Fig. 2

Sensitivity and specificity of anti-HER-2 × HBsAg. a The breast cancer cells were labeled with trastuzumab, anti-HER-2₀₀₁ × HBsAg, anti-HER-2₀₀₄ × HBsAg, anti-HBsAg (anti-HB) and relevant isotype antibodies (iso) as indicated, followed by secondary superparamagentic antibodies. Measured changes in relaxivities reflect specificity and sensitivity of labeling. All experiments were conducted three times. The data presented are representative for all. The SK-BR-3 and the patients’ breast cancer cells (BC001 - the data for the patient 001 are representative to all 10 patients) cells were heavily labeled with trastuzumab, anti-HER-2 antibodies, and anti-HER-2 × HBsAg constructs. The MCF-7 and the patients’ human breast epithelial cells (HBE) were not labeled at the statistically significant relaxivity change. The isotype antibodies did not label the breast cancer and healthy cells. b Sera of patients with high titers of anti-HBsAg antibodies were rapidly cryoimmobilized, crushed, thawed, immunoprecipitated with superparamagnetic molecular baits as indicated, released into electrophoresis, and immunoblotted. Lanes’ of superparamagnetic immunoprecipitation labels. 1. Immuno-naïve patient, not immunized, not infected, tested with the clinical diagnostics. 2. Vaccinated patient tested with anti-HER-02₀₀₁ x HBsAg; 3 Vaccinated patient tested with isotype antibody; 4. Vaccinated patient tested with anti-HER-02₀₀₄ x HBsAg; 5. Vaccinated patient tested with isotype antibody; 6. Vaccinated patient tested with Engerix; 7. Vaccinated patient tested with isotype antibody; 8. Vaccinated patient tested with Recombivax; 9. Vaccinated patient tested with isotype antibody; 11. Vaccinated patient tested with anti-HBsAg isotype antibody 10, 12 molecular weight standards. Three experiments provided the same data. All molecular baits used in this study were pulling out the same anti-HBsAg molecule as determined by Hepatitis B clinical diagnostic assays. (c) The blots from (b) were quantified at high sensitivity and revealed only anti-HBsAg antibodies in the patients sera and no other molecules immunoprecipitated and labeled. (d–m) The breast cancer cells were labeled with trastuzumab (d), anti-HER-2₀₀₄ × HBsAg (e) mix of both tagged with different flurochromes, (f) anti-HER-2₀₀₁ × HBsAg followed by anti-HBsAg fluorescent antibodies (g) or after initial labeling with anti-HER-2₀₀₁ × HBV, the cells were labeled with trastuzumab. (h, i) Isotype antibodies for trastuzumab (j) anti-HER-2₀₀₁ (k) anti-HER-2₀₀₄ (l) anti-HBsAg (m) did not reach statistically significant counts of the labelled cells