BsAb specificity for Listeria and RBC antigens as revealed by Fluorescence Microscopy.

<p><b>A</b>. mBsAb interaction with cell surface proteins of <i>Listeria monocytogenes</i> (<b>a and b</b>), mBsAb binding capacity to human RBCs (<b>c and d</b>). <b>B</b>. Affinity of pBsAb with target surface molecules of <i>Listeria monocytogenes</i> cells (<b>a and b</b>) and RBCs (<b>c and d</b>). At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

Dot blot analysis.

<p><b>A</b>) <b>a</b> and <b>b</b> show recognition of <i>Listeria</i> surface antigen and RBC ghost proteins by mBsAb respectively. <b>c</b> and <b>d</b> exhibit controls where PVDF membrane was coated with <i>E. coli</i> and <i>Salmonella</i> Ags respectively and allowed to react with mBsAb (specific to <i>Listeria monocytogenes</i> and RBCs). A negative result was obtained in controls. <b>B</b>) <b>a</b> and <b>b</b> exhibit binding of pBsAb to Listeria surface protein and RBC membrane proteins respectively. PVDF membrane coated with <i>E. coli</i> and <i>Salmonella</i> Ags shows negative result when allowed to react with pBsAb (<b>c</b> and <b>d</b> respectively). At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

Interaction of <i>Listeria monocytogenes</i> cells with BsAb employing fluorescence-activated cell sorting analysis.

<p>The cells with log fluorescence intensities were gated BsAb and <i>Listeria</i> cells. (<b>a</b>) <i>Listeria monocytogenes</i> cells only, (<b>b</b>) Anti-RBC Ab incubated with <i>L. monocytogenes</i> cells (<b>c</b>) Bispecific antibody of polyclonal origin (pBsAb) incubated with <i>Listeria</i> cells for 1 hr. At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

An Alternative Chemical Redox Method for the Production of Bispecific Antibodies: Implication in Rapid Detection of Food Borne Pathogens

<div><p>Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen <i>Listeria monocytogenes</i> (<i>L. monocytogenes</i>) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the <i>L. monocytogenes</i> and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of <i>L. monocytogenes</i> cells. The agglutination process necessitated the specific presence of <i>L. monocytogenes</i> and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches.</p></div

Western blot analysis.

<p><b>A</b>) <b>a</b> and <b>b</b> show the reactivity of mBsAb to RBC ghost proteins and <i>Listeria</i> surface protein respectively while <b>c</b> and <b>d</b> exhibit incapability of mBsAb to react with <i>E. coli</i> and <i>Salmonella</i> Ags respectively. Similarly,pBsAb reacted well with RBC ghost proteins and <i>Listeria</i> InlB protein (<b>Ba</b> and <b>Bb</b> respectively). On the other hand negative result is observed for <i>E. coli</i> and <i>Salmonella</i> proteins when pBsAb is used for recognition (<b>Bc</b> and <b>Bd</b> respectively). At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

Schematic agglutination representation of erythrocytes and <i>Listeria monocytogenes</i> cells mediated by bispecific antibodies.

<p>Schematic agglutination representation of erythrocytes and <i>Listeria monocytogenes</i> cells mediated by bispecific antibodies.</p

Bispecific antibody mediated haemagglutination.

<p>mBsAb (<b>A</b>) as well as pBsAb (<b>B</b>) rendered agglutination of RBCs and <i>Listeria</i> cells on a glass slide. A given volume (50 μl of 200 ng/ml) of BsAb specific against both RBCs and <i>Listeria</i> cell surface Ag was mixed with <i>Listeria</i> cells-negative (<b>Aa, Ba</b>) or <i>Listeria</i> cells-positive (<b>Ab, Bb</b>). After 1 hr of incubation, the RBC agglutination results were observed under a microscope (right panel; magnification, 20X). At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

Detection of <i>Listeria monocytogenes</i> using agglutination assay in inoculated meat (beef, chicken and mutton) samples.

<p>mBsAbs as well as pBsAbs rendered agglutination of RBCs when incubated with <i>Listeria</i> inoculated meat. The wells marked as ‘B’ are controls lacking <i>Listeria</i> cells. At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

Demonstration of hemagglutination employing mBsAb (A) and pBsAb (B) against human red blood cells and <i>Listeria</i> cells.

<p>The wells contain a constant number of RBC (20 μl of 60% hematocrit) and <i>Listeria</i> cells (10³ cells) plus serial two-fold dilutions of BsAb. The spread pattern in the experimental series indicates positive hemagglutination through well 5 and 4 respectively for mBsAb and pBsAb incubated wells. The wells marked as a, b and c were incubated with anti-Lis Ab, anti-RBC Ab and a mixture of these two respectively. At least three independent experiments were performed for each sample and data are representative of three independent experiments with similar observations.</p

Specificity determination of in-house prepared BsAb by ELISA.

<p>Either Listeria Ag or RBC antigen was coated followed by recognition with BsAb so that its one arm was fully captured with the antigen. Further RBC Ag or Listeria Ag (which was not used for coating) was added for stipulated time period. The plate was thoroughly washed and allowed to interact with Rabbit anti RBC antibody or Rabbit anti <i>Listeria</i> antibody. Finally, the signal was detected using HRP-tagged goat anti-rabbit Ab. In case of controls instead of BsAb, anti-RBC Ab or anti-Lis Ab or mixture of the two was added. <b>A</b>) RBC antigen was coated and anti-Lis mAb was used for detection. mBsAb was employed. <b>B</b>) Listeria Ag coated and anti-RBC mAb detected the signal. mBsAb was used. <b>C</b>) RBC antigen was coated and anti-Lis pAb was used for detection. pBsAb was exploited. <b>D</b>) Listeria Ag coated and anti-RBC pAb detected the signal. pBsAb was used. Only BsAbs could give significant signals. On the other hand, the controls gave very feeble signals validating the formation of BsAb. At least three independent experiments were performed and data are represented as mean of at three independent experiments ± SD value. (<i>P</i> values; BsAb vs anti-RBC control <0.05, BsAb vs anti-Lis control <0.05, BsAb vs anti-RBC and anti-Lis mixture <0.05).</p