Assay Dev Worksheet

5/19/2018 Assay Dev Worksheet

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Metrics for Assay Development

Z-Prime*:

Z-Factor*:

%CV: Standard Deviation Mean

see Sample Data tab for sample calculations run on one plate

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

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-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 begreater than or equal to 0.5. A perfect assay would have a Z-prime value approaching 1.0.

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 Z-

factor should be greater than or equal to 0.5. For an activation screen, simply replace the 100% inhibition control with the 100% activation

control.

1

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)|

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.

100X (
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10838414&itool=iconabstrhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10838414&itool=iconabstr


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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]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10838414&itool=iconabstrhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10838414&itool=iconabstr


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aver compound: 281,438,753

stdev compound: 28,288,637

%CV: compound: 10.05 = positive (minimum signal) control in replicates of 16

average max: 296,570,427

stdev max: 19,047,390 = hit

%CV: max 6.42

average min: 3,641,523 = samples in singlets

stdev min: 279,783

%CV: min 7.68

Z-Prime 0.8021

Plate Number 1: Raw Data

2 3 4 5 6 7 8 9 10 1

A 311,656,960 328,751,968 332,511,424 315,507,376 301,482,880 328,462,432 288,825,984 298,116,480 313,453,216 315,982,65

B 3,475,320 255,769,936 255,502,912 253,605,424 276,429,088 294,972,448 275,311,520 303,163,488 283,551,904 310,766,52

C 309,456,672 323,358,528 323,038,400 232,964,736 289,795,008 320,255,456 295,834,144 278,681,696 291,314,944 288,246,12

D 3,666,836 279,370,976 294,233,632 276,973,312 286,428,032 310,241,248 291,923,072 279,344,384 291,234,000 309,635,40

E 296,700,896 302,729,856 241,412,448 306,755,616 286,623,712 266,880,144 295,056,080 300,557,248 276,790,592 270,444,72

F 3,689,461 254,641,664 309,326,304 305,497,184 307,003,360 247,722,624 295,260,736 224,608,368 280,461,184 264,156,62

G 287,128,128 254,995,440 288,276,080 310,989,024 264,909,104 248,477,168 283,565,344 272,627,968 285,747,536 252,116,73

H 3,310,254 292,184,448 264,813,568 261,532,592 293,055,376 314,299,168 278,752,720 305,556,272 295,159,360 253,844,14

I 279,876,832 277,444,288 252,189,856 307,165,808 262,678,880 248,099,312 284,415,152 273,395,712 285,210,000 269,343,87

J 4,068,030 322,293,920 311,095,712 266,371,808 314,565,328 275,090,704 277,618,176 296,868,832 298,148,416 281,966,64

K 318,311,584 268,908,416 292,449,952 293,463,296 296,951,872 299,088,320 285,279,056 252,124,912 275,906,432 256,193,88

L 4,226,086 343,157,248 270,817,824 304,609,920 260,027,440 293,330,176 285,564,496 294,528,112 246,791,040 273,731,71

M 277,811,744 255,789,088 291,662,144 270,942,752 264,024,944 262,092,784 269,327,680 263,622,032 267,025,728 263,031,74N 3,535,866 316,963,392 312,290,784 273,491,200 308,384,320 285,457,088 280,518,432 251,692,768 264,238,752 303,971,55

O 268,862,272 309,980,160 280,652,480 283,310,032 282,845,280 287,944,544 274,180,576 273,268,512 277,995,808 271,971,52

P 3,582,390 296,102,592 305,433,600 306,261,792 301,637,696 312,120,672 285,651,456 306,439,952 302,507,712 304,973,15

2 3 4 5 6 7 8 9 10 1

Inhibition = (mean of Max Control - Experimental Value)/(mean of Max Control - mean of Min. Control) x 100

= negative (maximum signal) control in replicates of 16

Plate Number 1: % Inhibition

Columns 3-22 = (enzyme + sample compounds in 2% DMSO )

Column 2 = Max signal reference control (enzyme + 2% DMSO compound car

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

Column 23 = Min signal reference control (enzyme + control inhibitor in 2% DM

Raw data from Plate number 1 expressed as percent inhibition relative to maximum and minimum reference signal controls:


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A -5 -11 -12 -6 -2 -11 3 -1 -6

B 100 14 14 15 7 1 7 -2 4

C -4 -9 -9 22 2 -8 0 6 2

D 100 6 1 7 3 -5 2 6 2

E 0 -2 19 -3 3 10 1 -1 7

F 100 14 -4 -3 -4 17 0 25 5

G 3 14 3 -5 11 16 4 8 4

H 100 1 11 12 1 -6 6 -3 0

I 6 7 15 -4 12 17 4 8 4

J 100 -9 -5 10 -6 7 6 0 -1

K -7 9 1 1 0 -1 4 15 7

L 100 -16 9 -3 12 1 4 1 17

M 6 14 2 9 11 12 9 11 10

N 100 -7 -5 8 -4 4 5 15 11

O 9 -5 5 5 5 3 8 8 6

P 100 0 -3 -3 -2 -5 4 -3 -2

Example of a 384 well control plate with marginal precision.

See explanation below.231,884

44,951

19

Plate Number 2: Control Data

4 5 6 7 8 9 10 11 12A 165,440 151,280 159,090 189,240 155,160 167,800 209,720 211,000 158,880 159,

B 204,290 251,870 209,620 174,590 211,420 206,680 181,700 172,240 193,200 157,

C 236,170 269,460 217,760 233,980 258,020 207,690 205,830 193,510 201,190 233,

D 277,120 366,190 199,720 248,920 253,980 277,500 259,870 266,480 246,160 215,

E 269,530 288,390 192,800 222,400 253,120 230,640 179,270 213,650 193,400 221,

F 258,830 210,720 204,170 213,600 215,910 272,270 209,680 236,440 222,240 196,

G 241,880 217,070 215,200 242,820 202,990 213,250 206,730 267,720 205,470 184,

H 218,880 224,130 221,960 207,880 224,420 212,220 264,570 222,810 181,760 163,

I 168,880 174,720 175,840 202,480 242,150 209,180 207,540 202,790 200,400 162,

J 189,500 259,520 252,800 256,890 215,810 235,380 204,740 189,930 241,360 154,

K 229,440 273,760 234,880 225,480 281,340 236,490 291,540 238,430 228,120 221,

L 287,910 273,140 340,850 249,790 379,310 301,120 325,540 274,480 293,860 278,

M 215,790 209,290 229,160 255,560 253,970 242,440 272,480 254,570 281,510 221,

N 191,560 243,150 225,030 226,180 262,720 257,720 266,180 248,090 216,750 245,

O 223,370 213,860 244,120 293,310 347,360 227,940 229,490 289,880 309,010 210,

Columns 4 through 20 contain identical uninhibited control enzyme, generating the max

Entire Plate Average

Entire Plate StDev

Entire Plate %CV


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P 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 50,139 39,938 29,310 53,906 32,796 41,856 34,586 42,005 36,288

17 21 18 13 22 14 18 15 19 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

Averages, standard deviations, and coefficients of variation are calculated for each row, column, and the entire plate to assess precision. Go

for in vitro assays are


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12 13 14 15 16 17 18 19 20 21

291,488,416 308,339,008 300,278,944 300,815,200 251,313,296 234,508,768 300,115,840 310,220,544 325,040,608 310,237,184 297,9

259,292,800 251,329,264 262,376,704 277,575,392 257,104,992 275,673,344 284,927,424 307,666,496 264,116,160 295,002,048 314,6

273,669,568 257,564,496 286,623,744 314,830,944 255,599,920 296,730,880 269,748,192 321,834,784 304,001,344 324,322,656 312,7

298,121,680 2,492,887 245,322,192 277,470,496 281,351,552 281,028,448 296,640,960 296,311,712 291,405,280 295,120,640 315,3

278,364,704 272,935,168 234,546,224 264,051,664 278,628,800 275,647,456 259,109,792 310,667,904 317,833,632 327,944,320 320,9

278,278,528 239,832,800 261,795,472 248,814,000 255,769,936 245,506,768 279,096,080 310,602,176 304,704,736 277,654,144 280,5

284,116,160 228,826,064 244,974,288 242,726,864 296,688,624 278,456,720 257,161,344 294,502,848 300,372,800 261,276,672 304,8

270,915,568 279,242,640 262,669,712 254,630,016 307,412,720 298,282,208 279,505,616 237,678,272 270,362,416 255,494,256 296,1

250,151,824 271,795,472 244,410,064 252,189,856 271,368,656 241,489,376 279,597,568 295,243,040 300,188,576 276,295,808 313,4

296,808,912 288,237,424 243,131,264 264,236,624 281,337,440 245,731,664 261,882,432 261,392,960 254,827,536 289,848,640 289,0

261,905,808 282,890,832 266,519,888 257,206,384 250,875,728 276,373,792 281,813,888 319,628,064 326,055,552 288,396,992 287,6

243,632,976 284,100,512 228,719,152 255,769,936 265,038,656 264,552,256 238,782,224 272,223,616 288,114,048 305,719,776 308,9

271,414,304 268,000,464 274,847,744 243,572,368 231,934,416 270,120,384 270,618,176 282,732,960 289,820,032 311,095,744 300,1

250,106,560 267,609,712 281,949,184 263,645,696 296,521,152 271,524,832 289,477,184 245,938,512 301,951,744 257,471,328 342,7

283,114,528 272,375,104 277,365,696 250,127,856 287,634,336 292,068,224 256,554,784 297,121,152 261,975,888 308,838,336 229,6

304,704,736 280,173,856 318,380,608 304,262,816 292,039,168 290,220,992 301,240,960 284,540,928 320,259,840 308,578,752 303,8

12 13 14 15 16 17 18 19 20 21

er)

O )

:


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2 -4 -1 -1 15 21 -1 -5 -10 -5

13 15 12 6 13 7 4 -4 11 1

8 13 3 -6 14 0 9 -9 -3 -9

-1 100 17 7 5 5 0 0 2 0

6 8 21 11 6 7 13 -5 -7 -11

6 19 12 16 14 17 6 -5 -3 6

4 23 18 18 0 6 13 1 -1 12

9 6 12 14 -4 -1 6 20 9 14

16 8 18 15 9 19 6 0 -1 7

0 3 18 11 5 17 12 12 14 2

12 5 10 13 16 7 5 -8 -10 3

18 4 23 14 11 11 20 8 3 -3

9 10 7 18 22 9 9 5 2 -5

16 10 5 11 0 9 2 17 -2 13 5 8 7 16 3 2 14 0 12 -4

-3 6 -7 -3 2 2 -2 4 -8 -4

14 15 16 17 18 19 20 row average row stdev row %CV

169,720 205,950 280,670 182,970 223,710 169,470 172,230 184,241 33,283 18

178,160 207,390 238,650 254,260 258,730 171,840 250,680 207,223 33,049 16

54,780 190,310 242,160 318,120 195,370 201,160 242,110 217,684 53,378 25

254,340 237,770 338,100 227,440 318,880 379,730 346,730 277,302 53,519 19

233,600 220,930 226,360 304,480 219,300 259,360 277,240 235,623 34,941 15

215,390 206,130 249,160 282,240 240,520 268,400 319,890 236,616 34,037 14

241,830 266,340 277,190 236,900 247,040 222,750 227,650 230,425 25,530 11

171,580 275,280 280,320 273,060 252,380 237,540 269,320 229,522 36,202 16

207,390 221,930 235,390 148,270 146,410 159,420 180,800 190,964 29,016 15

168,290 208,270 217,560 216,690 231,530 244,640 219,570 218,052 30,191 14

310,240 175,730 252,080 285,920 261,920 261,630 237,730 249,794 32,581 13

301,890 169,290 226,120 290,650 312,960 226,190 316,220 285,158 48,687 17

268,610 173,690 237,890 256,510 249,710 278,560 293,510 246,721 30,218 12

278,150 146,730 237,680 279,370 223,520 235,640 279,010 238,981 33,935 14

307,240 157,280 237,690 223,650 207,470 212,320 237,200 245,389 47,893 20

um signal


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276,280 139,530 145,520 183,050 187,840 155,690 213,680 216,443 43,174 20

227,343 200,159 245,159 247,724 236,081 230,271 255,223 column average

67,885 39,367 39,952 48,146 43,201 56,082 48,989 column stdev

30 20 16 19 18 24 19 column %CV

ontents.

d % CVs

rmal


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23

row

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

23rowletters


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6 A

100 B

-1 C

100 D

-8 E

100 F

-4 G

100 H

1 I

100 J

-10 K

100 L

-3 M

100 N11 O

100 P


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Plate 1:

2 3 4 5 6 7 8

A -11 -12 -6 -2 -11 3

B 14 14 14 7 1 7

C -9 -9 21 2 -8 0

D 6 1 7 3 -5 2E -2 19 -3 3 10 1

F 14 -4 -3 -4 16 0

G 14 3 -5 11 16 4

H 1 11 12 1 -6 6

I 6 15 -4 11 16 4

J -9 -5 10 -6 7 6

K 9 1 1 0 -1 4

L -16 9 -3 12 1 4

M 14 2 9 11 12 9

N -7 -5 8 -4 4 5

O -5 5 4 5 3 8

P 0 -3 -3 -2 -5 4

Example of Identifying Outliers (hits)

Well Position % Inhibition

A3 -11

B3 14C3 -9

D3 6

E3 -2

F3 14

G3 14

H3 1

I3 7

J3 -9

HITS ARE SCORED AS BEING GREATER THABOVE THE MEAN OF THE POPULATION OF

IN THIS EXPERIMENT:

(this is the sample data from the prior page)

-20

0

20

40

60

80

100

120

0 40 80 120 16

Percen

tInhibition

Well Positio

Scatter Plot showing % INHIBITION of 320


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K3 9

L3 -16

M3 14

N3 -7

O3 -5

P3 0

A4 -12B4 14

C4 -9

D4 1

E4 19

F4 -4

G4 3

H4 11

I4 15

J4 -5

K4 1

L4 9

M4 2N4 -5

O4 5

P4 -3

A5 -6

B5 15

C5 22

D5 7

E5 -3

F5 -3

G5 -5

H5 12

I5 -4

J5 10

K5 1

L5 -3

M5 9

N5 8

O5 5

P5 -3

A6 -2

B6 7

C6 2

D6 3

E6 3

F6 -4G6 11

H6 1

I6 12

J6 -6

K6 0

L6 12

M6 11

N6 -4


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O6 5

P6 -2

A7 -11

B7 1

C7 -8

D7 -5

E7 10F7 17

G7 16

H7 -6

I7 17

J7 7

K7 -1

L7 1

M7 12

N7 4

O7 3

P7 -5

A8 3B8 7

C8 0

D8 2

E8 1

F8 0

G8 4

H8 6

I8 4

J8 6

K8 4

L8 4

M8 9

N8 5

O8 8

P8 4

A9 -1

B9 -2

C9 6

D9 6

E9 -1

F9 25

G9 8

H9 -3

I9 8

J9 0K9 15

L9 1

M9 11

N9 15

O9 8

P9 -3

A10 -6

B10 4


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C10 2

D10 2

E10 7

F10 5

G10 4

H10 0

I10 4J10 -1

K10 7

L10 17

M10 10

N10 11

O10 6

P10 -2

A11 -7

B11 -5

C11 3

D11 -4

E11 9F11 11

G11 15

H11 15

I11 9

J11 5

K11 14

L11 8

M11 11

N11 -3

O11 8

P11 -3

A12 2

B12 13

C12 8

D12 -1

E12 6

F12 6

G12 4

H12 9

I12 16

J12 0

K12 12

L12 18

M12 9

N12 16O12 5

P12 -3

A13 -4

B13 15

C13 13

D13 100 hit

E13 8

F13 19


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G13 23

H13 6

I13 8

J13 3

K13 5

L13 4

M13 10N13 10

O13 8

P13 6

A14 -1

B14 12

C14 3

D14 17

E14 21

F14 12

G14 18

H14 12

I14 18J14 18

K14 10

L14 23

M14 7

N14 5

O14 7

P14 -7

A15 -1

B15 6

C15 -6

D15 7

E15 11

F15 16

G15 18

H15 14

I15 15

J15 11

K15 13

L15 14

M15 18

N15 11

O15 16

P15 -3

A16 15

B16 13C16 14

D16 5

E16 6

F16 14

G16 0

H16 -4

I16 9

J16 5


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K16 16

L16 11

M16 22

N16 0

O16 3

P16 2

A17 21B17 7

C17 0

D17 5

E17 7

F17 17

G17 6

H17 -1

I17 19

J17 17

K17 7

L17 11

M17 9N17 9

O17 2

P17 2

A18 -1

B18 4

C18 9

D18 0

E18 13

F18 6

G18 13

H18 6

I18 6

J18 12

K18 5

L18 20

M18 9

N18 2

O18 14

P18 -2

A19 -5

B19 -4

C19 -9

D19 0

E19 -5

F19 -5G19 1

H19 20

I19 0

J19 12

K19 -8

L19 8

M19 5

N19 17


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O19 0

P19 4

A20 -10

B20 11

C20 -3

D20 2

E20 -7F20 -3

G20 -1

H20 9

I20 -1

J20 14

K20 -10

L20 3

M20 2

N20 -2

O20 12

P20 -8

A21 -5B21 1

C21 -9

D21 0

E21 -11

F21 6

G21 12

H21 14

I21 7

J21 2

K21 3

L21 -3

M21 -5

N21 13

O21 -4

P21 -4

A22 0

B22 -6

C22 -6

D22 -6

E22 -8

F22 5

G22 -3

H22 0

I22 -6

J22 3K22 3

L22 -4

M22 -1

N22 -16

O22 23

P22 -2

average 5


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stdev 10


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9 10 11 12 13 14

-1 -6 -7 2 -4 -1

-2 4 -5 13 15 12

6 2 3 8 13 3

6 2 -4 -1 99 17-1 7 9 6 8 21

24 5 11 6 19 12

8 4 15 4 23 17

-3 0 14 9 6 11

8 4 9 16 8 18

0 -1 5 0 3 18

15 7 14 12 5 10

1 17 8 18 4 23

11 10 11 8 10 7

15 11 -2 16 10 5

8 6 8 5 8 6

-3 -2 -3 -3 6 -7

N OR ~EQUAL TO 3 STANDARD DEVIATIONS COMPOUNDS TESTED

0 200 240 280 320

(A3-P22)

ompounds tested in a single plate


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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:



8 per column (in every other well)

OR replicates of 16 per column

Controls in replicates of 8 per column

Controls in either replicates of

Standard Setup for Reference Controls on Assay Plates:

Standard Protocol for transferring 96 well plate contents to 384

Using a 96 channel pipettor (Quad Mapping)

Documents

Assay Dev Worksheet