Metrics for Assay Development

Z-Prime*:

1-(

3 X (Standard Deviation of the Maximum Signal Control) + 3 X (Standard Deviation of the Minimum Signal Control)
|(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 separation band between
maximum and minimum controls and (b) the standard deviations are low . For a good assay, z-prime values for each plate should be
greater than or equal to 0.5. A perfect assay would have a Z-prime value approaching 1.0.

Z-Factor*:

1-(

3 X (Standard Deviation of Batch of Compounds Tested) + 3 X (Standard Deviation of the 100% Inhibition Control)
|(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% inhibition. It will have a
higher value when (a) there is a wide separation band between sample compound values and 100% inhibition control values and (b) the
standard deviations are low. Z-factor can be used to assess data from an entire screen. For a screen to have a reasonable hit rate, the Zfactor should be greater than or equal to 0.5. For an activation screen, simply replace the 100% inhibition control with the 100% activation
control.
%CV:

100

Deviation
( Standard
)
Mean

The coefficient of variation is the standard deviation divided by the mean of a replicate set of values. It can be expressed as a %CV by
multiplying by 100. Low %CV indicates high precision, high %CV indicates poor precision.

see Sample Data tab for sample calculations run on one plate

* 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]

aver compound:
stdev compound:
%CV: compound:
average max:
stdev max:
%CV: max
average min:
stdev min:
%CV: min

281,438,753
28,288,637
10.05
296,570,427
19,047,390
6.42
3,641,523
279,783
7.68

Z-Prime

= negative (maximum signal) control in replicates of 16

= positive (minimum signal) control in replicates of 16
= hit
= samples in singlets

Example of 384 well Raw Data in a typical enzyme inhibition screen

Column 2 = Max signal reference control (enzyme + 2% DMSO compound carrier)
Column 23 = Min signal reference control (enzyme + control inhibitor in 2% DMSO )
Columns 3-22 = (enzyme + sample compounds in 2% DMSO )

0.8021

Plate Number 1: Raw Data

A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P

2
311,656,960
3,475,320
309,456,672
3,666,836
296,700,896
3,689,461
287,128,128
3,310,254
279,876,832
4,068,030
318,311,584
4,226,086
277,811,744
3,535,866
268,862,272
3,582,390

3
328,751,968
255,769,936
323,358,528
279,370,976
302,729,856
254,641,664
254,995,440
292,184,448
277,444,288
322,293,920
268,908,416
343,157,248
255,789,088
316,963,392
309,980,160
296,102,592

4
332,511,424
255,502,912
323,038,400
294,233,632
241,412,448
309,326,304
288,276,080
264,813,568
252,189,856
311,095,712
292,449,952
270,817,824
291,662,144
312,290,784
280,652,480
305,433,600

5
315,507,376
253,605,424
232,964,736
276,973,312
306,755,616
305,497,184
310,989,024
261,532,592
307,165,808
266,371,808
293,463,296
304,609,920
270,942,752
273,491,200
283,310,032
306,261,792

6
301,482,880
276,429,088
289,795,008
286,428,032
286,623,712
307,003,360
264,909,104
293,055,376
262,678,880
314,565,328
296,951,872
260,027,440
264,024,944
308,384,320
282,845,280
301,637,696

7
328,462,432
294,972,448
320,255,456
310,241,248
266,880,144
247,722,624
248,477,168
314,299,168
248,099,312
275,090,704
299,088,320
293,330,176
262,092,784
285,457,088
287,944,544
312,120,672

8
288,825,984
275,311,520
295,834,144
291,923,072
295,056,080
295,260,736
283,565,344
278,752,720
284,415,152
277,618,176
285,279,056
285,564,496
269,327,680
280,518,432
274,180,576
285,651,456

9
298,116,480
303,163,488
278,681,696
279,344,384
300,557,248
224,608,368
272,627,968
305,556,272
273,395,712
296,868,832
252,124,912
294,528,112
263,622,032
251,692,768
273,268,512
306,439,952

10
313,453,216
283,551,904
291,314,944
291,234,000
276,790,592
280,461,184
285,747,536
295,159,360
285,210,000
298,148,416
275,906,432
246,791,040
267,025,728
264,238,752
277,995,808
302,507,712

11
315,982,656
310,766,528
288,246,128
309,635,408
270,444,720
264,156,624
252,116,736
253,844,144
269,343,872
281,966,640
256,193,888
273,731,712
263,031,744
303,971,552
271,971,520
304,973,152

Raw data from Plate number 1 expressed as percent inhibition relative to maximum and minimum reference signal controls:
% Inhibition = (mean of Max Control - Experimental Value)/(mean of Max Control - mean of Min. Control) x 100
Plate Number 1: % Inhibition
2

10

11

A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P

-5
100
-4
100
0
100
3
100
6
100
-7
100
6
100
9
100

-11
14
-9
6
-2
14
14
1
7
-9
9
-16
14
-7
-5
0

-12
14
-9
1
19
-4
3
11
15
-5
1
9
2
-5
5
-3

-6
15
22
7
-3
-3
-5
12
-4
10
1
-3
9
8
5
-3

-2
7
2
3
3
-4
11
1
12
-6
0
12
11
-4
5
-2

-11
1
-8
-5
10
17
16
-6
17
7
-1
1
12
4
3
-5

3
7
0
2
1
0
4
6
4
6
4
4
9
5
8
4

-1
-2
6
6
-1
25
8
-3
8
0
15
1
11
15
8
-3

-6
4
2
2
7
5
4
0
4
-1
7
17
10
11
6
-2

-7
-5
3
-4
9
11
15
15
9
5
14
8
11
-3
8
-3

Example of a 384 well control plate with marginal precision.

Columns 4 through 20 contain identical uninhibited control enzyme, generating the maximum signal
See explanation below.
Entire Plate Average
Entire Plate StDev
Entire Plate %CV

231,884
44,951
19

Plate Number 2: Control Data

A
B
C
D
E
F
G
H
I
J
K
L
M
N
O

4
165,440
204,290
236,170
277,120
269,530
258,830
241,880
218,880
168,880
189,500
229,440
287,910
215,790
191,560
223,370

5
151,280
251,870
269,460
366,190
288,390
210,720
217,070
224,130
174,720
259,520
273,760
273,140
209,290
243,150
213,860

6
159,090
209,620
217,760
199,720
192,800
204,170
215,200
221,960
175,840
252,800
234,880
340,850
229,160
225,030
244,120

7
189,240
174,590
233,980
248,920
222,400
213,600
242,820
207,880
202,480
256,890
225,480
249,790
255,560
226,180
293,310

8
155,160
211,420
258,020
253,980
253,120
215,910
202,990
224,420
242,150
215,810
281,340
379,310
253,970
262,720
347,360

9
167,800
206,680
207,690
277,500
230,640
272,270
213,250
212,220
209,180
235,380
236,490
301,120
242,440
257,720
227,940

10
209,720
181,700
205,830
259,870
179,270
209,680
206,730
264,570
207,540
204,740
291,540
325,540
272,480
266,180
229,490

11
211,000
172,240
193,510
266,480
213,650
236,440
267,720
222,810
202,790
189,930
238,430
274,480
254,570
248,090
289,880

12
158,880
193,200
201,190
246,160
193,400
222,240
205,470
181,760
200,400
241,360
228,120
293,860
281,510
216,750
309,010

13
159,760
157,470
233,010
215,210
221,120
196,880
184,390
163,770
162,790
154,400
221,760
278,370
221,000
245,200
210,430

190,260

263,870

234,880

245,360

229,800

216,920

256,090

264,080

255,320

221,360

223,053

243,151

222,368

230,530

249,218

232,203

235,686

234,131

226,789

202,933

37,422
17

50,139
21

39,938
18

29,310
13

53,906
22

32,796
14

41,856
18

34,586
15

42,005
19

36,288
18

Column and Row data on a Single Plate: This is an analysis of the row and column values of a control plate, where each well has the same contents.
Averages, standard deviations, and coefficients of variation are calculated for each row, column, and the entire plate to assess precision. Good % CVs
for in vitro assays are <5%, for cell or organismal based, 10-12%. Well C14 indicates a bubble or debris present in the well, causing an abnormal
reading.

arrier)
DMSO )

12
291,488,416
259,292,800
273,669,568
298,121,680
278,364,704
278,278,528
284,116,160
270,915,568
250,151,824
296,808,912
261,905,808
243,632,976
271,414,304
250,106,560
283,114,528
304,704,736

13
308,339,008
251,329,264
257,564,496
2,492,887
272,935,168
239,832,800
228,826,064
279,242,640
271,795,472
288,237,424
282,890,832
284,100,512
268,000,464
267,609,712
272,375,104
280,173,856

14
300,278,944
262,376,704
286,623,744
245,322,192
234,546,224
261,795,472
244,974,288
262,669,712
244,410,064
243,131,264
266,519,888
228,719,152
274,847,744
281,949,184
277,365,696
318,380,608

15
300,815,200
277,575,392
314,830,944
277,470,496
264,051,664
248,814,000
242,726,864
254,630,016
252,189,856
264,236,624
257,206,384
255,769,936
243,572,368
263,645,696
250,127,856
304,262,816

16
251,313,296
257,104,992
255,599,920
281,351,552
278,628,800
255,769,936
296,688,624
307,412,720
271,368,656
281,337,440
250,875,728
265,038,656
231,934,416
296,521,152
287,634,336
292,039,168

17
234,508,768
275,673,344
296,730,880
281,028,448
275,647,456
245,506,768
278,456,720
298,282,208
241,489,376
245,731,664
276,373,792
264,552,256
270,120,384
271,524,832
292,068,224
290,220,992

18
300,115,840
284,927,424
269,748,192
296,640,960
259,109,792
279,096,080
257,161,344
279,505,616
279,597,568
261,882,432
281,813,888
238,782,224
270,618,176
289,477,184
256,554,784
301,240,960

19
310,220,544
307,666,496
321,834,784
296,311,712
310,667,904
310,602,176
294,502,848
237,678,272
295,243,040
261,392,960
319,628,064
272,223,616
282,732,960
245,938,512
297,121,152
284,540,928

20
325,040,608
264,116,160
304,001,344
291,405,280
317,833,632
304,704,736
300,372,800
270,362,416
300,188,576
254,827,536
326,055,552
288,114,048
289,820,032
301,951,744
261,975,888
320,259,840

21
310,237,184
295,002,048
324,322,656
295,120,640
327,944,320
277,654,144
261,276,672
255,494,256
276,295,808
289,848,640
288,396,992
305,719,776
311,095,744
257,471,328
308,838,336
308,578,752

22
297,972,768
314,687,744
312,716,704
315,338,016
320,990,656
280,518,432
304,803,584
296,132,448
313,453,216
289,093,840
287,662,496
308,968,352
300,127,200
342,756,704
229,651,424
303,853,536

12

13

14

15

16

17

18

19

20

21

22

s:

2
13
8
-1
6
6
4
9
16
0
12
18
9
16
5
-3

-4
15
13
100
8
19
23
6
8
3
5
4
10
10
8
6

-1
12
3
17
21
12
18
12
18
18
10
23
7
5
7
-7

-1
6
-6
7
11
16
18
14
15
11
13
14
18
11
16
-3

15
13
14
5
6
14
0
-4
9
5
16
11
22
0
3
2

21
7
0
5
7
17
6
-1
19
17
7
11
9
9
2
2

15
205,950
207,390
190,310
237,770
220,930
206,130
266,340
275,280
221,930
208,270
175,730
169,290
173,690
146,730
157,280

16
280,670
238,650
242,160
338,100
226,360
249,160
277,190
280,320
235,390
217,560
252,080
226,120
237,890
237,680
237,690

17
182,970
254,260
318,120
227,440
304,480
282,240
236,900
273,060
148,270
216,690
285,920
290,650
256,510
279,370
223,650

18
223,710
258,730
195,370
318,880
219,300
240,520
247,040
252,380
146,410
231,530
261,920
312,960
249,710
223,520
207,470

19
169,470
171,840
201,160
379,730
259,360
268,400
222,750
237,540
159,420
244,640
261,630
226,190
278,560
235,640
212,320

-1
4
9
0
13
6
13
6
6
12
5
20
9
2
14
-2

-5
-4
-9
0
-5
-5
1
20
0
12
-8
8
5
17
0
4

-10
11
-3
2
-7
-3
-1
9
-1
14
-10
3
2
-2
12
-8

-5
1
-9
0
-11
6
12
14
7
2
3
-3
-5
13
-4
-4

0
-6
-6
-6
-8
5
-3
0
-6
3
3
-4
-1
-16
23
-2

18
16
25
19
15
14
11
16
15
14
13
17
12
14
20

A
B
C
D
E
F
G
H
I
J
K
L
M
N
O

ximum signal

14
169,720
178,160
54,780
254,340
233,600
215,390
241,830
171,580
207,390
168,290
310,240
301,890
268,610
278,150
307,240

20 row average row stdev

row %CV
172,230
184,241
33,283
250,680
207,223
33,049
242,110
217,684
53,378
346,730
277,302
53,519
277,240
235,623
34,941
319,890
236,616
34,037
227,650
230,425
25,530
269,320
229,522
36,202
180,800
190,964
29,016
219,570
218,052
30,191
237,730
249,794
32,581
316,220
285,158
48,687
293,510
246,721
30,218
279,010
238,981
33,935
237,200
245,389
47,893

276,280

139,530

145,520

183,050

187,840

155,690

213,680

227,343

200,159

245,159

247,724

236,081

230,271

255,223 column average

67,885
30

39,367
20

39,952
16

48,146
19

43,201
18

56,082
24

me contents.
. Good % CVs
abnormal

216,443

48,989 column stdev

19 column %CV

43,174

20

row
23 letters
279,291,264
A
3,231,863
B
298,662,144
C
3,727,063
D
320,780,128
E
3,816,925
F
308,449,120
G
3,305,155
H
293,527,840
I
3,343,713
J
325,870,144
K
3,801,948
L
305,497,152
M
3,886,712
N
263,243,952
O
3,596,751
P

row
23 letters

6
100
-1
100
-8
100
-4
100
1
100
-10
100
-3
100
11
100

A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P

Scatter Plot showing % INHIBITION of 320 compou

120

Percent Inhibition

100
Plate 1:
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P

80
2

60
40
20
0
-20
0

3
-11
14
-9
6
-2
14
14
1
6
-9
9
-16
14
-7
-5
0

40

4
-12
14
-9
1
19
-4
3
11
15
-5
1
9
2
-5
5
-3

5
-6
14
21
7
-3
-3
-5
12
-4
10
1
-3
9
8
4
-3

80

6
-2
7
2
3
3
-4
11
1
11
-6
0
12
11
-4
5
-2

120

7
-11
1
-8
-5
10
16
16
-6
16
7
-1
1
12
4
3
-5

8
3
7
0
2
1
0
4
6
4
6
4
4
9
5
8
4

160

Well Position(A3

Example of Identifying Outliers (hits)

(this is the sample data from the prior page)
Well Position
A3

% Inhibition
-11

B3

14

C3
D3
E3
F3
G3
H3
I3
J3

-9
6
-2
14
14
1
7
-9

IN THIS EXPERIMENT:
HITS ARE SCORED AS BEING GREATER THAN OR ~EQU
ABOVE THE MEAN OF THE POPULATION OF COMPOUN

K3
L3
M3
N3
O3
P3
A4
B4
C4
D4
E4
F4
G4
H4
I4
J4
K4
L4
M4
N4
O4
P4
A5
B5
C5
D5
E5
F5
G5
H5
I5
J5
K5
L5
M5
N5
O5
P5
A6
B6
C6
D6
E6
F6
G6
H6
I6
J6
K6
L6
M6
N6

9
-16
14
-7
-5
0
-12
14
-9
1
19
-4
3
11
15
-5
1
9
2
-5
5
-3
-6
15
22
7
-3
-3
-5
12
-4
10
1
-3
9
8
5
-3
-2
7
2
3
3
-4
11
1
12
-6
0
12
11
-4

O6
P6
A7
B7
C7
D7
E7
F7
G7
H7
I7
J7
K7
L7
M7
N7
O7
P7
A8
B8
C8
D8
E8
F8
G8
H8
I8
J8
K8
L8
M8
N8
O8
P8
A9
B9
C9
D9
E9
F9
G9
H9
I9
J9
K9
L9
M9
N9
O9
P9
A10
B10

5
-2
-11
1
-8
-5
10
17
16
-6
17
7
-1
1
12
4
3
-5
3
7
0
2
1
0
4
6
4
6
4
4
9
5
8
4
-1
-2
6
6
-1
25
8
-3
8
0
15
1
11
15
8
-3
-6
4

C10
D10
E10
F10
G10
H10
I10
J10
K10
L10
M10
N10
O10
P10
A11
B11
C11
D11
E11
F11
G11
H11
I11
J11
K11
L11
M11
N11
O11
P11
A12
B12
C12
D12
E12
F12
G12
H12
I12
J12
K12
L12
M12
N12
O12
P12
A13
B13
C13
D13
E13
F13

2
2
7
5
4
0
4
-1
7
17
10
11
6
-2
-7
-5
3
-4
9
11
15
15
9
5
14
8
11
-3
8
-3
2
13
8
-1
6
6
4
9
16
0
12
18
9
16
5
-3
-4
15
13
100 hit
8
19

G13
H13
I13
J13
K13
L13
M13
N13
O13
P13
A14
B14
C14
D14
E14
F14
G14
H14
I14
J14
K14
L14
M14
N14
O14
P14
A15
B15
C15
D15
E15
F15
G15
H15
I15
J15
K15
L15
M15
N15
O15
P15
A16
B16
C16
D16
E16
F16
G16
H16
I16
J16

23
6
8
3
5
4
10
10
8
6
-1
12
3
17
21
12
18
12
18
18
10
23
7
5
7
-7
-1
6
-6
7
11
16
18
14
15
11
13
14
18
11
16
-3
15
13
14
5
6
14
0
-4
9
5

K16
L16
M16
N16
O16
P16
A17
B17
C17
D17
E17
F17
G17
H17
I17
J17
K17
L17
M17
N17
O17
P17
A18
B18
C18
D18
E18
F18
G18
H18
I18
J18
K18
L18
M18
N18
O18
P18
A19
B19
C19
D19
E19
F19
G19
H19
I19
J19
K19
L19
M19
N19

16
11
22
0
3
2
21
7
0
5
7
17
6
-1
19
17
7
11
9
9
2
2
-1
4
9
0
13
6
13
6
6
12
5
20
9
2
14
-2
-5
-4
-9
0
-5
-5
1
20
0
12
-8
8
5
17

O19
P19
A20
B20
C20
D20
E20
F20
G20
H20
I20
J20
K20
L20
M20
N20
O20
P20
A21
B21
C21
D21
E21
F21
G21
H21
I21
J21
K21
L21
M21
N21
O21
P21
A22
B22
C22
D22
E22
F22
G22
H22
I22
J22
K22
L22
M22
N22
O22
P22

0
4
-10
11
-3
2
-7
-3
-1
9
-1
14
-10
3
2
-2
12
-8
-5
1
-9
0
-11
6
12
14
7
2
3
-3
-5
13
-4
-4
0
-6
-6
-6
-8
5
-3
0
-6
3
3
-4
-1
-16
23
-2
average

stdev

10

of 320 compounds tested in a single plate

<< Hit

160

9
-1
-2
6
6
-1
24
8
-3
8
0
15
1
11
15
8
-3

200

10
-6
4
2
2
7
5
4
0
4
-1
7
17
10
11
6
-2

11
12
13
14
-7
2
-4
-1
-5
13
15
12
3
8
13
3
-4
-1
99
17
9three standard6deviations above
8 the mean 21
11
6
19
12
15
4
23
17
14
9
6
11
9
16
8
18
plate
5
0
3
18
mean
14
12
5
10
8
18
4
23
11
8
10
7
-2
16
10
5
8
5
8
6
-3
-3
6
-7

240

osition(A3-P22)

ATER THAN OR ~EQUAL TO 3 STANDARD DEVIATIONS

ATION OF COMPOUNDS TESTED

280

320

96 Well Plates:
Vertical Columns = 1-12
Horizontal Rows = A-H

384 Well Plates:

Vertical Columns = 1-24
Horizontal Rows = A-P

96 Well Plates:
Columns 1 and 12 left empty

384 Well Plates:

Columns 1 and 2 left empty
Columns 23 and 24 left empty

8 per column (in every other wel

OR replicates of 16 per column

Standard Protocol for transferring 96 well plate contents to 384

Using a 96 channel pipettor (Quad Mapping)
96 Well Plates:
Vertical Columns = 1-12
Horizontal Rows = A-H

384 Well Plates:

Vertical Columns = 1-24
Horizontal Rows = A-P

Standard Setup for Reference Controls on Assay Plates:

96 Well Plates:
Columns 1 and 12 left empty
Controls in replicates of 8 per column
384 Well Plates:
Columns 1 and 2 left empty
Columns 23 and 24 left empty
Controls in either replicates of
8 per column (in every other well)
OR replicates of 16 per column