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1.  Indirect Hemagglutinating Antibody Response to Herpesvirus hominis Types 1 and 2 in Immunized Laboratory Animals and in Natural Infections of Man 
Applied Microbiology  1974;28(3):392-399.
Indirect hemagglutinating (IHA) antibody responses to Herpesvirus hominis types 1 and 2 (HVH-1 and HVH-2) were compared to complement-fixing and neutralizing antibody responses in immunized laboratory animals (rabbits, guinea pigs, and hamsters) and in natural infections of man. With the immunized animals, type specificity was seen only in the IHA test and only with antisera produced in hamsters and in the rabbits immunized with HVH-2. In human nongenital infections (considered to be caused predominately by HVH-1), IHA and neutralizing antibodies developed at about the same rate and reached approximately the same levels for HVH-1 and HVH-2. IHA titers tended to be higher than neutralizing antibody titers for both virus types. In genital infections (considered to be caused predominately by HVH-2), there was a rapid IHA antibody response to HVH-2, and the early HVH-2 antibody demonstrable by IHA, but not by neutralization tests, was found to be immunoglobulin M in nature. In genital infections, IHA titers for HVH-2 were markedly higher than neutralization titers, but there was no pronounced difference in neutralizing the IHA antibody titers for HVH-1. Several patients with genital infections fialed to develop IHA antibody for HVH-1. The IHA test possessed no greater sensitivity than did complement fixation or neutralization tests for serodiagnosis of HVH infections. Despite the fact that a number of patients with genital infections produced IHA antibody only for HVH-2, the test was no more effective than the neutralization test in providing a type-specific serodiagnosis of infection, due largely to the fact that the rapid IHA antibody response to HVH-2 prevented demonstration of a further, significant antibody titer increase in a number of cases.
PMCID: PMC186731  PMID: 4371513
2.  Detection of Coronavirus 229E Antibody by Indirect Hemagglutination 
Applied Microbiology  1972;24(5):703-707.
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.
PMCID: PMC380648  PMID: 4674373
3.  Analysis of antibody assay methods and classes of viral antibodies in serodiagnosis of cytomegalovirus infection. 
Journal of Clinical Microbiology  1978;8(2):153-159.
Forty-nine serum pairs with antibody to cytomegalovirus (CMV) were evaluated for rises in antibody titer (greater than or equal to fourfold) by indirect hemagglutination (IHA) and complement fixation (CF), using a freeze-thaw antigen (FT) and a glycine extract antigen (GE). In this sample CF-FT detected more rises in antibody titer than did CF-GE. IHA detected the least number. The apparent reason for stationary antibody titers with CF-GE and IHA was the presence of high antibody titers in the first serum specimen. Separation of immunoglobulin classes of 20 serum pairs by sucrose gradient centrifugation indicated that these antibodies with IHA were of the immunoglobulin M (IgM) class and those with CF-GE were of the IgG class. By separation of immunoglobulin classes, rises in IgG CMV antibody titers were seen with IHA, rises not observed in the whole serum because of high IgM antibody titers in the first serum specimen. Absence of rises in antibody titers with CF-FT was due in part to too early sampling of the second serum specimen (less than 21 days) and in part to an apparent inability of some individuals to respond with antibody reactive with FT antigen. CF-GE and CF-FT antibodies of the IgM class were detected in some sera, usually in specimens collected more than 10 days after the onset of symptoms. Although reactive with CMV antigen, the specificity of these IgM antibodies in relation to rheumatoid factor requires clarification.
PMCID: PMC275176  PMID: 212446
4.  Reactivity of Envelope, Capsid, and Soluble Antigens of Herpesvirus hominis Types 1 and 2 in the Indirect Hemagglutination Test 
Infection and Immunity  1974;10(1):102-106.
Envelope, capsid, and soluble antigens of Herpesvirus hominis (HVH) types 1 and 2 were compared to crude antigens (disrupted HVH-infected cells) for potency, type-specificity, and diagnostic value in the indirect hemagglutination (IHA) test. The envelope appeared to be the predominant component reacting in the IHA test, but recurrent HVH infections increased the reactivity of human sera with capsid antigens. The soluble antigens reacted in the IHA test with HVH immune animal sera, but very few convalescent-phase human sera showed reactivity with soluble antigens. Overall, none of the subunit antigens showed greater type-specificity than did crude antigens in IHA tests with immune animal or convalescent-phase human sera. Recurrent infections with type 1 or type 2 viruses tended to broaden heterotypic reactivity of the patients' sera with both crude and subunit antigens, even in patients showing only a single type of antibody by IHA inhibition. Subunit antigens were no more sensitive than crude antigens in demonstrating significant IHA antibody titer rises for serodiagnosis of herpesvirus infections, and they generally had to be used at lower working dilutions than crude antigens.
PMCID: PMC414963  PMID: 4366916
5.  Serological Diagnosis of Human Melioidosis with Indirect Hemagglutination and Complement Fixation Tests 
Applied Microbiology  1970;20(5):825-833.
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.
PMCID: PMC377056  PMID: 5530276
6.  Serodiagnosis of Imported Schistosomiasis by a Combination of a Commercial Indirect Hemagglutination Test with Schistosoma mansoni Adult Worm Antigens and an Enzyme-Linked Immunosorbent Assay with S. mansoni Egg Antigens 
Journal of Clinical Microbiology  2002;40(9):3432-3437.
A commercial indirect hemagglutination (IHA) test using erythrocytes coated with Schistosoma mansoni adult worm antigens (WA) and an enzyme-linked immunosorbent assay (ELISA) with S. mansoni egg antigens (SEA) were assessed for their use in serodiagnosis of imported schistosomiasis (hereafter these tests are designated WA/IHA and SEA/ELISA, respectively). The sensitivity of the tests was evaluated with sera from 75 patients with proven S. mansoni infection, 25 with proven S. haematobium infection, and 10 with clinical Katayama fever. The specificity was assessed with sera from 283 patients with various parasitic, bacterial, viral, and fungal infections and sera containing autoimmune antibodies. Sensitivities of the WA/IHA with a cutoff titer of 1:160 (WA/IHA160) in detecting S. mansoni, S. haematobium, S. mansoni and S. haematobium combined, and clinical Katayama fever were 88.0, 80.0, 86.0, and 70.0%, respectively, with a specificity of 98.9%. The WA/IHA with a cutoff of 1:80 (WA/IHA80) showed sensitivities of 94.7, 92.0, 94.0, and 90.0%, respectively, with a specificity of 94.7%. The comparable values of SEA/ELISA were 93.3, 92.0, 93.0, and 50.0%, respectively, with a specificity of 98.2%. Combined use of ELISA and WA/IHA80 gave sensitivities of 100% for S. mansoni, S. haematobium, and S. mansoni and S. haematobium combined and 90% for Katayama fever. The specificity of this combination in detecting schistosomiasis was 92.9%. Combination of SEA/ELISA with WA/IHA160 gave sensitivities of 98.7, 96.0, 98.0, and 80% with a specificity of 97.2%. Our findings suggest that WA/IHA and SEA/ELISA are each sensitive and specific serological tests that are easy to use for the diagnosis of imported schistosomiasis. The combined use of these two tests enabled the serological diagnosis of schistosomiasis to be achieved with very high degrees of both sensitivity and specificity.
doi:10.1128/JCM.40.9.3432-3437.2002
PMCID: PMC130766  PMID: 12202589
7.  Laboratory diagnosis of Mycoplasma pneumoniae infection. 3. Detection of IgM antibodies to M. pneumoniae by a modified indirect haemagglutination test. 
Epidemiology and Infection  1989;103(3):613-623.
The indirect haemagglutination (IHA) test was compared with the complement-fixation (CF) test for the measurement of antibodies to Mycoplasma pneumoniae. A modification of the IHA was used to measure M. pneumoniae IgM antibodies. Sera were obtained from various groups of patients who were either culture or antigen positive for M. pneumoniae in nasopharyngeal aspirates or who had fourfold or greater increase in CF antibody or a titre greater than or equal to 320. The results of these comparisons showed that the modified IHA test was specific and more sensitive (89% as opposed to 64%) than the CF test. The modified IHA test for the detection of IgM antibody was highly effective in the recognition of recent or current infection with the mycoplasma. It was also of equal sensitivity to an indirect enzyme immunoassay for the detection of IgM antibodies to M. pneumoniae.
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PMCID: PMC2249550  PMID: 2514114
8.  Diagnostic value of indirect hemagglutination in the seroepidemiology of Shigella infections. 
Journal of Clinical Microbiology  1976;3(2):143-148.
To evaluate the usefulness of the indirect hemagglutination (IHA) test in the epidemiological investigation of shigellosis, single serum specimens were tested from 50 patients with Shigella dysenteriae 1 (Shiga bacillus) infections, 103 asymptomatic contacts of these cases, 267 adult and 100 student control, and serum specimens collected during two outbreaks caused by S. sonnei and one outbreak due to S. flexneri 6. In patients with S. dysenteriae 1, 74% demonstrated titers of greater than or equal to 1:40, with 50% showing titers of greater than or equal to 1:160, whereas in the controls 10.4% had titers of greater than or equal to 1:40 and only 0.3% had titers of greater than or equal to 1:160. IHA titers in serum specimens collected from patients with S. sonnei and S. flexneri 6 were too low to be considered diagnostic for individual patients, but were useful in analysis of group results. Groups of ill individuals yielded titers significantly higher than non-ill groups; however, titers from ill groups were usually less than 1:40. The IHA test for S. dysenteriae 1 antibodies serves as a valuable adjunct to the diagnosis of Shiga bacillus dysentery. In our laboratory, an IHA titer of 1:40 or 1:80 is a "borderline positive." Shiga bacillus dysentery is strongly indicated when IHA titers are greater than or equal to 1:60.
PMCID: PMC274250  PMID: 767361
9.  Guinea Pig Herpes-Like Virus Infection I. Antibody Response and Virus Persistence in Guinea Pigs After Experimental Infection 
Infection and Immunity  1973;7(3):426-431.
Guinea pigs, experimentally infected with guinea pig herpes-like virus produced antibodies which were detectable by indirect hemagglutination (IHA), complement-fixation (CF), and neutralization tests. The IHA test appeared to be a more sensitive method than the CF or neutralization test for determining antibody response in guinea pigs immediately after infection, but the IHA method was not suitable for the detection of antibody in animals receiving small doses of virus. Antibody titers obtained by CF tests were generally higher than those obtained by the neutralization test, and they followed the same time course when individual animals were studied serially. Intracardiac inoculation produced the best antibody response in guinea pigs when compared with other routes of infection. Guinea pigs infected by the intraperitoneal, intranasal, or oral route showed rising antibody titers but the levels were low. Infectious virus was isolated from and persisted in all inoculated animals in the presence of antibody regardless of the route of inoculation. Recovery of infectious virus required cultivation or cocultivation of tissue cells containing virus. The administration of antilymphocyte sera delayed the appearance of IHA antibody but had no effect on antibodies determined by the CF- and neutralization tests.
PMCID: PMC422695  PMID: 4351586
10.  Detection of cytomegalovirus antibody with latex agglutination. 
Transfusion-acquired cytomegalovirus (CMV) infections should be prevented in seronegative immunocompromised patients by providing blood products from donors who are also seronegative. Latex agglutination was investigated as a simple and rapid method for detecting antibody against CMV. Latex beads were coated with CMV antigen, incubated for 8 min at room temperature with 25 microliter of sera, and examined for agglutination. The sensitivity and specificity of latex agglutination was compared with that of indirect hemagglutination (IHA, Cetus Corp., Emeryville, Calif.) and enzyme immunoassay (EIA) with sera from 604 random blood donors or patients. Of 327 serum samples shown to be seronegative by EIA and IHA, 327 had a latex agglutination titer of less than 1:4 (specificity, 100%). Of 236 serum samples with detectable antibody by EIA and IHA, 228 had a latex agglutination titer of 1:4 or greater (sensitivity, 97%). Plasma collected with EDTA, heparin, or citrate was satisfactory for latex agglutination. Latex agglutination results correlated quantitatively with those of EIA, and the test also detected fourfold or greater rises in antibody with paired sera from six patients with posttransfusion CMV infections. Latex agglutination is a sensitive and specific assay that is rapid and simple to perform and should be effective in selecting seronegative blood donors to prevent posttransfusion CMV infections in seronegative recipients.
PMCID: PMC268323  PMID: 2991330
11.  Gonococcal serology. A comparison of three different tests. 
Three serological tests for the detection of gonococcal antibodies were compared: an enzyme-linked immunosorbent assay (ELISA), an indirect haemagglutination reaction (IHA), and a gonococcal complement-fixation test (GCFT). The ELISA was performed with gonococcal pili of a Rotterdam strain (1443) as antigen, the IHA with pilus antigen of an American strain (2686, Buchanan), and the GCFT with whole gonococci of a single strain (46695, Oliver) as antigen. The tests were performed on sera from the same groups of Dutch patients; samples of sera were taken at the first examination and generally 11-22 days later. The ELISA and the IHA were more sensitive than the GCFT. The specificity of the tests was equal in low-risk groups, but the GCFT was slightly more specific in high-risk groups. The ELISA and the IHA did not differ in sensitivity and specificity. The agreement between the ELISA and IHA for patients with uncomplicated gonorrhoea was low (chi = 0.44), but the agreement between the GCFT and the two pilus assays was less (chi = 0.26 and 0.20). The sensitivities were highest for sera from patients with oropharyngeal gonorrhoea or with gonococcal complications; again the ELISA and the IHA were more sensitive than the GCFT.
PMCID: PMC1046130  PMID: 6130818
12.  Evaluation of the Indirect Hemagglutination Assay for Diagnosis of Acute Leptospirosis in Hawaii 
Journal of Clinical Microbiology  2000;38(3):1081-1084.
Timely diagnosis of leptospirosis is important to ensure a favorable clinical outcome. The definitive serologic assay, the microscopic agglutination test (MAT), requires paired sera and is not useful for guiding early clinical management. The only screening test approved for use in the United States, the indirect hemagglutination assay (IHA), has not undergone extensive field evaluation. To assess the performance of the leptospirosis IHA in Hawaii, serum from patients evaluated for leptospirosis between 1992 and 1997 were tested with the IHA at the Hawaii State Laboratories Division and with the MAT at the Centers for Disease Control and Prevention. Leptospirosis was considered confirmed by a fourfold rise in MAT titer and/or a positive culture. A total of 92 (41%) of 226 specimens from 114 persons with confirmed leptospirosis were found positive by IHA. Only 18 (15%) of 119 specimens obtained within 14 days of onset were IHA positive, compared to 74 (69%) of 107 specimens collected more than 14 days after onset (P <0.001). Repeat testing ultimately resulted in 78 (68%) of the confirmed cases having at least one specimen found positive by IHA. Thirteen different presumptive infecting serogroups were identified among 251 specimens with an MAT titer of ≥200 and obtained from persons with confirmed or probable leptospirosis. Fifty (68%) of 73 specimens with Icterohaemorrhagiae as the presumptive infecting serogroup were found positive by IHA, compared to 44 (47%) of 93 specimens with Australis as the presumptive infecting serogroup (P, 0.01). The IHA test was positive for 3 (1%) of 236 specimens from 154 persons without leptospirosis. The sensitivity of the leptospirosis IHA in Hawaii was substantially below figures reported previously, particularly early in the course of illness, limiting its usefulness for diagnosing acute infection. Since the presumptive infecting serogroup affected IHA results and the prevalence of serovars varies with geography, the performance of the IHA should be assessed locally. More sensitive leptospirosis screening tests are needed in Hawaii.
PMCID: PMC86345  PMID: 10699001
13.  Evaluation of MUREX SUDS Toxo test. 
Journal of Clinical Microbiology  1987;25(11):2049-2053.
The SUDS Toxo test (MUREX Corp., Norcross, Ga.) was compared with the indirect hemagglutination test (IHA) and the indirect fluorescent-antibody test (IFA) by examining 404 serum specimens, including 64 (15.8%) specimens with IFA titers of greater than or equal to 1:2. When SUDS was compared with IHA, sensitivity (96.4%), specificity (97.9%), and negative predictive value (99.4%) indicated that there were similar reactivities between the two tests. When an IFA titer of greater than or equal to 1:16 was considered significant and IHA and SUDS were compared with IFA, IHA was slightly less sensitive but had a higher positive predictive value than did SUDS; however, there was no statistical difference between the tests. When SUDS was compared with IFA, in which a titer of greater than or equal to 1:16 was considered significant, the high negative predictive value (100%), excellent sensitivity (100%) and specificity (98.3%), and ease of performance made SUDS an attractive alternative to IHA for screening single serum specimens for toxoplasmosis.
PMCID: PMC269409  PMID: 3320079
14.  Evidence of Burkholderia pseudomallei-Specific Immunity in Patient Sera Persistently Nonreactive by the Indirect Hemagglutination Assay▿ 
The indirect hemagglutination assay (IHA) is the most frequently used serological test to confirm exposure to Burkholderia pseudomallei. Patients with culture-confirmed disease often have a nonreactive IHA at presentation and occasionally fail to seroconvert on serial testing. We investigated whether using antigens derived from the cultured isolates of persistently IHA-nonreactive patient sera improved the sensitivity of the IHA. In addition, we assessed the antigen-specific lymphocyte response in this group of patients to a panel of B. pseudomallei antigens, including those derived from their own cultured isolates. Eleven patients with culture-proven melioidosis were identified as having persistently IHA-nonreactive sera. A modified IHA using erythrocytes sensitized with patient isolate-derived antigen tested against convalescent-phase serum was performed. The majority (82%) of sera showed a negative (≤1:5) result, one was borderline (1:20), and one was positive at the cutoff value (1:40). IHA-nonreactive sera were also tested by enzyme immunoassay (EIA), with 73% (8/11) demonstrating IgG positivity. In addition, lymphocytes isolated from persistently IHA-nonreactive patient sera demonstrated significantly increased proliferation in response to B. pseudomallei antigens compared to controls. These studies demonstrate the presence of B. pseudomallei-specific antibody by EIA and B. pseudomallei-specific lymphocytes in patient sera categorized as persistently nonreactive according to the IHA. New immunoassays are required and should incorporate B. pseudomallei antigens that are immunoreactive for this subset of IHA-nonreactive patient sera.
doi:10.1128/CVI.00077-11
PMCID: PMC3147347  PMID: 21677111
15.  Indirect hemagglutination test for human antibody to typhus and spotted fever group rickettsiae. 
Journal of Clinical Microbiology  1975;2(5):430-437.
An indirect hemagglutination (IHA) test is described that uses glutaraldehyde-stabilized erythrocytes treated with a rickettsial erythrocyte-sensitizing substance obtained from Rickettsia typhi or Rickettsia rickettsii. The serological reagent was stable for at least 3 months at room temperature and 6 months at 4 C. It exhibited group specificity and no group cross-reactivity. At a minimum dilution of 1:40, acute and early convalescent epidemic and murine typhus antisera showed 86% positive reactors, whereas similar spotted fever antisera had 74% positive reactors. In comparison with the indirect fluorescent antibody test, the IHA procedure gave lower titers but showed comparable detection of seroconversion with most paired sera. The IHA test demonstrated significantly higher titers than the complement fixation test and was more sensitive than either the complement fixation or Weil-Felix test in identifying seroconversion. No agglutination was observed when sensitized erythrocytes were tested with rodent sera known to contain rickettsial antibodies.
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PMCID: PMC274203  PMID: 811685
16.  Typing Herpesvirus hominis Antibodies and Isolates by Inhibition of the Indirect Hemagglutination Reaction 
Applied Microbiology  1974;28(3):400-405.
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.
PMCID: PMC186732  PMID: 4371294
17.  Screening for toxoplasmosis in pregnancy. 
Journal of Clinical Pathology  1981;34(6):659-664.
The prevalence of antibody against Toxoplasma gondi in a population of 715 pregnant women has been evaluated by two methods: indirect haemagglutination antibody (IHA) and indirect fluorescent antibody (IFA) test and all positive sera were checked by the dye test. Five hundred of the study population were questioned on diet and on animal contact to elucidate a possible relation to the prevalence of antibody. Results are expressed in international units (IU) of antibody against T gondi. Of the 715 sera, 171 were positive by IHA and 173 by IFA. One hundred and sixty-seven sera were positive by both tests, ninety-eight (58%) correlating exactly, as to the concentration of antibody. The ten sera which were not positive by both tests all had detectable antibody at the minimum concentration only (12 IU). The dye test confirmed all sera positive by both tests with the exception of three. It also confirmed one of four sera positive by IHA antibody alone and two of six positive by IFA alone. All sera that proved dye test-negative had low antibody concentrations (12 IU) by IHA or IFA. The IHA test, which is commercially available in kit form, would be suitable for use as a screening test during pregnancy. The estimated annual rate of antibody acquisition over the age range 16-40 years is 1.2% per annum with the highest rate in the 36-40 age group (2.5% per annum) and the lowest in the 26-30 age group (0.4% per annum). The clinical history was not significantly different between those with and those without antibody against T gondi but significantly more women in the 36-40 age group had a history of animal contact than those in the 26-30 age group. No conclusive evidence of recent or current infection was found.
PMCID: PMC493645  PMID: 7019265
18.  Studies on the effect of growth medium composition on the antigenicity of Mycoplasma bovis. 
The Journal of Hygiene  1980;84(1):29-36.
Serum proteins adsorbed from the culture medium were detected in Mycoplasma bovis antigens, the number and type of proteins depending on the serum used in the medium. The alpha-globulins cross-reacted with alpha-globulins from different types of sera but the gamma-globulins did not. The removal of non-specific medium antibodies by absorption showed that they affected the gel diffusion and growth precipitation tests, producing cross-reactions between M. bovis and M. bovigenitalium, but that the complement fixation, tube agglutination, and growth inhibition tests were not similarly affected. The presence of serum proteins in the antigens changed their specific reactivity in all the tests. The production of antibodies to serum proteins was increased by the use of an adjuvant, but it appeared that production of specific antibodies to the mycoplasmas was not.
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PMCID: PMC2133824  PMID: 6766155
19.  Comparison of six methods for the detection of antibody to cytomegalovirus. 
Journal of Clinical Microbiology  1985;22(6):1014-1019.
Five commercial assays were compared to a standardized complement fixation (CF) test for the detection of antibody to cytomegalovirus. Two hundred and thirty serum specimens were analyzed. In addition, nine pairs of acute- and convalescent-phase sera were tested by two of the commercial assays. The assays were compared as to sensitivity, specificity, and positive and negative predictive value, as well as incidence of false-positive and -negative results. Samples which did not agree in all the assays were retested and tested with an indirect fluorescent-antibody assay. Of 228 specimens, 103 (45.2%) were positive by CF. Of the 230 samples, 2 (0.9%) were inconclusive by CF and readable in the other assays. Of the 230 specimens, 97 (42.2%) were positive by an enzyme immunoassay (EIA; Litton Bionetics), 100 (43.5%) were positive by a second EIA (Abbott Laboratories), 104 (45.2%) were positive by a third EIA (M. A. Bioproducts). One hundred and eight (47.0%) were positive by indirect hemagglutination (IHA; Cetus Corporation), and 110 (47.8%) were positive by latex agglutination (LA; Hynson, Westcott and Dunning). Sensitivity and specificity were similar with all the assays (93 to 100%). The greater numbers of positive results by IHA and LA were confirmed by repeat CF testing at less than 1:8 dilution, and by indirect fluorescent-antibody assay. Acute- and convalescent-phase serum pairs showed a significant rise in antibody titer when tested by anticomplement immunofluorescence, IHA, and LA. There was good agreement among the assays, with LA having the highest sensitivity.
PMCID: PMC271869  PMID: 2999186
20.  Micro-indirect hemagglutination test for detection of antibody against transmissible gastroenteritis virus of pigs. 
A micro-indirect hemagglutination (IHA) test was developed for detecting antibody against transmissible gastroenteritis (TGE) virus of pigs. TGE virus propagated in swine kidney cell cultures was highly purified and concentrated by the combination of ammonium sulfate precipitation, treatment with fluorocarbon, and sucrose density gradient centrifugation. Tanned sheep erythrocytes were sensitized with purified virus for use in the IHA test. The results of testing 104 serum samples collected from pigs in the field indicated that the IHA antibody titers were approximately five times higher than those obtained by a serum neutralization test and that there was good correlation between the antibody titers determined by the two tests. High IHA antibody titers developed in pigs experimentally exposed to virulent TGE virus. Sensitized sheep erythrocytes were stable under long-term storage at 4 degrees C (at least for 50 days). The conclusions made are that the IHA test described is more sensitive than the serum neutralization test for the detection of TGE antibody and may be of value for serodiagnosis of TGE.
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PMCID: PMC274712  PMID: 197119
21.  Micro Indirect Hemagglutination Test for Cytomegalovirus 
Applied Microbiology  1971;21(1):104-107.
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.
PMCID: PMC377126  PMID: 4322278
22.  Cytomegalovirus antigenic heterogeneity can cause false-negative results in indirect hemagglutination and complement fixation antibody assays. 
Journal of Clinical Microbiology  1985;22(5):768-771.
Cord sera and antepartum maternal sera from three congenitally cytomegalovirus (CMV)-infected infants and their mothers were CMV seronegative (titer, less than 8) in a complement fixation (CF) assay with a glycine-extracted CMV AD169 antigen; sera from two of the infants and mothers were also seronegative in a commercial indirect hemagglutination (IHA) assay with AD169 antigen. In tests with their own CMV isolates propagated and made into glycine-extracted CF antigen, all were seropositive. When 108 random cord sera were assayed for CF antibody with AD169, Davis, and A antigens (A is a locally derived antigen from one of the above infants), 44 were seropositive and 54 were seronegative for all three antigens. Of the remaining 10 sera, 4 were positive for A only, 3 were positive for A and Davis only, 2 were positive for Davis and AD169 only, and 1 was positive for AD169 only. All 10 were positive when a mixture of all three antigens was used. The IHA assay with AD169 antigen was positive with only 4 of these 10 sera. These results suggest that up to 6% of sera may be misclassified as seronegative in the CF and IHA assays if only a single antigen is used.
PMCID: PMC268523  PMID: 2997271
23.  Evaluation of the Indirect Hemagglutination Assay for Diagnosis of Acute Leptospirosis 
Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources and expertise to perform the microscopic agglutination test. There is a need for rapid and simple serological tests which facilitate the early diagnosis of leptospirosis, while antibiotic therapy may be most effective. A commercially available indirect hemagglutination assay (IHA; MRL Diagnostics, Cypress, Calif.) was evaluated with multiple serum specimens from 107 patients being investigated for leptospirosis. By using a combination of enzyme-linked immunosorbent assay (ELISA) methods for immunoglobulin M (IgM) and IgG antibodies and the microscopic agglutination test, 54 patients were found to have leptospirosis and 53 were found not to have leptospirosis. The sensitivity of IHA for the detection of acute leptospirosis was 100%, the specificity was 94%, the positive predictive value was 95%, and the negative predictive value was 100%. IHA was negative when 13 antinuclear antibody-positive sera, 24 serum specimens from patients with syphilis, and 16 serum specimens false positive by the Venereal Disease Research Laboratory test were tested. IHA was shown to detect both IgM and IgG classes of antibodies in human sera. Serum specimens from 27 dogs investigated for leptospirosis were studied: 3 samples gave nonspecific hemagglutination, but for all remaining samples, the results of IHA and an IgM ELISA were concordant. Performance of IHA was simple, and IHA requires no specialized equipment. It represents a useful assay for laboratories which require a leptospiral diagnostic capability but lack the expertise to perform specialist investigations.
PMCID: PMC124798  PMID: 9431911
24.  Cross-reactions between Actinobacillus (Haemophilus) pleuropneumoniae strains of serotypes 1 and 9. 
Journal of Clinical Microbiology  1990;28(3):535-539.
Actinobacillus pleuropneumoniae strains of serotypes 1 and 9 were studied for their serological properties by means of agglutination, coagglutination (CoA), indirect hemagglutination (IHA), Co-IHA, ring precipitation (RP), and immunodiffusion (ID) tests. Particulate and soluble antigens of unheated and heat-treated bacterial cells were used in various serological tests. Agglutination, CoA, and RP tests demonstrated common antigens between strains of serotypes 1 and 9. Quantitative estimation of serotype-specific antigenic activity by CoA, RP, and ID tests proved useful in differentiating strains of serotypes 1 and 9. IHA and Co-IHA tests using sheep erythrocytes sensitized with unheated or heat-treated whole-cell saline extract and the ID test using boiled whole-cell saline extract as antigen distinguished the strains of serotypes 1 and 9. In studies of absorption of rabbit antisera with heterologous whole-cell antigens there was no absorption of antibodies in tube agglutination and IHA tests, suggesting that serotype 1 and 9 strains belong to two distinct serogroups. It appears that the cross-reactivity between serotype 1 and 9 strains could be due to common epitopes associated with cell wall antigens.
PMCID: PMC269658  PMID: 1691210
25.  Mycoplasma agalactiae subsp. bovis in pneumonia and arthritis of the bovine. 
The pneumonic lungs of 42 cattle from 26 feedlots were examined for the presence of mycoplasma, pathogenic bacteria and viruses. Four animals representative of two lots failed to yield mycoplasma. One of these yielded the virus of infectious bovine rhinotracheitis and Pasteurella hemolytica, the other yielded only P. P. multocida. Nine animals in eight lots yielded Mycoplasma sp.: five of these were M. bovirhinis, two were M. arginini and two were untypable. All of these animals yielded one or more of P. hemolytica, P. multiocida, infectious bovine rhinotracheitis virus or bovine virus diarrhea virus. Twenty-five of 29 animals in 16 lots yieled M. agalactiae subsp. bovis from lung tissues. The same organism was recovered from the arthritic joints of 12 of these animals. Eight of the 25 animals yielded no other pathogen and all of these had not received any treatment. Nine of the 25 M. agalactiae subsp. bovis positive animals also yielded one or more of P. hemolytica, P. multocida, Corynebacterium pyogenes or infectious bovine rhinotracheitis virus. Bacteriological and virological studies were not completed for the remaining eight of the 25 positive animals. In five lots of cattle which had not received medication for pneumonia and for arthritis only M. agalactiae subsp. bovis was recovered. Twenty-five grossly normal lungs obtained from normal cattle at the time of slaughter were cultured and all were negative. The possible role of M. agalactiae subsp. bovis in pneumonia and arthritis was discussed.
PMCID: PMC1277697  PMID: 832194

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