The competitive ELISA method (also known as inhibition or blocking ELISA) quantitatively measures the interference of an analyte with an expected signal, thereby determining the analyte concentration in the sample.

Antigen-antibody binding is a reversible reaction: A + B ⇋ AB (A: antibody, B: antigen, AB: antigen-antibody complex) At equilibrium, the dissociation constant is expressed as: Kd = [A][B] / [AB], where [AB] is the concentration of the antigen-antibody complex, [A] is the concentration of free antibody, and [B] is the concentration of free antigen.

(1) When antibodies are scarce and antigens are in excess, after equilibrium [AB] > [A].

(2) When antigens are scarce and antibodies are in excess, after equilibrium [AB] < [A].

(3) When antigen and antibody concentrations are appropriately matched, the system reaches a half-saturation state at equilibrium, where [AB] = [A], and thus:Kd = [A][B] / [AB] = [B]

At half-saturation, if the antibody concentration is low, the antigen consumed by the reaction is minimal, and thus the free antigen concentration [B] approximates the total antigen concentration [B]total. Therefore, identifying the total antigen concentration that achieves half-saturation after equilibrium allows for calculation of the dissociation constant.

At the half-saturation state, if the antibody concentration is low and only a small amount of antigen is consumed in the reaction, the concentration of free antigen [B] after the reaction is approximately equal to the total antigen concentration [B] total. At this point, Kd = [B] ≈ [B] total. Therefore, as long as the antibody concentration is low, identifying the total antigen concentration  [B] total that reaches the half-saturation state after the reaction equilibrium allows the dissociation constant to be determined.

The basic process of this method involves selecting a small amount of target molecule and a labeled signal molecule that occupies 80% of the binding sites of the target molecule. A gradient-diluted test sample is added and allowed to react with both molecules. The signal value of the target molecule–signal molecule complex is then measured to generate a dose-response curve (typically sigmoidal in shape). The IC50 value is derived from this curve: the smaller the IC50, the stronger the affinity of the test sample for the target molecule.

-- No sample production required; capable of measuring crude or unpurified samples.

-- High reproducibility: reduced variability between repeated samples and analyses.

-- High flexibility: applicable to direct, indirect or sandwich ELISA formats.

-- More robust: less sensitive to sample dilution and matrix effects compared with sandwich ELISA.

-- Professional technical team with extensive experience.

-- High measurement efficiency, label-free and real-time monitoring.

-- Advanced instrumentation.

-- Broad application range: suitable for protein, nucleic acid, polypeptides, and nanomaterial interaction analysis.