Loss of Rubella Antibody from Immune Globulin Treated with Kaolin

Sera and immune globulin (IG) preparations are customarily treated with kaolin before titration of their rubella hemagglutination-inhibiting (HI) antibody in order to rid them of nonspecific inhibitors of hemagglutination. The treatment was shown in this investigation to have no adverse effect on the antibody level of the sera but was found to remove considerable amounts of gamma-globulin from IG preparations. Evidence of this removal was obtained by serological tests, by spectrophotometric determination of protein concentration and by disc electrophoresis. In contrast to kaolin, heparin-manganese chloride (MnCl(2)) treatment of IG preparations had essentially no effect on the level of antibody globulin by all the criteria used. Heparin-MnCl(2)-treated IG lots were in these respects similar, if not identical, to their untreated counterparts. Since nonspecific inhibitors associated with the beta-lipoprotein fraction of serum are removed by the method employed to fractionate the IG samples, it seems unnecessary to treat the latter in any way for the HI test. No difficulty was encountered in this investigation with several untreated IG lots.     

Rubella—Technical Problems in the Performance of Hemagglutination-Inhibition (HI) Tests

The rubella hemagglutination inhibition (hi) test is the most sensitive serologic method available for diagnosis of infection and for determination of immunity status. At present, however, it is not a well-standardized procedure and several modifications are in use. Comparison of results obtained on the same specimens by different laboratories has shown wide variations. The sensitivity and reliability of the technique is decidedly influenced by each of the variables of the test system; these include erythrocytes, antigen, methods employed for removal of non-specific inhibitors and natural agglutinins from test sera, the pH of the diluents and the temperature of incubation. The presence or absence of hi antibody cannot be determined reliably by screening a single low dilution of serum. Commercially available reagents and kits for rubella hi tests vary widely in their reliability. The test should be performed only by experienced persons who understand the principles of the technique and who are aware of the pitfalls and of the importance of using adequate controls in each run.     

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.     

Assessment by immunoenzyme analysis of the immune status of donors with reference to the viruses of rubella, measles and herpes simplex

The results of testing the blood sera obtained from donors at a blood transfusion center in Moscow for the presence of antibodies to rubella, measles and herpes simplex viruses, carried out by means of the enzyme immunoassay with the use of the corresponding test systems, are presented. Antibodies to rubella, measles and herpes simplex viruses have been detected, respectively, in 81.5, 96.7 and 100% of blood sera. The proportion of sera with low, medium and high antibody titers has proved to be virtually the same with respect to antibodies to rubella and herpes simplex viruses, the sera with medium antibody titers constituting 59%. At the same time tests for measles antibodies have shown the prevalence of sera with low titers (49.2%) with the highest percentage of seronegative donors (18.5%, as compared with 3.3% in rubella and the absence of negative sera in herpes simplex).     

Single-serum diagnosis of recent rubella infection with the use of hemagglutination inhibition test and enzyme-linked immunosorbent assays

Single-serum diagnosis of recent rubella infection was attempted with the use of hemagglutination inhibition (HI) test and two commercially available enzyme-linked immunosorbent assays (ELISAs). The period during which IgM antibody was detectable by IgM capture ELISA was 4 to 30 days after the onset of rash. Rubella IgG ELISA values relative to HI titer were lower in the sera from the patients with recent infection than in the sera from the subjects with remote infection. IgM ELISA and the combined use of IgG ELISA and HI test are two useful methods of single-serum diagnosis of recent rubella infection.     

应用ELISA法检测风疹病毒IgG抗体

实验证明,将0.1％脱氧胆酸钠制备的风疹病毒粗制抗原,用于ELISA法检测风疹病毒IgG抗体,效果较满意,方法的特异性好,与常规血凝抑制试验(HI)的相关性也好,所测抗体的几何平均值为HI的4倍。用本法初步调查了北京市不同年龄人群的风疹感染率,证明随年龄增长风疹感染率迅速上升,18岁以上人群达94％。检测河北省沧州地区孕妇的风疹IgG阳性率为99％。用於风疹病人的血清学诊断,获得较好结果。     

Trypsinized Human Group O Erythrocytes as an Alternative Hemagglutinating Agent for Japanese Encephalitis Virus

Trypsinized human group O erythrocytes were found to be a suitable alternative to gander cells in hemagglutination (HA) and hemagglutination inhibition (HAI) tests for Japanese encephalitis (JE) virus. In the HAI test, no cross-reactions against JE virus were observed with immune sera containing antibody to taxonomically related or unrelated viruses, with mouse brain antigen, or with nonantibody serum inhibitors; specific antibody rise could be detected in an immunized rabbit. Gander and trypsinized human group O cells gave comparable titers in the HAI test, but the latter were preferable since (i) they required less challenging HA antigen, being more sensitive to agglutination by JE virus, and (ii) all human and some animal sera investigated were devoid of natural agglutinins for these cells, thereby eliminating or reducing the need for prior adsorption with packed cells.     

A trial application of the latex test for evaluation of antibody level in rabbit immune sera]

A latex test was elaborated which served for evaluation of quality of rabbit immune sera for antigen 0 of selected Gram-negative bacteria. Sensitivity and specificity of this test in comparison with passive hemagglutination and immunoenzymatic DOT-ELISA reactions was evaluated. These studies were performed on immune sera for antigen O of Salmonella groups B, C1, C2, D and E, Yersinia pseudotuberculosis and in antigen preparations for above listed microorganisms both in homologous and heterologous systems. It was found that sensitivity of the latex test is 9 to 160 times lower than that of passive hemagglutination and 7 to 307 lower than for DOT-ELISA. Sensitivity of the latex test and passive hemagglutination reaction was evaluated on the basis of results of cross reaction between studied antigens and unabsorbed rabbit sera, establishing so called sensitivity indexes, which were informing how many times heterologous titer is lower than homologous titer. So evaluated sensitivity of the latex test was close to sensitivity of the passive hemagglutination reaction. It was found that slide latex test is characterized by satisfactory sensitivity and good sensitiveness and may be used for evaluation of antibody level 0 antigens of Salmonella and Yersinia. The value of this test is characterized by high repeatability of results, as well as low work and time-consuming.     

Method for Extracting Viral Hemagglutination-Inhibiting Antibodies from the Nonspecific Inhibitors of Serum

Various methods are used to remove nonspecific inhibitors from sera before titering viral hemagglutination-inhibiting antibodies. These methods have several undesirable features; some are tedious and time-consuming, some remove antibody along with nonspecific inhibitors, and different techniques are usually required to remove the nonspecific inhibitors for different viruses. This communication describes a single method that uses diethylaminoethyl-Sephadex to extract the immunoglobulin G antibodies for several viruses from nonspecific inhibitors. The procedure is fast, simple to perform, and removed the nonspecific inhibitors for influenza, Western equine encephalitis, dengue-2, and rubella viruses.     

A survey of Clostridium perfringens enterotoxin antibody in human and animal sera in western Canada

Sera from human, cattle, sheep, swine, and horse populations in western Canada were tested for the presence of Clostridium perfringens enterotoxin antibody by the passive hemagglutination (PHA) test, supplemented by an immunodiffusion test and by counterimmunoelectrophoresis. A total of 224 human, 345 cattle, 165 sheep, 620 swine, and 768 horse serum samples were examined. Low-titer reactions in the PHA test were detected in human, cattle, horse, and swine sera, in that order, with no titers demonstrated in sheep. The titers in human sera ranged up to 1:128 and three of these samples were also positive in the other two serological tests. The significance of this antibody is not clear, but it is suggested that the low prevalence of the antibody may reflect a low prevalence of enterotoxigenic C. perfringens strains in western Canada. Such serological surveys may be applicable to epidemiological studies involving enterotoxigenic C perfringens.     

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.     

A rubella haemagglutination inhibition test not requiring removal of non-specific inhibitors. 1. Elaboration of the test

The rubella haemagglutination inhibition (HI) test can be simplified and the probelm of non-specific inhibitors circumvented by allowing the haemagglutinin to react with erythrocytes before addition of the serum specimen. The elaboration of the test procedure is described, and preliminary studies indicate that the new method may prove useful both in the diagnosis of rubella infection and for determination of immunity status.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.     

Production of an diagnostic erythrocyte agent from Pseudomonas aeruginosa exotoxin A

The sensitization of formolized sheep red blood cells with exotoxin A by means of chromium chloride or glutaraldehyde is more effective with respect to their sensitivity in the passive hemagglutination test than loading by means of amidol, tannin and rivanol. The use of chromium chloride decreases the consumption of exotoxin A 2, 8, 16 and 16 times in comparison with the use of amidol, tannin, rivanol or glutaraldehyde respectively. The high specificity of erythrocyte diagnosticum obtained from exotoxin A by means of chromium chloride is indicated in the study of hyperimmune sera to 22 different antigens of enteric bacteria and staphylococci in the passive hemagglutination test and to 10 different enterobacterial and staphylococcal antigens in the antibody neutralization test.     

Comparison of the Reliability and Sensitivity of Three Serological Procedures in Detecting Antibody to Yersinia pestis (Pasteurella pestis)

Three serological procedures, the agar-gel precipitin inhibition, the complement fixation, and the indirect hemagglutination tests, were used to detect and measure antibody to Yersinia pestis in the sera from 383 individuals. Although all three tests were useful in detecting plague antibody, the most reliable and sensitive test procedure was indirect hemagglutination.     

Serology of Rubella—Comparison of Fluorescent Antibody,Complement Fixation and Neutralization Tests for Diagnosis of Current Infections and Determination of Sero-immunity

Neutralization, complement fixation (CF) and indirect fluorescent antibody (FA) assays for rubella virus were compared for sensitivity in the serologic diagnosis of infection, for demonstrating antibody in the sera of infants with suspected rubella syndrome, and in the detection of antibody elicited by past infection (determination of immunity status). The combination of CF and FA tests was shown to be the most useful for serologic diagnosis of infection, largely eliminating the need for the slower and more cumbersome interference neutralization test.Neutralizing antibodies were found to appear rapidly in the course of infection, antibodies demonstrable by immunofluorescent staining appeared slightly later, and CF antibodies were rarely demonstrable in sera collected earlier than 14 days after onset of illness. Antibodies detected by all three techniques showed good correlation in infants with clinical evidence of rubella syndrome and corresponding maternal sera. The indirect FA technique compared favorably with the neutralization test for the detection of antibody elicited by past infection (determination of immunity status) and offered distinct advantages in ease of technical performance and more rapid results. In both current and past infections, FA titers tended to be higher than neutralizing antibody titers.     

Improved Dextran Sulfate-Calcium Chloride Method for the Removal of Nonspecific Inhibitors with Modifications for Nonspecific Agglutinin Removal in the Rubella Hemagglutination Inhibition Test

The dextran sulfate precipitation method for removal of nonspecific inhibitors from sera to be tested in the rubella hemagglutination-inhibition test was modified by shortening the dextran sulfate-serum mixture incubation period from 2 hr to 30 min. A comparative study using both treatment times showed no significant difference in rubella antibody titers. Reducing the treatment time even further also gave comparable results, indicating the select 30-min incubation period is not marginal for undertreatment. It was shown that 41.5% of the 159 serums tested had nonspecific agglutinins at levels from 1:8 to as high as 1:64. In each specimen, all demonstrable nonspecific agglutinin was removed by adsorption with 10%, 0- to 2-day-old chick red blood cells rather than the usually recommended 50% adult chicken erythrocytes.     

Comparison of Methods for Removal of Nonspecific Inhibitors of Arbovirus Hemagglutination

Human sera were treated with kaolin, acetone, and dextran sulfate to determine the best method for removing nonspecific hemagglutination inhibitors. Results indicated that on surveys for group A, group B, and some group C arbovirus HI antibodies, dextran sulfate treatment of sera could be used effectively. This method, however, has limited usefulness for detecting HI antibody for a number of arboviruses, particularly some members of the Bunyamwera supergroup since nonspecific inhibitors for these antigens were not completely removed. HI antibodies in sera drawn early after dengue and Venezuelan equine encephalitis infection were detected more readily after dextran sulfate treatment than after kaolin treatment. Kaolin, but not dextran sulfate, was shown to remove antibody from IgM fractions of sera.     

Studies on live rubella vaccine. VI. Seroepidemiological surveillance and immunization of adolescents in high school.

The rubella seroimmunity status of a total of 1,204 students aged 12 to 19 in a junior and a senior high school in Osaka district was surveyed. Among these, 487 students (40.5%) were found to be seronegative (less than 1 : 8) by the hemagglutination inhibition (HI) test. A total of 287 students were immunized with live rubella vaccine, Biken lot No.7233. This caused an increased titer in all except one of the 262 seronegative students, while among 25 students with an initial HI antibody titer of 1 : 8 it caused more than 4-fold increase in 20 and 2-fold increase in 5. The vaccine caused no clinical manifestations, such as fever, rash or arthralgia.     

Modified Hemagglutination-Inhibition Test for Rubella Employing Human Group O Erythrocytes

A hemagglutination-inhibition (HI) test for rubella is described which utilizes human group O, rather than 1-day-old chick, erythrocytes. The test was found to be as sensitive and reproducible for detection of rubella antibody as HI tests employing chick erythrocytes. Advantages to the use of human erythrocytes are (i) they are more available, (ii) it is unnecessary to absorb natural agglutinins from human test sera, and (iii) heparin-MnCl₂-treated sera do not agglutinate human erythrocytes, as is sometimes the case with chick erythrocytes. Factors influencing the reliability of the test are discussed.