ELISA METHOD TO DETECT ABO BLOOD GROUP IN EXTERNAL SECRETION FLUIDS

Background: Usually it takes a large number of volume sample to determine blood group from external secretion fluids. But, in certain condition, samples are only available in very small amount. The objective of this study is to detect the presence of ABO blood group substances in mucosal fluid using ELISA technique, thus only requires small amount of samples.Objective: To develop an ELISA technique using the current anti-ABO antibodies for determination of blood group by hemagglutination technique and second peroxidase label antibody specific for mouse IgG, originally used for another ELISA technique.Methods: 100 μl of diluted human intestinal mucosal fluid were incubated overnight in 4oC in ELISA microplate wells, followed by addition anti-ABO antibodies. Then after incubation, a second revealing antibody anti mouse IgG labeled with peroxidase was added. After a brief incubation, substrate H2O2 and chromogenic TMB were added.Results: Positive reaction is marked by development of blue colour, which, on termination enzymatic reaction by addition 100 μl H2SO4 change to yellow.Conclusion: An ELISA method for detecting ABO substance in mucosal fluid can be developed from antibodies not specifically made for this technique, but specific only for the target.

Transmural gradient of leukocyte-endothelial interaction in the rat gastrointestinal tract

Gastrointestinal injury usually starts in the superficial mucosa. We investigated whether leukocyte-endothelial interactions were greater in the gastrointestinal mucosa than the submucosa and muscularis in control tissue and after upregulation of adhesion molecules with endotoxin and after chemical insult with nonsteroidal anti-inflammatory drugs. Inactin-anesthetized rats were given either endotoxin, flurbiprofen, or nitric oxide (NO)-flurbiprofen, after which ICAM-1 and P-selectin expression was measured with the dual-label antibody technique. Leukocyte-endothelial interactions in the different gastric layers were assessed after endotoxin using intravital microscopy. Endotoxin caused a two- to threefold increase in ICAM-1 expression in the stomach and duodenum. There was, however, a gradient in expression across the gut wall with the level of expression in the superficial mucosa (per g) being only 10–25% of that in the deeper layers in both control and endotoxin-treated animals. Constituitive expression of P-selectin in control animals was barely detectable. Endotoxin caused a modest increase in mucosal P-selectin but a very significant increase in the deeper layers. Flurbiprofen caused a slight upregulation of ICAM-1 in the gastric mucosa and duodenum, whereas NO-flurbiprofen had no affect on expression. Intravital microscopy revealed no adhesion and virtually no leukocyte rolling in the vessels of the gastric mucosa despite endotoxin treatment. There was, however, some adhesion and significant leukocyte rolling in the submucosa and muscularis. Thus the superficial gastric and duodenal mucosal microcirculations have a much lower density of ICAM-1 and P-selectin and less leukocyte-endothelial interactions than occurs in the deeper layers of the gut wall even during stimulated upregulation with endotoxin.

Analyte and label binding assay read by flow cytometry

A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.