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Appendix A

Note: Although this software and accompanying documentation is dated 2004-2005, it is still valid in 2013. Questions can be sent to CDC-INFO.

Sample Data and Output

A collection of sample data files and program output is included on the distribution diskette. These files may be used to test the program and check results after selecting various options in the different modules. The sample data file is MA112190.DAT and appears as:

COMMENT:FRASCH ASSAY PB2 1:75 PLATE 1 UNKNOWNS ARE PB2 1:75
1.716 1.757 1.769 1.840 1.462 0.960 0.562 0.313 0.171 0.090 0.041 1.440
1.438 1.462 1.458 1.800 1.493 1.013 0.590 0.346 0.173 0.092 0.044 1.409
0.967 1.014 0.998 1.790 1.484 0.984 0.589 0.334 0.175 0.096 0.047 1.255
0.586 0.606 0.627 1.801 1.472 0.970 0.588 0.322 0.169 0.085 0.043 1.239
0.319 0.347 0.346 1.794 1.445 0.958 0.587 0.312 0.175 0.086 0.045 0.233
0.167 0.186 0.190 1.746 1.424 0.948 0.573 0.312 0.168 0.090 0.046 0.255
0.094 0.103 0.099 1.791 1.531 0.955 0.584 0.325 0.170 0.091 0.040 0.001
0.047 0.051 0.051 1.822 1.478 0.906 0.536 0.305 0.158 0.082 0.040 0.001

COMMENT:FRASCH ASSAY PB2 1:75 PLATE 2 UNKNOWNS PB2 1:75
1.830 1.843 1.914 1.898 1.637 1.045 0.620 0.341 0.178 0.091 0.046 1.446
1.655 1.681 1.690 1.884 1.628 1.077 0.630 0.350 0.184 0.091 0.047 1.461
1.078 1.103 1.059 1.896 1.652 1.094 0.641 0.343 0.182 0.099 0.050 1.207
0.655 0.658 0.635 1.898 1.644 1.091 0.623 0.348 0.173 0.095 0.048 1.203
0.363 0.361 0.353 1.884 1.653 1.054 0.626 0.352 0.186 0.099 0.050 0.223
0.193 0.198 0.188 1.914 1.652 1.084 0.624 0.338 0.175 0.090 0.043 0.225
0.091 0.095 0.094 1.841 1.612 1.079 0.624 0.349 0.172 0.068 0.046 0.001
0.048 0.047 0.045 1.769 1.603 1.053 0.643 0.326 0.179 0.093 0.046 0.001

The template used to abstract the data is located in file FINAL.TPL. Module 2 may be used to examine the template format and the dilution schedules (refer to Module 2 and Appendix B). Users may run the data abstraction module (Module 3) to produce the .STD, .UNK, .QC, and .LBL files. These are also included on the distribution diskette: MA112190.STD, MA112190.UNK, MA112190.QC, and MA112190.LBL.

Modules 4 may then be used to estimate the four parameters of the logistic-log fit. A sample file of standard curve fitting information is MA112190.FIT contains these results. A portion of that file is reproduced here:

File = C:\ELISAWIN\DOWNLOAD\MA112190.STD- Plate: 1 Project ID = Test Template / Standards ID = Std 1Date: 11/19/96 Time: 11:22:53

FRASCH ASSAY PB2 1:75 PLATE 1 UNKNOWNS ARE PB2 1:75 Results of approximate least squares fitThe procedure used 7 iterations.

Starting values for the iterative process:2.28081 -2.32942 .03409 1.00000

Dil. Rel. Dil. Known O.D. Pred. O.D. Residual % Error Weight
0.001666667 100.00 1.716 1.7559 -0.040 2.325 1.0000
0.001666667 100.00 1.757 1.7559 0.001 0.063 1.0000
0.001666667 100.00 1.769 1.7559 0.013 0.741 1.0000
0.000833333 50.00 1.438 1.4315 0.007 0.453 1.0000
0.000833333 50.00 1.462 1.4315 0.031 2.087 1.0000
0.000833333 50.00 1.458 1.4315 0.027 1.818 1.0000
0.000416667 25.00 0.967 1.0095 -0.042 4.391 1.0000
0.000416667 25.00 1.014 1.0095 0.005 0.448 1.0000
0.000416667 25.00 0.998 1.0095 -0.011 1.148 1.0000
0.000208333 12.50 0.586 0.6097 -0.024 4.047 1.0000
0.000208333 12.50 0.606 0.6097 -0.004 0.613 1.0000
0.000208333 12.50 0.627 0.6097 0.017 2.757 1.0000
0.000104167 6.25 0.319 0.3300 -0.011 3.458 1.0000
0.000104167 6.25 0.347 0.3300 0.017 4.890 1.0000
0.000104167 6.25 0.346 0.3300 0.016 4.615 1.0000
0.000052083 3.12 0.167 0.1732 -0.006 3.697 1.0000
0.000052083 3.12 0.186 0.1732 0.013 6.896 1.0000
0.000052083 3.12 0.190 0.1732 0.017 8.856 1.0000
0.000026042 1.56 0.094 0.0959 -0.002 2.049 1.0000
0.000026042 1.56 0.103 0.0959 0.007 6.868 1.0000
0.000026042 1.56 0.099 0.0959 0.003 3.105 1.0000
0.000013021 0.78 0.047 0.0603 -0.013 28.335 1.0000
0.000013021 0.78 0.051 0.0603 -0.009 18.270 1.0000
0.000013021 0.78 0.051 0.0603 -0.009 18.270 1.0000

The final coefficients (a, b, c, d) are:

2.13195
-2.10001
0.01841
1.19810

Variances of coefficients =

0.00245
0.00328
0.00000
0.00211

Regular and adjusted sums of squares =

0.007834
0.007834

Approximate multiple R^2 =

0.9991

Logistic-log and spline fit plots may be viewed using Module 5

Module 6 may then be entered to calculate patient serum sample or unknown antibody concentrations. In this sample data set a calibration factor of 1500 was chosen. A complete listing of these results using the unweighted least squares fit is included in file MA112190.RPT. A sample of the report file is reproduced here:

Title:
File: C:\ELISAWIN\DOWNLOAD\MA112190 Project ID = Test Template / Standards ID = Std 1Date: 11/19/96 Time: 11:23:32
:FRASCH ASSAY PB2 1:75 PLATE 1 UNKNOWNS ARE PB2 1:75 Results from Least Squares fit. Coefficient file selected: C:\ELISAWIN\DOWNLOAD\MA112190.CFT

  A
2.132
B
-2.100
C
0.018
D
1.198
     
Optical densities may range between: 0.032 (l asym) and 2.132 (u asym)The range of standard dilutions is: 76800 - 600 The range of standard OD's is 0.047 - 1.769Calibration factor is: 1500
PLATE: 1 UNK# 1 / ID = Unk 1
# OD DILUTION Avg. OD CONCENTR. Avg.
CONC.
STD
DEV.
CV
01 1.840 300   964.043        
02 1.800 300 1.820 850.047 907.045 80.607 8.887
03 1.462 600    792.541         
04 1.493 600 1.477 839.400 815.970 33.134 4.061
05 0.960 1200    692.807         
06 1.013 1200 0.987 754.256 723.532 43.451 6.005
07 0.562 2400    680.189         
08 0.590 2400 0.576 720.796 700.493 28.713 4.099
09 0.313 4800    708.477         
10 0.346 4800 0.329 789.232 748.855 57.102 7.625
11 0.171 9600    739.668         
12 0.173 9600 0.172 749.174 744.421 6.722 0.903
13 0.090 19200    689.887         
14 0.092 19200 0.091 710.248 700.068 14.398 2.057
15 0.041 38400    286.910        
16 0.044 38400 0.043 364.736 325.823 55.031 16.890
Mean (Excludes N/C & Cen):       708.276   166.979 23.575

 

PLATE: 1 QUALITY CONTROL
# OD DILUTION Avg. OD CONCENTR. Avg.
CONC.
STD
DEV.
CV
01 1.440 1200    1523.070         
02 1.409 1200 1.424 1441.331 1482.200 57.798 3.899
03 1.255 1800    1666.735         
04 1.239 1800 1.247 1623.825 1645.280 30.342 1.844
05 0.233 19200    2067.105         
06 0.255 19200 0.244 2276.285 2171.695 147.913 6.811
Mean (Excludes N/C & Cen):       1766.392   330.317 18.700

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