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A reverse passive agglutination method was developed to detect soluble antigens of Legionella spp. By this method Legionella antigens were detected in urine specimens from 14 of 15 antigenuric patients with clinically diagnosed Legionnaires disease and in none of 263 urine samples from healthy subjects or patients with urinary tract infections. Intra-genus cross-reactivity was observed only between L. pneumophila serogroups 2, 3, and 6. The Legionella reverse passive agglutination method was also evaluated with reference to reagent concentrations, test conditions, and subjectivity of reading test results. The method is rapid and does not require special equipment.

A reversed passive hemagglutination test was developed to assay relative concentrations of soluble antigen of Legionnaires disease (Legionella pneumophila serogroup 1) in human urine samples. The test is highly sensitive, being able to detect as little as 0.0002 microgram of total antigen. Preliminary results with this test on serial urine and serum samples from a patient with legionellosis show that measurable amounts of antigen are present in urine during the course of the illness. However, no antigen could be detected in the serum of the patient.

A method of detecting gonococcal antigens by solid-phase radioimmunoassay with radioactively labelled antibody is described. A specificity test has been developed that enables this method to be used to detect gonococcal antigens in urine sediments. When sediments from samples of urine from male patients with gonorrhoea were tested, 31 (74%) of 42 gave positive results, clearly distinguishing them from sediments from urine samples from men with non-specific urethritis, none of which was positive. Ten of 14 urine sediments from urine samples from women with gonorrhoea gave positive results, as did 3 of 18 sediments from urine samples from women patients without gonorrhoea. These experiments demonstrate that gonococcal antigens can be detected in urine by radioimmunoassay; the method could be useful in diagnosis if, after refinement, its sensitivity and specificity were to be increased.

Forensic investigations often demand a clear definition of secretor status. Lewis-typing of secretion stains may help to verify non-secretor results and to identify mixtures of secretions from Le (a-b-) persons and secretors (or non-secretors). Furthermore it gives an additional check on secretor status, determined by ABO-grouping. Few problems may arise, when testing prepared saliva or semen stains. Therefore our interest was focussed on the possibility of Lewis-typing in stains appearing in forensic case work such as cigarette tips, stamps and envelope flaps, semen stains and vaginal swabs, nasal secretion, sweat and urine stains. All stains with the exception of sweat and urine were successfully Lewis-typed. In saliva stains Lewis substances could be determined even after 5 years and in semen stains for at least up to 40 days.

A survey of hepatitis B patients, asymptomatic hepatitis B antigen (HBsAg) carriers, and control subjects was conducted to determine the relationship between antigenemia and antigen excretion in saliva, urine, and stool. Radioimmunoassay was used to detect HBsAg. Specificity-confirmed HBsAg was detected in the saliva of 6 (30%) of 20 antigenemic patients, 1 (5%) of 20 nonantigenemic patients, 14 (34%) of 41 carriers, and 0 of 112 controls. HBsAg was detected in urine only after 100-fold concentration of first-morning specimens. Specificity-confirmed HBsAg was present in the urine of 7 (16%) of 43 carriers; unconfirmed HBsAg was found in the urine of 5 (13%) of 38 patients and 5 (5%) of 112 controls. Unconfirmed HBsAg was detected in concentrated stool specimens from 5 (46%) of 11 patients and 3 of 8 carriers and controls. Longitudinally collected specimens from antigenemic subjects showed no consistent patterns of antigen excretion.

A nuclear antigen was detected in the mouse liver nonhistone protein fraction by using antibodies to whole liver cells. The antigen was purified to homogeneity from perchloric acid extracts of liver tissue. It gave a single band corresponding to tool wt 21,000 in sodium dodecyl sulfate gel electrophoresis. Amino acid and carbohydrate analysis showed predominance of the acidic amino acids, lack of proline, and absence of carbohydrate. Immunofluorescence staining of liver sections confirmed the nuclear localization of the antigen. Its tissue distribution was studied by using radioimmunoassay. Of the various tissues extracted for analysis, the liver contained the highest amounts of the antigen, about 1 mug/mg of solubilized liver protein. Other tissues examined showed 2-4 percent of the amount of antigen present in the liver. Two transplantable hepatomas in C3H/HeJ and C57L/J mice, respectively, and three spontaneous C3H hepatomas showed greatly decreased levels of the antigen compared to normal liver. The amount of antigen in hepatomas varied from nondetectable to 2 percent of the amount of antigen found in the livers of the mice. The antigen was also found in the blood. The antigen was found in high concentrations (up to 13 mg/ml) in the urine of normal mice. This suggests identity with the previously known mouse urinary protein (MUP). In addition to the extremely high urinary output, the properties found to be shared by MUP and the nuclear antigen included similar serum concentrations (2-60 mug/ml), a sex difference with lower values in females, same molecular size as determined by gel filtration, and immunological identity. The nuclear localization of MUP and its disappearance from hepatomas suggest that it may have an important regulatory function.

We report the development and testing of an enzyme-linked immunosorbent assay with excellent sensitivity for the detection of Haemophilus influenzae type b (HI(b)) antigen in clinical specimens from patients with HI(b) meningitis. The assay, an indirect sandwich technique, uses polystyrene balls as a solid phase and an alkaline phosphatase-labeled goat anti-rabbit globulin conjugate. Specimens are incubated with polystyrene balls armed with burro anti-HI(b) antiserum, and recognition antibody is visualized by addition of alkaline phosphatase-labeled anti-globulin, together with the enzyme substrate p-nitrophenyl phosphate. Concentrations of antigen are determined from standard curves prepared by using purified HI(b) capsular antigen polyribophosphate. The assay reproducibly detects polyribophosphate at concentrations between 1 and 5 ng/ml. Cross-reactions have not as yet been encountered in simulated and authentic clinical specimens containing other species including Escherichia coli, Klebsiella pneumoniae, group B Streptococcus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, and Listeria monocytogenes. In preliminary tests with 11 spinal fluid specimens, 2 serum specimens, and 5 urine specimens from patients with culture-proved HI(b) meningitis, antigen was detected in all specimens in concentrations ranging from 1 to 7,000 ng/ml. Antigen was not detected in any of 62 clinical specimens which were culture negative for HI(b), including 11 spinal fluid specimens from patients with bacterial meningitis caused by microorganisms other than HI(b). The enzyme-linked immunosorbent assay technique described here is considerably simpler than radioimmunoassay and, based on concurrent tests with 14 positive clinical specimens, may be more sensitive than counterimmunoelectrophoresis. It seems, therefore, to hold considerable promise for clinical use in rapid detection of systemic HI(b) infections.

Salmonella typhi antigens D, Vi, and d were detected in the urine of 59 out of 61 (97%) bacteriologically confirmed typhoid fever patients by slide coagglutination with monovalent antisera coupled to protein A-rich staphylococci. These antigens were also detected in the urine of an additional 22 patients, 16 of whom subsequently demonstrated seroconversion by S. typhi O antibody agglutination, but from whom the bacterium was not isolated. The remaining 13 patients had negative urine coagglutination results, no isolation of S. typhi from blood or stool specimens, and no demonstration of seroconversion. These results suggest that the method of slide coagglutination of urine can be used to screen patients with suspected typhoid fever with a high degree of reliability. The method may also have potential importance in the diagnosis of typhoid when the bacterium is not isolated.

Several substances in urine were found to inhibit the radioimmunoassay of added gonococcal antigens. The supernatants of two-thirds of urine samples from male patients with either gonorrhoea or non-specific urethritis (NSU) were inhibitory. The inhibition caused by many, but not all, samples was reduced or completely abolished by the addition of soybean trypsin inhibitor (STI); STI-sensitive inhibition is thought to be due to proteolytic enzymes, probably from pus cells. Their inhibitory effect was shown to be due to their action on gonoccocal antigens and not on antibodies in the assay system. Some supernatants contained other inhibitors unaffected by STI; some of these were dialysable and others were not. Sediments from the urine of patients with NSU or gonorrhoea were often strongly inhibitory, but treatment with STI annulled all but very slight inhibition. STI-treated sediments could, therefore, be used in an assay designed to detect gonococcal antigens.

Urine specimens from 23 children and 9 adults who were undergoing treatment for malignancy as well as urines from 40 normal individuals were concentrated and examined for evidence of papovavirus infection. Papovavirus particles were detected in 6 of 64 urines examined by electron microscopy. Three of the particle-positive urines induced BK virus-specific immunofluorescence after inoculation of WI38 cells, and three isolations of BK virus were made by inoculation of urines from virus-excreting patients into Vero cells. BK virus-specific hemagglutination-inhibiting and immunofluorescence neutralizing antibodies were found in a majority of urines from adult patients, in about a fifth of pediatric patients, and less often in normal urines. Urines of virus-excreting patients generally had antibodies. In indirect fluorescent antibody tests, BK virus-specific antibodies of the immunoglobulin G class were found in five urine specimens from patients; immunoglobulin A antibodies were not detected in any urine. These data suggest that activation of BK virus is related to immunosuppression and not to transplantation itself and that the occurrence of virus-specific antibodies in urine may be indicative of virus multiplication in the urinary tract.

A solid-phase radioimmunoassay designed to detect serotype 6 Pseudomonas aeruginosa antigens was evaluated for its ability to rapidly diagnose urinary tract infections. Twelve P. aeruginosa serotypes were easily differentiated in the assay from eight other gram-negative bacterial species. During log-phase growth, the assay detected antigens in culture when approximately 10(6) or more serotype 6 P. aeruginosa organisms were present. Both cell-associated and solubilized antigens were detected. The assay detected antigens in 13 of 17 urine specimens which grew greater than 10(5) P. aeruginosa, 3 of 38 which grew other gram-negative rods, and none of 83 with no growth. Two of the three positive specimens from the other gram-negative rod group probably also contained P. aeruginosa. No preincubation of the urine specimens was required, and results were available within 2.5 h. The assay represents an improvement over other procedures for rapidly diagnosing urinary tract infections in that it allows diagnosis by species and should be adaptable to semiautomation.

An enzyme-linked immunosorbent assay for the detection of Haemophilus influenzae type b antigen was developed. It was able to detect purified polyribose phosphate at concentrations of greater than or equal to 1 ng/ml in cerebrospinal fluid. This was 50 times more sensitive than counterimmunoelectrophoresis with the same antiserum. The sensitivity for polyribose phosphate in urine was similar, but that in serum was about 10 times less. Nonspecific reactions were observed with blood-stained cerebrospinal fluid and some sera. These were differentiated from true positive reactions by a blocking test with unconjugated immune serum. A wide range of organisms was tested for cross-reactivity in the assay. With the exception of a protein A-rich strain of Staphylococcus aureus, they gave absorbances of < 8% of that of the homologous system. In a series of five cases of proven H. influenzae type b meningitis, the sensitivity of the assay with cerebrospinal fluid was confirmed to be at least 2(5) times greater than that of counterimmunoelectrophoresis. The results indicate that the enzyme-linked immunosorbent assay is highly sensitive and specific in detecting H. influenzae type b antigen. The necessity to perform the blocking assay on all sera limits its usefulness for the examination of these specimens. However, it should prove valuable for the detection of the antigen in cerebrospinal fluid and urine.

The direct immunofluorescence method for the detection of antibody-coated bacteria in urine sediments was used to test urine samples from obstetrical patients with the clinical diagnosis of acute pyelonephritis or cystitis. Antibody-coated bacteria were present in the urine from 12 of 15 patients with acute pyelonephritis, but they were not observed in the urine from 13 patients with cystitis. The clases of antibody coating the bacteria were IgG, IgA, and, in some cases, IgM. A correlation between a high titer of antibody in serum and the presence of antibody-coated bacteria in the urine was noted. These results confirm that the immunofluorescence test can be useful, as previously reported, in distinguishing infection of the kidney from infection of the bladder.

Using a sheep antiserum to human glomerular basement membrane (GBM), studies of urine from healthy adults showed the presence of two cross-reactive antigens. These antigens were purified partially by preparative electrophoresis and electrofocusing, and separated on G-200; both appeared to be acidic, of high molecular weight, and carbohydrate rich. Their immunologic relationship to human GBM solubilized by several techniques was deduced from lines of identity with the native GBM digests in double diffusion analyses. These antigens will combine with homologous anti-GBM antibodies and block their fixation to human kidney sections, and will evoke heterologous anti-GBM antibody production in the rabbit. Fractionation studies of normal human serum indicated the presence of trace amounts of basement membrane antigens in the circulation. Although the serum antigens appear immunologically identical to the urinary antigens, the precise anatomic structures from which both are derived is not certain. Demonstration of immunoreactive basement membrane antigens in the circulation provides a plausible source of immunogen for the potential development of anti-GBM antibody-mediated glomerulonephritis as well as a clue to a mode for reestablishment of tolerance in such an autoimmune disorder.