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\centerline{\bf Description of ELISAeq Computer Program}
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This Computer program is written in GW-BASIC we have named ELISAeq.
The main function of this program is to derive the linear \& hyperbolic
interpolation formula using the known standard and to determine the
Ag/Ab (Antibody or antigen) concentration of unknown samples. The
ELISAeq have three main sections I) Input Section (Lines 270--2000)
II) Subroutine section (lines 2400--3800)  and III) Calculation \&
Result section (lines 2010--2400). The detail description of each
section is as below.

\noindent{\bf Input Section}

This section of the program allow the user to input information. Lines
270--720 allow to feed the optical density (OD) data from ELISA plate.
The OD data can be feed directly from microplate reader by interfacing its output
port to the serial port of microcomputer using RS 232-C interface, or by using keyboard or
file. Lines 820--1280 allow to display the OD data in $8\times 12$ format on
screen, to modify the OD data, to store this OD data for future use, and
also allow to print the OD data in $8\times 12$ on printer. Lines 1330--2000
allow to feed the dilution template, which is the information about known
standard such as the rows containing the standard samples, the concentration
of these known standards and whether the samples are in singlet, duplicates,
triplicates etc. This dilution template can be feed by using keyboard or by
using the file, and can be stored for future use.

\noindent{\bf Subroutine Section}

This section consist three subroutines I) Linear regression II) Hyperbolic
regression and III) Error trapping. Linear regression subroutine
(lines 3340--3540) derive a linear interpolation formula using the
linear regression and least square curve fitting
method . The hyperbolic regression
subroutine (lines 3550--3830) derive a hyperbolic
interpolation formula using the hyperbolic regression method. Error trapping
(lines 3840--3930) subroutine handle the errors occurs, when the program is running.

\noindent{\bf Calculation \& Result section}

This section perform the required calculation by utilizing the subroutine
section. Lines 2020--2410 allow to calculate the average value of OD if the
samples on the plate are in duplicate, triplicate etc, and also allow to
display \& print the average value. The program utilized the standard
data to determine the Sl-range (semilogarithmic linear range)
and derive a linear interpolation formula for this range by using linear
regression and least square curve-fitting method. The hyperbolic
interpolation formula was also derived by using hyperbolic regression method
from standard data. The OD of unknown samples was checked
and Ab/Ag concentration determined by linear interpolation formula whose
OD is in the Sl-range and by hyperbolic interpolation formula whose OD is
beyond the Sl-range. It also calculate the signal, capacity
and flatness of hyperbolic curve, which play a vital role in ELISA
optimization. The program  allow to display and print
the results.
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Fig.~1. Format for ELISA titration samples are put serially diluted
in consecutive duplicated wells.

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Fig.~2. Shows an example printout from one ELISA plate.
For each plate, the user has the option to print the average  OD values,
the sl-range, the linear interpolation formula, the hyperbolic interpolation
formula and to calculate Ab/Ag concentration of unknown samples.
The rows containing standard samples is also shown in the printout.
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Fig.~3. Experimental data ($\bullet$) and regression curves for ELISA of
IgG$_{2b}$. The semilogarithmic linear range (sl-range) of the curve also
shown.
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