Improvement and optimization of a T-cell-dependent antibody response (TDAR) method for BALB/c mice using keyhole limpet hemocyanin (KLH) as specific antigen

Figures & data

Figure 1. OD values obtained using different KLH coating concentrations. The results showed that when the concentration of coated antigen reached 80 µg/ml, the OD value did not increase significantly when higher concentrations were employed. For this assay, test sera dilution was fixed at 1:800 (in 1% BSA/PBS) and the HRP-conjugated goat-anti-mouse IgG antibody dilution was fixed at 1:16,000 (in 1% BSA/PBS).

Table 1. OD_sample/OD_negative ratios.

Sample	IgG/HRP
	1:1000	1:2000	1:4000	1:8000	^a1:16,000	1:32,000
1:50	2.3	2.7	3.2	5.9	10.0	11.5
1:100	2.2	2.5	3.1	5.7	12.6	11.9
1:200	2.5	2.9	3.0	4.7	10.4	10.4
1:400	1.9	2.3	2.8	4.0	9.9	9.8
1:800	1.8	2.3	2.6	3.7	10.3	9.4
1:1600	1.8	2.1	2.7	3.9	9.9	6.9
1:3200	2.1	2.1	2.8	3.6	7.3	5.2
1:6400	1.9	2.0	2.8	2.3	4.8	3.6

^aWhen the dilution of HRP-goat anti-mouse IgG was 1:16,000, the OD_sample/OD_negative ratio was the largest, and all the ratios were greater than 2.1.

Figure 2. Demonstration of the linearity and the linear range of the serum samples.

Table 2. Within-group RSD.

	OD Value
	1:200	1:400	1:800
1	1.964	1.416	0.948
2	2.064	1.549	1.116
3	1.726	1.379	1.048
4	1.979	1.512	0.973
5	2.122	1.146	1.148
6	1.765	1.346	0.918
Mean	1.936	1.391	1.025
SD	0.146	0.131	0.085
RSD (%)	7.51	9.40	8.33

Table 3. Between-group RSD.

	OD Value
	1:200	1:400	1:800
1	1.979	1.562	0.915
2	1.746	1.422	1.246
3	1.618	1.133	1.138
4	1.836	1.515	1.248
5	2.177	1.047	1.414
6	1.748	1.420	1.015
Mean	1.850	1.350	1.163
SD	0.182	0.192	0.164
RSD (%)	9.83	14.22	14.10

Table 4. OD value analysis of anti-KLH IgG in peripheral blood of mice.

Group	KLH IgG
Negative control (no CTX, + KLH)	1.406 ± 0.146
Parallel control (+ CTX, no KLH)	^a0.171 ± 0.029
CTX-treated (+ CTX, + KLH)	^a^,^b0.737 ± 0.185

Values shown are mean OD values ± SD (n = 16 mice/group).

^aValue significantly different from negative control, p < 0.05.

^bValue significantly different from parallel control, p < 0.05.

Table 5. Analysis of anti-KLH IgG in peripheral blood of mice.

Groups	KLH IgG
	+	−	Total	Effective rate%
Negative control	15	1	16	93.75
Parallel control	0	16	16	0
CTX	14	2	16	87.50
Total	29	19	48	60.42

Values shown are those from n = 16 mice/group.