Influenza A Neuraminidase Antibody Assay with Sensitized Erythrocytes

Erythrocytes sensitized with purified neuraminidase (Hong Kong) antigens were used for assay of influenza A neuraminidase antibodies. The neuraminidase indirect hemagglutination test was equal to the neuraminidase hemagglutination-inhibition (enhancement) test and appeared to be better than the neuraminidase inhibition test for detection of fourfold or greater antibody rises in paired sera from influenza patients or vaccinees. It was better than both tests for detection of neuraminidase antibody. The neuraminidase indirect hemagglutination test is simple to perform and has the advantage of direct antigen-antibody assay.     

Influence of albumin on rubella virus hemagglutination and the hemagglutination-inhibition test.

The HEPES-saline-albumin-gelatin (HSAG) diluent found optimal for agglutination of fowl erythrocytes by rubella virus antigen is also optimal for agglutination of trypsin-treated human group O cells. Albumins from different commercial sources, however, can have varying inhibitory effects on rubella hemagglutination titers. This can have a significant effect on the hemagglutination-inhibition test since antibody titers measured by this procedure are related to the amount of antigen used.     

Improved hemagglutination and hemagglutination-inhibition tests for Akabane virus using formalinized goose erythrocytes

When formalinized instead of fresh goose erythrocytes were used in the hemagglutination (HA) test system of the Akabane virus, the agglutinability of the erythrocytes increased and became less salt-dependent. The improved method based on these findings should facilitate the hemagglutination-inhibition (HI) test and may be useful for epidemiological studies of the Akabane virus.     

Rubella Hemagglutination Inhibition: Removal of Nonspecific Agglutination Due to Manganous Chloride

Nonspecific inhibitors of rubella hemagglutination can be removed by treatment of sera with heparin-manganous chloride for use in the hemagglutination-inhibition test. After removal of nonspecific inhibitors by this procedure, an excess of manganous chloride may remain. This may cause the cells to agglutinate, thus obscuring the reading at low serum dilutions. This disadvantage can be overcome by the addition of sodium carbonate, which forms an insoluble compound with manganous chloride and does not interfere with antibody determination. The procedure presents a further refinement of the hemagglutination inhibition test for rubella by increasing specificity and sensitivity; it permits detection of antibody levels as low as 1:4 in sera.     

Yellow Fever Vaccine. V. Antibody Response in Monkeys Inoculated with Graded Doses of the 17D Vaccine

A dosage equal to or greater than approximately 3.4 Dex (decimal exponent, log(10)) weanling mouse intracerebral 50% lethal dose (LD(50)) was sufficient to elicit a yellow fever antibody response, as determined by the plaque neutralization (PN) test, in better than 90% of vaccinated rhesus monkeys. Lower dosages were progressively less effective in terms of PN titers and the PN and hemagglutination-inhibition serological conversion rates observed. A dose of between 3.4 and 4.2 Dex weanling mouse intracerebral LD(50), or one-tenth to one times the dosage recommended for man, provided an optimal antibody response in monkeys. In rhesus monkeys, in contrast to the findings for man, pre-existing yellow fever antibody did not interfere with the antibody response to yellow fever vaccine. The PN test was felt to be a more sensitive and specific indicator of yellow fever antibody in rhesus monkeys after vaccination than the hemagglutination inhibition or complement fixation tests.     

Analysis of the Diluents Used for Rubella Virus Hemagglutination and Hemagglutination-Inhibition Tests

A new diluent (ADGP) for the rubella virus hemagglutination (HA) and hemagglutination-inhibition (HI) tests is described. It was found that the HA and HI titers and the erythrocyte agglutination pattern were improved in ADGP compared to previously described diluents. The influence of the components of ADGP and of various test conditions on optimal HA and HI results were examined.     

Detection of antibodies by microtitrator techniques

Summary and conclusions 1. An evaluation was made of methods and equipment used for microtitration.2. The simplicity, accuracy, reproducibility, time saving and economy of microtitersystems well substaniate their use over other outdated methods.3. Microtiter systems have been found to be useful in agglutination, complement fixation, conglutination and hemagglutination procedures in disease processes caused by bacteria, rickettsia, viruses, fungi, protozoans, metozoans and certain allergic manifestations.4. A method combining the use of microtitration and an Autoanalyzer has been presented. The microtitraters provided a rapid, accurate and economical method for the preparation of dilutions in plastic cups, which were then fed through either a complement fixation or a hemagglutination manifold for analysis. These were passed through a flow-cell colorimeter and the results, compared with standards, were recorded. This produced an exceedingly sensitive and accurate endpoint.5. The combined Microtiter-auto-analyzer method provided a simple and precise procedure for continuously titrating hemagglutins and complement fixing antibodies with a reproducibility of ±2.3%.Presented before the Section on Immunology (F4), IX International Congress for Microbiology, Moscow, U.S.S.R. July, 1966.     

Statistical Evaluation of Diluents and Automatic Diluting and Pipetting Machines in Influenza Serology

The use of three diluents (i.e., 0.01 m phosphate-buffered saline, PBS; PBS with 0.2% gelatin, PBS/GEL; and PBS with 0.4% bovine plasma albumin) and three methods (i.e., the standard tube macro-procedure, TUBE; the manual microtechnique, MANUAL; and the semiautomatic microtechnique, AUTO) were statistically compared for their reproducibility and sensitivity in determining hemagglutinin (HA) and hemagglutination-inhibition (HI) antibody titers. In the HA test, analyses of between-cell variances of the different methods showed the AUTO microtiter procedure to be more reproducible than the standard TUBE method. The MANUAL microtiter procedure was the least reproducible. In the HI test, the TUBE method was the most reproducible. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter procedure and PBS/GEL diluent appeared to be the method and diluent of choice. Evaluation of another instrument, the autopipetter, which standardizes the volume of diluent to be added in the microtechnique, suggests that the reproducibility of the AUTO microtiter procedure might be further increased.     

Effect of serum-antigen incubation times on the expression of non-specific inhibitors of rubella hemagglutination.

Under certain conditions, serum very low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) can inhibit rubella hemagglutination. The level of this non-specific hemagglutination-inhibition (HI) activity increases as the incubation period between serum and antigen is increased. Treatment of serum with heparin-MnCl2 does not precipitate HDL, and may not effect complete removal of all VLDL. This treatment method, therefore, should be considered a source of false-positive reactions, especially when extended serum-antigen incubation periods are used to enhance HI activity and to detect low levels of IgM.     

Serum i antigen: A new human blood-group glycoprotein

A glycoprotein has been found in normal serum which inhibits the hemagglutination caused by human anti-i cold agglutinins. This glycoprotein binds to a Sepharose anti-i affinity column at 4°C and can be quantitatively eluted at 37°C. The eluted glycoprotein is specific in that it has potent hemagglutination-inhibition activity against anti-i antibodies but does not inhibit the hemagglutination by anti-I, -A, -B, -H or by the lectins Con A, PHA or Vicia graminea. The anti-i affinity method should provide a simple procedure for large scale purification and subsequent characterization of the i-antigen.     

Use of Trypsin-Modified Human Erythrocytes in Rubella Hemagglutination-Inhibition Testing

A method of treating human erythrocytes with trypsin has been modified and found to be an efficient and practical indicator system for the rubella hemagglutination-inhibition test. Both the trypsin-treated human cells and the widely used, newborn chicken erythrocytes were used in comparative testing of 464 selected diagnostic rubella serums. Results with each cell system were essentially the same. The trypsin treatment procedure has been found to be relatively simple, and with our limited testing has not presented any problems with reproducibility. Other advantages include the ready availability of human cells, greater intralaboratory standardization of the test by using the same donors over a long period of time, and elimination of adsorption of test sera with red blood cells.     

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Hemagglutination and fluorescent antibody (FA) are compared for the direct detection of virus devoid of host cells. A determination was made of the minimal number of tissue plaque-forming units of Venezuelan equine encephalomyelitis virus that could be detected by the hemagglutination technique. Similar concentrations of the virus in bovine albumin borate saline, Brain Heart Infusion broth (Difco), and demineralized water were tested by the FA technique. Somewhat higher concentrations of the virus in bovine albumin borate saline were used in the hemagglutination-inhibition test. The quantitative hemagglutination procedure employed for these studies was carried out at 37 C for 75 min with variations in concentration of goose red cells. As a result of lowering the red cell concentration, smaller concentrations of virus were detected. The direct FA staining procedure applied to slide preparations containing known numbers of tissue culture plaque-forming units of virus was negative. Adsorbed viral antigen on agglutinated goose erythrocytes was visualized by direct and indirect FA techniques.     

Toxoplasmosis in Lower Mammals*

SYNOPSIS. Sera from 270 domestic and wild mammals were tested for antibodies against Toxoplasma gondii by the indirect hemagglutination method. The specificity of reactions was determined by the hemagglutination-inhibition test. Positive results at a titer of 1:64 were given by 10 of 138 cattle, 3 of 74 swine, 1 of 8 cats and none of 3 horses. Of the wild animals tested, only 1 of 9 woodchucks was positive; 17 raccoons, 14 opossums and 7 skunks were negative.     

Prevalence rate and age of acquisition of antibodies against JC virus and BK virus in human sera

A total of 480 serum samples from donors including 384 children up to 10 years of age were examined by the hemagglutination-inhibition (HI) test for the rates of prevalence and age of acquisition of HI antibodies against JC virus and BK virus. Among 136 serum samples from various age groups, there were five (4%) with no detectable antibodies against BK or JC virus, 75 (55%) with antibodies against both viruses, 41 (30.1%) with antibodies against only BK virus and 26 (19%) with antibodies against only JC virus. The prevalence of antibodies against JC and BK viruses was 70.5% and 80.8%, respectively, and the mean HI titers (4 x 2n,n greater than or equal to 1) were 4.90 and 4.30. About 50% of the children had acquired antibodies against BK virus by 3 years of age and against JC virus by 6 years of age. These results indicate that dual latent infections with both viruses are common, although independent infections with either virus are predominant in the human population.     

Arbovirus surveillance in Florida: wild vertebrate studies 1965-1974.

Wildlife species from 38 of Florida's 67 counties were surveyed over a 10 year period for the presence of antibody to the five major arboviruses circulating in the state. The routine screening of 7891 sera from wild birds and mammals via the hemagglutination-inhibition (H1) test with selected reactors subjected to serum neutralization testing has 1) provided information regarding geographic distribution and seasonality of circulation of these viruses 2) identified enzootic foci of infection and those species of wildlife most commonly infected and 3) documented the potential value of certain wild mammals as indicators of St. Louis Encephalitis and Venezuelan equine encephalomyelitis virus activity prior to the detection of human cases. Limited studies of Tamiami and Tensaw virus on sera from mammals collected for other purposed provided additional baseline information on the activity of these viruses in Florida mammals. Isolations of eastern equine encephalomyelitis virus were made from the heart of a loggerhead shrike (Lanius excubitor), Tensaw virus from the brain of a gray fox (Urocyon cinereoargenteus), and Keystone virus from the heart of a bluejay (Cyanocitta cristata).     

Possible Application of the IHI Test in the Species Identification of Hair

Immunological species differentiation of keratein from hair has previously been demonstrated by the precipitin ring test in tubes (Pillemer et al. 1939) and by the indirect hemagglutination test (Simonsen 1970). In the present study the possibility of species identification of s-carboxymethylkeratein (SGMK) from single hairs was examined. SCMK and rabbit anti-SCMK sera from man, horse, dog and ox were prepared according to methods described by Gillespie (1962) and Simonsen (1970, 1971). Suitable antisera were used for the indirect hemagglutination-inhibition (IHI) test (Stavitsky 1954). The antisera were absorbed with heterologous SCMK and the inhibition test performed using SCMK extracted from 5 cm stretches of hairs by reduction and alkylation in 1 ml fluid volumes. To each vial containing 0.5 ml of antiserum in a serial 2-fold dilution row of the respective antisera was added 0.05 ml of a homologous or a heterologous single-hair SCMK. After incubation at 37 °C for 30 min. SCMK-coated goat erythrocytes were added and the test read after incubation at 20 °C for 18 hrs.     

Screening and preliminary characterization of hemagglutinins in Vietnamese marine algae

Extracts from 44 species of Vietnamese marine algae, including 15 Chlorophyta, 18 Rhodophyta and 11 Phaeophyta species, were examined for hemagglutination activity with a variety of different animal and human erythrocytes that were untreated or treated with enzymes. Almost all extracts showed activity toward at least one type of erythrocytes, although those from three Chlorophyta and two Rhodophyta species showed no hemagglutination with any type of erythrocytes examined. Strong activity was detected in extracts from two Chlorophyta (Anadyomene plicata and Avrainvillea erecta) and four Rhodophyta species (Gracilaria eucheumatoides, Gracilaria salicornia, Kappaphycus alvarezii, and Kappaphycus striatum) with enzyme-treated rabbit and sheep erythrocytes. The hemagglutinins of seven Chlorophyta and eight Rhodophyta species were examined for sugar-binding specificity, pH- and temperature-stability, and divalent cation-independency of hemagglutination using ammonium sulfate-precipitates prepared from their extracts. In a hemagglutination-inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides, except the Codium arabicum and Gracilaria euchematoides hemagglutinins, whose activities were inhibited by both N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. On the other hand, all of the hemagglutinins activities were inhibited by some glycoproteins. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, and suggest the presence of lectins specific for high mannose N-glycans, complex N-glycans, or O-glycans. The activities of these algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations. These results indicate that Vietnamese marine algae are a good source of novel and useful lectins.     

Further Studies on the Anti-Immunoglobulin G Hemagglutination-Inhibition Test for Influenza

Incorporating species-specific anti-immunoglobulin G (IgG) serum into the hemagglutination-inhibition (HI) test for influenza markedly increased sensitivity without loss of specificity. The effectiveness of anti-IgG serum for augmenting antibody titers may be influenced by the variable potency of commercial anti-IgG preparations. Maximal enhancement of HI titers was achieved when anti-IgG serum and virus-antiserum mixtures were incubated at 23 C for 10 min. Precision and reproducibility of the test were within acceptable limits. Other conditions likely to affect the test were investigated.     

Isolation and identification of dengue viruses by combined use of C6/36 cells and the immune adherence hemagglutination test

Dengue viruses were isolated in a mosquito cell clone, C6/36, and their serotypes were identified by the immune adherence hemagglutination (IAHA) test. Even when viruses were not cytopathogenic, the IAHA test detected virus growth in the cells.     

Lectin-like activity of Lactobacillus acidophilus strain JCM 1026

The lectin-like activity of the Lactobacillus acidophilus strain JCM 1026 was studied by hemagglutination and hemagglutination-inhibition assays. L. acidophilus strain JCM 1026 was found to hemagglutinate human and animal erythrocytes. Neuraminidase-treatment of human-type O erythrocytes enhanced the activity. Treatment of the bacterial cells with proteinase K reduced hemagglutinating activity significantly. Although several mono- and disaccharides did not inhibit hemagglutination, several different glycoproteins did. These data indicate that a proteinaceous lectin-like component(s) recognizing carbohydrate-containing molecules is located on the cell surface of L. acidophilus.