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Application of freeze-dried, one-day-old chick erythrocytes to viral hemagglutination and hemagglutination-inhibition tests.

Erythrocytes collected from 1-day-old chicks were stabilized by fixation with formaldehyde and by freeze-drying after treatment with carbon monoxide. Suspensions of freeze-dried erythrocytes in distilled water or physiological saline had a homogeneous bright reddish-purple color. Freeze-dried erythrocytes were compared with fresh erythrocytes for hemagglutination and hemagglutination-inhibition tests for various viruses including rubella, Japanese encephalitis, influenza, mumps, Newcastle disease, and Sendai viruses. After storage at 4 degrees C for 1 year or more, freeze-dried erythrocytes maintained their original appearance and sensitivity to hemagglutination antigens.

Animals

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.

Animals

Application of freeze-dried monkey erythrocytes to measles viral hemagglutination and hemagglutination-inhibition tests.

Erthrocytes collected from monkey species including grivet, rhesus, and cynomolgus monkeys were stabilized by fixation with glutaraldehyde of a low concentration and were freeze-dried in vacuo. These freeze-dried erythrocytes were compared with fresh erythrocytes for measles viral hemagglutination and hemagglutination inhibition and could be used in both tests instead of fresh erythrocytes. They maintained their initial appearance and sensitivity to measles viral hemagglutinins after storage at 4 degrees C for 4 months more.

Animals

Solid phase immunosorbent technique (SPIT) for the demonstration of specific IGM antibodies by hemagglutination inhibition and passive hemagglutination.

The adsorption of protein to glass and plastic has been well documented. It has also been demonstrated that the absorbed antigen or antibodies retain their activity in as much as they are able to react with a specific antibody respective specific antigen(1). Furthermore the adsorbed antignene or antibody complex may also react with a later added antigen. Anti IgM is adsorbed to the solid phase and will retain IgM from the patient's serum. If the patient's serum contains specific IgM this will react with the specific antignene. If the antigen is a hemagglutinin it will combine with the specific IgM and therefore added erythrocytes will not hemagglutinate. If the antigen is coated on erythrocytes the coated erythrocytes will combine with the specific IgM and hemagglutination will take place. The reaction is schematically demonstrated in Fig. 1.

Erythrocytes

Serology of Legionnaires disease: comparison of indirect fluorescent antibody, immune adherence hemagglutination, and indirect hemagglutination tests.

An immune adherence hemagglutination (IAHA) test for the measurement of antibodies to Legionella pneumophila was developed and evaluated for the diagnosis of Legionnaires disease. Its sensitivity was compared to that of the indirect fluorescent antibody (IFA) test and a recently developed indirect hemagglutination (IHA) test. The sensitivity of the three tests appeared to be similar, with the IFA test giving slightly higher titers. Both the IHA and IAHA tests appear useful for the serodiagnosis of Legionnaires disease; the IAHA test has the advantage that it can be used with many other serological antigens.

Antibodies, Bacterial

Improved rubella hemagglutination inhibtion test: inactivation of non-immunoglobulin hemagglutination inhibitors by phospholipase C.

A method using phospholipase C (PL-C) for removing nonspecific inhibitors (NSI) of rubella virus hemagglutinin is described. PL-C was found to hydrolyze NSI without altering the hemagglutination inhibition (HI) activity of the specific antibody and could be used to remove NSI in the rubella HI test by using formalinized erythrocytes, which resisted the enzymatic action; fresh erythrocytes were lysed by PL-C. The HI test using PL-C treated sera gave true measurements of actual rubella antibody content, and HI titers of PL-C treated sera were identical or equivalent (+/-1 dilution) to those of sera treated with dextran sulfate and CaCl2 (DS-C). Thus, the PL-C method gave results as reproducible and reliable as the DS-C method and was more convenient.

Antibodies, Viral

Hemagglutination and hemagglutination-inhibition studies with a strain of Nebraska calf diarrhea virus (bovine rotavirus).

A hemagglutinin has been prepared from Nebraska calf diarrhea virus (NCDV) propagated in BS-C-1 cell line. After cesium chloride centrifugation, the hemagglutinin of the bovine rotavirus was found to be associated with intact virions (density 1.355 g/ml) but not with virions lacking an outer capsid layer (density 1.375 g/ml). In hemagglutination-inhibition (HAI) tests, the hemagglutinin reacted specifically with NCDV serum, and HAI seroconversions were detected in some sera tested. Cross-reactions were observed in complement fixation tests between the human and bovine rotaviruses but were not demonstrated by HAI, suggesting that the hemagglutinin detects a specific rather than a group antibody response.

Animals

Indirect hemagglutination test for detection of antibody against bovine leukemia virus.

An indirect hemagglutination test was developed for detecting antibody against Bovine Leukemia Virus. Medium from Bovine Leukemia Virus infected monolayer fetal lamb kidney cell line cultures was concentrated by a combination of ammonium sulfate precipitation and freezedrying. Tanned sheep red blood cells were exposed to p24 and GP antigens, separately and together. The GP antigen did not attach to the sheep red blood cells, while the p24 antigen did. On testing 150 samples from suspect animals with the crude antigen-coated sheep red blood cells, the indirect hemagglutination test detected almost the same number (74) of positive animals as the agar gel immunodiffusion test with GP antigen (79), and by each of these test a far greater number of cases of leukosis were detected than by hematologic examination (58) or agar gel immunodiffusion test with p24 antigen (44). These results suggest that the indirect hemagglutination test may detect almost the same number of positive as the agar gel immunodiffusion test with GP antigen. Moreover, its results can be read two days earlier, and an impression of the antibody level in the sera is obtained. In routine or large-scale work the indirect hemagglutination test is too troublesome and time-consuming compared to the agar gel immunodiffusion test. The indirect hemagglutination test may be improved if it becomes possible to sensitize sheep red blood cells with GP antigen.

Animals

Hemagglutinating activity of Fusobacterium nucleatum.

Gingival isolates of oral Fusobacterium nucleatum strains (gram-negative anaerobic fusiform bacilli) have shown the characteristic ability to hemagglutinate a variety of erythrocytes (RBC) of human and animal origin. Other members of the genus tested (F. necrophorus, F. varium, and F. mortiferum) displayed little if any ability to hemagglutinate RBC. The hemagglutination (HA) activity could be observed in the F. nucleatum strains with the whole cells and in most instances with sonicated preparations of the organisms. The HA activity was observed in cell wall preparations of the organism and appeared dependent upon a heat-labile protein component of the cell wall. In decreasing order, the RBC that would hemagglutinate with the smallest concentration of HA preparations were rabbit, monkey, human, sheep, horse, and ox. No differences in HA activity of the preparations with cells from the various human blood types were noted. Absorption of the HA preparation of one strain with human cells removed HA moiety was bound to the cells via a Ca2+ binding site interaction since ethylenediaminetetraacetic acid and ethylene glycol-bis-N,N'-tetraacetic acid inhibited binding, and HA could be reestablished by the addition of Ca2+ but not Mg2+. Rabbit antisera to the F. nucleatum strains inhibited HA activity when tested with the HA preparation in the standard test, whereas anti-Leptotrichia buccalis sera or normal rabbit sera had no effect. A tanned-cell passive HA test with rabbit anti-F. nucleatum sera displayed reactivity between the homologous strains but little reactivity with the other Fusobacterium species tested.

Animals

[Hemagglutinating activity of enterotoxic strains of E. coli isolated from calves].

The manose-resistant hemagglutination activity of 272 E. coli strains isolated from calves sick or dead of colibacteriosis, 43 of which were enteropathogenic, was assessed. Strain enteropathogeneity was determined after the method of Smith and Halls (1967). It was established that 38 (88.3%) of the 43 enteropathogenic strains cause full or partial hemagglutination of horse or pig erythrocytes. Manose-resistant hemagglutination was not observed in the non-enteropathogenic E. coli strains. Manose-resistant hemagglutination of enterotoxic E. coli strains is in relation with their enteropathogeneity.

Animals

Rubella antibody assay by the immunoperoxidase technique: comparison with the hemagglutination inhibition test for determination of immune status.

The rubella immune status of 128 young women was determined with two assays for rubella antibody, the immunoperoxidase technique and the hemagglutination inhibition test. Comparison of the results of these techniques showed agreement in 125 cases (110 positive and 15 negative). Positive results were obtained for one woman only by the immunoperoxidase assay and for two women only by the hemagglutination inhibition test; all three of these women had a minimal titer of rubella antibody of 1:16. The sensitivity of the immunoperoxidase test was comparable to that of the hemagglutination inhibition test, and the specificity of the former test was verified. The peroxidase-labeled antibody technique is easier to perform than the hemagglutination inhibition test and is less time-consuming since sera do not require prior treatment and results can be obtained in as little as 90 min.

Antibodies, Viral

A passive hemagglutination test for diagnosis of trench fever due to Rochalimaea quintana.

A passive hemagglutination test devised for diagnosis of trench fever was easily performed and highly sensitive and specific. Tanned sheep erythrocytes were sensitized with soluble antigen from Rochalimaea quintana. The test detected antibody in six of seven cases of primary infection and in four cases of late, relapsed trench fever. Titers of antibody ranged from 1:20 to 1:640. Although both IgM and IgG antibody to R. quintana were detected by passive hemagglutination, IgG appeared to be the major reactive antibody. Antigens involved in the reaction were two types of proteins, one inactivated at 50 C and 60 C and the other at 80 C and 100 C. Of 322 control samples of sera that were tested, only one reacted positively; thus, the test had a specificity of greater than 99%. The single positive reaction was in serum from a patient with Q fever. This finding suggests that, in an area where Q fever is endemic, this disease must be ruled out in the interpretation of a positive passive hemagglutination test. Sera should be tested routinely against tanned, unsensitized erythrocytes, since an occassional sample of serum may agglutinate unsensitized cells. Because of its sensitivity and specificity, as well as its simplicity of performance, the passive hemagglutination test shows promise as a useful procedure for serologic identification of both acute and past infection with R. quintana.

Antibodies, Bacterial

Improved indirect hemagglutination test for cytomegalovirus using human O erythrocytes in lysine.

A modified indirect hemagglutination test for cytomegalovirus antibodies is described in which glutaraldehyde-fixed human O cells, rather than sheep cells, are used. Nonspecific hemagglutination was reduced by use of the optimal tannic acid concentration for each cell batch and the addition of 0.1 M lysine to the phosphate-buffered saline in which the fixed, tanned, sensitized cells were resuspended for use in the test. Three of 349 sera showed nonspecific hemagglutination by this technique. Antigen made by freezing phosphate-buffered saline (pH 7.2) over an infected monolayer can be used at dilutions of 1:8 to 1:15. Fixed, tanned, sensitized cells ready for use in the test can be stored in liquid nitrogen for up to 8 months. Use of cryoprotectants and washing after thawing is unnecessary. Simplification of the assay permits one person to screen 300 sera in 1 day or to determine the immune status of a potential donor or recipient in 45 min after the test is set up. The modified assay compares favorably in sensitivity with the complement fixation test and with previously described methods for performing the indirect hemagglutination test.

ABO Blood-Group System

Use of sodium polyanetholesulfonate-CaCl2 for removal of serum nonspecific inhibitors of rubella hemagglutination: comparison with other polyanion-divalent cation combinations.

By using trypsin-treated human type O cells as indicators, we compared the abilities of four polyanion-divalent cation combinations (heparin-MnCl(2); high-and low-molecular-weight dextran sulfate-CaCl(2); and sodium polyanetholesulfonate [SPS]-CaCl(2)) for removal of serum non-immunoglobulin (lipoprotein) inhibitors of rubella hemagglutination. The combination of SPS-CaCl(2) was found to be the most effective, precipitating completely the pre-beta and beta-lipoproteins and reducing the alpha-lipoprotein levels by more than 50%. Hemagglutination patterns after this treatment were clear and stable, and, when normal sera were tested, hemagglutination-inhibition (HI) titers were comparable to those obtained after standard heparin-MnCl(2) treatment. High-molecular-weight dextran sulfate-CaCl(2) removed serum lipoproteins almost as effectively as SPS-CaCl(2). However, problems of nonspecific agglutination and the heavy hemagglutination patterns resulting made this combination unacceptable for routine purposes. Neither low-molecular-weight dextran sulfate-CaCl(2) nor heparin-MnCl(2) removed the pre-beta lipoproteins completely, and occasionally traces of beta-lipoprotein also remained after treatment. The presence of pre-beta lipoproteins in normal sera after treatment may be of no consequence in the HI test since we have found that the very-low-density lipoprotein fractions obtained by ultracentrifugal methods from normal sera (those corresponding to the pre-beta fractions obtained by electrophoresis) had no HI activity. However, very-low-density lipoprotein fractions from all hyperlipemic sera tested had HI activity (titers ranging from 1:16 to 1:1,024) which, in the majority of cases, was not eliminated after heparin-MnCl(2) treatment. In every case, treatment with SPS-CaCl(2) removed this nonspecific activity completely. Since hyperlipemic sera may occasionally be encountered in routine rubella HI antibody testing, we recommend the use of SPS-CaCl(2) rather than heparin-MnCl(2) for pretreatment of sera.

Antibodies, Viral

Quantitation of antibodies to varicella-zoster virus by immune adherence hemagglutination.

Immune adherence hemagglutination was compared with the complement fixation test as a means of measuring antibodies to varicella-zoster virus. Analysis of acute- and convalescent-phase sera from patients infected with varicella-zoster or with herpes simplex virus showed the immune adherence hemagglutination test to be more sensitive than the complement fixation test, and greater cross-reactivity between the two viruses appeared to be associated with the increased sensitivity. The two assay methods were used to measure antibodies to varicella-zoster virus in 265 sera obtained from patients of different ages as well as sera from 26 patients with leukemia. There were 35 cases where antibodies were detected by immune adherence hemagglutination but not by complement fixation, whereas in five cases the converse was found. Our findings support the contention that immune adherence hemagglutination is the method of choice for detecting antibodies to varicella-zoster virus.

Acute Disease