Antigens in pigeon breeders' disease

It has been shown that many pigeon materials contain one or more of four pigeon proteins—pigeon γ-globulin (PGG), pigeon serum albumin (PSA), pigeon β-globulin (PBG) and a protein cross-reacting immunologically with PGG termed XPGG. It is thought that the ability of seemingly unrelated sources of pigeon material, e.g. pigeon droppings, serum, egg yolk and white to react with sera from cases of pigeon breeders' disease is due to the presence of PGG, PSA, PBG or XPGG in these materials.

Only in pigeon droppings were important antigens other than PGG, PSA, PBG and XPGG found and it is possible that pigeon droppings are the only `complete' source of antigens concerned with the disease unless specified conditions arise.

     

Antibodies against purified pigeon IgA in pigeon breeders' disease.

It is demonstrated that previously described PDF1-A antigens from pigeon dropping extract contain pigeon IgA. The sera of patients with pigeon breeders' disease contain precipitating antibodies against pigeon IgA, in contrast to the sera of pigeon breeders suffering from unrelated forms of pulmonary dysfunction. The degree of complement consumption by PDF1A antigens, pigeon serum, and pigeon IgA turned out to be correlated. No correlation was found between precipitating anti-pigeon IgA antibodies and complement consumption by pigeon IgA in patients' sera.     

Precipitating and non-precipitating complement consuming IgG subclass antibodies in pigeon breeders' disease

The sera of patients with pigeon breeder's disease usually contain precipitating serum factors as well as human complement consuming factors as shown by incubation of the serum with pigeon dropping antigens. Although a single serum factor, possibly an IgG antibody, might account for both phenomena, affinity chromatography experiments revealed that the sera of patients with pigeon breeders' disease contain non-recipitating, human complement consuming, serum factors besides precipitating serum factors which are also capable of complement consumption. The non-precipitating serum factors most likely belong to the IgG3 immunoglobulin subclass, whereas the precipitating antibodies belong to the subclasses IgG1 and IgG2.     

Enzyme immunoassay for detection of antibody to Epstein-Barr virus-specific early antigen.

Antibody to Epstein-Barr virus-induced early antigen could be measured in sera using enzyme-linked antibody. The sensitivity of this assay was comparable to that of the indirect immunofluorescent technique. Measurement of early antigen was accomplished on a producer cell line which was specifically treated to block late gene expression. It is now possible to measure Epstein-Barr virus-induced early antigen and viral capsid antigen antibodies by using the same cell line.     

Enzyme immunoassay with high sensitivity and accuracy for specific antibody to neocarzinostatin

A quantitative enzyme immunoassay (EIA) for specific antibody to neocarzinostatin (NCS) is described which uses enzyme-labeled anti-rabbit IgG antibody, solid-phase NCS and standard purified specific antibody to NCS. The dose of the standard was determined by sandwich EIA for rabbit IgG. The lower detection limit was 3 ng of the specific antibody per tube. The accuracy of the assay was excellent and a comparative study with the sandwich EIA for rabbit IgG showed good correlation. The antiserum to NCS of the highest titer was found to contain 0.6 mg and 40 mg per ml of specific antibody to NCS and of normal IgG, respectively. The accuracy of the assay results and the purity of the standard was established by 2 recovery tests for anti-NCS antibody.     

Enzyme immunoassay for the measurement of histamine

This paper reports a competitive solid-phase enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone treatment and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of an anti-histamine monoclonal antibody which was used to coat the wells of a microtitration plate. After incubation and washing, peroxidase activity associated with the solid phase is measured. With this method the histamine concentration in blood or various tissues may be determined easily, safely and reproducibly. Histamine concentrations from 0.3 to 20 ng/ml may be measured with the procedure reported here.     

Enzyme immunoassay for the detection ofGiardia lamblia

An enzyme immunoassay (EIA) was developed for direct detection ofGiardia lamblia antigens in fecal specimens. The EIA was evaluated by testing specimens from 1,331 subjects in the USA and Egypt. For the 353 specimens from human subjects in the USA there was a 97% overall agreement between the results of the EIA and direct microscopic examination, yielding a sensitivity and specificity of 92% and 99% respectively. Due to adverse field conditions the EIA did not perform as well in the specimens collected and tested in Egypt. The sensitivity and specificity for 585 human specimens from Egypt were 74% and 97% respectively.     

Enzyme immunoassay for detection of antibody to toxins A and B of Clostridium difficile.

An enzyme immunoassay (enzyme-linked immunosorbent assay [ELISA]) to detect hamster antibody to toxins A and B of Clostridium difficile was developed in which toxin preparations are used to coat the solid phase. The specificity of the assay was supported by blocking tests with the toxin preparations and proteins from a nontoxigenic strain. Sera from immunized and control hamsters were tested by this technique, and results were compared with those from a cytotoxicity neutralization assay. Antibody to toxins A and B assayed by ELISA showed a close quantitative correlation with antibody titers obtained by cytotoxicity neutralization. The ELISA assays described appear to provide a sensitive, specific, and practical method to define the prevalence of antibody to C. difficile toxins. These assays could be readily applied to human sera to examine and study the immune response of patients with C. difficile-induced disease.     

Enzyme and DTT treatment of adherent RBCs for antibody identification by a solid-phase immunoassay system

Treatment of RBCs with protease enzymes or dithiothreitol (DTT) causes denaturation of several RBC antigens and is regularly used in antibody identification. In this study,we have standardized enzyme and DTT treatment of adherent RBCs in the magnetic-mixed passive hemagglutination assay (M-MPHA) for antibody identification. We have also tried drying these treated RBCs. The optimal enzyme and DTT treatment conditions for intact adherent RBCs were determined, in addition to the optimal condition for drying RBCs. All tested agents with the exception of chymotrypsin could be applied to RBC drying, but the optimal condition for drying was different from that for intact RBCs. Enzyme or DTT treatment of adherent RBCs and their application in M-MPHA provide a simple and convenient method for antibody identification. Furthermore, the technique of drying treated RBCs provides an even stronger antibody identification tool, since dried RBCs can be stored for a long period.     

Enzyme immunoassay for antibodies to thyroxine in human serum using synthesized antibody as a calibrator

We prepared human IgG labeled with rabbit antibodies to thyroxine, and used it for the assay of antibodies to thyroxine in human serum as a calibrator. The assay system consisted of thyroxine labeled with beta-D-galactosidase and a microcolumn containing goat antibodies to human IgG immobilized on Sepharose 4B. Each serum sample or standard serum containing human IgG labeled with anti-thyroxine antibodies was incubated with the enzyme-labeled thyroxine, and the reaction mixture was passed through the microcolumn. The column was washed to remove the unbound label, and enzyme activity of the bound label was assayed. The minimum detectable amount of the anti-thyroxine antibodies was 0.3 ng/assay tube. The analytical recoveries of human IgG labeled with the antibodies added to human serum were from 103% to 108%. We measured anti-thyroxine antibodies in human sera from 187 patients with autoimmune thyroid diseases. The antibodies were detected in 32 serum samples, and the values in positive samples varied from 0.31 to 2.31 micrograms/ml. On the other hand, in 58 sera from patients with non-thyroid diseases, the antibodies were detected in only two samples.     

Enzyme immunoassay for diagnosis of gonorrhea.

An enzyme immunoassay (EIA; Gonozyme [Abbott Laboratories]) for gonococcal antigen was assessed for the rapid diagnosis of gonorrhea. Patients attending two sexually transmitted disease clinics were tested by EIA and culture on Thayer-Martin medium. EIA was highly effective in detecting gonococcal infection among symptomatic men, with 70 of 75 (93.3%) culture-positive men having positive tests and no false-positive reactions. The performance of the test was not as good in detecting cervical gonorrhea; the best result obtained was a sensitivity of 87% (33 of 38) for EIA compared with culture. EIA false-positives occurred at a relatively low rate for women, with the test having a specificity of ca. 97%. The test clearly is capable of detecting gonococcal antigen in cervical and urethral specimens, but its role in routine diagnosis is not clear. Its performance seems equal to that of the Gram stain for men, but it seems to be less sensitive than culture for cervical gonorrhea--a drawback in high-risk populations. The low false-positive rate could be an important issue in screening low-prevalence populations.     

Enzyme immunoassay for detection of antibodies against herpes simplex virus with the use of different viral antigens

Enzyme-linked immunosorbent assays (ELISA) for detection of antibodies to herpes simplex virus type 1 (HSV-1) have been performed by using different immunosorbents prepared by passive adsorption of four HSV-1 antigen preparations to the wells of polystyrene microtitre plates in order to compare the the sensitivity, specificity and reproducibility of the tests. The following antigen preparations have been used: virus-infected native Vero cells and their lysates, membrane glycoproteins and virus nucleocapsid proteins. The optimal conditions have been established for each assay system: the concentrations of adsorbed antigen and the "threshold" values of the optical density. For each antigen tested except of the nucleocapsid (NC) proteins, comparable antibody titres were found in the sera of 6 patients with different forms of herpes infections and in 86 healthy subjects. In the sera of 6 herpetic patients the antibody titres were higher against NC antigen than against other antigen preparations.     

Enzyme immunoassay analysis of antibody specificities present in the circulating immune complexes of selected pathological sera

Using immobilized anti-C3 antibody and an enzyme immunoassay, sera from 26 patients (eight with systemic lupus erythematosus (SLE), four with Hashimoto's thyroiditis, eight haemophiliacs and six with post-hepatitis cirrhosis) containing high levels of circulating immune complexes (IC) were selected. The IC were precipitated with 2.5% polyethylene glycol, washed, treated with acid buffer, neutralized and tested using an enzyme immunoassay in parallel with the original sera for antibody activity against a panel of antigens: human myosin and thyroglobulin, mouse actin and tubulin, calf thymus DNA and trinitrophenyl coupled to bovine serum albumin (TNP/BSA). It was found that all the isolated IC may contain IgG, IgA and IgM antibodies reacting with actin tubulin and TNP/BSA and also, depending upon the disease, antibodies reacting with some of the other antigens of the panel. By comparison to the antibodies present in the original sera, higher titers of antibodies were found in the isolated IC while some antibody specificities not detected in a given serum were occasionally noted in the isolated IC. The antibodies present in the IC seem to possess characteristics similar to those of polyreactive human natural autoantibodies. It is concluded that natural autoantibodies participate actively in the formation of IC found in pathological sera.     

Enzyme immunoassay for detection of antibody-coated bacteria.

To quantitatively evaluate factors potentially affecting antibody coating of bacteria in urine, we developed an assay with enzyme-linked rather than fluorescein-conjugated immunoglobulin. Using the enzyme immunoassay (EIA) in an in vitro system in which concentrations of serotype O44 Escherichia coli and antibody titer to E. coli Orr O44 O antigen were known, we compared specimens run in parallel with a fluorescent antibody (FA) assay. At greater than or equal to 10(5) bacteria per ml, antibody titer to homologous O antigen correlated directly with absorbance in the EIA. Both tests had sensitivities exceeding 95% in specimens containing greater than or equal to 10(5) bacteria per ml, but the FA test detected 23 of 27 positive specimens with less than 10(5) bacteria per ml compared with 21 of 43 detected by EIA (P = 0.002). However, nonspecific fluorescence caused false positives in 8% of negative tests run by FA compared with 1% of simultaneous EIA tests (P = 0.05). pH alterations and pretreatment of bacteria with antibiotics did not affect either test. Heterologous E. coli strains showed no cross-reactivity with O44 antiserum, but all Staphylococcus aureus isolates tested caused false positives in both assays, and one Klebsiella strain repeatedly caused a false-positive FA assay. The EIA appears to be a simple, quantitative, and specific technique for detection of antibody-coated bacteria in this experimental system.     

Enzyme immunoassay of histamine in foods

A competitive enzyme immunoassay of histamine in foodstuffs has been developed using a monoclonal antibody against a histamine‐benzoquinone adduct. In this assay, histamine present in food samples was treated with 1,4‐benzoquinone to form histamine‐benzoquinone by a simple and quick reaction and a histamine‐benzoquinone‐horse‐radish peroxidase conjugate was used as the labelled hapten. The apparent association constant (K_a) of the antibody used was 3.6 ×10⁶ l/mol and Gibbs’ energy of the immune complex formation has been estimated to find the optimal incubation time of the assay. The method enabled determination of histamine in fish, cheese, wine and beer at a concentration as low as 7 ng/ml with an accuracy of ± 15%. The recovery of the immunoassay was 88.9–114%. Cross‐reactivities of histidine, tyramine, tryptamine and its derivatives were lower than 0.001% and did not affect the assay.     

Enzyme immunoassay for the detection of streptomycin and dihydrostreptomycin in milk

Polyclonal antisera against streptomycin were prepared by using a streptomycin‐oxime derivative coupled to bovine serum albumin for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested in a competitive assay using a streptomycinenzyme conjugate (prepared by coupling a streptomycin‐hydrazone derivative to horseradish peroxidase) in a double antibody solid phase technique. The only detectable cross‐reactivity of the assay system with other aminoglycoside antibiotics and other substances similar in structure was shown to be with dihydrostreptomycin of about 148.7%. The detection limit in buffer solution was 0.6 ng ml^‐1 for streptomycin and 0–4 ng ml^‐1 for dihydrostreptomycin. Employing rapid sample preparation procedures, streptomycin and dihydrostreptomycin were detected in milk at levels as low as 6 and 0.8 ng ml^‐1respectively.     

Enzyme immunoassay for the detection of group A streptococcal antigen.

A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 10(4) or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 10(3) CFU per test. Heterologous microorganisms tested at 10(6) CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen.     

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.