Indirect Hemagglutination Test for Detection of Antibodies to Cytomegalovirus

An indirect hemagglutination test has been adapted for use with cytomegalovirus. The test is highly sensitive and reproducible. Both immunoglobulin M and immunoglobulin G antibodies can be detected by this method. The hemagglutination reaction can be inhibited by small amounts of homologous antigen. This principle permits early identification of virus isolated from diagnostic specimens.     

Micro Indirect Hemagglutination Test for Cytomegalovirus

In an effort to obtain the flexibility and ease of performance of a rapid, serological test for detection of cytomegalovirus antibody, the indirect hemagglutination (IHA) technique was investigated by using a microserological system. Antigens were prepared from tissue cultures of infected human fibroblasts. The specificity of the cytomegalovirus antibody response detected by the IHA test correlated well with the standard neutralization test. The IHA method was more sensitive than the complement fixation test in detecting antibody in congenitally infected newborns. There appeared to be some heterologous antibody response with Herpesvirus hominis or varicella virus infections. The IHA test pattern was found to be very stable with excellent persistence of agglutination.     

Microtiter Indirect Hemagglutination Procedure for Identification of Streptococcal M-Protein Antibodies

A number of investigators have attempted to utilize the hemagglutination system for detection of streptococcal type-specific antibody in human sera. Cross-reactions have made the procedure unreliable without cumbersome and time-consuming manipulation of the test sera. A method is described in which a microtiter indirect hemagglutination technique, using sensitized sheep erythrocytes, is sensitive, specific, and reliable for titration of type-specific antibody after naturally acquired or induced streptococcal infection.     

Typing Herpesvirus hominis Antibodies and Isolates by Inhibition of the Indirect Hemagglutination Reaction

Inhibition of the indirect hemagglutination reaction (IHA inhibition) was compared to several other methods for type-specific identification of Herpesvirus hominis (HVH) antibodies and isolates. The method appears to have the greatest value for typing antibodies for HVH type 1 and HVH type 2 in human sera; identification of antibody type was relatively simple and results were definitive. The IHA-inhibition test permitted serological diagnosis of HVH type 2 infection in three young adults with meningoencephalitis, thus extending the mounting evidence that nervous system involvement with this virus type is not limited to neonatal infections. II/I indexes of neutralizing or IHA antibody gave an accurate indication of the presence of HVH type 2 antibody in those sera containing type 2 antibody by IHA inhibition, but they indicated the presence of HVH type 2 antibody in one-half or more of the sera shown to contain only HVH type 1 antibody by IHA inhibition. For typing HVH isolates, the IHA-inhibition test gave results identical to those obtained by direct fluorescent-antibody staining using cross-absorbed conjugates, but the IHA-inhibition test was much more cumbersome and time-consuming to perform than was direct fluorescent-antibody staining. A microneutralization technique for virus typing also gave results identical to those obtained with direct fluorescent-antibody staining and IHA inhibition. However, typing HVH isolates by plaque size or the differential effect of incubation temperature was found to be less definitive and accurate.     

Use of Stable, Sensitized Cells in Indirect Micro Hemagglutination Test for Amebiasis

Human "O" cells were fixed with pyruvic aldehyde, treated with tannic acid, and fixed with glutaraldehyde. The cells were sensitized with amoeba antigen and stored in a refrigerator. The sensitized cells were used periodically for the indirect hemagglutination test with a battery of sera from patients with intestinal amebiasis and confirmed and unconfirmed amebic liver abscess, and also from negative controls. The same battery was tested with cells sensitized with different batches of antigen and also with fresh sheep cells. None of the cells showed any reaction with negative control sera. The fixed cells remained sensitive and stable throughout the study. Reproducibility of the titers with the fixed cells within each day and from day to day was satisfactory. The titers with fixed human "O" cells were slightly lower than were the titers with fresh sheep cells. The advantages of using stable, sensitized cells are pointed out.     

Stable Mycoplasma Antigen Preparations for Indirect Hemagglutination Tests

In immunological studies of mycoplasmas, the use of glutaraldehyde for the fixative makes it possible to use erythrocytes from commercially available defibrinated sheep blood. It eliminates the necessity of having to screen blood from individual sheep to obtain a suitable source of erythrocytes, as when employing tannic acid for fixation and sensitization. The chemical bonding of soluble mycoplasma proteins to glutaraladehyde-fixed sheep erythrocytes by bis-diazotized 3,3'dimethoxy derivative, benzidine, yields preparations that are satisfactory antigens for performing the indirect hemagglutination test by the microtiter technique. The antigenic preparations are satisfactory for use after storage at 4 or -10 C for many months. Incorporation of 5% glycerine in the final suspending milieu makes it possible to obtain uniform suspensions of the fixed and sensitized sheep erythrocytes after freezing and after repeated freezing and thawing. Proteins from Mycoplasma arthritidis and M. hominis have been coupled to glutaraldehyde-fixed erythrocytes by diazotization. The last mentioned preparation detected the presence of antibodies in titers greater than 1:10 in 37% of 237 pregnant women whose ages ranged between 20 and 30 years. There was no correlation between the presence of specific antibodies in the blood and the isolation of M. hominis from the cervical canal.     

A Modification of the Indirect Immunofluorescence Test for Detection of Ehrlichia phagocytophila Antibodies

A modified technique for production of antigen and performance of the test is described. A suspension of infected neutrophils was directly applied to multiwell slides. Multichannel pipettes may be used for dilution and application of sera. The modification inreases the capacity both by production of the antigen and by performance of the test. This paper also gives a quantitative determination of the antibodies.     

Method for Typing Antisera to Herpesvirus hominis by Indirect Hemagglutination Inhibition

A test for typing antisera to Herpesvirus hominis that uses the method of indirect hemagglutination inhibition is described. The test, which is based upon the differential absorption of herpes antisera by preparations of type 1 and type 2 antigens, is rapidly and easily performed. The results permit some conclusions to be drawn regarding the antigenic relationships between the two virus types. Some of the practical limitations of the test are discussed.     

Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella-zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

Evaluation of a Hemagglutination Test for Human Leptospirosis

An indirect hemagglutination test for the diagnosis of leptospirosis is described; the test uses a soluble antigen from serotype patoc to sensitize sheep erythrocytes which are then fixed with glutaraldehyde. Evaluation of this procedure indicates that it is more reliable than the conventional macroscopic agglutination test and, in contrast with both microscopic and macroscopic agglutination tests, is positive only with sera from persons with current leptospiral illness. The test is simple and convenient and sensitized fixed cells may be stored for at least a year. In comparison with the macroscopic and microscopic tests, only a single antigen is required.     

Serological Activity of Protein A of Staphylococcus aureus: the Precipitinogen as an Antigen for Determining Antibodies by the Passive Hemagglutination Test

Tanned sheep erythrocytes have been considered incapable of sensitization with the precipitinogen of protein A of Staphylococcus aureus, so that the activity of this antigen in serological reactions has so far been studied by means of the agar diffusion test (ADT) only. The precipitinogen of the protein A in this study was found to become attached to the tanned erythrocytes and to sensitize them for the passive hemagglutination test (PHT). It was determined that, in contrast to nonspecific reactivity between normal human serum and the precipitinogen in the ADT, the reaction in the PHT was of specific nature. Of seven species studied, all normal human, dog, and hog sera tested were positive in the PHT. However, the hemagglutinin titers of the sera of the two animal species by far exceeded those of the human sera. The data emphasized the usefulness of the highly sensitive PHT for assaying antibodies to protein A.     

Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies

Background

Ocular infection with Chlamydia trachomatis can cause trachoma, which is the leading cause of blindness due to infection worldwide. Despite the large-scale implementation of trachoma control programmes in the majority of countries where trachoma is endemic, there remains a need for a vaccine. Since C. trachomatis infects the conjunctival epithelium and stimulates an immune response in the associated lymphoid tissue, vaccine regimens that enhance local antibody responses could be advantageous. In experimental infections of non-human primates (NHPs), antibody specificity to C. trachomatis antigens was found to change over the course of ocular infection. The appearance of major outer membrane protein (MOMP) specific antibodies correlated with a reduction in ocular chlamydial burden, while subsequent generation of antibodies specific for PmpD and Pgp3 correlated with C. trachomatis eradication.

Methods

We used a range of heterologous prime-boost vaccinations with DNA, Adenovirus, modified vaccinia Ankara (MVA) and protein vaccines based on the major outer membrane protein (MOMP) as an antigen, and investigated the effect of vaccine route, antigen and regimen on the induction of anti-chlamydial antibodies detectable in the ocular lavage fluid of mice.

Results

Three intramuscular vaccinations with recombinant protein adjuvanted with MF59 induced significantly greater levels of anti-MOMP ocular antibodies than the other regimens tested. Intranasal delivery of vaccines induced less IgG antibody in the eye than intramuscular delivery. The inclusion of the antigens PmpD and Pgp3, singly or in combination, induced ocular antigen-specific IgG antibodies, although the anti-PmpD antibody response was consistently lower and attenuated by combination with other antigens.

Conclusions

If translatable to NHPs and/or humans, this investigation of the murine C. trachomatis specific ocular antibody response following vaccination provides a potential mouse model for the rapid and high throughput evaluation of future trachoma vaccines.     

Evaluation of Two ELISA and Two Indirect Hemagglutination Tests for Serodiagnosis of Pulmonary Hydatid Disease

To establish a definite diagnosis for pulmonary hydatid disease, combination of radiology and serology is useful. In this study, 19 preoperative sera from patients with surgically confirmed pulmonary hydatidosis, 40 sera from patients with other parasitosis and pulmonary diseases, and 20 sera from healthy donors were evaluated using 4 different serological tests, i.e., the commercial ELISA (ELISA-kit) test, the ELISA (ELISA-lab) test prepared in our laboratory, the commercial indirect hemagglutination assay kit (IHA-kit) test, and the IHA test using sensitized sheep red blood cells with tannic acid (IHA-TA). The ELISA-kit was the most sensitive (84.2%) and the most specific test (100.0%). The ELISA-kit also demonstrated the highest positive (100.0%) and negative (95.2%) predictive values. The sensitivity of the ELISA-lab test, that we prepared, was found to be 73.6%, whereas the IHA-kit test and the IHA-TA test were found to be 73.6% and 68.4%, respectively. The specificity of these tests was 96.6%, 98.3%, and 83.3%, respectively. When all 4 tests were assessed together, it was found that the sensitivity had risen to 94.7%. When the ELISA-kit was assessed with the IHA-kit and IHA-TA together, it was found that the sensitivity was 89.5% and 84.2%, respectively. Likewise, the combination of the ELISA-lab and IHA-kit or IHA-TA allowed us to achieve a sensitivity of 84.2% in cases of pulmonary echinococcosis. In conclusion, the diagnosis would be imminent if least 2 tests were applied together.     

Passive Haemagglutination Test for Anti-rhinovirus Antibodies

The use of chromic chloride as a coupling reagent has made it possible to coat red cells with rhinovirus protein. This is shown by immunofluorescence, electron microscopy and immunocolloidal experiments.     

Improved Hemagglutination Test for Identifying Type A Strains of Pasteurella multocida

A simple, improved, indirect hemagglutination test is described for the recognition of Type A strains of Pasteurella multocida. It involves the treatment of mucoid cultures with testicular hyaluronidase. Hydrolysis of the capsular hyaluronic acid presumably releases the specific antigen for adsorption to erythrocytes.     

Detection of Chlamydial Antibodies in Animal Sera by Double Diffusion in Gel

Postinoculation sera collected from pigeons, turkeys, guinea pigs, sheep, a calf, a rabbit, and a horse experimentally infected with various strains of Chlamydia psittaci yielded a high incidence of positive reactions when tested by double diffusion in gel. Antigen was a deoxycholate extract of SA-2 strain of C. trachomatis. Good correlation was obtained with results of complement fixation tests, whereas double diffusion in gel was less sensitive. Immunoelectrophoresis of the antigen revealed presence of two antigens in the extract.     

Serological Diagnosis of Human Melioidosis with Indirect Hemagglutination and Complement Fixation Tests

An indirect hemagglutination (IHA) test and a complement fixation (CF) test were evaluated from test results on sera from 212 human melioidosis patients of which 119 were culturally proved cases. Significant antibody titers (IHA titers of 1:40 or greater and CF titers of 1:4 or greater) were demonstrated with either test in all except five patients. IHA and CF titers ranged as high as 1:20,480 and 1:1,024, respectively. Antibodies were usually demonstrated by both tests 1 week after onset of disease. Transient seronegative reactions during the course of disease were seen in sera of approximately 19% of the patients with either IHA and CF but rarely with both tests. High titers in either test were obtained by the third week of disease and reached maximum levels in 4 to 5 months. Titers usually were detectable for 9 or more months. Antibodies were detected by IHA and CF tests in 80 to 100% of the sera obtained at various time intervals from 9 months to 2 or more years after disease onset. Antibody persistence occurred in patients who had a short disease course, as well as in patients with prolonged, complicated infections. The IHA test had excellent specificity when evaluated with normal human sera and diverse antimicrobial sera from hyperimmunized rabbits and human patients. The CF antigen appeared to contain common antigens with some but not all types of Pseudomonas aeruginosa. The specificity of the CF antigen could be enhanced without appreciable effect on its sensitivity by use of a titer of 1:8 in lieu of 1:4 as a criterion for a significant reaction. Either test could be used advantageously for the laboratory diagnosis of melioidosis.