Anti-band 3 and anti-spectrin antibodies are increased in Plasmodium vivax infection and are associated with anemia(1)
Malaria-induced anemia is thought to arrise from hemolysis of infected red blood cells (RBCs), as well as from clearance of non-infected red blood cells (nRBCs). In P. falciparum infections, removal of nRBCs is possibly boosted by increased levels of self-antibodies against nRBCs proteins. An inverse association was found in between hemoglobin levels and anti-phosphatidylserine antibodies in humans with late post-anemia due to P. falciparum infection. In vitro erythrophagocytosis of nRBCs was mediated by anti-erythrocyte antibodies purified from anemic patients with vivax malaria, possibly through a decrease in cell deformability.
Anemic patients infected with P. vivax had higher levels of IgG against erythrocyte proteins than the infected non-anemic patients or healthy controls.
A representative nRBC 2D-SDS-PAGE map was obtained.
Sera from the infected groups recognized a greater number of spots than the sera from the healthy control group. Spots recognized exclusively by sera from anemic malaria patients included band 3, spectrin (both alpha and beta chains), cytoplasmic actin 1, protein 4.1, protein band 4.2, ankyrin 1, and dematin.
Anemic patients had significantly higher levels of anti-band 3 antibodies in comparison to the non-anemic ones. A similar result was also observed for anti-spectrin antibodies. The levels of hemoglobin decreases with the increase in the level of anti-band 3 and anti-spectrin antibodies.
Nineteen of them were hypothetical proteins of the parasite and their mimetic human RBC proteins were associated with plasma membranes: ankyrin, actin, and spectrin. Several mechanisms are involved in the production of self-reactive antibodies in malaria including polyclonal activation of B cells induced by parasite antigens, dysregulation of B lymphocytes, and stimulation of specific B lymphocytes by molecular mimicry. A surge in anti-spectrin antibodies following the intraperitoneal injection of spectrin in rats affected the homeostasis of RBC by accelerating their elimination and stimulating erythropoiesis.
Since spectrin is present on the intracellular side of the erythrocyte membrane, it remains unclear how anti-spectrin antibodies bind to a cytoplasmic protein. The reaction of anti-spectrin antibodies with RBCs has been previously described by studies investigating the clearance of aged erythrocytes. The destruction of erythrocytes releases spectrin in the plasma, where it could bind to circulating anti-spectrin antibodies, thereby eliciting complement deposition onto these complexes. RBC engulfment by macrophages is affected by cell stiffness and molecular interactions in the cell surface.
Detection of IgG against nRBCs proteins: Serum levels of IgG against nRBCs extracts were determined by ELISA. Briefly, each well of a 96-well flat-bottomed polystyrene microplate was coated with 0.1 ng of RBC protein extract in 0.1 M carbonate buffer, pH 9.6, and then incubated overnight at 4°C. After five washes with PBS containing 1% (w/v) bovine serum albumin (BSA), the plate was blocked with nonfat powdered milk for two hours at 37 °C. Next, plates were washed and incubated, in duplicate, with serum samples diluted 1:200 in PBS/BSA for 2 hours at 3 °C. Wells were rewashed and incubated with horseradish peroxidase conjugated polyclonal anti-human IgG diluted 1:8000 in PBS/BSA for 90 minutes at 37 °C. Finally, the plates were washed again, and the reaction was revealed using 0.5 mg/mL o-phenylenediamine dihydrochloride substrate in 0.05 M phosphate-citrate buffer, pH 5.0. The reaction was stopped with 3M H2SO4. Optical density (OD492 nm) was determined in a microplate reader . The levels of specific IgG were expressed as OD492nm.