Establishment and preliminary application of a rapid fluorescent focus inhibition test (RFFIT) for rabies virus

The World Health Organization (WHO) standard assay for determining levels of the rabies virus neutralization antibody (RVNA) is the rapid fluorescent focus inhibition test (RFFIT), which is used to evaluate the immunity effect after vaccination against rabies. For RFFIT, CVS-11 was used as the challenge virus, BSR cells as the adapted cells, and WHO rabies immunoglobulin (WHO STD) as the reference serum in this study. With reference to WHO and Pasteur RFFIT procedures, a micro-RFFIT procedure adapted to our laboratory was produced, and its specificity and reproducibility were tested. We tested levels of RVNA in human serum samples after immunization with different human rabies vaccines (domestic purified Vero cell rabies vaccine (PVRV) and imported purified chick embryo cell vaccine (PCECV)) using different regimens (Zagreb regimen and Essen regimen). We analyzed the levels of RVNA, and compared the immune efficacy of domestic PVRV and imported PCECV using different immunization regimens. The results showed that the immune efficacy of domestic PVRV using the Zagreb regimen was as good as that of the imported PCECV, but virus antibodies were generated more rapidly with the Zagreb regimen than with the Essen regimen. The RFFIT procedure established in our laboratory will enhance the comprehensive detection ability of institutions involved in rabies surveillance in China.     

A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus.

Levels of neutralizing antibody to lymphocytic choriomeningitis (LCM) virus in the sera of 66 infected persons were assayed by a rapid fluorescent focus-inhibition test (RFFIT). The test was more sensitive than the mouse-neutralization (MN) test and could be completed in less than 24 h. The RFFIT titers were compared with titers obtained by the indirect fluorescent-antibody (IFA) and complement-fixation (CF) tests. Neutralizing antibody detected by the RFFIT remained positive after IRA, CF and MN antibodies had disappeared. The RFFIT for detection of LCM antibody is specific and reproducible and seems especially useful for determining the incidence and epidemiology of LCM virus infections.     

High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R² = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.     

Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies

Neutralizing antibodies (nAbs) prevent the entry of viruses into permissive cells. Since nAbs represent correlates of protection against the Rabies lyssavirus, the presence of sufficient nAbs indicates effective vaccination. Accordingly, Rabies lyssavirus-specific nAb titers need to be determined in routine diagnostics to identify individuals being at risk of Rabies lyssavirus infections due to insufficient immunity. The current gold standard for the quantification of Rabies lyssavirus-specific nAbs is the rapid fluorescent focus inhibition test (RFFIT). However, RFFITs are expensive and labor-intensive since multiple microplate wells must be evaluated one-by-one by trained personnel through microscopic inspection, which limits the number of samples that can be processed. To overcome this disadvantage, we established a novel assay for Rabies lyssavirus-specific nAbs relying on an in-cell-ELISA (icELISA)-based neutralization test (icNT). The icNT differs from the RFFIT in the readout phase, and can be automatically quantified in minutes using broadly available microplate readers. During the establishment, icNT parameters such as antibody concentrations, permeabilization procedures, blocking reagents, infectious doses, and the duration of infection were optimized. Afterwards, a dose-dependent detection of Rabies lyssavirus neutralization was demonstrated using the WHO Standard Rabies Immunoglobulin reference. A panel of 200 sera with known RFFIT titers revealed very good sensitivity and specificity of the icNT. Furthermore, the icNT showed very good intra- and inter-assay precision. By recognizing Rabies lyssavirus-specific antigens, the assay can be applied immediately to automatically quantify the concentration of Rabies lyssavirus nAbs in routine diagnostics or for various basic research questions such as screening for antiviral compounds.     

Automation of a Hemagglutination-Inhibition Test for Parainfluenza 3 Antibodies in Bovine Sera

An automated hemagglutination-inhibition (HI) test for the "shipping fever" strain (SF-4) of parainfluenza 3 antibody in bovine sera was developed and compared to manual tube and microtiter test procedures. The automated system operating at 60 samples per hr provided the most test results per specified time period, and the manual tube test provided the least. The manual microtiter test and the automated system at 40 samples per hr, falling between the two above procedures, were comparable in the number of sera that could be titrated in 1 day by one technician. There was little difference between automated and manual test reproducibility when measured at the twofold titer one-dilution difference level. However, the automated system titrated a higher number of sera at the same titer on repeat runs than either of the manual test procedures. The automated one-quartile difference reproducibility (each twofold dilution subdivided into 4 units-"quartiles") was equal to the manual test one-dilution difference reproducibility. The standard deviation of the per cent variation from the mean of paired serum titers for 40-sample-per-hr runs ranged from +/-3.49 to +/-5.36%. The manual and automated systems were of comparable sensitivity in their detection of negative sera.     

A Comparison of Two Serological Methods for Detecting the Immune Response after Rabies Vaccination in Dogs and Cats being Exported to Rabies-free Areas

Levels of rabies virus neutralizing antibody in sera from dogs and cats were titrated to endpoint by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and retested by the RFFIT and the Fluorescent Antibody Virus Neutralization test (FAVN). The two tests were compared for their ability to detect the 0.5 international units/ml (I.U.) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. No difference was observed in sensitivity or specificity for either method in tests of 168 sera from unvaccinated animals or 70 sera from vaccinated animals with high levels of neutralizing antibody (an initial RFFIT titre of > or = 1.0 I.U.). Test to test variation occurred for results obtained by both RFFIT and FAVN for 95 sera from vaccinated animals with low to moderate levels of neutralizing antibody (RFFIT titre < 1.0 I.U.). No significant differences were detected for the 95 sera in the frequency for one methodology more often than the other to have a positive response (> or = 0.5 I.U.), nor were significant differences detected for the symmetry (P = 0.43) or the marginal homogeneity (P = 0.39) of results obtained by the two methods. Both methods can adequately identity unvaccinated animals, but false positive and false negative results are possible for either method when a single test is used to measure the antibody response of low-responding vaccinated animals. Nucleotide sequence analysis identified several amino acid differences in stocks of the challenge rabies virus from different laboratories. The small differences in neutralizing antibody titre that may result from mutations in the challenge virus are not important for evaluating immunity induced by vaccines which are themselves prepared from a variety of different rabies virus strains, but differences in the challenge virus, rather than differences in methodology, may account for at least some of the discrepant results reported in inter-laboratory surveys. Comparative studies of serological methods for measuring rabies antibodies should use well-characterized unpassaged virus stocks obtained from a single reference laboratory.     

Chicken embryo related (CER) cell line for quantification of rabies neutralizing antibody by fluorescent focus inhibition test.

Levels of rabies virus neutralization antibody in sera from vaccinated dogs and cattle were either measured by mouse neutralization test (MNT) or by rapid fluorescent focus inhibition test (RFFIT), performed on CER monolayers. The two tests were compared for their ability to detect the 0.5 International Units/ml (I.U.) recommended by the World Health Organization (WHO) as the minimum response for proof of rabies immunization. A significant correlation was found between the two tests (n=211; r=0.9949 in dogs and 0.9307 in cows, p<0.001), good sensitivity (87.5%), specificity (94.7%) and agreement (96.6%) as well. RFFIT method standardized on CER cell system for neutralizing antibodies detection turns the diagnosis easier and less expensive, specially when a great number of samples must be tested from endemic areas as commonly found in Brazil.     

Circulating immune complexes in Graves' disease.

Circulating immune complexes in Graves' disease sera were detected by the 125I Clq deviation test. High titers of immune complexes were detected and correlated significantly with the microsomal antibody but not with the thyroglobulin antibody titer nor with serum thyroxine levels. Serum fractionation studies in a patient with high titer of immune complexes revealed these to be heterogeneous in size, sedimenting in 19S, intermediate and 7S regions. The data suggest a role for immune complexes in the pathogenesis of Graves' disease.     

Preparation of a respiratory syncytial virus human reference serum for use in the quantitation of neutralization antibody.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, young children and the elderly. Yet, the development of a vaccine to protect against RSV infection still remains an unmet need. At present, immune responses to experimental vaccines under investigation are usually evaluated by ELISA and/or by neutralization assays against RSV. However, both types of assays are generally performed somewhat differently at different laboratories. An important step towards standardization of serology is the use of a standard human reference serum enabling normalization of results generated within and between laboratories. To fill this need, we prepared and characterized a human reference serum against the A2 strain of respiratory syncytial virus. The serum represents a pool of more than 400 individual human sera obtained from commercial sources. The sera were screened and selected on the basis of individual RSV neutralization titers. A final neutralization titer of 973 (95% C.I., 884-1072) was assigned to the final reference serum pool after it was tested three times in the presence of 10% guinea pig complement and a titer of 286 (95% C.I., 243-337) was assigned to the serum when it was tested in the absence of an exogenous complement source. Sterilely reconstituted lyophilized aliquots of the serum exhibited a stable neutralization titer for at least 1 month at room temperature and at 4 degrees C, as well as after 5 weekly freeze-and-thaw cycles at -20 degrees C. In the lyophilized state, the neutralization titer of the lyophilized reagent was stable for at least 6 months, the last time point tested. Two additional smaller pools of serum with high and medium neutralization titers of 2692 and 575, respectively, were also produced in parallel for use as positive controls and were designated as control sera. The reference serum can be used to normalize neutralization and/or other RSV-specific assay results from different laboratories and the control sera can be used for quality control purposes or as part of a panel to test operator proficiency. Individual lyophilized aliquots of the reference and control sera may be obtained from the US National Institute of Allergy and Infectious Diseases (NIAID) Reference Reagent Repository.     

Serological responses of coyotes to two commercial rabies vaccines.

Between August 1993 and September 1994 we documented serological responses of coyotes (Canis latrans) vaccinated with two commercial rabies vaccines licensed for use in domestic dogs. Serologic responses were documented by testing for rabies virus neutralizing antibodies with the rapid fluorescent focus inhibition test (RFFIT) at 30, 90, 180, 270, and 365 days post-vaccination. All coyotes vaccinated with Imrab 3 (Rhone-Merieux, Inc.), and 75% of those vaccinated with Dura-Rab 3 (Immunovet, Inc.) seroconverted, as evidenced by the presence of antirabies antibody titers > or = 1:5 in one or more of the five post-vaccination samples. The percent of coyotes showing a titer > or = 1:5 was generally greater and titer levels appeared higher and more persistent among animals vaccinated with Imrab 3 than Dura-Rab 3. Presence of titers via RFFIT tests demonstrates the antibodies produced in coyotes by these rabies vaccines functionally bind and neutralize rabies virus in vitro, but these results do not constitute a demonstration of protection required for licensure for use in coyotes.     

Demonstration of rabies virus-specific antibody in the sera of free-ranging Iowa raccoons (Procyon lotor).

Between 1984 and 1988, a study was conducted to evaluate the frequency of rabies virus neutralizing antibodies in raccoons (Procyon lotor) in two counties in Iowa. Nine hundred eighty five raccoons were trapped and tagged in Guthrie and Cerro Gordo counties during the spring, summer and fall of each year. Sex, age and weight were recorded for each animal and a blood sample was collected. Serum samples were tested for the presence of serum neutralizing antibodies (SNA) by the rapid fluorescent focus inhibition test (RFFIT), mouse serum neutralization test (MSN), and an indirect fluorescent antibody (IFA) technique for detecting immunoglobulin G. Fifty-one raccoons (5%) were found to have SNA by the RFFIT. Thirty-six serum samples (24 with RFFIT antibody titer greater than 3.0, and 12 less than 3.0) were also tested by the MSN, with results correlating well with the RFFIT results (r = 0.86, P less than 0.01, Kappa = 0.93). In 35 raccoons with SNA by the RFFIT, six individuals had immunoglobulin G binding activity by the IFA test. These results provided serologic evidence of exposure of raccoons to rabies virus in an area free of enzootic raccoon rabies.     

The mouse neutralization test in comparison with the rapid fluorescent focus inhibition test: differences in the results in rabies antibody determinations

Sixteen lots of rabies immune globulin (RIG) and six lots of rabies immune horse serum (RIS) from different producers were examined for rabies antibody by the standard mouse neutralization test (MNT) and the rapid fluorescent focus inhibition test (RFFIT). An equine rabies standard serum was assayed in parallel. In comparisons of RIS with this standard the MNT and RFFIT gave comparable results. In comparisons of RIG the antibody values in the MNT was two to ten times higher than that in the RFFIT in 15 out of 16 lots. The MNT and RFFIT are thus not fully comparable when measuring rabies antibodies in RIG. The choice of the titration method is obviously important in the measurement of the antibody concentration in RIG or RIS in IU against an equine rabies reference preparation. The described differences could have consequences for the use of RIG.     

Anticomplement immunofluorescence for the titration of antibody to varicella-zoster virus

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella-zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type-1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone-fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.     

Analysis of antibody response in goats to caprine herpesvirus 1

Serum samples of goats experiencing natural and experimental infections and/or reactivation of caprine herpesvirus 1 (CpHV.1) were analysed with neutralization and Western blotting (WB) tests. WB immunological patterns resulted differently and related to neutralizing titers. In serum samples having neutralizing titer 1:2-1:4, antibodies to two proteins of Mw of 150 and 34 kDa were present. Antibodies against several proteins, two of those being characterized by monoclonal antibodies as gB and gC, were visualized by WB in sera having titer > or = 1:8. The neutralizing antibody titers and the pattern of antibody reactivity were hypothesized to modulate the reactivation and re-excretion process of CpHV.1.     

The Second International Standard for anti-poliovirus sera types 1, 2 and 3.

The Second International Standard for anti-poliovirus sera types 1, 2 and 3 was established by the WHO Expert Committee on Biological Standardization in 1991 on the basis of an extensive collaborative study. Nine laboratories from eight countries participated and all used neutralizing antibody assays. The standard is a human serum pool which contains antibodies to all three poliovirus types and replaces the three previously established monovalent standards which were all hyperimmune monkey sera. The standard was assigned an activity of 25 IU of anti-poliovirus serum (type 1) human: 50 IU of anti-poliovirus serum (type 2) human; and 5 IU of anti-poliovirus serum (type 3) human. The study also showed significant interlaboratory differences in relative potency are observed when human sera are compared to hyperimmune monkey sera. It was therefore recommended that National laboratory references are established from human sera.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.     

Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied.     

小鼠仙台病毒标准化血清的制备及鉴定

目的制备标准化抗小鼠仙台病毒（Sendai Virus,SV）免疫血清,为清洁级及SPF级实验小鼠质量检测提供批量、稳定、敏感的质控血清。方法使用动物来源的仙台病毒（sv）感染BALB／c小鼠,获得高效价免疫血清,经IFA、IEA、ELISA等方法检测血清效价。结果制备多量抗SV血清,经多种方法检定,IFA效价达1：640,IEA效价达1：320,ELISA效价达1：102400～1：204800。结论本研究中制备的sV抗血清达到同批次大量高滴度的水平,可作为检测实验小鼠中sv病原的标准化质控血清。     

Comparison of Methods for Coccidioidomycosis Complement Fixation

A Laboratory Branch Task Force of the National Communicable Disease Center has proposed a standardized complement fixation procedure (LBCF) and an adaptation of this to microtitration techniques (MT) as uniform methods for performing complement fixation (CF) tests. A common procedure should make CF results from one laboratory more comparable to another. In addition, it would be preferable if the common procedure reproduced the titer levels of a testing procedure which is to be replaced, particularly when valid clinical interpretations have been derived from the latter. Replicated sets of sera were tested by the LBCF, MT, and the standard Smith CF procedure for coccidioidomycosis. Results with all three procedures were highly reproducible within an acceptable one-tube variation of a twofold dilution series, but the frequency of one-tube variations was greater with the MT method than with the other two. There was no statistical difference in the titers obtained with the Smith and LBCF procedures, but there was a significant difference when the MT results were compared to those with the Smith method. The LBCF method should be acceptable as a standardized and uniform CF procedure for coccidioidomycosis, subject to comparative testing between different laboratories.