Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C

Figures & data

Table 1. Determination of isotype and affinity constant of selected monoclonal antibodies.

	Isotype
		ELISA
MAb	Isostrip	IgG1	IgG2a	Affinity constant
1F3E3	IgG2a	0.14	2.13	1.78 × 10^9
1F2C2	IgG2a	0.06	1.89	7.10 × 10^8
1F3B3	IgG2a	0.12	1.12	7.00 × 10^7
1F4E11	IgG2b	0.08	0.03	2.10 × 10^8
Neg. Ctrl.	NT	0.03	0.03	—

ELISA results represent OD values obtained at 450 nm.

Culture medium was used as negative control (Neg Ctrl).

Affinity constant (K_a) of each MAb was determined using formula described in Materials and methods.

NT, not tested.

Figure 1. Western blot reactivity profile of selected anti-tetanus toxin monoclonal anti-bodies. Tetanus toxoid was separated over a 10% polyacrylamide gel, electroblotted to PVDF membranes, and then probed with anti-TeNT MAb. SM, protein size marker.

Table 2. Reactivity of specific monoclonal antibodies to tetanus toxoid, toxin, fragment C, and light chain.

MAb	Toxoid	Toxin	Fragment C	Light chain
1F3E3	3.00	3.00	3.00	0.03
1F2C2	3.00	3.00	2.57	0.06
1F3B3	1.31	0.59	0.06	0.96
1F4E11	3.00	3.00	2.43	0.33
Neg Ctrl	0.08	0.03	0.03	0.03

The results represent OD values obtained at 450 nm by ELISA.

Culture medium was used as negative control (Neg Ctrl).

Figure 2. Representative cross-inhibition profiles of tetanus toxin-specific monoclonal antibody. Increasing concentrations of competitor MAb were used to inhibit binding of the enzyme-conjugated detector MAbs 1F3E3 (a), 1F2C2 (b) and 1F4E11 (c) to tetanus toxin. The results show the percentage inhibition of the binding of detector MAb to tetanus toxoid.

Table 3. Immunoglobulin heavy and light chain variable region genes usage by the TeNT-specific hybridoma clones.

MAb	VH	JH	DH	Vκ	Jκ
1F3E3	IGHV1-9*01	IGHJ3*01	IGHD3-2*01	IGV19-93*01	IGKJ2*01
1F2C2	IGHV3-6*02	IGHJ3*01	IGHD5-5*01	IGKV6-17*01	IGKJ1*01
1F3B3	IGHV1-39*01	IGHJ1*01	IGHD2-10*01	IGKV4-59*01	IGKJ2*01
1F4E11	IGHV1-4*01	IGHJ3*01	IGHD5-7*01	IGKV12-41*01	IGKJ1*01

Figure 3. Inhibition of tetanus toxin binding to ganglioside GT1b by monoclonal antibodies. An appropriate level of tetanus toxin in the presence or absence of increasing concentrations of MAb was allowed to bind to ganglioside GT1b. The percentage inhibition of TeNT biding was then calculated as described in the Materials and methods.