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HLA class I-directed IgG antibodies have traditionally been detected with a complement-dependent lymphocytotoxicity (CDL) technique. We have evaluated two solid-phase enzyme-linked immunoassays (EIA) and compared them with the CDL antihuman globulin (AHG) dithiothreitol-treated (DTT) PRA in their ability to discriminate between the presence or absence of HLA class I-directed IgG antibodies in serum from patients awaiting transplantation. The EIA were: (1) an EIA that uses solubilized HLA class I antigens (sHLA-I) isolated from a 240-member platelet donor pool, and (2) the PRA-STAT ELISA kit. For the first comparison, we used 691 serum samples from 272 patients taken before they had been transplanted. The data show a significant (P < 0.0001) linear correlation (r = 0.77 between the AHG DTT PRA and the sHLA EIA. They also demonstrate that the mean sHLA-I EIA ratio significantly increases (P < 0.01) above background levels with each stepwise increase in AHG DTT PRA level. Discordant results were 1.0% (7/691) for sHLA-I EIA+ PRA- and 6.3% (44/691) for PRA+ sHLA-I EIA-. However, a lower correlation was noted between the AHG DTT PRA and the PRA-STAT (Nextran) PRA results (n = 230; r = 0.42). The sHLA-I EIA is able to determine whether or not HLA Class I IgG antibodies are present in serum from transplant candidates and is an appropriate adjunct to the traditional CDL PRA assay, whereas the PRA-STAT is not.

The objective of this study was to evaluate the use of sHLA in a solid-phase EIA as a rapid and sensitive way to identify potential IgG HLA class-I-typing reagents. To evaluate the efficacy of the sHLA EIA, we used the assay to screen 259 HLA-A, -B, and -C antisera that our laboratory had procured using the standard NIH LCA. A positive result obtained by the sHLA EIA, which was defined as an EIA ratio of 3 SD above the mean of 91 anti-HLA-negative sera, revealed that 91% (79 of 87) of the A-locus-typing reagents were positive, 96% (150 of 156) of the B-locus antisera were positive, and only 75% (12 of 16) of the C-locus reagents were positive. The typing reagents that were negative by EIA (n = 18) fell into two categories. First, 38% (7 of 18) were negative by sHLA EIA, as they were IgM-typing reagents (NIH LCA reactivity ameliorated by DTT). The second group of the 11 remaining typing reagents had a mean EIA ratio of 1.0 +/- 0.246 (mean +/- 1 SD), which was significantly (P0.001) higher than the mean of the 91 negative controls that were used to establish the negative cutoff. The overall sensitivity of the sHLA EIA to detect HLA class-I-directed IgG was 97.2%.

An increase in atrial pressure has been shown to cause an increase in the concentration of atrial peptides (atriopeptin) in plasma. We therefore hypothesized that a reduction in atrial pressure would decrease the concentration of atriopeptin in plasma. In formulating this hypothesis we assumed that changes in the concentration of other circulating hormones or changes in cardiac nerve activity during hemorrhage would not affect the secretion of atriopeptin. To test the hypothesis, we bled sham-operated conscious dogs at a rate of 0.8 ml.kg-1.min-1 to decrease right and left atrial pressures. Hemorrhage was continued until a total of 30 ml of blood per kilogram body weight had been removed. Identical experiments were performed on conscious cardiac-denervated dogs. The concentration of plasma atriopeptin was decreased in each group of dogs after 10 ml of blood per kilogram of body weight had been removed, but the decrease achieved statistical significance only in the cardiac-denervated dogs. Further hemorrhage, however, produced no further decreases in circulating atriopeptin in either group even though atrial pressures continued to decline as more blood was removed. A comparison of the atriopeptin response to hemorrhage revealed no significant difference between the sham-operated and cardiac-denervated dogs, thus providing no evidence for a specific effect of cardiac nerves on atriopeptin secretion during hemorrhage. Our results demonstrate that the relationship between atrial pressure and plasma atriopeptin that has been observed repeatedly during atrial stretch is not evident during relatively slow, prolonged hemorrhage. There is, however, a small decline in circulating atriopeptin during the initial stage of hemorrhage that could be of biological significance.