Reverse Passive Haemagglutination Test Research Articles

Outbreak of acute gastroenteritis caused by human group C rotavirus in a youth educational center in Okayama Prefecture

In May 2000, an outbreak of acute gastroenteritis caused by human group C rotavirus (CHRV) occurred in a youth educational center located in the southern area of Okayama Prefecture. A total of 172 schoolchildren and teachers, who consisted of 51 persons belonging to F school and 121 persons belonging to K school, joined in an educational program at the center from May 24 to 26. Eighty-seven individuals (50.6%) of them showed clinical symptoms of gastroenteritis from May 24 to 30, and the outbreak peaked on May 27. The major clinical symptoms were abdominal pain (87.4%), diarrhea (50.6%), nausea or vomiting (21.8%), fever > 37 degrees C (12.6%), and headache (14.9%). The clinical symptoms of the patients in F school were more severe than those in K school. Thirty-two fecal specimens were collected from the patients and examined for gastroenteritis viruses by electron microscopy, ELISA, reverse passive hemagglutination test, and reverse transcription (RT)-PCR. As a result, CHRVs were detected in 21 specimens (65.6%) by RT-PCR. The possible route of the CHRV infection was thought to be a person to person transmission by following reasons: (i), CHRVs were detected in stools from patients who became ill on the first day of the program; (ii), CHRVs were not detected in stools from cooks; (iii), no possible causal food was found by epidemiological analysis of the outbreak. Furthermore, phylogenetic analysis of the VP7 gene among CHRVs isolated in Okayama revealed that the virus detected in this study was more closely related to the virus isolated from a sporadic case of gastroenteritis in 1996 than that isolated from an outbreak occurred in 1999.

Neutralization assay for human group C rotaviruses using a reverse passive hemagglutination test for endpoint determination.

A novel neutralization assay for human group C rotavirus (CHRV) was developed by using a reverse passive hemagglutination (RPHA) test for endpoint determination. In this assay, the neutralization (N)-RPHA test, serial twofold dilutions of sera were mixed with a solution of CHRV that yielded an RPHA test titer of 8 at 3 days after infection. The mixtures were incubated at 37 degrees C for 1 h and were inoculated onto CaCo-2 cell monolayers in a 96-well microplate. Maintenance medium containing 100 microgram of pancreatin per ml was placed in each well. The plate was sealed with sticky plastic film and was incubated at 37 degrees C for 3 days under continuous rotation. Then, the RPHA test titer of each well was determined. The neutralization titer was expressed as the reciprocal of the maximum dilution of the serum that exhibited a fourfold (75%) or greater reduction in the RPHA test titer (8 to 2 or less). Seroconversion of neutralizing antibody was demonstrated by this method in four sets of paired serum specimens from patients with diarrheal disease caused by CHRV. The seroprevalence of CHRV in the general population in Okayama Prefecture was 26.8% by immunofluorescence and 25.5% by the N-RPHA test. The N-RPHA test described here is the first system used to assay for a neutralization antibody against CHRV and is applicable in both clinical and epidemiological settings.

Rapid diagnosis of typhoid fever.

A Reverse Passive Haemagglutination Test (RPHA) was designed for the detection of Salmonella typhi antigen and rapid diagnosis of typhoid fever. Two per cent fresh sheep RBC's were coated with 32 micrograms/ml of immunoglobulin. The minimal detectable level of the antigen was 1250 micrograms/ml. Cross reactions were observed with the samples of patients suffering from Salmonella paratyphi A and pseudomonas infections. The RPHA established was used for the detection of S. typhi antigen in culture broths from 100 patients with clinically suspected typhoid fever with culture and/or widal positive, 50 patients with septicemia caused by bacteria other than S. typhi and 50 normal, afebrile healthy controls. It was found that the sensitivity and specificity of this assay was 70% and 92% respectively.

Survey of human group C rotaviruses in Japan during the winter of 1992 to 1993.

Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.

Hepatitis B virus infection in pregnant women and its transmission to infants.

HBsAg was screened by Reverse Passive Haemagglutination Test (RPHA) and was confirmed by ELISA test in 157 pregnant females and their newborns. Anti-HBc and IgM anti-HBc was done in these cases by enzyme immuno-assay. The overall prevalence of HBsAg in mothers was 16 out of 157 (10 per cent) and in cord blood of newborns 5 per cent. The transplacental transmission was found in eight of 16 (50 per cent) HBsAg positive mothers. Anti-HBc was present in 12 out of 16 (75 per cent) HBsAg positive mothers and, of these, seven (58 per cent) neonates acquired HBsAg infection. IgM anti-HBc was present in seven out of eight (88 per cent) HBsAg positive neonates, suggesting active in utero infection. Fourteen out of 16 (88 per cent) neonates born to HBsAg positive mothers were alive and healthy, one was stillborn and one had a congenital anomaly.

Reverse passive haemagglutination test in the diagnosis of infectious bursal disease.

The reverse passive haemagglutination test (RPHA) was used to detect infectious bursal disease (IBD) virus antigen in various organs of experimentally infected chickens and field cases. The results of RPHA were compared with those of agar gel immunodiffusion (AGID) and latex agglutination (LAT) tests. IBD antigen was detected in 86.4%, 80.4% and 80.5% of different organs by RHPA, LAT and AGID respectively. Although the differences are not statistically significant, the LAT is recommended because of cost and speed of obtaining results.

Relation of Blood Groups And HBsAg Positivity in Blood Donors Living in Gaziantep Region

HBsAg presence has been searched in blood donors by Reverse Passive Hemagglutination(RPH) test in the years of 1987-1993 in Atatürk Blood Bank in Gaziantep. 2902(4.8 %) of 59287 blood donors were found as HBsAg-Positive. Relation between blood groups of healthy blood donors and blood groups of HBsAg carriers was evaluated. statistically using X 2 and significance of the difference between two percentage tests. HBsAg positivity rate was found lower than the expected ratio in A and B blood groups, in expected value in 0 blood group, and higher than expected ratio in AB blood group(p<0.005).

Detection of Japanese encephalitis virus antigens in the CSF using monoclonal antibodies

Background: Antigen detection in the CSF is an invaluable tool in the diagnosis of viral infections of the nervous system, especially in the early phase of the illness. Very little information is available on the use of antigen detection in the diagnosis of Japanese encephalitis (JE). Objectives: Evaluation of two monoclonal antibody-based antigen detection methods in the diagnosis of JE in comparison with the detection of virus-specific IgM antibodies in the CSF. Study design: In this study 115 patients with a clinical diagnosis of JE were investigated. A reverse passive haemagglutination test for the detection of soluble JEV antigens, an immunofluorescent assay for the detection of cell-associated antigen and an IgM capture ELISA for the detection of virus specific IgM antibodies in the CSF were used. Results: Laboratory confirmation of JE was possible in 92/115 patients. Virus-specific IgM was detected in 75/92 and JEV antigen was detected in 52/92 patients. Soluble antigen was detected in 37/52, cell-associated antigen in 30/52. There was no significant difference in the sensitivity of the two antigen detection systems used. Conclusions: Diagnosis by antigen detection could be done less frequently than by demonstration of virus-specific IgM antibodies in the spinal fluid. However, antigen detection proved useful during the first week of illness when IgM antibodies were not detected in the CSF.

Detection and characterisation of circulating cysticercal antigens in human cerebrospinal fluid

With biotin labelled and unlabelled immunoglobulin fraction of anticysticercal antibodies raised in rabbits, tandem-enzyme linked immunosorbent assay (T-ELISA), capture-dot immunobinding assay (C-DIA) and reverse passive haemagglutination (RPHA) tests were developed for the detection of cysticercal antigens. The sensitivity levels were respectively, 9 ng ml −1, 2 ng ml −1 and 45 ng ml −1. All three methods were of equal specificity as none of the antigens of Mycobacterium tuberculosis, Japanese encephalitis virus and Echinococcus granulosus reacted with anticysticercal IgG. Cysticercal antigens were detected in the cerebrospinal fluid (CSF) of confirmed neurocysticercosis at sensitivity levels of 91·6% by T-ELISA, 83·33% by C-DIA and 75% by RPHA and specificity levels of >93%. Western analysis of these antigens in CSF showed mainly antigens of 64–68 kDa and 24–28 kDA. By crossed immunoelectrophoresis (CIE) with an intermediate gel technique, five circulating antigens were found to be released from scolex and fluid.

Occurrence of Anti-CMV-, Anti-HIV-1, and HBsAg-Positive Individuals in a Group of Blood Donors and Patients on Chronic Dialysis in Lebanon

Occurrence of Anti-CMV-, Anti-HIV-1, and HBsAg-Positive Individuals in a Group of Blood Donors and Patients on Chronic Dialysis in Lebanon

Changing aetiologic patterns of acute viral hepatitis in Taiwanese children

During 1981-86, 76 children were diagnosed as having acute viral hepatitis at the Department of Pediatrics, National Taiwan University Hospital, which is a major referral centre for hepatitis in children in northern Taiwan. The majority (64%) of children had acute hepatitis B which had occurred mainly during infancy. Perinatal transmission from a hepatitis B e negative surface antigen (HBsAg) carrier mother or infection through blood transfusion from a donor who had escaped notice by a less sensitive screening test (reverse passive haemagglutination test) for HBsAg were the two important modes of transmission of hepatitis B virus. The number of cases of acute hepatitis B declined after 1984, with the beginning of the nation-wide hepatitis B vaccination programme. Due to an outbreak of hepatitis A in northern Taiwan in 1982, the number of cases of hepatitis A peaked that year. Subsequently, cases of acute hepatitis A decreased remarkably. Better socio-economic conditions and improved hygiene might have contributed to the marked decrease of viral hepatitis A. The frequency of non-A, non-B hepatitis remained stable during the study period. It is possible to conclude that the aetiologic pattern of acute hepatitis in Taiwanese children changed during the past 6 years: clinical cases of hepatitis A and B decreased, probably because of more effective control of hepatitis A and B virus infections, whereas the control of non-A, non-B virus apparently requires further efforts.

A reverse passive haemagglutination test for detection of Japanese encephalitis virus antigens in cerebrospinal fluid

A rapid sensitive and specific reverse passive haemagglutination test (RPHA) was developed for the detection of Japanese encephalitis virus (JEV) antigens in human cerebrospinal fluid (CSF). Sheep red cells were sensitized with five monoclonal antibodies (109, 112, 203, 204 and 301) reactive with envelope glycoprotein of JEV. Viral antigens were detected in CSF from 35 of 58 (60%) clinical cases of JE when the five MAb coated cells were used in combination. An IgM capture ELISA detected JEV specific antibodies in CSF among 52 of these 58 cases (90%). While 29 specimens contained both antigen and IgM antibodies, 23 had only IgM antibodies and 6 had only antigen. RPHA proved valuable for detection of viral antigens in CSF samples obtained within 10 days after onset of clinical symptoms. Amongst the five MAbs used, the individual antigen detection rates were 44, 12, 43, 12 and 36%, for MAbs 109, 112. 203. 204 and 301. respectively.

Avidin-biotin enhanced reverse passive hemagglutination method for detection of HIV antigens

A new assay for HIV antigens, the avidin-biotin enhanced reverse passive hemagglutination (AB RPHA) test is described which is suitable for detection of infection in cell culture experiments. This assay is simple to perform and economical, and has sensitivity equal to or greater than that of commercial ELISA assays. The coated red cells used for this assay may be stored in the frozen or lyophilized state.

Comparative study between Reverse Passive Hemagglutination test and Hematest II on screening of fecal occult blood for colorectal cancer in human dry dock.

Comparative study between Reverse Passive Hemagglutination test and Hematest II on screening of fecal occult blood for colorectal cancer in human dry dock.

Detection of Chlamydia psittaci from various animal species by reverse passive haemagglutination

A reverse passive haemagglutination test (RPH) has previously been developed to detect the genus-specific antigen of Chlamydia. Clinical samples were obtained from various sites of different animal species. The RPH test detected chlamydial antigen from clinical cases of conjunctivitis in cats, abortion in sheep and psittacosis in birds. Although not as sensitive as cell culture isolation, this test has the advantages of rapidity and of dtecting antigen from dead chlamydiae.

Comparative study between Hemoccult II test and Reverse Passive Hemagglutination test on mass screening for colorectal cancer

Comparative study between Hemoccult II test and Reverse Passive Hemagglutination test on mass screening for colorectal cancer

Microassay of bovine serum and urinary myoglobin by reverse passive hemagglutination.

A sensitive microassay by reverse passive hemagglutination (RPHA) was carried out for the measurement of bovine myoglobin (Mb). Sheep erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-bovine Mb antibodies purified by affinity chromatography. The RPHA test was done by a microtiter method and minimal detectable Mb levels were 10ng/ml in serum and 2ng/ml in urine. Mb levels of 15 normal calves and 15 normal cows ranged from <10 to 80ng/ml in serum and from <2 to 16ng/ml in urine. Sixteen calves with nutritional myopathy showed markedly increased Mb levels in serum, 640 to 51, 200ng/ml, and in urine, 640 to 256, 000ng/ml. Serum Mb levels in calves recovered from nutritional myopathy decreased to nearly normal ranges within 3 to 5 days. Although each of the 51 cows at the onset of parturient paresis showed high levels of serum Mb ranging from 320 to 20, 480ng/ml, while about 30 per cent of the cows showed normal serum CPK activities. Serum Mb levels in cows recovered from parturient paresis rapidly decreased to the normal levels after 2 to 4 days. Serum Mb in cases slaughtered continued at high levels until the last treatment of parturient paresis. It would be concluded that RPHA method is useful for Mb determination in evaluating the myolytic state of bovine myopathy and for early detection of the disease.

Detection of an Antigen (AN6520), Possibly Related to Non‐A, Non‐B Hepatitis, by Monoclonal Antibodies. I

The antibody to AN6520 antigen, which was isolated from the liver of a patient with non-A, non-B hepatitis (NANBH), has been detected frequently in convalescent sera from patients with NANBH by the passive hemagglutination (PHA) test. In a further study, we established hybridoma cells secreting antibodies against AN6520 antigen and obtained ascitic fluids with PHA titers ranging from 1:10(5) to 1:10(7). In immunodiffusion with AN6520 antigen, all monoclonal antibodies were found to form an identical precipitin line. These lines were also identical to those formed by rabbit antiserum against AN6520 antigen and by convalescent sera from patients with NANBH. With one of the monoclonal antibodies, 1-F12, solid-phase radioimmunoassay (SP-RIA) for detecting AN6520 antigen was developed as well as blocking RIA for anti-AN6520 antibody detection. The antigen assay was 50 times more sensitive than the reverse passive hemagglutination (R-PHA) test, with a sensitivity threshold of the 1 ng/ml of antigen solution; the antibody assay was 10 times more sensitive than PHA. The results with this blocking RIA were mostly in agreement with the data obtained by PHA. Furthermore, the antigen in human sera, which had never been detected by R-PHA test, could be detected by SP-RIA.

Confirmation of human cytomegalovirus by reverse passive hemagglutination with monoclonal antibodies reactive to the major glycosylated peptide (GP-66)

Sheep erythrocytes coated with three monoclonal antibodies, each reactive to a different epitope of the 66-kilodalton cytomegalovirus (CMV) matrix protein, were used in a reverse passive hemagglutination test with CMV-infected cell lysate to identify and confirm the CMV. The test is specific only for CMV, since 5 laboratory strains of CMV (AD169, Davis, Espilat, C-87, and Towne) and 10 clinical isolates reacted well, but uninfected MRC-5 cell lysate, lysates of herpes simplex virus types 1 and 2, varicella-zoster virus, and adenoviruses did not react. The reactive CMV lysate was confirmed by the pretreatment of CMV lysate with the three monoclonal antibodies followed by the addition of antibody-coated erythrocytes. The reverse passive hemagglutination test and the confirmatory blocking test are performed at the same time, requiring 2 h to complete. Since V-bottom microtiter 96-well plates and a 25-microliter pipette can be used to perform the test, it is ideal for CMV confirmation, especially when the equipment to read the fluorescent-antibody test or enzyme-linked immunosorbent assay is not available.

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination.

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.