Native epitopes of human IgA.

We have studied the epitopes of the human IgA molecule by using 27 monoclonal antibodies in two simple gel diffusion methods. By testing pairs of monoclonal antibodies for coprecipitation of an IgA myeloma protein, we have clearly identified sterically independent epitopes on the molecule. By testing the non-coprecipitating monoclonal antibodies with soluble IgA-monoclonal antibody immune complexes, we have identified the overlapping epitopes. Eleven proto-epitopes were mapped on the IgA molecule. Since we have not used solid-phase methods, we have presumably identified native epitopes of the IgA molecule.

The effect of long endurance running on natural killer cells in marathoners.

Ten experienced marathoners were exercised 3 h in the laboratory. Blood samples were collected at 0 h baseline, 1 h exercise, and 5 min, 1.5 h, 6 h, and 21 h recovery and were analyzed for total number of lymphocytes expressing membrane antigens found on natural killer (NK) cells. NK activity was also measured. Four of the seven subpopulations of lymphocytes studied, Leu-11+19+, Leu-11+19-, Leu-11+7-, and Leu-19+11-, showed significant within-subject effects over time, using repeated measures ANOVA. Simple contrasts with baseline values showed that, at 1.5 h and 21 h recovery, total number of lymphocytes bearing three different combinations of NK markers, Leu-11+19+, Leu-11+19-, and Leu-11+7-, were significantly decreased when compared with baseline values. At 1.5 h recovery, NK activity was significantly decreased below baseline levels for four of the six effector NK cell/target K562 myelogenous leukemia cell (E:T) ratios tested. At 6 h recovery, NK activity was still decreased significantly with the 12.5:1 and 3:1 E:T ratios. By 21 h recovery, NK activity did not differ significantly from baseline levels. Cortisol levels at 5 min post-exercise were negatively correlated with NK activity at 1.5 h recovery (r = -0.62, P = 0.05, 50:1 E:T ratio; r = -0.66, P = 0.04, 25:1 E:T ratio). Further research is needed to elucidate the effect these changes have on host immunosurveillance and immunoresponsiveness in vivo.

Neuroendocrine and stress hormone changes during mirthful laughter.

Positive emotional activities have been suggested as modifiers of neuroendocrine hormones involved in the classical stress response. To detect changes in these components during a mirthful laughter experience, the authors studied 10 healthy male subjects. Five experimental subjects viewed a 60 minute humor video and five control subjects did not. Serial blood samples were measured for corticotropin (ACTH), cortisol, beta-endorphin, 3,4-dihydrophenylacetic acid (dopac)--the major serum neuronal catabolite of dopamine, epinephrine, norepinephrine, growth hormone, and prolactin. Repeated measures analysis of variance showed that cortisol and dopac in the experimental group decreased more rapidly from baseline than the control group (p = 0.011, p = 0.025, respectively). Epinephrine levels in the experimental group were significantly lower than the control at all time points (p = 0.017). Growth hormone levels in the experimental group significantly increased during baseline (p = 0.027) and then decreased with laughter intervention (p less than 0.0005), whereas, the controls did not change over time (p = 0.787). ACTH, beta-endorphin, prolactin, and norepinephrine levels did not significantly increase. The mirthful laughter experience appears to reduce serum levels of cortisol, dopac, epinephrine, and growth hormone. These biochemical changes have implications for the reversal of the neuroendocrine and classical stress hormone response.

Effects of long-endurance running on immune system parameters and lymphocyte function in experienced marathoners.

The extent and duration of changes in leukocyte subsets, lymphocyte subpopulations, spontaneous blastogenesis, cortisol, and catecholamines were measured in ten experienced marathoners, who ran 3 h to exhaustion in a laboratory setting. Blood samples were taken at baseline, 1 h of exercise, and 5 min, 1.5 h, 6 h, and 21 h of recovery. The 3-h endurance run was associated with significant leukocytosis, granulocytosis, neutrophilia, monocytosis, and eosinopenia during recovery. All of these parameters except for eosinophils returned to normal by 21 h of recovery. Total lymphocyte count increased 31% at 1 h of exercise, then decreased 19% at 1.5 h of recovery when compared with baseline values. T cell count showed no significant changes, but B cell lymphocytosis was measured at 5 min and 6 h of recovery. T helper/T suppressor ratio (H/S) was significantly elevated 39% at both 1.5 h and 21 h of recovery due to the decrease in number of T suppressor cells. Spontaneous blastogenesis was significantly increased 52% by 1 h of exercise and remained elevated throughout recovery. The increase in cortisol from baseline to 1.5 h of recovery correlated positively with the increase in both total leukocyte count (r = 0.78, P = 0.008) and granulocyte count (r = 0.81, P = 0.005). Our results suggest that exhaustive endurance exercise in marathon runners is associated with many significant perturbations in immune system parameters, most of which return to normal levels at 21 h of recovery.

Measuring human IgG4 by a two-site immunoradiometric assay with monoclonal antibodies.

By using 2 monoclonal antibodies, we developed a solid-phase 2-site immunoradiometric assay for measuring human IgG4. The measuring range (0.05-20 micrograms/ml) covered more than 2 orders of magnitude. The sensitivity level should make this assay especially useful when IgG4 concentrations are too low to be detected by conventional methods.

A monoclonal antibody may show cross-reactivities in Ouchterlony assays but not in other assays.

A monoclonal antibody to human IgG was tested with myeloma proteins of the four IgG subclasses. When tested by immunofluorometric assay, enzyme-linked immunosorbent assay, hemagglutination and hemagglutination inhibition assays, the antibody reacted with IgG3 but not with the other three IgG subclasses. When tested by Ouchterlony assays in the presence of polyethylene glycol, the antibody formed lines with all four IgG proteins. The line with IgG3 was sharp and stable, but the lines with the other three IgG subclasses tended to blur with time and with the lower PEG concentrations. These findings show that Ouchterlony assays can reveal cross-reactions of a monoclonal antibody that can be missed by more sensitive assays.

Evaluation of the powder-formulated enzyme multiplied immunoassay technique quantitative single test for gentamicin.

The usefulness of the enzyme multiplied immunoassay quantitative single test (EMIT QST) gentamicin assay was assessed for gentamicin analysis in patient sera. The EMIT QST reagents are in powder form in a single, premeasured vial and are run on a thermoregulated sample processor that controls mixing and timing steps. The results of the clinical evaluation showed that the standard curve was stable throughout a 26-day study period. Within-run precision on 20 replicates at 4.0 micrograms/ml yielded a coefficient of variation (CV) of 5.6%; between-run precision on 66 analyses at 6.0 micrograms/ml over a 152-day period yielded a CV of 4.0%. Mean recovery through the range of the standard curve with 10 spiked patient samples was 102%. Comparative analysis with radioimmunoassay of 95 patient samples showed a correlation of 0.97, with y = 0.93x - 0.03. It was concluded that the EMIT QST gentamicin assay is an appropriate, rapid methodology for patient gentamicin analysis.

Simple, rapid 125I-labeled cyclosporine double antibody/polyethylene glycol radioimmunoassay used in a pediatric cardiac transplant program.

We modified the Sandoz cyclosporine radioimmunoassay because of our need for frequent clinical monitoring of cyclosporine drug levels in allo- and xenograft pediatric cardiac transplant patients. With application of a commercially available [125I]cyclosporine label in place of [3H]cyclosporine and a second antibody/polyethylene glycol (PEG) method of separation in place of charcoal separation, we simplified and enhanced the speed and precision of assay performance. Studies of 140 whole blood samples comparing this new method to the [3H]cyclosporine radioimmunoassay (RIA) method of Berk and colleagues yielded a coefficient of correlation of 0.96 (p less than 0.00001) with means of 626 and 667 ng/ml for [3H]RIA and [125I]RIA, respectively, and a regression equation of y = 28 + 1.02x. The major advantages are that total assay time is reduced to approximately 1 h; [125I]cyclosporine label is used, avoiding the problems associated with liquid scintillation counting; and precision is enhanced by separating bound and free fractions with second antibody/PEG. These modifications should provide for greater ease of assay performance and improved clinical utility of cyclosporine monitoring not only in the pediatric but also in the adult transplant patient.

Two monoclonal antibodies to two different epitopes of human growth hormone form a precipitin line when counterdiffused as soluble immune complexes.

We have tested whether soluble immune complexes obtained by mixing human growth hormone (hGH) with one anti-hGH monoclonal antibody (MAb) can form a precipitin line when diffused against another MAb in a polyethylene glycol containing gel. By testing seven anti-hGH MAbs one against the other in this assay, we have found that 10 pairs of MAbs out of the 21 possible combinations formed a line. Apparently, the first MAb formed soluble hGH dimers that were linked by the second MAb into precipitating linear complexes. Since each precipitin line was formed by the cooperative reaction of two MAbs, this sequential reaction of MAbs may be used in methods for the positive selection of MAbs that are suitable for two-site immunoassays.

One antigen may form two precipitin lines and two spurs when tested with two monoclonal antibodies by gel diffusion assays.

A monoclonal antibody reactive with human immunoglobulin (Ig)G4 and a monoclonal antibody reactive with IgG1, IgG2 and IgG4 were tested with an IgG4 myeloma protein by double diffusion in a polyethylene glycol-containing gel. In a three-well Ouchterlony pattern, the IgG4 myeloma protein formed lines of double partial identity (double spur) with the two monoclonal antibodies. The two spurs lengthened and thickened with decreasing concentrations of polyethylene glycol indicating that soluble immune complexes diffused past the precipitin lines and formed the spurs. In a two-well pattern, the myeloma protein formed two lines with mixtures of the two monoclonal antibodies indicating that the immune complexes formed by the two antibodies distributed bimodally in the gel as if two types of complexes were formed. These unpredicted findings indicate that the process of antigen-antibody precipitation in gels needs to be analyzed further by using monoclonal antibodies.

Pinworm prevalence in California elementary school children, and diagnostic methods.

The pinworm prevalence among 302 children tested in 1980 in five Southern California elementary schools was determined to be 11.6%. The range was 7.3-15.4%. In a 1982 study involving 158 children in six schools, the prevalence was 21.6% (range 11.1-38.9%). The results of these studies are compared with those of a similar prevalence study done in the same area and in some of the same schools during the 5-year period 1960-1964. The prevalence for the earlier period, involving 700 children in six schools, was 34.6%, and ranged from 29.2-43.0%. A new, flexible, plastic pinworm slide was used in the 1980 and the 1982 studies. In the 1982 study, this diagnostic method was evaluated and compared for efficiency and use against the standard cellulose tape/glass slide. The two are equally effective in picking up eggs, and in reading quality. The plastic slide is easier to use and does not break.

Immunoassays: a roadmap for the 1980s.

The use of immunoassay has greatly expanded the capability of the clinical laboratory. This paper describes the basic principles behind immunoassay as well as a number of specific applications of those principles. Radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, chemiluminescence, and nephelometry are some of the subjects discussed. The discussion of fundamental principles forms a basis for exploration of the future of immunoassay.