Development and evaluation of a new immunohistochemistry-based test for the detection of rabies virus neutralizing antibodies

Figures & data

Figure 1. Showing the 50% end point dilution in the Rapid Fluorescent Focus Inhibition Test. Note approximately 50% BHK 21 cells in the microscopic field are infected as evidenced by presence of fluorescent foci representing rabies nucleoprotein (×400).

Figure 2. Showing 50% end point dilution in the immunohistochemistry based test. Note the presence of dark brown inclusions representing rabies nucleoprotein in approximately 50% of BHK 21 cells (×400).

Table 1. Results for repeatability/intra-assay precision

Sample     #	Sample     ID	Test results (IU/mL)					Median titer (IU/mL)	Percent of the results within ± 2- fold of the median           (%)
		Titer 1	Titer 2	Titer 3	Titer 4	Titer 5
1	102/11	12.1	10.2	11.2	9.3	10.2	10.2	100
2	112/11	12..0	12.2	14.2	12.8	10.2	11.5	100
3	114/11	6.0	5.0	5.0	7.0	8.0	6.0	100
4	120/11	20.0	19.0	24.0	23.0	19.0	20.0	100
5	132/11	30.0	37.0	40.0	28.0	37.0	37.0	100
6	140/11	50.0	44.0	50.0	44.0	37.0	44.0	100
7	142/11	5.8	5.8	7.4	8.4	8.4	7.4	100
8	150/11	15.0	15.0	10.0	17.0	17.0	15.0	100
9	162/11	40.0	50.0	58.0	54.0	58.0	54.0	100
10	167/11	4.5	4.0	6.5	4.0	4.0	4.0	100
11	170/11	19.0	20.0	23.0	17.0	20.0	20.0	100
12	HRIG	121.0	128.0	110.0	112.0	120.0	120.0	100

Figure 3. Bland Atman plot showing the agreement between the 2 tests. Please see section “results” for explanation.

Figure 4. Scatter plot graph of results obtained between the 2 tests. Note the good correlation among samples with high titers (>20 IU/mL). There was no correlation with samples having titer less than 20 IU/mL.

Table 2. Comparison of antibody titers observed by RFFIT and immunohistochemistry test

Test	The mean of 50% endpoint dilutions of test sera (n = 120)	The mean of 50% end point dilutions of reference serum	Titer in IU/mL
RFFIT	1: 3048	1: 4094	22.32
IHC	1: 3056	1: 4098	22.38

Table 3. Results for intermediate/inter-assay precision

Sample#	Sample ID	Test results (IU/mL)			Median titer (IU/mL)	Percent of the results within ± 2-fold of the median (%)
		Titer 1	Titer 2	Titer 3
1	13/12	10.2	8.4	11.2	10.2	100
2	17/12	45.0	52.0	44.0	45.0	100
3	23/12	140.0	132.0	132.0	132.0	100
4	34/12	7.0	10.0	6.0	7.0	100
5	43/12	25.0	21.0	17.0	21.0	100
6	52/12	25.0	20.0	19.0	20.0	100
7	54/12	45.0	45.0	30.0	45.0	100
8	62/12	8.0	10.2	8.3	8.3	100
9	66/12	19.0	19.0	16.0	19.0	100
10	72/12	50.0	63.0	50.0	50.0	100
11	80/12	5.8	4.5	3.4	4.5	100
12	HRIG	120.0	124.0	98.0	120.0	100

Table 4. Results for the lower limit of quantitation

Sample iD	Dilution factor*	Observed Titers (IU/mL)											Observed median titer (IU/mL)	Percent of the results within ± 2-fold of the   median (%)
		Titer   1	Titer   2	Titer   3	Titer   4	Titer   5	Titer   6	Titer   7	Titer   8	Titer   9	Titer   10	Titer   11
HRIG	1000	0.12	0.09	0.1	0.2	0.3	0.2	0.12	0.2	0.12	0.2	0.2	0.2	100
	500	0.22	0.3	0.4	0.3	0.3	0.22	0.24	0.3	0.3	0.2	0.2	0.3	73
	100	1.2	1.4	1.0	1.4	1.4	0.9	1.2	1.3	1.2	1.2	0.8	1.2	100
	50	2.4	2.0	2.8	2.0	1.8	2.4	1.9	2.4	2.2	2.4	2.3	2.35	100
	25	4.8	4.5	4.8	4.0	4.6	4.5	4.2	3.8	4.2	4.6	4.8	4.55	100
	10	12.5	14.5	10.5	11.4	12.5	10.5	12.5	12.8	10.5	12.5	10.7	11.95     1111.211.2	100
	5	0.4	0.6	0.4	0.4	0.4	0.4	0.3	0.4	0.3	0.5	0.4	0.4	100

N/A, Not Applicable. *Samples were pre-diluted in rabies antibody negative human serum to the desired dilution factor before testing

Table 5. Results of specificity of immunohistochemistry based neutralization test

Sample #	Pretreatment conditions		Observed titer        (IU/mL)	Fold change in titer              (IU/mL)§*
142/11	Homologous Competitor (Inactivated Rabies Pitman Moore Strain)	1:2 Diluted   Media Control	8.9	N/A
		1:2 Diluted     Competitor	<0.1	(–) 178.0
		1:4 Diluted     Competitor	0.5	(–) 17.8
		1:8 Diluted     Competitor	2.0	(–) 4.5
	Heterologous Competitor (Inactivated Measles Virus)	1:2 Diluted   Media Control	10.0	N/A
		1:2 Diluted     Competitor	10.3	(+) 1.3
		1:4 Diluted     Competitor	11.4	(+) 1.4
		1:8 Diluted     Competitor	8.9	(–) 1.1

HRIG was treated with either increasing concentrations of rabies vaccine or inactivated measles virus and RVNA titers tested. There is a marked decrease in titers after treating with homologous inhibitor compared with heterologous inhibitor.