Evaluation of the Ca 1 monoclonal antibody in distinguishing oral squamous cell carcinoma from non-malignant or premalignant oral lesions

The Ca antigen has been reported to be present on the surface of malignant but not, with few exceptions, non-malignant cells. We investigated the potential usefulness of the monoclonal Ca 1 antibody in differentiating oral squamous cell carcinoma from non-malignant or premalignant oral neoplasms. Paraffin-embedded sections from 33 biopsy specimens of 12 hyperplastic and 21 neoplastic oral lesions were examined by immunohistochemical staining. Seven of the 33 specimens showed positive staining for Ca antigen. Fifteen of 21 specimens of oral squamous cell carcinoma were negative for Ca antigen, and one case of focal keratosis was positive. The results indicate that the use of the Ca 1 antibody to distinguish oral squamous cell carcinoma from non-malignant or premalignant oral lesions is highly unreliable.     

Exposure of the Rh0(D) antigen on the surface and cytoplasmic domains of the red cell membrane

Inside-out (IO) and right-side-out (RO) vesicles derived form human red blood cells were tested for their ability to bind 125I-labelled IgG anti-RHO(D). The binding of anti-RHO(D) to RO vesicles from RHO(D)-positive cells was quantitatively similar to that exhibited by intact cells when compared on a membrane surface area basis. There was no significant binding of labelled antibody to IO vesicles from RhO(D)-positive cells or to either RO or IO vesicles derived from RhO(D)-negative cells. The RhO(D) antigen was immunologically accessible on only the plasma side of the membrane in RhO(D)-positive red cells, as has been shown for blood group antigens defined by carbohydrate determinants. No immunologically reactive RhO(D) antigen was present on either RO or IO vesicles derived from RHO(D)-negative red cells.     

Interaction of IgG and its fragments with red cells

The red cell uptake of ¹³¹I following incubation of red cells with [¹³¹I] IgG prepared from normal donors was shown to be IgG and not trace contaminants such as transferrin, lipids or iodide. The criteria used were immunodiffusion, DEAE chromatography, gel filtration and exchange with unlabelled lipoproteins and plasma. The uptake of [¹³¹I]IgG was pH and ionic strength dependent and was influenced by the proportion of cells to IgG present during the reaction. With constant cell concentration the uptake of [¹³¹I]IgG increased progressively as more IgG was added to the cells and approached an asymptotic value suggesting that there was saturation of red cell binding sites. When the IgG concentration was kept constant the uptake of IgG was inversely proportional to the red cell concentration. No difference in the molar binding of IgG, Fab or Fc was found indicating that the non-antibody binding of IgG does not preferentially involve any part of the IgG molecule. The molar quantities of carefully prepared [¹³¹I]IgG bound to red cells were similar to those obtained with [¹³¹I]BSA. The non-antibody red cell binding of IgG was contrasted with the antibody type of IgG binding.     

Immunoelectron microscopy of Kell and Cellano antigens on red cell ghosts

The ultrastructural arrangement of Kell (K1) and Cellano (K2) antigens on red cells was studied using ferritin-conjugated rabbit antihuman IgG to stain ghosts derived from antibody-sensitized intact red cells. Estimates of the number of sites under equilibrium binding were obtained by counting both ferritin grains and ferritin clusters on electron micrographs. Values for Kell sites on heterozygous red cells ranged from 2300 to 5900 and for Cellano sites from 2000 to 5000 with no discernible dosage effects. Both the Kell and Cellano antigens were clustered, which was due to conjugate staining of the ghost membranes and does not reflect the topological arrangement of these antigens on the native membrane. The ferritin grain patterns found with the Kell and Cellano antigens was similar to those observed with the Rh antigens except for the markedly reduced antigen site density.     

Antiidiotypic IgG crossreactive with Rh alloantibodies in red cell autoimmunity

IgG autoantibodies eluted from RBCs of antiglobulin positive normal blood donors contained at least two antibody populations, an IgG autoantibody (Ab 1), and an IgG population (Ab 2) that agglutinated RBCs coated with some Rh(D) alloantibodies. Eight of 24 autoantibody eluates tested agglutinated 3 of 10 anti-Rh(D) sensitized RBCs. The agglutinating activity was inhibited specifically by preincubation of the autoantibody eluate with the reactive anti-D. The reaction did not require the Fc domain of the anti-Rh(D), since autoantibody eluates agglutinated RBCs coated with F(ab')2 prepared from the reactive anti-D sera. These findings indicate that the RBCs of some antiglobulin- positive blood donors contain an immunoglobulin auto-antiidiotype (Ab 2) against the RBC autoantibody (Ab 1) which is demonstrable through its cross-reactivity with selected Rh(D) alloantibodies. Identification of auto-antiidiotypes in RBC autoimmunity lends support to the idiotype- antiidiotype network hypothesis of immune regulation and is consistent with the bizarre and complex serology of autoimmune hemolytic anemia. The absence of clinical hemolysis in antiglobulin-positive normal blood donors suggests that immunoglobulin idiotype-antiidiotype interactions may play a role in modulating the effects of RBC autoimmunity.     

Evaluation of the Ca 1 monoclonal antibody in distinguishing oral squamous cell carcinoma from non-malignant or premalignant oral lesions

The Ca antigen has been reported to be present on the surface of malignant but not, with few exceptions, non-malignant cells. We investigated the potential usefulness of the monoclonal Ca 1 antibody in differentiating oral squamous cell carcinoma from non-malignant or premalignant oral neoplasms. Paraffin-embedded sections from 33 biopsy specimens of 12 hyperplastic and 21 neoplastic oral lesions were examined by immunohistochemical staining. Seven of the 33 specimens showed positive staining for Ca antigen. Fifteen of 21 specimens of oral squamous cell carcinoma were negative for Ca antigen, and one case of focal keratosis was positive. The results indicate that the use of the Ca 1 antibody to distinguish oral squamous cell carcinoma from non-malignant or premalignant oral lesions is highly unreliable.     

Anti-D prozone and membrane sulfhydryl modification

An IgG anti-D prozone is produced by progressive inactivation of the D antigen following red cell exposure to increasing concentrations of thimerosal and phenol present as antibody excess is achieved. Partial inactivation of the D antigen by routinely added thimerosal, an organic mercurial, and phenol is associated with an unstable D antigen-antibody complex resulting in an increased rate of dissociation of anti-D and with a decreased reactivity of the cell-bound anti-D in the antiglobulin reaction. Complete D inactivation occurs with concentrations in excess of 0.43 microM thimerosal. If attributable to inactivation of a surface-exposed sulfhydryl group, it suggests that less than 5 percent of these are involved in D-antigen activity. The data do not exclude the possibility that D inactivation may result from alteration of sulfhydryl groups other than those exposed at the surface.     

The antiglobulin reaction with native and enzyme-modified red cells

The quantity of anti-IgG bound at equilibrium to cell-bound anti-D failed to increase with enzymatic modification of intact red blood cells. This contrasts with results obtained by immunoelectronmicroscopy with unsealed red blood cell ghosts. Ghosts derived from enzyme-modified, anti-D-sensitized intact red blood cells show an increased ratio of anti-IgG to anti-D. The ratio declined with protease modification of intact cells, possibly because of steric hinderance to anti-IgG binding within the cellular agglutinates. At comparable levels of cell-bound anti-D and anti-IgG, enzyme-modified cells were more agglutinable in the antiglobulin reaction than unmodified cells. As in saline hemagglutination, enzymatic enhancement of agglutinability appears to be unrelated to the amount of antibody bound, but rather may be related to other factors such as alterations of the biophysical properties of the red blood cell membrane.     

Rh0(D) : Antigen Content of Human Spermatozoa*

¹³¹I-labelled anti-Rh₀ (D) antibody was made to react with suspensions of spermatozoa and red blood cells from Rh₀ (D) positive and negative donors at 37° for 1 hour. Spermatozoa from Rh₀ (D) positive donors did not take up more ¹³¹I anti-Rh₀ (D) than was bound by spermatozoa from Rh₀ (D) negative donors. The mean value for Rh₀ (D) positive donors was 0.0142±0.0061×10^-2 μg. nitrogen per 10⁶ spermatozoa, and the comparable value for Rh₀ (D) negative donors was 0.0189±0.0130×10^-2 μg. nitrogen per 10⁶ spermatozoa. Red blood cells from Rh₀ (D) positive donors took up thirty to forty times more ¹³¹I anti-Rh₀ (D) antibody than was taken up by spermatozoa from Rh₀ (D) positive donors, whereas red blood cells from Rh₀ (D) negative donors took up only 0.5 to 1.7 times more ¹³¹I anti-Rh₀ (D) antibody than was taken up by spermatozoa from Rh₀ (D) negative donors. Pre-treatment of spermatozoa with trypsin increased by two to three times the quantity of ¹³¹I anti-Rh₀ (D) antibody bound to spermatozoa from both Rh₀ (D) positive and negative donors. The quantitative increase was similar for spermatozoa from Rh₀ (D) positive and negative donors.

No evidence for the presence of Rh₀ (D) antigen on human spermatozoa was obtained using ¹³¹I anti-Rh₀ (D).