Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

Table 1 Characteristics of monoclonal anti-HBs antibodies

Clone	Isotype	Kaff (M-1)	Recognized epitope
34D7-S3	IgG1	5.24 × 108	Conformational
33D7-S4	IgG1	3.27 × 108	Conformational
38F3-S6	IgG1	3.75 × 108	Conformational
37C5-S7	IgG1	4 × 108	Linear
35C6-S11	IgG1	2.87 × 108	Linear
35G9-S15A	IgG1	1.73 × 108	Conformational
47G3-S15C	IgM	NI	Conformational
31D4-H12	IgG1	2.2 × 108	Linear

NI: Not identified.

Table 2 Mutations and subtype of recombinant mutant HBs antigens expressed in COS7 cells

Clone	Mutations	Subtype
Gly D	Standard sequence	adw
Gly y	Standard sequence	ayw
91-4696	S113T/T143S	adw
SA6	Q129R/G130N/A166V	adw
SA7	M133T	ayw
1056	P120S/S143L	ayw
PA17D	D144E/multiple mutations	NI
M5	Y100S/T118V/R122K/M133I/	ayw
	Y134N/P142S/S143L/G145K
BA2.4	Y100C/P120T	ayw
SA4	M133T/y161F	adw
BA3.2	T123N/C124R	ayw

NI: Not identified.

Table 3 Reactivity of mutant HBs antigens secreted by transfected cells as determined by three commercial ELISA kits

Expressed	Bioelisa		ETI-MAK-4		Hepanostika
HBsAg	A450	Index	A450	Index	A450	Index	Conc.
Gly D	> 3	> 5.5	2.9	5.1	> 3	> 11	> 200
Gly Y	2.97	5.5	2.97	5.2	> 3	> 11	> 200
91-4696	> 3	> 5.5	2.87	5	> 3	> 11	> 200
SA6	2.96	5.5	2.74	4.8	> 3	> 11	> 200
SA7	> 3	> 5.5	2.87	5	> 3	> 11	> 200
1056	1.5	2.8	1.8	3.2	2.32	8.6	162
PA17D	1	1.8	2.7	4.7	> 3	> 11	> 200
M5	1.59	2.9	0.74	1.3	1.39	5.1	92
BA2.4	1.66	3.1	2.08	3.6	2.33	8.6	162
SA4	1.67	3.1	1.34	2.3	2.12	7.8	148
BA3.2	0.54	1	0.65	1.1	1	3.7	62
pRK5	0.17	0.31	0.08	0.15	0.1	0.37	0
Medium	0.18	0.33	0.11	0.19	0.1	0.37	0

Results are expressed as absorbance (A) measured at 450 nm, index (ratio of absorbance of mutant sample to absorbance obtained for 1.25 ng/mL of standard adw HBsAg) and concentration (Conc.) presented as ng/mL. Concentration was measured only by the Hepanostika kit using the standard adw HBsAg to construct the standard curve.

Table 4 Reactivity patterns of monoclonal anti-HBs antibodies with expressed HBs antigen mutants

Monoclonal antibody	GlyD	GlyY	91-4696	SA6	SA7	1056	PA17D	M5	BA2.4	SA4	BA3.2
34D7-S3	++	++	++	++	++	+	-	-	+	+	-
33D7-S4	++	++	++	++	++	+	-	-	+w	+	-
38F3-S6	+++	+++	+++	+++	+++	+++	+w	-	-	+	-
37C5-S7	+++	+++	+++	+++	+++	+w	++	+++	++	+w	+
35C6-S11	+++	+++	+++	+++	+++	+w	+++	+	+	+w	+w
35G9-S15A	+++	+++	+++	+++	+++	-	-	-	+w	+w	-
31D4-H12	++	++	++	++	++	-	-	-	+w	+w	-
47G3-S15C	+++	+++	+++	+	+	-	-	+	+	-	-

Results are expressed as ratio of absorbance (A) of a given mutant to that obtained for 1.25 ng/mL of standard recombinant adw HBsAg and presented as : -: ≤ negative control (pRK5 plasmid); +^w: > negative control < 1; +: 1-3; ++: 3-6; +++: > 6.

Table 5 Cross inhibition of monoclonal antibodies binding to HBs antigen

UnlabeledmAbs	HRP-conjugated mAbs
	34D7-S3	33D7-S4	38F3-S6	37C5-S7	35C6-S11	35G9-S15A	31D4-H12
34D7-S3	4	4	4	2	3	4	1
33D7-S4	3	4	4	2	4	3	2
38F3-S6	3	3	4	1	3	4	2
37C5-S7	1	1	1	4	1	2	3
35C6-S11	2	4	4	2	4	3	2
35G9-S15A	4	3	3	1	3	3	2
47G3-S15C	3	3	3	1	3	3	2
31D4-H12	2	1	1	3	1	2	3

1: 0%-20% inhibition; 2: 20%-50% inhibition; 3: 50%-80% inhibition; 4: 80%-100% inhibition.