35. The interaction between an antigen (Ag) and a single-chain antibody (Ab) was studied using Scatchard analysis. The result is shown below.

In the plot, the Y-axis represents [Ab–Ag]/[Ag] and the X-axis represents [Ab–Ag].

The affinity of interaction and the total concentration of antibody, respectively, can be determined from

(A) slope and Y-intercept
(B) Y-intercept and slope
(C) X-intercept and slope
(D) slope and X-intercept

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Introduction

The Scatchard analysis antigen antibody interaction is a fundamental concept in
immunology and biochemistry. It is frequently asked in exams such as CSIR-NET, GATE, JAM,
and MSc entrance examinations. This method allows the determination of binding
affinity and total antibody concentration from a simple linear plot.

Understanding Scatchard Analysis

Scatchard analysis is based on the following equation:

[Ab–Ag] / [Ag] = K_a(n − [Ab–Ag])

Where:

• K_a = association constant (affinity)
• n = total number of antibody binding sites
• [Ab–Ag] = concentration of bound antigen

Axes in the Scatchard Plot

• Y-axis: [Ab–Ag] / [Ag]
• X-axis: [Ab–Ag]

The graph is a straight line with a negative slope, characteristic of Scatchard plots.

Key Parameters Obtained from the Graph

Slope

Slope = −K_a

The magnitude of the slope gives the affinity of interaction.

X-intercept

X-intercept = n

This represents the total concentration of antibody binding sites.

Y-intercept

Y-intercept = K_a × n

This is a combined parameter and does not directly give affinity or antibody concentration alone.

Option-Wise Explanation

Option (A): Slope and Y-intercept ❌

Slope gives affinity, but Y-intercept represents K_a × n, not total antibody concentration.

Option (B): Y-intercept and slope ❌

Same parameters as Option (A) in reverse order. Still does not directly give antibody concentration.

Option (C): X-intercept and slope ❌

Although both parameters are correct, the order is incorrect. The question asks for affinity first.

Option (D): Slope and X-intercept ✅

Slope gives affinity, and X-intercept gives total antibody concentration. This matches the requirement.

Final Answer

Option (D): slope and X-intercept

Quick Exam Tip

Remember:
Slope = −Affinity (K_a)
X-intercept = Total antibody binding sites
Y-intercept = K_a × binding sites

Conclusion

In Scatchard analysis antigen antibody interaction, the binding affinity is
obtained from the slope of the plot, while the total antibody concentration
is obtained from the X-intercept. Therefore, the correct answer is
Option (D). Understanding this graphical interpretation is essential for
scoring well in immunology and biochemistry examinations.