Dados do Trabalho

Título

BSA can interfere in the serodiagnosis of canine visceral leishmaniasis by suspension array technology

Introdução

Canine Visceral Leishmaniasis (CVL), caused by Leishmania infantum, is a significant public health concern since animals serve as reservoirs for human disease. A diagnostic method capable of accurately and rapidly distinguishing dogs with symptomatic and asymptomatic CVL from healthy and vaccinated dogs is crucial for controlling both canine and human disease. In addition to exploring new candidate antigens, the development of innovative antibody detection methodologies is essential to create faster and more efficient CVL tests.

Objetivo (s)

To achieve this objective, we have been working on a multiplex methodology using flow cytometry to detect Leishmania-specific antibodies in canine serum samples. During this process, we identified the necessity to conjugate small peptides with carrier proteins or tags to facilitate efficient binding of these antigens to non-magnetic beads used in multiplex assays.    

Material e Métodos

A  peptide derived from the L. infantum A2 protein (VQ34 - Campos et al, 2018) was employed for coupling to non-magnetic beads in three different formulations: 1) unconjugated 2) conjugated with BSA or 3) conjugated with a beta-alanine–lysine(x 4)—cysteine tag (Wakeman et al. 2021). L. infantum promastigotes total antigen (LiAg) was also used. Each peptide formulation and LiAg was individually coupled to 5.5μm polystyrene nonmagnetic carboxylated beads (Bangs Laboratories Inc, USA).  After serological reaction with canine serum (infected asymptomatic or symptomatic, healthy controls or vaccinated dogs) the samples were acquired in a CytoFLEX flow cytometer (Beckman Coulter, Hialeah, FL, USA).  

Resultados e Conclusão

Total LiAg antigen coated beads demonstrated a negative reaction to LiAg in the sera of healthy dogs, regardless of vaccination status. Conversely, all symptomatic dogs displayed high labeling percentages (above 97%), whereas a more diverse response was observed in the asymptomatic group. Our results with the VQ34 formulations demonstrated that VQ34-TAG displayed enhanced sensitivity, without altering the responses observed in the control group, when compared to  unconjugated VQ34. However,  VQ34-BSA conjugation revealed the presence of anti-BSA antibodies in the serum of healthy dogs, whether vaccinated against CVL or not, as well as in the serum of infected dogs. These findings suggest that the use of BSA as a carrier in bead coupling assays should be avoided in serological tests with canine samples, as it can significantly interfere with the specificity and sensitivity of the tests.

Palavras Chave

Canine Visceral Leishmaniasis; Non-magnetic Beads Suspension Array Technology; Flow Cytometry; Serology