检测β⁃CTX和N⁃MID水平的双标记时间分辨免疫荧光分析方法的建立和评价

doi:10.13481/j.1671-587X.20210330

Abstract

Abstract: Objective

To develop a double-labeled time-resolved immunofluorescence analysis （TRFIA） method for the determination of the levels of β-cross-linked C-terminal telopeptide of type Ⅰ collagen（β-CTX） and N-terminal middle molecular fragment of osteocalein（N-MID） of osteocalcin，and to evaluate its detection performance.

Methods

The 4E5和2B7 monoclonal antibodies（MAb）of β-CTX and N-MID antigens were constructed， and then coated as coating antibodies in 96-well plates， and the antibodies were labeled with europium ion（Eu³⁺） and samarium ion（Sm³⁺），respectively. A double antibody sandwich TRFIA method was established and prepared into a kit. The detection performance was evaluated by the sensitivity， accuracy （dilution recovery）， specificity， precision， stability， and clinical sample alignment of the kit.

Results

The prepared double-labeled TRFIA Kit had a detection sensitivity of 0.025 μg·L^－1 for β-CTX and a linear range of 0.025－5.000 μg·L^－1；the detection sensitivity for N-MID was 0.5 μg·L^－1， and the linear range was 0.5－200.0 μg·L^－1； the average dilution recovery rate of β-CTX was 102.13%， and the average dilution recovery rate of N-MID was 103.02%；there were no obvious cross-reaction with other common bone test indicators and the specificity was high.The intra-assay coefficient of variation（CV） of β-CTX was 5.81%－7.82%， and the inter-assay CV was 5.97%－8.02%； the intra-assay CV of N-MID was 6.05%－8.32%， and the inter-assay CV was 6.14%－8.56%； the TRFIA Kit could be stored stably at 4 ℃ for half a year and stably stored at 37 ℃ for 7 d.

Conclusion

The established β-CTX and N-MID double-labeled TRFIA method has the advantages of high sensitivity， high specificity， high accuracy， convenience and speed， and is suitable for the detection of large-scale clinical samples.

Key words: β-cross-linked C-terminal telopeptide of type Ⅰ collagen, N-terminal middle molecular fragment of osteocalein, osteoporosis, double antibody sandwich, double-labeled time-resolved immunofluorescence analysis

CLC Number:

R331

Qian MAO, Cuicui CHEN, Huankun LIANG, Penge LIU, Shuhai ZHONG, Laiqing LI. Establishment and evaluation of a double⁃labeled time⁃ resolved immunofluorescence analysis method for detecting levels of β⁃CTX and N⁃MID[J].Journal of Jilin University(Medicine Edition), 2021, 47(3): 770-776.

Figures/Tables 5

Fig. 1

Tab.1

Tab.2

Tab.3

Tab.4

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Sample	Dilution	Concentration [ρ_B/(μg·L^－1)]	Determined [ρ_B/(μg·L^－1)]	Recovery rate (η/%)
β-CTX standard 1	1∶2	8.4	8.53	101.6
	1∶4	4.2	4.34	103.3
	1∶8	2.1	2.18	103.8
β-CTX standard 2	1∶2	9.6	9.47	98.6
	1∶4	4.8	4.92	102.6
	1∶8	2.4	2.51	104.6
β-CTX standard 3	1∶2	10.4	10.69	102.8
	1∶4	5.2	5.27	101.3
	1∶8	2.6	2.62	100.6
N-MID standard 1	1∶2	110.4	110.18	99.8
	1∶4	55.2	57.79	104.7
	1∶8	27.6	29.06	105.3
N-MID standard 2	1∶2	164.8	162.99	98.9
	1∶4	82.4	85.53	103.8
	1∶8	41.2	42.89	104.1
N-MID standard 3	1∶2	180.8	180.26	99.7
	1∶4	90.4	95.19	105.3
	1∶8	45.2	47.73	105.6

Item	Sample		Average [ρ_B/(μg·L^－1)]	Standard deviation	CV (η/%)
Item	Test	Concentration	Average [ρ_B/(μg·L^－1)]	Standard deviation	CV (η/%)
β-CTX	Intra-assay	Low	0.25	0.02	7.82
		Median	1.80	0.12	6.47
		High	3.70	0.21	5.81
	Inter-assay	Low	0.30	0.03	7.56
		Median	2.30	0.14	5.98
		High	3.90	0.26	6.75
N-MID	Intra-assay	Low	2.50	0.21	8.32
		Median	55.60	3.36	6.05
		High	113.40	8.41	7.42
	Inter-assay	Low	3.80	0.30	8.02
		Median	66.50	4.08	6.14
		High	124.30	9.48	7.63

Group	β-CTX（4 ℃）					N-MID（4 ℃）
	(month)0	1	2	3	6	(month)0		1	2	3	6
Sample 1	0.23	0.23	0.21	0.19	0.17	20.23		20.10	20.06	20.01	19.32
Sample 2	0.50	0.49	0.48	0.49	0.49	40.68		40.67	40.51	40.30	39.98
Sample 3	1.68	1.60	1.53	1.58	1.47	80.74		80.70	79.91	79.12	78.93
Sample 4	4.87	3.35	3.24	3.30	3.28	126.30		126.02	125.61	125.30	124.01
Sample 5	4.87	4.80	4.82	4.75	4.69	186.50		186.00	185.30	185.90	184.24
Group	β-CTX（37 ℃）						N-MID（37 ℃）
(t/d) 0		1	3	5	7	(t/d) 0		1	3	5	7
Sample 1	0.25	0.25	0.24	0.25	0.24	22.60		22.60	22.50	22.52	22.46
Sample 2	0.48	0.48	0.48	0.48	0.47	50.40		50.60	49.60	49.01	48.53
Sample 3	2.06	1.95	1.87	1.90	1.82	80.72		80.63	79.81	79.21	78.64
Sample 4	3.54	3.50	3.51	3.45	3.38	110.96		110.52	109.60	109.10	108.73
Sample 5	4.93	4.90	4.85	4.80	4.75	160.48		160.10	159.72	158.14	158.23

Group	β-CTX
	Osteoporotic patient				Non-osteoporotic patient
	Teens	Pregnant women	Postmenopausal women	Elderly	Teens	Pregnant women	Postmenopausal women	Elderly
CLIA
Positive	15	20	25	30	0	0	0	0
Negative	0	2	1	1	15	22	26	31
Coincidence rate (η/%)	100.00	90.00	96.00	96.67	100.00	100.00	100.00	100.00
ELISA
Positive	15	20	25	30	0	0	0	0
Negative	0	2	1	1	15	22	26	31
Coincidence rate (η/%)	100.00	90.00	96.00	96.67	100.00	100.00	100.00	100.00
TRFIA
Positive	15	20	25	30	0	0	0	0
Negative	0	2	1	1	15	22	26	31
Coincidence rate (η/%)	100.00	90.00	96.00	96.67	100.00	100.00	100.00	100.00
Group	N-MID
Group	Osteoporotic patient				Non-osteoporotic patient
	Teens	Pregnant women	Postmenopausal women	Elderly	Teens	Pregnant women	Postmenopausal women	Elderly
CLIA
Positive	14	22	24	31	0	0	0	0
Negative	1	0	2	0	15	22	26	31
Coincidence rate (η/%)	92.86	100.00	91.67	100.00	100.00	100.00	100.00	100.00
ELISA
Positive	14	22	24	31	0	0	0	0
Negative	1	0	2	0	15	22	26	31
Coincidence rate (η/%)	92.86	100.00	91.67	100.00	100.00	100.00	100.00	100.00
TRFIA
Positive	14	22	24	31	0	0	0	0
Negative	1	0	2	0	15	22	26	31
Coincidence rate (η/%)	92.86	100.00	91.67	100.00	100.00	100.00	100.00	100.00

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Establishment and evaluation of a double⁃labeled time⁃ resolved immunofluorescence analysis method for detecting levels of β⁃CTX and N⁃MID

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