Metrics for Assay Development

Z-Prime*: 3 X (Standard Deviation of the Maximum Signal Control) + 3 X (Standard Deviation of the Minimum Signal Control)
1- ( |(Mean of the Maximum Signal Control - Mean of the Minimum Signal Control)| )

Z-prime is a dimensionless calculation used to assess the quality of a high-throughput assay. It compares the mean
value of the maximum signal control to the mean value of the minimum control, and will have a higher value when (a)
there is a wide separat

Z-Factor* 3 X (Standard Deviation of Batch of Compounds Tested) + 3 X (Standard Deviation of the 100% Inhibition Control)
1- ( |(Mean of the Compound Batch Tested - Mean of the 100% Inhibition Control)| )

Z-Factor is a dimensionless calculation used to assess the quality of a population of sample compounds tested. For an
inhibition screen, the Z-factor compares the mean value of all compounds tested to the mean value of the control which
represents 100% in

Standard Deviation
%CV:
100 x ### X( Mean )
( Stand
ard
Deviatio
The coefficient of variation is the standard deviation divided by the mean of a replicate set of values. It can be
n
expressed as a %CV by multiplying by 100. Low %CV indicates high precision, high %CV indicates poor precision.

S:N Mean of the maximum signal control - mean of minimum signal control
Stasnfdard deviation of the minimum signal control
 Signal to noise is a measure of the signal strength

S:B Mean of the maximum signal control

Mean of the minimum signal control

The signal to background is a measure of the assays' dynamic range.

SW (Mean of the Max Sig. Cont - Mean of the Min Sig. Cont)-3 X (Std. Dev. of Max sig. cont + Std. Dev. o
Standard deviation of the maximum signal control

The signal window (SW) is a measure of the assays' significant signal between min and max controls. An acceptable
value is > 2-fold.

* Zhang JH, Chung TD, Oldenburg KR. Related Articles, Links

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.
J Biomol Screen. 1999;4(2):67-73.
PMID: 10838414 [PubMed - as supplied by publisher]
 ###
Experiment: 1205LU cl7

DMSO
1 2 3 4 5 6 7 8 9 10 11 12
A 6E+05 5E+05 6E+05 5E+05 6E+05 6E+05 6E+05 5E+05 6E+05 6E+05 5E+05 5E+05
B 6E+05 4E+05 6E+05 4E+05 6E+05 5E+05 5E+05 5E+05 5E+05 5E+05 6E+05 4E+05
C 5E+05 5E+05 6E+05 6E+05 5E+05 5E+05 5E+05 5E+05 4E+05 5E+05 6E+05 3E+05
D 5E+05 5E+05 6E+05 5E+05 5E+05 4E+05 6E+05 5E+05 6E+05 5E+05 5E+05 6E+05
E 6E+05 4E+05 6E+05 5E+05 4E+05 5E+05 6E+05 5E+05 4E+05 5E+05 5E+05 3E+05
F 5E+05 4E+05 5E+05 5E+05 6E+05 5E+05 6E+05 5E+05 5E+05 4E+05 6E+05 5E+05
G 5E+05 3E+05 5E+05 6E+05 6E+05 5E+05 5E+05 5E+05 6E+05 6E+05 5E+05 5E+05
H 6E+05 4E+05 6E+05 5E+05 6E+05 5E+05 5E+05 5E+05 6E+05 6E+05 6E+05 6E+05
I 5E+05 3E+05 6E+05 5E+05 6E+05 4E+05 6E+05 6E+05 6E+05 6E+05 5E+05 5E+05
J 4E+05 4E+05 6E+05 6E+05 6E+05 3E+05 4E+05 6E+05 4E+05 5E+05 6E+05 5E+05
K 6E+05 6E+05 6E+05 5E+05 6E+05 5E+05 6E+05 4E+05 5E+05 4E+05 6E+05 5E+05
L 6E+05 4E+05 6E+05 6E+05 6E+05 4E+05 6E+05 5E+05 5E+05 5E+05 4E+05 5E+05
M 6E+05 6E+05 6E+05 5E+05 5E+05 6E+05 6E+05 5E+05 6E+05 5E+05 5E+05 5E+05
N 6E+05 5E+05 6E+05 5E+05 5E+05 4E+05 6E+05 5E+05 6E+05 5E+05 5E+05 4E+05
O 5E+05 5E+05 5E+05 5E+05 4E+05 5E+05 6E+05 5E+05 5E+05 4E+05 5E+05 5E+05
P 6E+05 4E+05 5E+05 4E+05 5E+05 4E+05 5E+05 4E+05 5E+05 4E+05 5E+05 3E+05

average 5E+05 5E+05 6E+05 5E+05 5E+05 5E+05 5E+05 5E+05 5E+05 5E+05 5E+05 5E+05
stdev 63415 82847 50933 42225 73225 72747 61878 47118 74946 60111 63642 87039
CV 0.12 0.18 0.09 0.08 0.14 0.15 0.11 0.09 0.14 0.12 0.12 0.18

DMSO DW1/2
Avg 5E+05 10716 up - un 5E+05
Median 5E+05 9880
St. Dev. 73072 6184 op + on 79256 3(op+on 2E+05
CV 0.14 0.58
Max 6E+05 66800
Min 3E+05 1240
S:N -6.893
S:B 0.0208
Sig. Win. 43
Z score 0.528
DW1/2 DMSO
13 14 15 16 17 18 19 20 21 22 23 24 average
19280 13600 11840 8640 8520 12480 9480 9360 9960 12840 10120 13200 549880
7880 8160 14640 13400 12000 8520 10840 4120 7360 5680 10720 6200 527480
9440 5960 7520 9400 7320 6840 9600 6400 12200 10280 15600 12960 495110
13040 6640 6240 9560 7920 8840 15960 11680 9720 8240 12440 11080 536933
12680 6960 4560 11200 19560 7800 13000 12520 7440 4840 11880 9960 476187
12480 5600 9120 10240 15480 7720 21440 10120 15440 7120 18560 7000 526227
10120 11440 19400 15480 13880 15520 10360 5280 12720 5920 12360 3840 507397
16960 11080 21040 8760 18680 6640 15960 7040 14200 7560 6960 7200 546367
11440 6600 9480 12000 17240 12480 1560 12000 13160 4560 14320 7480 532437
19280 9160 14520 6280 10240 12440 14480 7720 19480 4600 9240 7840 488940
7080 5440 7120 5960 8840 30920 66800 5800 10600 16200 15240 3040 526610
6800 9960 2720 4680 11920 9320 14520 11960 7640 5680 11200 7040 509063
10160 2800 6720 5160 9800 6640 7800 11120 16960 20160 17480 12360 532383
15160 6920 12280 12520 18440 11040 21160 13280 10760 6240 24640 4160 528850
7720 9560 7120 7640 15120 4480 9480 6360 18520 8720 2240 12320 490057
8400 3400 10280 4480 15400 6000 15680 3720 12040 1240 6880 8040 456810

11745 7705 10288 9087.5 13148 10480 16133 8655 12388 8117.5 12493 8357.5
4120 2940 5104 3286 4124 6201 14400 3210 3772 4777 5282 3300
0.35 0.38 0.50 0.36 0.31 0.59 0.89 0.37 0.30 0.59 0.42 0.39
 DW1/2
stdev CV average stdev CV
29923 0.05 11610 3023 0.26
76680 0.15 9127 3218 0.35
67132 0.14 9460 2938 0.31
60791 0.11 10113 2845 0.28
86330 0.18 10200 4213 0.41
70396 0.13 11693 5029 0.43
69070 0.14 11360 4610 0.41
69027 0.13 11840 5257 0.44
81440 0.15 10193 4430 0.43
107478 0.22 11273 4835 0.43
78757 0.15 15253 17889 1.17
88318 0.17 8620 3458 0.40
40552 0.08 10597 5329 0.50
65304 0.12 13050 6081 0.47
47740 0.10 9107 4469 0.49
62318 0.14 7963 4654 0.58
 DMSO DW1/2
1 590040 19280
2 649160 7880 By Column
3 512080 9440
700000
4 492160 13040
5 573480 12680
600000
6 535240 12480
7 496560 10120
500000
8 615480 16960

RLU
9 532720 11440
400000
10 388320 19280
11 552000 7080 300000
12 615840 6800
13 559320 10160 200000
14 606360 15160
15 504200 7720 100000
16 558480 8400
17 513440 13600 0
18 441720 8160 0 16 32 48 64 80 96 112 128 14
Well #
19 494280 5960
20 513200 6640
21 425240 6960
22 397000 5600
23 327200 11440
24 401640 11080
25 348800 6600
26 442880 9160
27 589880 5440
28 352440 9960
29 580000 2800
30 531920 6920
31 535320 9560
32 401240 3400
33 606920 11840
34 580080 14640
35 564360 7520
36 636520 6240
37 566840 4560
38 502840 9120
39 503320 19400
40 562680 21040
41 612120 9480
42 632280 14520
43 640440 7120
44 634920 2720
45 565320 6720
46 624800 12280
47 514160 7120
48 505880 10280
49 511440 8640
50 432200 13400
51 552920 9400
52 491680 9560
53 479400 11200
54 510840 10240
55 558720 15480
56 475120 8760
57 502760 12000
58 574800 6280
59 502760 5960
60 552520 4680
61 533240 5160
62 479200 12520
63 467480 7640
64 438160 4480
65 563920 8520
66 625560 12000
67 528280 7320
68 531680 7920
69 374720 19560
70 648760 15480
71 573400 13880
72 573040 18680
73 590280 17240
74 563200 10240
75 551320 8840
76 577200 11920
77 507280 9800
78 534880 18440
79 380960 15120
80 534480 15400
81 553560 12480
82 543800 8520
83 492560 6840
84 412400 8840
 85 494200 7800
86 548200 7720
87 519200 15520
88 545840 6640
89 442440 12480
90 302800 12440
91 491360 30920
92 387520 9320
93 568680 6640
94 426920 11040
95 491640 4480
96 433320 6000
97 555560 9480
98 504240 10840
99 509840 9600
100 598400 15960
101 599040 13000
102 625320 21440
103 455000 10360
104 505840 15960
105 634920 1560
106 422920 14480
107 590080 66800
108 560320 14520
109 552680 7800
110 587960 21160
111 561960 9480
112 473320 15680
113 539600 9360
114 488240 4120
115 471760 6400
116 544480 11680
117 486280 12520
118 547760 10120
119 539320 5280
120 492880 7040
121 559960 12000
122 603000 7720
123 430120 5800
124 476360 11960
125 474040 11120
126 500680 13280
127 481240 6360
128 433800 3720
129 565280 9960
130 536280 7360
131 446880 12200
132 602160 9720
133 405000 7440
134 478720 15440
135 570000 12720
136 640840 14200
137 587080 13160
138 374840 19480
139 478200 10600
140 501800 7640
141 560320 16960
142 606880 10760
143 528800 18520
144 487320 12040
145 551040 12840
146 504440 5680
147 478040 10280
148 509400 8240
149 522920 4840
150 447320 7120
151 564880 5920
152 555800 7560
153 572000 4560
154 466560 4600
155 359280 16200
156 541000 5680
157 474840 20160
158 498640 6240
159 438120 8720
160 405720 1240
161 512960 10120
162 605880 10720
163 571000 15600
164 532760 12440
165 480080 11880
166 571560 18560
167 466200 12360
168 630040 6960
169 464200 14320
170 614600 9240
171 592120 15240
172 422920 11200
173 468960 17480
174 501240 24640
175 502080 2240
176 476560 6880
177 534800 13200
178 418160 6200
179 319320 12960
180 578360 11080
181 307040 9960
182 501160 7000
183 514960 3840
184 557200 7200
185 541960 7480
186 481080 7840
187 541760 3040
188 485920 7040
189 543920 12360
190 446720 4160
191 474720 12320
192 333440 8040
 By Column

DMSO
DW 1/2

48 64 80 96 112 128 144 160 176 192

Well #
 DMSO DW1/2
1 590040 19280
2 513440 13600 By row
3 606920 11840
4 511440 8640 700000

5 563920 8520
6 553560 12480 600000

7 555560 9480
500000
8 539600 9360

RLU
9 565280 9960
400000
10 551040 12840
11 512960 10120
300000
12 534800 13200
13 649160 7880 200000
14 441720 8160
15 580080 14640 100000
16 432200 13400
17 625560 12000 0
18 543800 8520 0 16 32 48 64 80 96
well #
112 128 144 160
19 504240 10840
20 488240 4120
21 536280 7360
22 504440 5680
23 605880 10720
24 418160 6200
25 512080 9440
26 494280 5960
27 564360 7520
28 552920 9400
29 528280 7320
30 492560 6840
31 509840 9600
32 471760 6400
33 446880 12200
34 478040 10280
35 571000 15600
36 319320 12960
37 492160 13040
38 513200 6640
39 636520 6240
40 491680 9560
41 531680 7920
42 412400 8840
43 598400 15960
44 544480 11680
45 602160 9720
46 509400 8240
47 532760 12440
48 578360 11080
49 573480 12680
50 425240 6960
51 566840 4560
52 479400 11200
53 374720 19560
54 494200 7800
55 599040 13000
56 486280 12520
57 405000 7440
58 522920 4840
59 480080 11880
60 307040 9960
61 535240 12480
62 397000 5600
63 502840 9120
64 510840 10240
65 648760 15480
66 548200 7720
67 625320 21440
68 547760 10120
69 478720 15440
70 447320 7120
71 571560 18560
72 501160 7000
73 496560 10120
74 327200 11440
75 503320 19400
76 558720 15480
77 573400 13880
78 519200 15520
79 455000 10360
80 539320 5280
81 570000 12720
82 564880 5920
83 466200 12360
84 514960 3840
 85 615480 16960
86 401640 11080
87 562680 21040
88 475120 8760
89 573040 18680
90 545840 6640
91 505840 15960
92 492880 7040
93 640840 14200
94 555800 7560
95 630040 6960
96 557200 7200
97 532720 11440
98 348800 6600
99 612120 9480
100 502760 12000
101 590280 17240
102 442440 12480
103 634920 1560
104 559960 12000
105 587080 13160
106 572000 4560
107 464200 14320
108 541960 7480
109 388320 19280
110 442880 9160
111 632280 14520
112 574800 6280
113 563200 10240
114 302800 12440
115 422920 14480
116 603000 7720
117 374840 19480
118 466560 4600
119 614600 9240
120 481080 7840
121 552000 7080
122 589880 5440
123 640440 7120
124 502760 5960
125 551320 8840
126 491360 30920
127 590080 66800
128 430120 5800
129 478200 10600
130 359280 16200
131 592120 15240
132 541760 3040
133 615840 6800
134 352440 9960
135 634920 2720
136 552520 4680
137 577200 11920
138 387520 9320
139 560320 14520
140 476360 11960
141 501800 7640
142 541000 5680
143 422920 11200
144 485920 7040
145 559320 10160
146 580000 2800
147 565320 6720
148 533240 5160
149 507280 9800
150 568680 6640
151 552680 7800
152 474040 11120
153 560320 16960
154 474840 20160
155 468960 17480
156 543920 12360
157 606360 15160
158 531920 6920
159 624800 12280
160 479200 12520
161 534880 18440
162 426920 11040
163 587960 21160
164 500680 13280
165 606880 10760
166 498640 6240
167 501240 24640
168 446720 4160
169 504200 7720
170 535320 9560
171 514160 7120
172 467480 7640
173 380960 15120
174 491640 4480
175 561960 9480
176 481240 6360
177 528800 18520
178 438120 8720
179 502080 2240
180 474720 12320
181 558480 8400
182 401240 3400
183 505880 10280
184 438160 4480
185 534480 15400
186 433320 6000
187 473320 15680
188 433800 3720
189 487320 12040
190 405720 1240
191 476560 6880
192 333440 8040
 By row

DMSO
DW1/2

4 80 96 112 128 144 160 176 192

well #
 1 2 3 4 5 6 7 8 9
A 590040 513440 606920 511440 563920 553560 555560 539600 565280
B 649160 441720 580080 432200 625560 543800 504240 488240 536280
C 512080 494280 564360 552920 528280 492560 509840 471760 446880
D 492160 513200 636520 491680 531680 412400 598400 544480 602160
E 573480 425240 566840 479400 374720 494200 599040 486280 405000
F 535240 397000 502840 510840 648760 548200 625320 547760 478720
G 496560 327200 503320 558720 573400 519200 455000 539320 570000
H 615480 401640 562680 475120 573040 545840 505840 492880 640840
I 532720 348800 612120 502760 590280 442440 634920 559960 587080
J 388320 442880 632280 574800 563200 302800 422920 603000 374840
K 552000 589880 640440 502760 551320 491360 590080 430120 478200
L 615840 352440 634920 552520 577200 387520 560320 476360 501800
M 559320 580000 565320 533240 507280 568680 552680 474040 560320
N 606360 531920 624800 479200 534880 426920 587960 500680 606880
O 504200 535320 514160 467480 380960 491640 561960 481240 528800
P 558480 401240 505880 438160 534480 433320 473320 433800 487320

700000
COLUMN

600000

500000

400000

300000

200000

100000

0
0 5 10 15 20 25
100000

0
0 5 10 15 20 25
 10 11 12 13 14 15 16 17 18 19
551040 512960 534800 19280 13600 11840 8640 8520 12480 9480
504440 605880 418160 7880 8160 14640 13400 12000 8520 10840
478040 571000 319320 9440 5960 7520 9400 7320 6840 9600
509400 532760 578360 13040 6640 6240 9560 7920 8840 15960
522920 480080 307040 12680 6960 4560 11200 19560 7800 13000
447320 571560 501160 12480 5600 9120 10240 15480 7720 21440
564880 466200 514960 10120 11440 19400 15480 13880 15520 10360
555800 630040 557200 16960 11080 21040 8760 18680 6640 15960
572000 464200 541960 11440 6600 9480 12000 17240 12480 1560
466560 614600 481080 19280 9160 14520 6280 10240 12440 14480
359280 592120 541760 7080 5440 7120 5960 8840 30920 66800
541000 422920 485920 6800 9960 2720 4680 11920 9320 14520
474840 468960 543920 10160 2800 6720 5160 9800 6640 7800
498640 501240 446720 15160 6920 12280 12520 18440 11040 21160
438120 502080 474720 7720 9560 7120 7640 15120 4480 9480
405720 476560 333440 8400 3400 10280 4480 15400 6000 15680

700000

600000

A
B
C 500000
D
E
F
G 400000

H
I
J 300000
K
L
M
N 200000

O
P

100000

20 25 30 0
0 2 4 6
 P

100000

20 25 30 0
0 2 4 6

35000

33000

31000

29000

27000

25000

23000

21000

19000

17000

15000
0
17000

15000
0
 20 21 22 23 24
9360 9960 12840 10120 13200
4120 7360 5680 10720 6200
6400 12200 10280 15600 12960
11680 9720 8240 12440 11080
12520 7440 4840 11880 9960
10120 15440 7120 18560 7000
5280 12720 5920 12360 3840
7040 14200 7560 6960 7200
12000 13160 4560 14320 7480
7720 19480 4600 9240 7840
5800 10600 16200 15240 3040
11960 7640 5680 11200 7040
11120 16960 20160 17480 12360
13280 10760 6240 24640 4160
6360 18520 8720 2240 12320
3720 12040 1240 6880 8040

ROW
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

6 8 10 12 14 16 18
 20
21
22
23
24

6 8 10 12 14 16 18

35000
ROW
Ro
33000 w
52
Ro
31000 w
53
Ro
29000 w
54
Ro
27000 w
55
Ro
25000 w
56
Ro
23000 w
57
Ro
21000 w
58
Ro
19000 w
59
Ro
17000 w
60
15000
0 2 4 6 8 10 12
 w
59
Ro
17000 w
60
15000
0 2 4 6 8 10 12
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

18
 20
21
22
23
24

18

Ro
w
52
Ro
w
53
Ro
w
54
Ro
w
55
Ro
w
56
Ro
w
57
Ro
w
58
Ro
w
59
Ro
w
60

12
 w
59
Ro
w
60

12