Development and performance evaluation of a SARS-CoV-2 neutralizing antibody assay using fluorescent immunochromatography

doi:10.3877/cma.j.issn.2095-5820.2025.04.003

Abstract

Abstract:

Objective

To develop and evaluate the immune response levels following SARS-CoV-2 vaccination or infection, a neutralizing antibody detection method based on fluorescent immunochromatography was developed and subsequently subjected to preliminary performance assessment.

Methods

The fluorescent immunochromatographic assay employs a competitive format. The test strip is coated with fluorescent microspheres-labeled receptor-binding domain and immobilized angiotensin-converting enzyme 2 along with sheep anti-mouse antibodies. This configuration enables quantitative detection of SARS-CoV-2 neutralizing antibodies in serum or plasma through fluorescence signals.

Results

The method quantifies neutralizing antibodies for SARS-CoV-2 at two concentrations (906 IU/ml and 1865 IU/ml), with within-batch coefficients of variation of 10.16% and 8.20%, respectively. The between-batch coefficients of variation are 10.43% and 9.48%, respectively. This method exhibits no cross-reactivity with other antibodies, indicating robust specificity. Analytical sensitivity showed a limit of detection concentration is 125 IU/ml, with an optimal measurement range spanning 125-3600 IU/ml. The total concordance rate with the pseudovirus neutralization test (pVNT) method is 87%, with a positive concordance rate of 85% and a negative concordance rate of 100%. In addition, the Kappa coefficient was 0.54 (P<0.001), indicating that the chromatography method had moderate consistency with the pVNT results.

Conclusion

This rapid and accurate fluorescent immunochromatographic assay provides a reliable tool for evaluating vaccine efficacy and treatment outcomes in SARS-CoV-2 patients.

Key words: SARS-CoV-2, neutralizing antibodies, methodological evaluation, immunofluorescence chromatography

Haoming Zhang, Zhiqi Zeng, Yong Liu. Development and performance evaluation of a SARS-CoV-2 neutralizing antibody assay using fluorescent immunochromatography[J]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2025, 13(04): 206-212.

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Figures/Tables 8

检测	理论浓度500 IU/ml		理论浓度250 IU/ml		理论浓度125 IU/ml		理论浓度62.5 IU/ml		理论浓度31.25 IU/ml
检测	实测浓度/IU/ml	准确度/%	实测浓度/IU/ml	准确度/%	实测浓度/IU/ml	准确度/%	实测浓度/IU/ml	准确度/%	实测浓度/IU/ml	准确度/%
第1次实测	468.34	-6.33	271.80	8.72	142.48	13.98	45.16	-27.74	55.12	76.38
第2次实测	538.50	7.70	219.34	-12.26	139.47	11.58	83.76	34.02	26.17	-16.26
第3次实测	557.26	11.45	234.96	-6.02	107.62	-13.90	71.50	14.40	43.54	39.33

病原体类型	样本编号	结果	NAb检测值/IU/ml	参考范围/IU/ml
甲型流感病毒	1	阴性	<125.00	0~3600
	2	阴性	<125.00
	3	阴性	<125.00
	4	阴性	<125.00
	5	阴性	<125.00
腺病毒	1	阴性	<125.00	0~3600
	2	阴性	<125.00
	3	阴性	<125.00
	4	阴性	<125.00
	5	阴性	<125.00
呼吸道合胞病毒	1	阴性	<125.00	0~3600
	2	阴性	<125.00
	3	阴性	<125.00
	4	阴性	<125.00
	5	阴性	<125.00

次数	质控品1	质控品2
1	1017.21	1708.48
2	930.44	1921.86
3	861.43	1809.81
4	933.10	1815.36
5	877.46	2045.19
6	1024.69	1678.44
7	1001.27	2142.04
8	1012.93	1822.98
9	992.14	1746.82
10	1177.46	1945.19
11	1014.69	1678.44
12	837.42	1713.62
13	951.01	1672.23
14	816.23	1952.76
15	1007.46	1856.60
16	938.23	2036.26
17	830.81	1708.33
18	1070.26	1998.12
19	894.51	2090.97
20	798.73	1735.88
$X ¯$	949.37	1853.97
CV/%	10.16	8.20

次数	质控品1	质控品2
1	1017.21	1708.48
2	930.44	1921.86
3	861.43	1809.81
4	933.10	1815.36
5	877.46	2045.19
6	1024.69	1678.44
7	1001.27	2142.04
8	1012.93	1822.98
9	992.14	1746.82
10	1177.46	1945.19
11	1014.69	1678.44
12	837.42	1713.62
13	951.01	1672.23
14	816.23	1952.76
15	1007.46	1856.60
16	938.23	2036.26
17	830.81	1708.33
18	1070.26	1998.12
19	894.51	2090.97
20	798.73	1735.88
$X ¯$	949.37	1853.97
CV/%	10.16	8.20

时间/d	质控品1	质控品2
1	939.00	1582.52
2	1011.99	1824.88
3	978.97	2035.82
4	1012.89	1770.62
5	908.13	1719.50
6	922.60	2135.40
7	1052.11	2076.21
8	912.13	1864.30
9	819.75	2017.17
10	913.62	1913.26
11	862.33	1552.01
12	799.56	1769.81
13	947.92	1693.46
14	1147.84	2052.32
15	1015.17	1722.48
16	870.21	1639.90
17	1037.42	1713.62
18	951.01	1912.23
19	716.23	1652.76
20	919.10	1773.67
$X ¯$	936.90	1821.10
CV/%	10.43	9.48

时间/d	质控品1	质控品2
1	939.00	1582.52
2	1011.99	1824.88
3	978.97	2035.82
4	1012.89	1770.62
5	908.13	1719.50
6	922.60	2135.40
7	1052.11	2076.21
8	912.13	1864.30
9	819.75	2017.17
10	913.62	1913.26
11	862.33	1552.01
12	799.56	1769.81
13	947.92	1693.46
14	1147.84	2052.32
15	1015.17	1722.48
16	870.21	1639.90
17	1037.42	1713.62
18	951.01	1912.23
19	716.23	1652.76
20	919.10	1773.67
$X ¯$	936.90	1821.10
CV/%	10.43	9.48

理论浓度/IU/ml	实测浓度/IU/ml	准确度/%	CV/%
62.5	96.40	54.24	46.45
	104.26	66.82
	36.97	-40.85
125	116.24	-7.01	12.73
	139.10	11.28
	109.58	-12.34
423	367.25	-13.18	12.79
	468.11	10.66
	455.90	7.78
906	818.87	-9.62	7.87
	924.16	2.00
	953.40	5.23
1865	1743.24	-6.53	6.37
	1682.92	-9.76
	1902.10	1.99
3600	3462.85	-3.81	4.00
	3355.24	-6.80
	3197.33	-11.19
7200	5833.92	-18.97	27.35
	10 229.34	42.07
	8042.57	11.70

检测方法	阳性		阴性		总符合率/%
检测方法	例数	符合率/%	例数	符合率/%	总符合率/%
pVNT	89	-	10	-	-
层析法	76	85	23	100	87

检测方法	pVNT阳性	pVNT阴性	合计
层析法阳性	76	0	76
层析法阴性	13	10	23
合计	89	10	99

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