6 ± 4.15	109.7 ± 4.93	102.8 ± 5.59
6.4	99.68 ± 3.86	102.9 ± 4.50	99.9 ± 3.71
Spiked T4 (ng mL⁻¹)
20	101.6 ± 8.03	96.6 ± 9.63	107.5 ± 8.70
40	102.0 ± 7.16	97.9 ± 7.74	105.0 ± 8.66
80	103.3 ± 9.39	102.9 ± 7.31	99.5 ± 4.80
160	103.6 ± 6.80	99.1 ± 7.58	95.4 ± 4.77
320	96.8 ± 6.10	101.5 ± 6.90	100.3 ± 4.74
640	100.0 ± 3.63	100.2 ± 5.77	96.3 ± 4.35

Values are mean of three determinations; RSD is the relative standard deviation.

The functioning of the KinExA instrument is provided in schematic representation Figure 1, detailed functioning of the KinExA instrument can be found in literature.^{16

–23} The sample solution comprises of a definite concentration of either anti-T3 or anti-T4 antibodies along variable concentrations of T3 or T4. After the completion of the sample equilibration time, the bead column is generated and the samples (T3 or T4) are drawn over the micro-PMMA beads coated with either T3-BSA or T4-BSA. The instrument run starts with the formation of microbead column and the generated response is a function of time. The instrument automatically performs several steps during the sample run. The anti-T3 or anti-T4 antibodies with vacant binding sites bind to T3 or T4 present on the coated beads. The exposure of the microcoated beads to the sample solution is very short to ensure that the anti-T3 or anti-T4 antibodies with occupied binding sites in the immune complex present in the sample solution does not get dissociated and hence are kinetically excluded. The sample aspiration step is followed by a buffer wash to remove any soluble reagents present in the microbead column. The next step after the buffer wash is the aspiration of fluorescently labeled secondary antibody over the microbead column, which binds to the already captured primary anti-T3 or anti-T4 antibody bound to T3 or T4 present on the PMMA microbeads.

The intensity of the fluorescence signal is inversely proportional to the antigen present in the sample; hence the concentration of T3 or T4 is quantified. The aspiration of fluorescently labeled secondary antibody step is followed by PBS buffer wash step to eliminate any spare fluorescently labeled antibody present on the bead column. The fluorescence intensity is quantified with the help of photodiode provided in the instrument. Figure 3A and B show the square-wave responses of T3 and T4 as a function of time. The lowest curves represent the highest concentrations in the calibration curve of either T3 (30 ng mL⁻¹) or T4 (1,000 ng mL⁻¹), whereas the highest curve represents the blank sample (zero concentration of T3 or T4 antigen) and only contains the antibodies for T3 and T4, respectively.

Fig. 3.

Real raw trend-line fluorescence responses obtained by the KinExA™ instrument for varying concentrations of (A) T3 and (B) T4.

The optimization of the KinExA method development parameters was carried out to determine the most suitable values for these parameters. During the KinExA analysis, we have three types of signals, net signal, signal 100%, and nonspecific binding. “Signal 100%” is that voltage signal produced when a solution containing only the anti-T3 or anti-T4 antibodies with no free T3 or T4 is passed over the coated PMMA beads. These anti-T3 or anti-T4 antibodies interact with T3-BSA or T4-BSA immobilized on the PMMA beads to produce “Signal 100%.” Another type of signal is “nonspecific binding” (NSB) and it is produced when the sample contains neither T3 or T4 antigen nor their antibodies. The signal is generated because of the PBS buffer and the fluorescently labeled secondary antibody. The third type of signal is the net signal and is defined as \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{Net \ Signal}} = \left[ { \left( {{ \rm{Signal }} \ 100 } \right) - \left( {{ \rm{NSB}}} \right) } \right] \end{align*} \end{document}

As is evident from the above equation, reproducibility of results depends on the low background noise. The reasons for high background noise are insufficient washing of the microbead column and retention of the chemicals on the surface of column wall. Sometimes fungal growth also occurs if the inner tubing remains unwashed for longer period. To alleviate such occurrences, various cleaning procedures as per the manufacturer's user manual were followed and included fast rinsing and overnight washing steps with KinExA washing solution.

Optimization of the experimental conditions was also performed for the developed sensors and these included concentration of T3-BSA or T4-BSA used for the coating of PMMA microbeads, anti T3 and anti T4 antibody concentrations, secondary labeled anti-mouse IgG antibody concentration, volume of samples drawn, and volume of labeled secondary antibody. Various concentrations of T3-BSA and T4-BSA ranging from (10–40 μg) to (10–50 μg), respectively, were used to verify the most appropriate concentration for bead coating with the help of the signal test. The results of the signal test showed that concentrations of 23 μg T3-BSA and 35 μg T4-BSA were the most appropriate concentrations for coating the beads. Similarly, the strength of anti-T3 or anti-T4 antibodies was also determined after investigating a range of concentrations ranging (10–70 ng mL⁻¹) for anti T3 antibodies and (10–40 ng mL⁻¹) for anti T4 antibodies, and the most suitable concentrations found with the help of signal test were 45 ng mL⁻¹ and 30 ng mL⁻¹, respectively. The optimum concentration of secondary labeled anti-mouse IgG antibody was found to be 150 ng mL⁻¹ for assay of both T3 and T4, after investigating a range of concentrations 70–200 ng mL⁻¹ of secondary labeled anti-mouse IgG antibody. The generated data from is KinExA instrument are represented in Figure 2A and B.

Conclusions

A new immunosensor assay for the estimation of T3 and T4 in serum based on KinExA-automated instrument was developed and validated. The developed methods possessed several benefits over the conventional ELISA technique in avoiding mass transport limitations and mobility effects, the LOD's achieved were quite low, and the analysis time was reduced significantly. The KinExA format also provided more surface area, 260 mm², compared to the 64 mm² for each microwell in the ELISA format, increasing the chances for the capture of free antibody. The KinExA format added to the convenience due to the automation of the instrument and thus can be valuable in medical diagnostic laboratories.

Footnotes

Acknowledgment

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding the research group project No. RG-1435–073.

Authors' Contributions

T.A.W. and I.A.D., conceived the project. T.A.W. and S.Z. designed and performed experiments, S.W.M analyzed data, and T.A.W., I.A.D., and S.Z., wrote the article.

Disclosure Statement

No competing financial interests exist.

Abbreviations Used

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