A comparative study of two different assay kits for the detection of secreted alkaline phosphatase in HPV antibody neutralization assays

Figures & data

Table 1. Comparison of different chemiluminescence-based SEAP assay kits

	VENDOR
Characteristics^a	Clontech	Roche	Life Technologies	Jaden Bioscience
Catalog no.	631738	11779842001	N10578	CM025
Kit size	1000 tests	500 tests	10 x 96 well plates	1000 tests
96-well plate format	Yes	Yes	Yes	Yes
Storage temperature	−20°C	4°C	4°C	4°C
List price (US $)	$838.00	$513.00	$1,124.00	$389.99
Sensitivity	100 fg	10 fg	NA^b	0.005 fg
Linear dynamic range^c	10^4	10^4–10^5	10^5	10^6 –10^7
Sample volume used (μL)	25	50^d	25	1–5
Approximate assay time (min)	60	60	30	60

^aThe information is based on those indicated in the production sheets or certificate of analysis, or on the website of each company. ^bNA: Not available. ^cRange of detection will vary depending on the instrument used to measure the activity. ^dFifty microliters of samples from 4-fold dilution are used.Website for each company: Clontech (http://www.clontech.com/US/Products/Fluorescent_Proteins_and_Reporters/Chemiluminescent_Reporters/Secreted_Alkaline_Phosphatase); Roche (http://www.roche-applied-science.com/shop/products/seap-reporter-gene-assay-chemiluminescent#tab-0); Life Technologies (http://www.lifetechnologies.com/us/en/home/life-science/protein-expression-and-analysis/enzymes-and-protein-activity-assays/epaassays-misc/novabrightchemiluminescent-reporter-gene-assays/novabright-chemiluminescent-seap-reporter-gene-assays.html); and Jaden Bioscience (http://www.jadenbio.com/assays/protein/ziva-seap.html)

Figure 1. ZiVa can better detect SEAP than GE. Pseudovirion particles of HPV types 16 and 18 were serially diluted, and used to infect 0.03 × 10⁶ 293TT cells. Two-fold serial dilutions were performed starting from 1:1000 to 1:1024000. Supernatants were harvested at 48 and 72 hr post-infection. Each PsV dilution was setup in duplicate wells. Mean ± standard deviation (SD) of 3 independent experiments are shown. (A) Infectivity levels (fold-increase) by PsV16 particles after 48 or 72 hr of incubation are shown. (B) Infectivity levels by PsV18 particles after 48 or 72 hr of incubation are shown. In both (A and B), the same supernatant samples were tested using ZiVa or GE SEAP detection kit. The plots show fold-increase in log₁₀ scale as a function of viral concentration (log₁₀). Some of the error bars are too small to be seen in the graphs.

Table 2. Ratios of ZiVa to GE in detecting SEAP

	ZiVa/GE ratio (48 hr)				ZiVa/GE ratio (72 hr)
PsV16 dilution	Exp 1	Exp 2	Exp 3	Mean(48 hr)	Exp 1	Exp 2	Exp 3	Mean (72 hr)
1,000	21	22	23	22	12	11	8	10
2,000	22	23	24	23	12	11	9	11
4,000	22	24	26	24	13	12	9	12
8,000	23	23	25	24	15	14	10	13
16,000	23	25	24	24	15	14	11	14
32,000	23	24	26	24	17	16	12	15
64,000	23	23	25	23	18	16	12	16
128,000	22	25	25	24	19	17	12	16
256,000	21	22	24	22	18	16	13	15
512,000	18	24	23	22	18	17	12	16
1,024,000	12	19	18	16	17	15	12	15
PsV18 dilution	Exp 1	Exp 2	Exp 3	Mean (48 hr)	Exp 1	Exp 2	Exp 3	Mean (72 hr)
1,000	23	24	27	24	15	14	10	13
2,000	23	24	26	25	16	15	11	14
4,000	24	25	28	26	17	17	12	15
8,000	24	24	28	26	18	17	12	16
16,000	24	22	27	24	18	18	12	16
32,000	23	22	24	23	19	18	12	16
64,000	12	13	24	16	19	18	12	16
128,000	12	11	13	12	19	18	12	16
256,000	9	7	7	8	17	17	12	15
512,000	1	4	1	2	12	14	11	12
1,024,000	2	1	2	1	15	15	8	12

Values in the ZiVa/GE ratios were calculated by dividing the values of mean fold-increase obtained using ZiVa by the values obtained with GE for the corresponding PsV dilution factors.

Figure 2. ZiVa can detect SEAP even using limiting numbers of 293TT cells. Different numbers of 293TT cells were plated at the start of the infections by PsV16 or PsV18. The number of cells ranged from 0.002 - 0.3 × 10⁶ cells per well, and 2-fold serial dilutions were performed. Each cell number was tested in triplicates. A fixed dilution factor of 1:100000 for PsV16 (A) and 1:10000 for PsV18 (B) were used to infect 293TT cells. At 48 or 72 hr post-infection, the supernatants were harvested. Cell numbers were plotted in log₁₀ scale. Mean ± SD of 3 independent experiments are shown. Some of the error bars are too small to be seen in the graphs.

Figure 3. ZiVa can detect SEAP at lower cell numbers using lower viral dilution concentrations. The experiments were conducted as described in Fig. 2, except higher viral dilution factors were used: 1:600000 for PsV16 and 1:60000 for PsV18. Mean ± SD of 3 independent experiments are shown. Some of the error bars are too small to be seen in the graphs.

Table 3. Comparison of 50% neutralization titer values measured by GE or ZiVa

	Anti-HPV16			Anti-HPV18
Sample ID	GE	ZiVa	ZiVa/ GE^a	GE	ZiVa	ZiVa/ GE
1	129	175	1.4	373	432	1.2
2	207	162	0.8	112	169	1.5
3	343	294	0.9	302	495	1.6
4	124	126	1.0	180	300	1.7
5	6999	5927	0.8	1112	1127	1.0
6	562	507	0.9	1301	2509	1.9
7	404	444	1.1	116	113	1.0
8	144	563	3.9	80	95	1.2
9	47	29	0.6	98	129	1.3
10	180	331	1.8	127	148	1.2
11	354	391	1.1	159	148	0.9
12	45	81	1.8	126	187	1.5
13	3602	2474	0.7	2279	2705	1.2
14	2491	2428	1.0	3440	4122	1.2
15	737	861	1.2	192	314	1.6
16	62	57	0.9	11	11	1.0
17	1357	1292	1.0	588	563	1.0
18	278	339	1.2	61	80	1.3
19	853	926	1.1	349	469	1.3
20	1139	1025	0.9	805	755	0.9
21	633	664	1.0	442	498	1.1
22	5159	4304	0.8	1261	1202	1.0
23	16	27	1.7	8	9	1.2
24	312	283	0.9	297	370	1.2
25	4063	4301	1.1	3463	3207	0.9
26	4098	3657	0.9	1900	1903	1.0
27	239	253	1.1	344	295	0.9
28	3378	4287	1.3	1846	1638	0.9
29	3795	3690	1.0	3704	3407	0.9
30	6256	5214	0.8	2784	3520	1.3
31	6060	6596	1.1	3213	2852	0.9
32	6725	4783	0.7	1724	1716	1.0
33	127	175	1.4	53	77	1.5
34	695	841	1.2	269	344	1.3
35	2339	2835	1.2	604	786	1.3
36	7341	6794	0.9	2292	2504	1.1
37	5394	4429	0.8	3186	2806	0.9
38	4409	4069	0.9	6670	6142	0.9
39	4791	6364	1.3	6013	6455	1.1
40	1898	1834	1.0	1282	1430	1.1
41	8565	9831	1.1	5761	6526	1.1
42	245	372	1.5	259	289	1.1
43	585	554	0.9	399	418	1.0
44	3930	3445	0.9	3484	2479	0.7
45	2727	2966	1.1	859	1052	1.2
46	4477	4767	1.1	879	774	0.9
47	9393	10044	1.1	1767	2038	1.2
48	305	222	0.7	649	821	1.3
49	1843	1958	1.1	670	711	1.1
50	8215	7633	0.9	2022	1996	1.0
51	5760	5838	1.0	894	821	0.9
52	1187	1035	0.9	1351	1292	1.0
53	5740	4756	0.8	369	378	1.0
54	492	534	1.1	45	46	1.0
55	8855	8281	0.9	7667	8745	1.1
56	1065	1043	1.0	458	561	1.2
57	5213	5355	1.0	292	281	1.0
58	1217	942	0.8	142	423	3.0
59	2624	1980	0.8	1825	2381	1.3
60	1675	1383	0.8	550	773	1.4
61	3106	2451	0.8	1977	2443	1.2
62	17828	24856	1.4	4312	5285	1.2
63	360	528	1.5	99	92	0.9
64	5054	109285	21.6	2209	3339	1.5
65	17124	14622	0.9	1910	1841	1.0
66	595	542	0.9	526	562	1.1
67	2361	2599	1.1	1905	1897	1.0
68	6893	7705	1.1	2264	2480	1.1
69	3321	2901	0.9	400	340	0.9
70	3068	3275	1.1	491	770	1.6
71	4170	3494	0.8	387	374	1.0
72	6826	6637	1.0	1376	1655	1.2

Fifty percent neutralization titers measured by ZiVa or GE from the corresponding samples are shown.

^aThe values of 50% neutralization titers measured by ZiVa were divided by those measured by GE.

Figure 4. HPV neutralizing antibody titers measured by ZiVa correlate with those of GE. Cell-culture supernatants from 293TT cell-based neutralization assay from the Uganda Immunogenicity Study were used to quantitate 50% neutralization titers in sera of Cervarix®-immunized individuals using ZiVa or GE. The supernatant samples collected after 72 hr of incubation were used to measure the SEAP activity. Spearman rank-correlational analyses were performed between the titers obtained by ZiVa or GE for both anti-HPV16 and -HPV18 antibodies.