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Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 9039270, 7 pages https://doi.org/10.1155/2020/9039270 Research Article Development of a Chemiluminescence Immunoassay for Quantification of 25-Hydroxyvitamin D in Human Serum 1,2 3 1 1,2,4 3 Shuang Han, Wuxian Qiu, Junlan Zhang, Zhonghu Bai , and Xiao Tong School of Biotechnology, Jiangnan University, Wuxi, China National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, China Aﬃliated Hospital of Jiangnan University, Department of Pediatrics, Wuxi, China ,e Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China Correspondence should be addressed to Zhonghu Bai; firstname.lastname@example.org and Xiao Tong; email@example.com Received 8 February 2020; Revised 28 March 2020; Accepted 27 May 2020; Published 1 August 2020 Academic Editor: Guido Crisponi Copyright © 2020 Shuang Han et al. /is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In this study, a chemiluminescence immunoassay (CLIA) for human serum 25-hydroxyvitamin D (25(OH)D) was established by a competition model. In serum, more than 99% of total circulating 25(OH)D binds to protein and less than 1% of 25(OH)D is in free form (Jassil et al., 2017). Before measuring concentration of 25(OH)D in serum, a releasing procedure should be conducted. A new reagent is used to release binding 25(OH)D to free form. Streptavidin (SA) was labeled to magnetic beads by a 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) method. Biotinylated VD was used as a competitor of 25(OH)D in samples. Anti-VD antibody (aby) was labeled to horseradish peroxidase (HRP) by EDC to react with 25(OH)D and biotinylated-VD molecules. /e pretreated samples or standards were added into the reaction tube with biotin-VD and anti-VD aby-HRP, free 25(OH)D in the sample competes with biotinylated VD for binding to anti-VD aby-HRP, an SA-labeled magnetic particle is added to isolate the signal-generating complex, and the signal is inversely proportional to the 25(OH)D concentration in the sample. /e method established shows good thermostability and performance. /e limitation of detection (LoD) is 1.43 ng/ mL. /e intra-assay coeﬃcient of variation (CV) is 3.66%–6.56%, the interassay CV is 4.19%–7.01%, and the recovery rate is 93.22%–107.99%. Cross-reactivity (CR) was remarkably low with vitamin D2, vitamin D3, 1, 25-dihydroxyvitamin D3, and 1, 25- dihydroxyvitamin D2. At the same time, the cross-reaction values with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 97% and 100%, respectively. /e developed method shows good correlation with the total VD product from Roche and DiaSorin. 1096 clinical patient samples were measured with developed reagent kit in this study. 7 types of disease were involved, and the concentration of 25(OH)D is less than 30 ng/mL in 94.98% of patients. is the most widely used indicator of vitamin D status in 1.Introduction either serum or plasma [13, 14]. /ere are methods on the Vitamin D (VD), also known as sunshine vitamin, plays an market for the analysis of 25(OH)D. A radiological im- important role in bone metabolism [1–3]. Vitamin D de- munoassay was developed by SchiolerV in 1988 , which ﬁciency can cause growth retardation and skeletal deﬁciency is time consuming and harmful to environment and oper- in infant and children [4, 5], osteopenia, and osteoporosis ator’s health. Several automated immunoassays were de- usually happen in adult who have low-level vitamin D in veloped too, such as Liaison Total Vitamin D, the IDS-iSYS circulation . Vitamin D deﬁciency also has a possible role 25-Hydroxy Vitamin D, the ARCHITECT 25-OH Vitamin in chronic diseases, such as cancer , autoimmune diseases D, and the ADVIA Centaur Vitamin D Total, and none of [8, 9], osteoarthritis , diabetes , and cardiovascular these immunoassays gave results equivalent to the liquid disease . /erefore, detection of vitamin D concentration chromatography-tandem mass spectrometry (LC-MS/MS) is a quite vital requirement of clinical diagnostics. 25(OH)D method . High-performance liquid chromatography 2 Journal of Analytical Methods in Chemistry storage buﬀer of the particle. EDC and NHS solutions were spectrometry was developed for 25(OH)D detection , but this method is quite expensive and requires special added to the Dynabeads to active the binding group on particle surface, suspended the particle and well mixed all training for the operator. In contrast, CLIA is a simple, sensitive, and cheap method for the high-throughput compounds. /e mixture was incubated with gently tilt quantiﬁcation of analyses in samples. In this study, a direct rotation at room temperature for 30 minutes. /e beads were competitive immunoassay was established on the CLIA washed two more times to remove the supernatant. /e platform. required amount of streptavidin was added into preactivated magnetic beads and incubated for another 30 minutes at room temperature with gently tilt rotation. At last, the 2.Materials and Methods particle was washed twice again and then suspended to 2.1. Reagents and Materials. Dynabeads MyOne carboxylic storage concentration with PBS buﬀer containing 0.5% BSA, acid beads, EZLink Sulfo-NHS-LC-Biotinylation Kit, 0.05% polysorbate-20, and 0.02% sodium azide. succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-car- boxylate (SMCC), and 4′-hydroxyazobenzene-2-carboxylic 2.3.2. Characterization of SA-Coated Magnetic Particle. acid (HABA) solution are obtained from /ermo Fisher; /e biotin-streptavidin system is used to test quality of the perﬂuorohexanoate(PFHxA), methanol, EDC, and NHS are in-house prepared magnetic particle. Two sets of reaction purchased from Sigma; an AKTA puriﬁer is purchased from tubes are prepared, 30 μL in-house prepared SA-coated GE healthcare; biotinylated vitamin D (BVD) is obtained magnetic particle is added, and 50 μL biotinylated HRP from DIASource; HRP is purchased from BBI solutions; (50 ng/mL) is added to one set and 50 μL HRP (50 ng/mL) is streptavidin is purchased from NeuroPeptide from China; a added to the other set. Both sets were incubated for 30 min at microscope is purchased from Olympus; an automicroplate C, and then the particles were washed 3 times with chemiluminescent analyzer is supplied by Baiming Bio- washing buﬀer. At last, 100 μL substrate reagent is added to technology from China; and an automagnetic beads generate signals. /e RLUs are 710,325 with biotinylated chemiluminescent analyzer is supplied by Zecheng Bio- HRP and 352 with HRP. /is result indicated that the in- technology from China. Mice used for antibody production house prepared magnetic particle is ready to use (see are obtained from Jiangnan University; VD is purchased Supplementary Table 1). from Conju-Probe. 2.3.3. Antibody Conjugates with HRP. Diﬀerent weights of 2.2. Antibody Immunization and Puriﬁcation anti-VD antibody and EDC are dissolved in separated 0.05 M sodium bicarbonate solutions. Mix them after the 2.2.1. Antibody Immunization. 25-hydroxyvitamin D was solute dissolved completely. HRP is dissolved in 0.1 conjugated to bovine serum albumin (BSA) by a SMCC Mphosphate buﬀer, pH 7.2. /en, the mixed solution is crosslinker before immunization. /ree female BALB/c mice added into the HRP solution and the pH is lowered to 5.8 were used as hosts, and each mouse weighed 20g and was 4 with diluted hydrochloric acid. It is incubated for 5 hours at weeks old. Hosts were immunized 3 times with 50 ug VD- room temperature. Finally, the impurities are removed with BSA in complete Freund’s adjuvant mixture, and the im- a desalination column. munization interval was 3 weeks, at a last boost with 100 ug VD-BSA in incomplete Freund’s adjuvant. Splenocytes were harvested from immunized mice in one week, then fusion 2.3.4. Characterization of Anti-VD Antibody-HRP with SP2/0 cells. Conjugates. /e anti-VD antibody-HRP conjugate is pre- pared in diﬀerent antibody/HRP ratios. Biotin-VD-SA magnetic particle coupling is used as a probe to test quality 2.2.2. Antibody Screening and Puriﬁcation. ELISA assay was of the anti-VD antibody-HRP conjugate. Several sets of employed to screen target anti-VD antibody. Biotinylated reaction tubes are prepared, 30 μL biotin-VD-SA magnetic 25(OH)D was used as a probe. HRP-conjugated anti-mouse particle coupling is added, and 50 μL diﬀerent anti-VD antibody was used to generate signals. 96-well plates were antibody-HRP is added with HRP as control into reaction coated with streptavidin (SA) as solid phase. One primary tubes, then incubated for 30 min at 37 C; the particle is screening from the supernatant of splenocyte-SP2/0 fusion washed 5 times with washing buﬀer. /en, 100 μL substrate cells was performed. 3 rounds of subcloning were conducted reagent is added. /e results indicate that anti-VD antibody- to obtain hybridoma-secreting anti-VD monoclonal anti- HRP conjugates are ready to use (see Supplementary body. /e anti-VD monoclonal antibodies were further Figure 1). validated as raw material to build 25(OH)D detection re- agent kit. 2.4. Reaction Condition Optimization. In serum, more than 99% of total circulating VD binds to protein and less than 1% 2.3. Reagent Component Preparation of VD is in free form. A VD-releasing process should be 2.3.1. Streptavidin-Coated Magnetic Bead Preparation . involved before measuring the concentration of total VD in Dynabeads MyOne was washed twice with 25 mM 2-mor- serum. Alkaline condition and DTT are used by Roche. In pholinoethanesulfonic acid (MES) buﬀer, pH 6.0, to remove this study, a new reagent has been developed to releasing VD Journal of Analytical Methods in Chemistry 3 which is more eﬃciency and time-saving; PFHxA and achieves high signals with a relative low variable coeﬃcient. methanol were used as key material in a PH 7.5 buﬀer. /e /e signal results are shown in Supplementary Figure 3 and Table 3. essential reagents required for this assay include biotin-VD, anti-VD antibody-HRP conjugates, and VD. Upon mixing the anti-VD antibody-HRP conjugates with a serum pre- 2.4.4. VD-Releasing Procedure Optimization. PFHxA and treated containing free VD, a reaction results between the methanol were used as key material to release the binding VD and anti-VD antibody. /en, the magnetic particle with 25(OH)D to free form. PFHxA is used as a main releasing binding biotinylated VD was added, competed binding to compound while methanol as a cosolvent, with a neutral the remnant anti-VD antibody-HRP conjugate. /e RLUs PH, and the releasing reagent has low eﬀect of following generated are inversely proportional to the VD concentra- reactions. /eoretically, the more the PFHxA is added, the tion. By utilizing several diﬀerent serum references of known more eﬃcient the releasing reagent will be; however, VD concentration, a concentration response curve can be according to results with more than 1% PFHxA in the generated, and the VD concentration of an unknown can be reagent, the ﬁnal RLU was decreased. /erefore, 1% PFHxA ascertained. In this study, factors such as VD-releasing was the chosen concentration. Data are shown in Sup- procedure, biotin-VD-magnetic particle concentration, anti- plementary Figure 4. /e concentration of methanol was VD antibody-HRP type and concentration, and incubation optimized, the CV value became better with methanol in time were optimized. the releasing buﬀer, 5% was the ﬁnal concentration, and data are shown in Supplementary Figure 5. Finally, the releasing time of 25(OH)D was optimized, data are shown 2.4.1. Method Procedure. We added 30 μL serum and 150 μL in Supplementary Figure 6. /e concentrations of samples VD-releasing reagent into a reacting tube, mixed, and in- hardly increase after 18 mins, so it is chosen as the ﬁnal cubated for 18 minutes at 37 C to release VD from binding releasing time. protein. 50 μL anti-VD antibody-HRP is added into a reacting tube and incubated for 15 minutes at 37 C, then added 30 μL magnetic particle coated with biotinylated VD 2.5. Performance Test Method and incubated for another 5 min at 37 C. After incubation, the magnetic particle was washed three times with washing 2.5.1. Limitation of Detection. 20 standards with 0 ng/mL buﬀer, and then 200 μL substrate reagent is added to gen- analyte are tested, and then MEAN-3SD value is calculated. erate signals. A signal reader is used to collect RLUs, and /e corresponding concentration is the limitation of concentration values are calculated if the calibrator curve is detection. available. 2.5.2. Precision and Recovery. /ree serum samples of dif- 2.4.2. Optimization of Biotin-VD and Anti-VD Antibody- ferent 25(OH)D concentrations were tested and duplicated HRP Concentration. Diﬀerent antibody/HRP ratio conju- separately in one experiment and repeated in 20 days, and gates present diﬀerent competing abilities. Signals of stan- the intra- and interassay CV are calculated. /e concen- dards increase along with the excess molar increase of HRP. tration of 25(OH)D solutions was added to three serum /e signal (0 ng/mL)/signal (150 ng/mL) value increases ﬁrst samples with diﬀerent analyte levels, and the recovery rate is and then remains at the same level, and the largest number is calculated. under condition of antibody/HRP ratio at 2 :1. /e results are shown in Supplementary Figure 2. A series concentration 2.5.3. Cross-Reactivity. /e speciﬁcity of the anti-VD anti- of biotinylated VD and anti-VD antibody-HRP was inves- body used to selected substances was evaluated by adding the tigated, and the signals and signal (0 ng/mL)/signal (150 ng/ interfering substance to a serum matrix at various concen- mL) value are acceptable when biotin-VD concentration is trations. /e cross-reactivity (CR) is deﬁned at the point 30 ng/mL and anti-VD antibody-HRP concentration is where the reduction in the signal corresponds to 50% of the 1,000 ng/mL. /e results of optimization are shown in signal achieved in the absence of analyte (B/B0 of 50%), as a Supplementary Table 2. percentage of the analyte concentration given the same fall in the signal. /e CR values were calculated as follows: CR (%) � IC50 of 25(OH)D/IC50 of competitor × 100%. 2.4.3. Optimization of Incubation Time. /e biotinylated VD and anti-VD antibody-HRP incubation time is studied, respectively. /e procedure remains unchanged described in 2.5.4. Accelerated Stability. /e whole kit including bio- the method procedure except for the incubation time. For tinylated VD, SA-coated magnetic particle, anti-VD anti- biotinylated VD, signals remain the same in our test time body-HRP, and VD standards was incubated at 37 C for 7 point. For anti-VD antibody-HRP, signals increase as the days, and the signals of standards and samples at diﬀerent incubation time is lengthened at ﬁrst but remain constant days were compared. while the reaction achieves dynamic equilibrium. Due to time-saving requirement of clinical, we choose 5 min in- cubation of biotinylated VD and 16 min incubation of anti- 2.5.5. Method Comparison. /e established method was VD antibody-HRP as the best incubation condition which compared with two on-market CLIA methods from Roche 4 Journal of Analytical Methods in Chemistry 80 50 y = 1.0958x – 0.2063 y = 0.7962x – 0.7793 2 2 R = 0.7957 R = 0.7942 0 0 020 40 60 0 204060 HPLC-MS/MS (ng/mL) HPLC-MS/MS (ng/mL) y = 0.9843x – 0.0563 R = 0.8964 020 40 60 HPLC-MS/MS (ng/mL) Figure 1: Comparison with UPLC-MS/MS. Table 1: Precision result. Table 2: Analytical recovery. Intra-assay CV (n � 20) Interassay CV (n � 20) VD concentration (ng/mL) Samples Samples CV Mean (ng/ CV Added concentration Tested Expected Recovery (%) Mean (ng/mL) SD SD (%) mL) (%) 0.00 9.76 10.54 107.99 1 9.91 0.65 6.56 9.56 0.67 7.01 5.63 15.88 15.48 97.48 1 11.25 20.98 19.90 94.85 2 24.93 1.13 4.53 24.37 1.02 4.19 22.50 31.18 31.97 102.53 3 50.52 1.85 3.66 50.18 2.14 4.26 45.00 58.99 54.99 93.22 0 23.53 24.56 104.38 5.625 29.59 29.21 98.72 2 11.25 35.10 34.87 99.34 and DiaSorin. /e sample size used to compare with Roche 22.5 47.20 45.76 96.95 is 244 and ranged from 3 ng/ml to 63.88 ng/ml. 202 serum 45 65.29 67.90 104.00 samples are used to compare with DiaSorin with range from 6.64 ng/ml to 89.00 ng/ml. 3.2. Method Performance. /e 4-parameter logic function method was used to ﬁt the standard curve of signal with 3.Result 25(OH)D concentration. /e following series concentra- tions of 25(OH)D were used as a standard curve, 0 ng/mL, 3.1. VD-Releasing Reagent Eﬃciency Test. Eﬃciency of the 10 ng/mL, 25 ng/mL, 50 ng/mL, 90 ng/mL, and 150 ng/mL. releasing reagent was tested by comparing test results with /e LoD is 1.43 ng/ml. Precision was tested, intra-assay CV HPLC-MS/MS concentration. /e correlation equation of the % is 3.66%–6.56%, interassay CV % is 4.19%–7.01%, and the developed method with HPLC-MS/MS is y � 0.984x−0.056, results are shown in Table 1. /e recovery rate is 93.22%– and correlation coeﬃcient is 0.9468. Results are shown in 107.99%, and the results are shown in Table 2. /e developed Figure 1. At the same time, samples were tested on the methods have high selectivity for 25(OH)D. Cross-reaction DiaSorin and Roche platforms side by side. /e correlation of values were less than 0.1% with vitamin D2, vitamin D3, 1, the DiaSorin method with HPLC-MS/MS is y � 0.796x − 0.779, 25-dihydroxyvitamin D3, and 1, 25-dihydroxyvitamin D2. and correlation coeﬃcient is 0.8911. /e correlation of the At the same time, the cross-reaction values with 25- Roche method with HPLC-MS/MS is y � 1.095x − 0.206, and hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 97% correlation coeﬃcient is 0.8920. Roche (ng/ml) Developed method (ng/mL) DiaSorin (ng/mL) Journal of Analytical Methods in Chemistry 5 –1 –2 –3 –4 –5 –6 Days Standard 1 Standard 4 Sample 1 Standard 2 Standard 5 Sample 2 Standard 3 Standard 6 Sample 3 Figure 2: Reagent kit accelerated stability. 80 90 y = 0.9847x + 3.401 y = 0.8755x – 0.0109 2 2 R = 0.9022 R = 0.9311 0 0 020 40 60 80 0 20 40 60 80 100 Roche (ng/mL) DiaSorin (ng/mL) Figure 3: Comparison with on-market products. Table 3: Distribution of clinical samples. Conc. of 25(OH)D Fracture Osteoporosis Osteoarthritis Autoimmune diseases Cancer Diabetes Cardiovascular disease <30 ng/mL 41 25 55 140 203 524 53 >30 ng/mL 0 4 1 16 6 27 1 Total 41 29 56 156 209 551 54 and 100%, respectively. Accelerated stability study was Test results show good agreement between the developed performed; the developed reagent showed great stability method and compared methods, and diﬀerence of the test under stressed temperature. /e RLUs changed less than values and mean values indicated slight amounts of bias 10% during 7-day periods under 37 C, see the results in between developed and compared methods. Figure 2. 3.4. Clinical Study. 1096 clinical patient samples were 3.3. Method Comparison. In this study, 244 serum samples measured with developed reagent kit in this study. Samples were measured by both the developed and Roche methods were treated using the same method we described in Section and 202 serum samples were measured by both the devel- 2.4.4. 7 types of disease were involved, and the concentration oped and DiaSorin methods. /e test results were regressed of 25(OH)D is less than 30 ng/mL in 94.98% patients. Data by the least square regression equation, and the correlation are shown in Table 3. Among patients have low-level 25(OH) coeﬃcient was computed too. Data are shown in Figure 3. D, nearly 50% are related to diabetes, about 20% patients Developed method (ng/mL) RLU difference of Day 0 (%) Developed method (ng/mL) 6 Journal of Analytical Methods in Chemistry 2% and LC-MS/MS-based nonimmunological direct detection 4% method used by PerkinElmer. Due to the diﬀerences of the 14% sample pretreatment method and speciﬁcity of biomaterial 5% employed in diﬀerent immunoassays, the agreement of test result is not very good. LC-MS/MS is usually considered as a 5% reference method, but the cost of instruments, consumables, and the low throughput limited its wide application. /is study established a chemiluminescence immunoassay for the quantiﬁcation of 25(OH)D in human serum with good performance and overall stability. /e method comparison result shows that this method has a good correlation with the 20% 25(OH)D kit from Roche and DiaSorin which are highly 50% admitted and widely used on the market. Samples results compared with HPLC-MS/MS in releasing reagent opti- mization section show that this method has a good agree- ment with the physical detection method which usually be considered as a reference method. In serum, 25(OH)D binds to protein as a complex; pretreatment of the sample should be performed before the test. In this study, a new releasing Fracture Cancer reagent was employed to maximize the release of 25(OH)D; Osteoporosis Cardiovascular disease besides, the PH of the releasing reagent is around 7.5 which Osteoarthritis Autoimmune diseases has low eﬀect on following reactions. In this study, in-house Diabetes produced biomaterial such as SA-coated magnetic particle, Figure 4: Distribution of clinical samples that 25(OH)D is less than anti-VD antibody, and anti-VD antibody-HRP conjugate 30 ng/mL. are critical to the reagent performance. /erefore, further studies should be conducted on the antibody-producing procedure and coupling conjugate procedure. Minimization 80 of batch diﬀerence of raw material should help to sustain performance of reagent kit. /e clinical study with devel- oped kits shows good agreement of the test result and disease, and the reagent kit was fulﬁlling for the clinical requirement. Data Availability /e data used to support the ﬁndings of this study are available from the corresponding author upon request. Conflicts of Interest /e authors declare that they have no conﬂicts of interest. Figure 5: Concentration of 25(OH)D of diﬀerent patients. Authors’ Contributions have cancers, and almost 14% got fracture. Data are shown Shuang Han and Wuxian Qiu equally contributed to this in Figure 4. Patients who have autoimmune disease, cancer, work. and diabetes have wider range of 25(OH)D level, patients who got fracture, osteoporosis, and osteoarthritis have Acknowledgments narrow range of 25(OH)D, and almost all are lower than 30 ng/mL. Data are shown in Figure 5. /is work was supported by Jiangsu Key R & D Program (BE2018621), Postgraduate Research Innovation Program of 4.Discussion and Conclusion Jiangsu Provence (KYCX18 1793), National First-Class Discipline Program of Light Industry Technology and En- According to the previous literature studies, there are many gineering (LITE2018-24), and the Fifteenth Batch of the “Six quantiﬁcation methods for 25(OH)D, such as antibody- Talent Peaks Project in Jiangsu Province” (SWYY-180). based isoluminol derivate direct competitive two-step chemiluminescent system used by DiaSorin; antibody-based Supplementary Materials acridinium ester competitive assay used by IDS plc, Abbott, and Siemens; 25-OH-vitamin D binding protein based Figure S1: characterization of anti-VD antibody-HRP electrochemiluminescence competitive assay used by Roche; conjugate (n � 3). Figure S2: performance of diﬀerent Concentration (ng/mL) Fracture Osteoporosis Osteoarthritis Autoimmune diseases Cancer Diabetes Cardiovascular disease Journal of Analytical Methods in Chemistry 7  T.J. SchiolerV, “Six direct radioimmunoassay of estradiol antibody/HRP ratio conjugates. Figure S3: optimization of evaluated,” Clinical Chemistry, vol. 34, no. 5, pp. 949–952, incubation time. Figure S4: optimization of PFHxA con- centration. Figure S5: optimization of methanol concen-  M.-k. Koivula, N. Matnlassi, and J. R. Paivilaitinen, “Four tration. Figure S6: optimization of VD-releasing time. Table automated 25-OH total vitamin D immunoassays and com- S1: characterization of the magnetic particle. Table S2: bi- mercial liquid chromatography tandem-mass spectrometry in otin-VD and anti-VD antibody-HRP concentration opti- Finnish population,” Clinical Laboratory, vol. 59, no. 3-4, mization. Table S3: precision results of optimization pp. 397–405, 2013. incubation time. 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Journal of Analytical Methods in Chemistry – Hindawi Publishing Corporation
Published: Aug 1, 2020
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