Reverse Passive Hemagglutination Research Articles

The detection of heat-aggregated IgG (as a model for immune complexes) by reverse passive haemagglutination using a human monoclonal rheumatoid factor coupled to erythrocytes

A human monoclonal IgM rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus (EBV)-immortalized cell line was purified by protein A-Sepharose adsorption and coupled by the chromic chloride method to human erythrocytes. The RF-coupled cells were incorporated in reverse passive haemagglutination (RPH) assays to detect immune complexes (IC) using heat-aggregated human IgG as a model system. The sensitivity of the RPH was comparable to an enzyme-linked immunosorbent assay (ELISA) using sheep C1q for the detection of ICs.

Solid phase reverse passive hemadsorption test for hepatitis B surface antigen

A solid phase reverse passive hemadsorption test (SP-RPHAd) for hepatitis B surface antigen detection is described. It was compared with a commercial reverse passive hemagglutination assay (Hepatest, Wellcome, U.K.). SP-RPHAd is four-fold less expensive than Hepatest and undiluted sera can be used instead of eight-fold diluted sera without risk of non-specific hemagglutination. Thus, the threshold of detection is lowered to about 5 ng/ml by SP-RPHAd.

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.

Application of solid phase aggregation of coated erythrocytes technique for detection of rinderpest antigen

A solid phase aggregation of coated erythrocytes (SPACE) technique was standardized and used for the detection of rinderpest antigen in 129 samples of 9 visceral organs; from 50 clinical samples such as gum scraping, tongue scraping, faeces, urine, nasal swabs; blood from 18 rinderpest infected cattle and 2 field samples. The comparative sensitivity of SPACE, reverse phase passive haemagglutination and counter-immuno-electrophoresis tests was found to be 86.8, 84.5 and 79.8%, respectively.

Fulminant hepatitis in children in Taiwan: The important role of hepatitis B virus

In a recent period of 64 months, fulminant hepatitis was diagnosed in 17 children at National Taiwan University Hospital. Eleven patients were younger than 12 months of age. Hepatitis A IgM antibody and delta-antibody were negative in all 17. Eleven (65%) patients had hepatitis B core IgM antibody, fulminant hepatitis B. Two to 5 months before onset of hepatitis. Five of the 11 children had received blood transfusions. Three of the five donors had hepatitis B e antibody (anti-HBe) and were hepatitis B virus DNA-negative hepatitis B surface antigen (HBsAg) carriers; another two were HBsAg negative, screened by a less sensitive reverse passive hemagglutination method. The mothers of all six infants younger than 6 months of age had HBsAg. HBe antigen and antibody were studied in five of these six mothers; all five had anti-HBe. We conclude that hepatitis B virus is the most important cause of fulminant hepatitis in children in Taiwan.

Sensitivity and specificity of capillary blood HBsAg as a surrogate marker for HBeAg in pregnant women

Infants at high risk of acquiring hepatitis B virus (HBV) infection from their hepatitis B e antigen (HBeAg)‐positive mothers are prime targets for early HBV immunization. The usefulness of fingerprick blood of pregnant women as a surrogate marker to identify infants who would need immunization soon after birth was evaluated. Using HBeAg from venous blood as the standard, the detection of hepatitis B surface antigen (HBsAg) by reverse passive haemagglutination in capillary blood yielded an overall sensitivity of 97% and a specificity of 96% for detecting HBeAg at a cutoff titre of 22.5. Pregnant women with a capillary HBsAg titre of 22.5 or greater are 24 times more likely to infect their babies, while the chances of transmitting HBV infection with a titre lower than the cutoff point are almost nil. When the cost of HBV vaccine eventually comes down to levels suitable for public health use, a cutoff titre of 22.5 is suggested in order to identify infants who should be vaccinated soon after birth.

Highly sensitive antigen detection procedures for the diagnosis of infectious bovine rhinotracheitis: Amplified ELISA and reverse passive haemagglutination

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of bovid herpesvirus 1 antigen was increased by up to 50-fold using the biotinavidin interaction to amplify the reaction, when compared with a simple sandwich ELISA. An alternative immunoassay, reverse passive haemagglutination (RPHA), had a similar sensitivity to the amplified ELISA, and was technically simpler to perform. Both the amplified ELISA and the RPHA could detect viral antigen in the nasal secretions of calves undergoing experimental primary infection with the virus from Day 3 to Day 7 after inoculation. Neither assay was as sensitive as virus isolation in cell culture and they failed to detect antigen in virus-positive samples from the calves from 8 days after inoculation, and from vaccinated calves undergoing challenge infection

A comparison of three rapid diagnostic methods for the detection of rotavirus infection in calves

Three techniques for the detection of rotavirus in faecal samples from calves with neonatal gastroenteritis were compared. A preliminary study indicated that reverse passive haemagglutination (RPHA) was at least as sensitive as the enzyme-linked immunosorbent assay (ELISA). These two immunoassays were compared with the detection of viral RNA by polyacrylamide gel electrophoresis (PAGE) on 209 field samples. Of the 77 samples in which at least one test gave a positive result, 69 were positive by both RPHA and PAGE, but only 49 were also positive by ELISA, indicating a lower sensitivity for the latter test. The overall agreement between RPHA and PAGE was 96%. The reasons for the discrepancies between the tests are discussed.

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.

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

Immunochemical Characterization of Hepatitis B e Antigen Subtypes, HBeAg/l and HBeAg/2, in Sera of Hepatitis B Virus Carriers

The interrelation between HBeAg subtypes, HBeAg/1 and HBeAg/2, in sera was examined immunochemically. The detection of HBeAg subtypes in immunodiffusion (ID) depended upon the amount of HBeAg determined by reversed passive haemagglutination (RPHA), ie, the titres of HBeAg in sera positive for both HBeAg/1 and HBeAg/2, positive for only HBeAg/1 and negative for both HBeAg/1 and HBeAg/2 were 2(9.8) +/- 1.5, 2(7.0) +/- 1.6, and 2(5.6) +/- 1.3, respectively. When the sera belonging to the latter two groups were concentrated up to 2(10) RPHA titre, the precipitin line corresponding to that of HBeAg/2-anti-HBeAg/2 was visualized in ID. Monoclonal anti-HBeAg antibody that absorbed only the precipitin line of HBeAg/1-anti-HBeAg/1 in ID was prepared for the characterization of HBeAg subtypes. A linear correlation (r = 0.91) between titres of HBeAg determined by the RPHA cells prepared with monoclonal and polyclonal antibodies was found in almost all HBeAg-positive sera. The reactivities of this monoclonal anti-HBeAg antibody to both HBeAg/1 and HBeAg/2 were demonstrated in affinity chromatography experiments using a Sepharose 4B column conjugated with this antibody. These results suggest that both HBeAg/1 and HBeAg/2 are constantly present in HBeAg-positive sera and that they are closely associated. Based upon these results, a hypothetical model for the elucidation of the immunological relationship between HBeAg/1 and HBeAg/2 is proposed.

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.

Glomerulonephritis Associated With Hepatitis B Virus Infection in Children

AbstractHepatitis B surface antigen (HBsAg) was identified by a reversed passive hemagglutination (RPHA) method in the serum of 13 of 15 patients with membranous glomerulonephritis (MGN), three of three Type III membranoproliferative GN (MPGN) proposed by Burkholder, three of 25 idiopathic minimal change nephrotic syndrome (IMCNS), three of 37 minor glomerular alterations with undetermined diagnosis (MGA) and none of 79 various other renal disease, in 266 Japanese children.Immunofluorescent studies showed granular deposition of Hepatitis B e antigen (HBeAg) in a similar pattern to that of IgG and β1C in the kidney of four patients with MGN, and of HBsAg in one with Type III MPGN. Eight of 13 MGN with positive serum HBsAg, two of three Type III MPGN and one of one MGA with positive serum HBs Ag had normalization of urinary findings during the observation period of one to eight years.Eight (5 MGN, 2 Type III MPGN and 1 MGA) of those with positive serum HBeAg showed seroconversion of HBeAg, and anti‐HBe appeared in five of eight as the urinary findings became normal. The normalization of urinary findings accompanied by seroconversion of HBeAg in the patients with MGA and with Type III MPGN suggests that HBeAg is the most important antigen involved in HB virus (HBV) associated GN including not only MGN but also Type III MPGN and MGA.

Large-scale survey of hepatitis B virus infection in families.

To investigate HBV transmission in families on three islands in Okinawa, Japan, prevalence of HBV markers in two groups of inhabitants was determined. One group consisted of members of families in which there was at least one HBsAg carrier (carrier families); the other group consisted of members of families in which there were no HBsAg carriers (non-carrier families). A total of 3,261 serum samples were collected from subjects on Iriomote Island, Hateruma Island, and Yonaguni Island. These samples were tested for HBsAg by reversed passive hemagglutination (RPHA) and for antibody to hepatitis B core antigen (anti-HBc) by radioimmunoassay. Overall prevalences of HBsAg and anti-HBc were 8.2 and 65.8 per cent respectively. The prevalence of anti-HBC among members of carrier families (80.8%) was significantly higher than that among members of non-carrier families (61.6%) (P less than 0.001). The prevalence of anti-HBc among members of carrier families was higher in all age groups, and was particularly so in children. Within carrier families, the prevalence of anti-HBc was significantly higher in families in which there was at least one HBsAg carrier with HBeAg (94.5%) than in families with no HBeAg-positive carriers (76.1%). This difference was especially marked in young children. These data suggest that in families with HBsAg carrier(s), the risk of transmitting HBV to members, particularly to young children, is higher than in families without carriers, and that the risk is further increased in families with HBeAg-positive carrier(s).

Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis.

An immunodiagnostic test for the detection of a soluble nonprotein mycobacterial antigen by reverse passive haemagglutination with IgM murine monoclonal antibody was developed. The test was used to analyse the cerebrospinal fluid of 89 patients with tuberculous meningitis (TBM) from India and 127 control subjects from India and the UK. The antigen was demonstrable in 88% of culture-positive and 73% of culture-negative TBM patients. However, it was also detected in 21% of Indian patients with pyogenic meningitis, and in 8% of Indian and 1% of UK control subjects. Antibodies binding to a soluble mycobacterial extract were detected at low titre in 68% of all subjects with TBM and in 37% of Indian cases of pyogenic meningitis. Because patients with TBM had raised levels of the antigen and of antibodies to the antigen, the possible role of immune complexes in the pathogenesis of the disease is briefly discussed.

Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: Comparison with ELISA

A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of ‘normal’ immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45°C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10 5 particles in a 25 μl vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination.

AN6520 Ag: an antigen purified from liver with non-A, non-B hepatitis.

An extract prepared from the liver of patient with chronic non-A, non-B (NANB) hepatitis was found to produce a precipitin line in immunodiffusion with a serum from a multiply transfused patient and those from patients convalescent from NANB hepatitis. The antigen was purified by gel filtration and density gradient centrifugation. The antigen had a buoyant density of 1.16-1.20 g/cm3 in cesium chloride, a sedimentation coefficient (S20,w) of 51.5, and a molecular weight of larger than 1.5 X 10(6) daltons. Electron microscopic examination revealed particles 29-34 nm in diameter (average 31.5 nm), which could be agglutinated by the specific antiserum. We developed a reverse passive hemagglutination (R-PHA) and a passive hemagglutination (PHA) technique for detection of the new antigen and antibody, respectively, and applied these to human sera. Antibody to the antigen was detected in 19/28 (67.9%) convalescent sera of NANB hepatitis. This prevalence was significantly higher than those found in convalescent sera of type A hepatitis patients (2/17 = 11.8%), type B hepatitis patients (2/15 = 13.3%), and normal blood donors (9/129 = 7.0%) (p less than 0.01); and the prevalence in hepatitis A and B patients did not differ significantly from that of normal donors. Furthermore, most (66.7%) of the cases of NANB hepatitis endemic in Shimizu City, Japan, showed clear seroconversion with respect to this antibody. These results suggest that the new antigen/antibody system is associated with NANB hepatitis.

Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes.

Two hemolysin genes of Vibrio parahaemolyticus WP1, a thermostable direct (TSD) hemolysin gene and a thermolabile hemolysin gene, were cloned into the pBR322 vector in Escherichia coli K-12 C600. A large amount of the TSD hemolysin produced in E. coli K-12 accumulated in the periplasmic space. The TSD hemolysin gene was localized on a 0.9-kilobase HindIII-BamHI fragment by identifying qualitatively the production of the TSD hemolysin by a reverse passive hemagglutination assay in the osmotic shock fluid. The thermolabile hemolysin gene was isolated on a 1.3-kilobase HindIII-PstI fragment by selection with the hemolysin on blood agar. Southern blot hybridization and colony hybridization experiments indicated that the TSD hemolysin gene was present in the chromosomal DNA of 15 Kanagawa phenomenon-positive strains but not in 14 negative strains, whereas the thermolabile hemolysin gene was detected in all strains. No homologous DNA sequences to TSD and thermolabile hemolysin genes were detected in the chromosomes of Vibrio cholerae, Vibrio vulnificus, non-O1 V. cholerae, and Vibrio anguillarum.

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.