Development and Technical and Clinical Validation of a Quantitative Enzyme-Linked Immunosorbent Assay for the Detection of Human Antibodies to Hepatitis B Surface Antigen in Recipients of Recombinant Hepatitis B Virus Vaccine

Pierre Cambron; Jeanne-Marie Jacquet; Bernard Hoet; Marc Lievens

doi:10.1128/CVI.00431-08

DOI: 10.1128/CVI.00431-08

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FIG. 1.
Linear regression between FBS and human-plasma anti-HBs free as diluent. Hum-plasma, human plasma.
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FIG. 2.
Observed values and the model predictions for linearity assessment. For groups 1 and 4, the prediction line for each sample is superimposed with the prediction line for the mean sample within each group.
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FIG. 3.
OD response of serial dilutions of three highly concentrated anti-HBs samples.
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FIG. 4.
Deming's regression of the anti-HBs in-house assay and AUSAB RIA with CVs of 11.86% for both the RIA and anti-HBs in-house assay. UL, upper limit; LL, lower limit.
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FIG. 5.
GMCs (mIU/ml) at each blood sampling time point in study A (elderly patients), study E (adolescents), and study F (infants) for the anti-HBs in-house assay versus reference assays. PI to PIV repesent postdose months 1 to 4, respectively, and m1, m2, etc., represent study months 1, 2, etc.

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TABLE 1.

Clinical studies retested with the anti-HBs in-house assay

Study	Population	Vaccine group^a	Reference assay	No. of patients in group to be tested	Time point(s)^b	Rationale
A	50-70 yr old	Experimental HepB formulation	RIA	72 (subset)	PI, mo 1	Verify concordance with AUSAB RIA in an elderly population which reaches lower seroprotection rate and GMT than in adults 18-40 yr old
		Engerix-B			PI, mo 1
					PII, mo 6
					PIII, mo 7
B	Adult nonresponders^c	Experimental HepB formulation	RIA	58 (all)	Pre	Verify concordance with AUSAB RIA in a difficult population and check specificity of anti-HBs in-house assay on prevaccination samples
		Engerix-B			PI, mo 1
					PII, mo 2
					PII, mo 6
					PIII, mo 7
C	11-18 yr old	Engerix-B	RIA	72 (subset)	PI, mo 1	Verify concordance with AUSAB RIA after vaccination with a vaccine from another manufacturer
		Recombivax^d			PII, mo 2
					PII, mo 6
					PIII, mo 7
D	18-40 yr old	Engerix old 1	EIA	72 (subset)	PII, mo 6	Verify concordance with AUSAB EIA following different formulations of the same vaccine
		Engerix old 2			PIII, mo 7
		Engerix
E	12-15 yr old	Ambirix 720/20	RIA, EIA	140 (all)	PIII, mo 7	Verify concordance with AUSAB RIA and AUSAB EIA
		Twinrix 360/10
F	Children <1 yr old	Infanrix hexa	RIA	50 (subset)	Pre	Verify concordance with AUSAB RIA after vaccination with HBV in combined pediatric vaccines
		Infanrix penta + Hiberix			PII, mo 6
					PII, mo 8
					PIII, mo 9
G	Children <1 yr old	Infanrix hexa	EIA	62 (all)	Pre, mo 1.5	Verify concordance with AUSAB EIA after vaccination with HBV in combined pediatric vaccines
		Infanrix penta + Hiberix			PIII, mo 7
					PIII, mo 15
					PIV, mo 6

↵a Engerix old 1, previous formulation, containing thiomersal as a preservative; Engerix old 2, previous formulation, using thiomersal during the production process.
↵b Pre, sample taken prior to vaccination; PI to PIV, postdose months 1 to 4; mo 1, mo 2, etc., study month 1, study month 2, etc.
↵c Nonresponders are healthy adults with an anti-HBs antibody concentration of <10 mIU/ml 2 to 5 months after having received at least four doses of HBV.
↵d Recombivax is a trademark of Merck & Co.

TABLE 2.
Calibration curve^a
Antibody concn (mIU/ml) 0.95th percentile
2.92 14.41
5.84 8.59
11.69 6.31
23.38 4.51
46.75 4.11
93.50 3.12
187.00 1.60
↵a The calibration curve is based on the distribution of the percentage of the absolute values of the relative bias (RE) for the complete set of standard points (0.95 percentile), where % RE = [(recalculated value − assigned value)/assigned value] × 100%.

TABLE 3.

Accuracy of anti-HBs in-house assay in terms of human anti-HBV immunoglobulin^a

Theoretical antibody concn (mIU/ml)	Measured antibody concn (mIU/ml)	% Recovery
3.3	3.4	100.4
6.3	5.6	90.4
6.3	5.8	93.4
10.4	9.8	94.2
10.4	9.4	90.3
20.8	19.9	95.5
20.8	18.9	90.8
41.5	42.0	101.2
41.5	41.7	100.4
82.6	81.1	98.2
82.6	80.1	96.9
163.9	129.9	79.3
163.9	150.0	91.5
476.2	446.0	93.7
909.1	840.6	92.5
2,000.0	1,967.9	98.4
3,333.3	3,304.4	99.1
5,000.0	5,048.4	101.0
10,000.0	9,885.2	98.9

↵a The geometric mean recovery was 94.9%. The 95% CI lower and upper limits were 92.2% and 97.6%, respectively.

TABLE 4.
Demonstration of assay linearity^a
Group Slope 95% CI
Lower limit Upper limit
1 −0.9990 −1.0318 −0.9662
2 −0.9910 −1.0348 −0.9472
3 −0.9802 −1.0180 −0.9423
4 −0.9976 −1.0722 −0.9230
↵a Ten samples with concentrations between 100 and 51,895 mIU/ml were grouped into four groups according to the predilution applied to reach the upper limit of the analytical range. Shown are the estimated slope for each predilution group with its 95% CI using a mixed model with log dilution, group, and samples as factors.

TABLE 5.

Specificity of the anti-HBs in-house assay compared to the AUSAB EIA

AUSAB EIA antibody concn (mIU/ml) before preincubation	Anti-HBs in-house assay antibody concn (mIU/ml) before preincubation	HBsAg preincubation with subtype(s)^a:
		ad + ay				adw2
		AUSAB EIA		Anti-HBs in-house assay		AUSAB EIA		Anti-HBs in-house assay
		Antibody concn (mIU/ml)	% Specificity	Antibody concn (mIU/ml)	% Specificity	Antibody concn (mIU/ml)	% Specificity	Antibody concn (mIU/ml)	% Specificity
20	15	<3.3	100	<3.3	100	<3.3	100	<3.3	100
94	61	<3.3	100	<3.3	100	<3.3	100	<3.3	100
119	125	<3.3	100	<3.3	100	<3.3	100	<3.3	100
346	352	<3.3	100	<3.3	100	<3.3	100	<3.3	100
382	428	<3.3	100	<3.3	100	<3.3	100	<3.3	100
1,556	1,111	<3.3	100	<3.3	100	<3.3	100	<3.3	100
1,957	2,410	<3.3	100	<3.3	100	<3.3	100	<3.3	100
2,237	2,575	<3.3	100	<3.3	100	<3.3	100	<3.3	100
49,680	42,660	<3.3	100	<3.3	100	86	99.8	49	99.9
54,490	61,065	<3.3	100	<3.3	100	651	98.8	103	99.8
<3.3	<3.3	<3.3	NA	<3.3	NA	<3.3		<3.3
<3.3	<3.3	<3.3	NA	<3.3	NA	<3.3		<3.3
<3.3	<3.3	<3.3	NA	<3.3	NA	<3.3		<3.3

↵a Preincubation was performed with either an excess of a mix of native ad and ay HBsAg subtypes or adw2 HBsAg subtype for 2 h at 37°C. Specificity represents the difference in concentrations observed before and after inhibition expressed as a percentage of the corresponding concentration measured without inhibition.

TABLE 6.

Calculation of the average concentration and CV in three samples with high anti-HBs concentrations

Sample	Dilution	OD	Anti-HBs antibody concn (mUI/ml)	Avg anti-HBs antibody concn (mUI/ml)^a	CV (%)
1	1	>3.000	>187
	10,000	1.652	772,731	754,411	3
	100,000	0.200	736,091
2	1	>3.000	>187
	10,000	1.438	644,921	649,409	1
	100,000	0.180	654,897
3	1	>3.000	>187
	10,000	1.705	833,075	823,976	2
	100,000	0.215	814,878

↵a Average concentration determined at dilutions of 10,000 and 100,000.

TABLE 7.

Contingency table between anti-HBs in-house assay and the AUSAB RIA with different cutoffs^a

Antibody concn cutoff (mIU/ml)	Agreement (95% CI)	Sensitivity (95% CI)	Specificity (95% CI)	Kappa	McNemar P value^b
2	97.71 (96.53-98.58)	99.02 (98.08-99.58)	87.13 (79.00-92.96)	0.881 (0.830-0.931)	0.3833
2.6	97.82 (96.66-98.67)	98.53 (97.45-99.24)	92.08 (84.99-96.52)	0.891 (0.843-0.938)	0.5034
3.3	97.61 (96.40-98.49)	98.04 (96.84-98.88)	94.06 (87.52-97.79)	0.883 (0.835-0.931)	0.0525
3.9	96.74 (95.37-97.79)	96.94 (95.52-98.01)	95.05 (88.82-98.37)	0.847 (0.793-0.900)	0.0003

↵a n = 919.
↵b Test of dissymmetry in concordance.

TABLE 8.
Bioequivalence of the slope for the anti-HBs in-house assay compared to the AUSAB RIA^a
Equivalence range lower specification Equivalence range upper specification Equivalence
0.50 2.00 Yes
0.67 1.50 Yes
0.80 1.25 Yes
0.90 1.11 Yes
↵a Shown are the results of Deming regression (ultrastructural model) with CVs of 11.86% for the RIA and the anti-HBS in-house assay. The regression represents a test of bioequivalence on the slope.

TABLE 9.

Seroprotection rates per time point and study for the anti-HBs in-house assay versus the AUSAB RIA and EIA reference assays

Study	No. of subjects/group^a	Time point^b	% Seroprotection in:
			Vaccine group 1			Vaccine group 2			Vaccine group 3
			In-house assay	RIA	EIA	In-house assay	RIA	EIA	In-house assay	EIA
A	72	PI, mo 1	15.3	20.8		0.0	2.7
	72	PII, mo 2	80.6	81.9		23.6	26.4
	72	PII, mo 6	95.8	93.1		59.7	56.9
	72	PIII, mo 7	98.6	98.6		81.9	83.3
B	51	PI, mo 1	47.1	49.0		63.6	63.6
	51	PII, mo 2	60.8	60.8		81.8	81.8
	50	PII, mo 6	48.0	42.0		85.2	81.5
	47	PIII, mo 7	72.3	72.3		96.2	96.2
C	100	PI, mo 1	2.0	2.0		9.7	12.5
	72	PII, mo 2	80.6	68.1		68.1	69.4
	72	PII, mo 6	93.1	93.1		95.8	95.8
	72	PIII, mo 7	98.6	97.2		100.0	100.0
D	72	PIII, mo 7	97.2		95.8	97.2		97.2	97.2	97.2
E	134	PI/II, mo 2	53.0	40.3	41.8	90.4	86.8	86.8
	145	PII/III, mo 7	99.3	98.6	99.3	100.0	100.0	100.0
F	50	Pre	13.8	16.9		12.2	12.2
	50	PII, mo 6	98.0	98.0		96.0	96.0
	50	PII, mo 8	94.0	94.0		100.0	100.0
	50	PIII, mo 9	100.0	100.0		100.0	100.0
G	34	Pre, mo 1.5	52.9		49.0	59.6		68.4
	47	PIII, mo 7	100.0		100.0	95.9		95.0
	53	PIII, mo 15	96.6		96.6	93.0		96.0
	57	PIV, mo 6	100.0		100.0	98.3		98.3

↵a If the number between the two groups was different, the lowest number was taken.
↵b Pre, sample taken prior to vaccination; PI to PIV, postdose months 1 to 4, respectively; mo 1, mo 2, etc., study month 1, study month 2, etc.

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Development and Technical and Clinical Validation of a Quantitative Enzyme-Linked Immunosorbent Assay for the Detection of Human Antibodies to Hepatitis B Surface Antigen in Recipients of Recombinant Hepatitis B Virus Vaccine

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Antibody concn (mIU/ml)	0.95th percentile
2.92	14.41
5.84	8.59
11.69	6.31
23.38	4.51
46.75	4.11
93.50	3.12
187.00	1.60

Group	Slope	95% CI
Group	Slope	Lower limit	Upper limit
1	−0.9990	−1.0318	−0.9662
2	−0.9910	−1.0348	−0.9472
3	−0.9802	−1.0180	−0.9423
4	−0.9976	−1.0722	−0.9230

Equivalence range lower specification	Equivalence range upper specification	Equivalence
0.50	2.00	Yes
0.67	1.50	Yes
0.80	1.25	Yes
0.90	1.11	Yes