Highly Targeted Detection of Priority Phytopathogen Pectobacterium brasiliense: From Obtaining Polyclonal Antibodies to Development and Approbation of Enzyme-Linked Immunoassay and Lateral Flow Immunoassay

Abstract

Pectobacterium brasiliense is a bacterial phytopathogen that causes soft and black rot and actively spreads worldwide. Our study is the first development of immunoassays for detecting P. brasiliense. We immunized rabbits and obtained serum with an extremely high titer (1:10⁸). Isolated polyclonal antibodies were tested by enzyme-linked immunosorbent assay (ELISA) using 18 closely related strains and 5 non-related bacterial pathogens. No cross-reactivity was found concerning the tested pathogens. The ELISA of P. brasiliense was developed in a double-antibody sandwich format with a detection limit of 1.5 × 10⁴ cells/mL. A lateral flow immunoassay (LFIA) for P. brasiliense was also developed in a double-antibody sandwich format with a detection limit of 1 × 10⁵ cells/mL. The results of P. brasiliense cells testing with LFIA in plant matrix showed a high correlation (R² = 0.932) between concentrations of added and revealed cells. When testing potato seed material, ELISA and LFIA confirmed 75 and 66% of positive samples according to real-time PCR, respectively. For negative samples, ELISA showed 84% coincidence, and LFIA coincided with PCR for 89% of samples. Thus, the developed immunoassays can be used to evaluate plant material in poorly equipped conditions or under field testing.

Keywords: ELISA; Pectobacterium brasiliense; lateral flow immunoassay; phytopathogen; polyclonal antibodies; potato soft rot.

Figure 1

Characteristics of serum (A) and isolated IgG fraction (B) specific to P. brasiliense using the indirect ELISA method with sorption of P. brasiliense bacteria in microplate wells at a concentration of 1 × 10⁸ cells/mL. Points at the curves are the average values of three replicates; error bars represent standard deviations.

Figure 2

Concentration dependences for P. brasiliense (1), P. carotovorum (2), P. odoriferum (3), and P. atrosepticum (4), obtained in a double-antibody sandwich ELISA. Points at the curves are the average values of four replicates; error bars represent standard deviations. The dotted curve approximates the concentration dependence for P. brasiliense, R² = 0.997. Difference between detection of different concentrations of P. brasiliense and detection of closely related species of Pectobacterium at high concentration (the value is indicated by the arrow) according to t-test: ns—p-value > 0.05, *—p-value > 0.01, ***—p-value < 0.001.

Figure 3

Test strips after analysis of samples containing different numbers of P. brasiliense cells (1—1 × 10⁸, 2—3.3 × 10⁷, 3—1.1 × 10⁷, 4—3.7 × 10⁶, 5—1.2 × 10⁶, 6—4.1 × 10⁵, 7—1.4 × 10⁵, 8—4.6 × 10⁴, 9—1.5 × 10⁴ cells/mL, 10—5.1 × 10³, 11—1.7 × 10³, 12—5.7 × 10² cells/mL, and 13—negative control) in buffer (A) and tuber extract from healthy potato (B) and the corresponding dependences of the intensity of the recorded signals of the test zones on the concentration of P. brasiliense (C). Points at the curves are the average values of two replicates; error bars represent standard deviations. The dotted curve approximates the concentration dependence for P. brasiliense in tuber extract, R² = 0.999. The reduction in color intensity at high concentrations is due to a hook effect specific to sandwich immunoassay formats, indicating that some bacteria–conjugate complexes cannot bind in the test zone occupied by bacteria.

Figure 4

The correlation of the concentrations of P. brasiliense added in a tuber extract and revealed by LFIA are shown on the X and Y axes, respectively. R² = 0.932.