Development and use of a micro haemagglutination inhibition (HAI) technique, based on Hepatest, for the detection and quantitation of hepatitis B surface antibody (Anti-HBs) in blood donors.

A simple micro haemagglutination inhibition technique, based on Hepatest (Wellcome Reagents Limited), is described. Its application in the screening of blood donors is shown, and its sensitivity is discussed in two forms of usage and also when compared to the previous immunoelectroosmophoresis technique used in this centre.     

A modification of Hepatest, using the Terasaki plate, for the detection of HBSAg in blood donors.

A simple modification of Hepatest (Wellcome Reagents) is described, which lends itself to the rapid large-scale screening of blood donors. The technique uses 4 mul of HBSAb coated turkey cells (Cayzer et al., 1974), and Terasaki plates (Hopkins and Das, 1973), in contrast to the 25 mul used in the microtitre tray technique. This results in a considerable saving on the cost of reagents, the cost per test being reduced to 16% of the cost of the recommended method. In our hands, this modification is as convenient to use as the manual microtitre tray technique. Positive sera from the screen test are also tested for non-specificity using horse IgG coated turkey control cells. The falso positive screen test rate has been reduced by the adoption of a modified buffer containing turkey serum.     

Haemagglutination assay for HBsAg — A new approach to reagent preparation

Human group O rhesus negative erythrocytes were spherocyted and fixed in glutaraldehyde, then coated initially with HBsAg and subsequently with anti-HBs. The resulting reagent was found to agglutinate in the presence of HBsAg positive serum, but not in HBsAg negative serum. The assay was evaluated for blood donor screening (39,962 donations) over a 6-month period, and was compared with Hepatest (RPHA) and AUSRIA-2 (RIA) for the study of two established HBsAg panels.     

Comparative study of three technics of inverse passive hemagglutination in the search for a surface antigen of the hepatitis b virus (Ag HBs) (author's transl)]

The following three different versions of reverse passive hemagglutination (RPHA) were evaluated for the detection of HBs antigen: Auscell I - Abbott, WH HBs - Wellcome and the hepanosticon - Organon technique and results obtained were compared with those obtained by the radio immuno assay (Ausria II of Abbott). In 493 sera studied, up to 16,8% were found positive by RPHA as compared to 17,2% positives by RIA. The percentage of false positives by the different methods varied from 4,9 to 7,3. Confirmatory tests, either absorption or neutralization, are necessary to ascertain accuracy of positive results in each of the 3 RPHA methods. The high quality of the Auscell and WH HBs confirmative test allows their sole use although they are slightly less sensitive than the RIA. We would recommand use of the Hepanosticon test whenever positive sera can be confirmed by RIA.     

Radioimmunoassay for detection of hepatitis B surface antigen

A modification of radioimmunoassay (RIA) for detection of HBsAg is described. The schedule for purification and the method of iodinization of purified HBsAg are presented. The sensitivity of RIA and other immunological methods for detection of HBsAg was analysed comparatively. RIA in the modification described detected HBsAg in a concentration of 50 mg/ml.     

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.     

Status and significance of testing for hepatitis B surface antigen and surface antibody

Following Blumberg's discovery of hepatitis B surface antigen (HBsAg), many attempts have been made to develop several in vitro diagnostic techniques for the detection of this antigen and its homologous antibody. The two-dimensional micro-Ouchterlony immunodiffusion has been the first technique used, rapidly replaced by procedures of increasing sensitivity characterized as second-generation and the currently available third-phase tests which include radioimmunoassay (RIA), reverse passive haemagglutination (RPHA), reverse passive latex agglutination (RPLA) and enzyme immunoassay (EIA). Among these, RIA appears to be the most sensitive and specific, whereas EIA, RPHA and RPLA have the advantage of long shelf-life of stable reagents, no need for sophisticated and expensive equipment and no hazard associated with the handling of radioactive isotopes. Moreover, the sensitivity of EIA should increase by objective reading with a colorimeter.The most sensitive method for the detection of surface antibody (anti-HBs) is again RIA, whereas passive haemagglutination (PHA) had the advantage of providing titres. Finally EIA, based on inhibition of a known amount of HBsAg, has at least the same sensitivity as PHA, but has the advantage that reagents are more stable and that it permits screening for both HBsAg and anti-HBs with the same reagents at the same time. The application of these highly sensitive techniques for the detection of HBsAg and anti-HBs has resulted in a consistent reduction in the incidence of post-transfusion hepatitis type B and in a better understanding of the aetiology, epidemiology and natural history of this infection.     

Reversed passive hemagglutination test fails to detect HBsAg in a number of HBeAg positive sera

In endemic areas of hepatitis B virus (HBV) infection, perinatal transmission from asymptomatic HBsAg carrier mothers to infants plays a major role in the transmission of HBV. HBeAg indicates a high level of viral replication and infectivity. Most of the infants born to HBeAg positive mothers become carriers. Prenatal screening of HBsAg would identify infected mothers and thus allow preventive administration of immunoglobulin and immunization to the newborns. Reversed passive hemagglutination assay (RPHA) is commonly used in Thailand for HBsAg screening. However this method has low sensitivity and gives false negative results. Therefore, infants born to HBsAg false negative mothers would not receive proper immunization. This study reveals the rate of false negative results for HBsAg by RPHA in high infectivity sera. Of 985 sera which were HBsAg positive by ELISA, 70 (7.1%) were negative for HBsAg by RPHA. Of these 70 false negative sera, 7 (10%) were HBeAg positive. Our results indicate that RPHA is a less sensitive method for detection of HBsAg than ELISA. RPHA can give false negative results even in sera with high HBV infectivity. Therefore, RPHA should be replaced by EIA for prenatal HBsAg screening or any other screening for HBV infection whenever possible.     

Comparative study of methods of detection of hepatitis 'B' surface antigen (HBsAg)

The serum samples were collected from 52 patients of acute viral hepatitis and 235 hospital staff from Kasturba Hospital for Infectious Diseases. HBsAg was detected in their sera by counter-immuno-electrophoresis (CIEP), reverse passive hemogglutination (RPHA) and by micro-enzyme-linked-immunosorbent assay (ELISA) technique. Among the patients, HBsAg was detected in 12 cases (23%) by CIEP, in 18 cases (34%) by RPHA and in 23 patients (45%) by ELISA. In the hospital staff, HBsAg was detected in 4 samples (1.7%) by CIEP, in 8 samples (3.5%) by RPHA and in 32 samples (13.5%) by ELISA. Thus ELISA was found to be the most sensitive technique in detecting HBsAg.     

Evaluation of turkey erythrocyte hemagglutination assay for the detection of hepatitis B antigen.

A recently described hemagglutination test for hepatitis B antigen (HBsAg) using turkey erythrocytes coated with horse antibody to HBsAg purified by affinity column chromatography was evaluated on a comparative basis with 100 HBsAg-positive and -negative serum samples. The turkey erythrocyte hemagglutination test (TEHA) was found to be less sensitive than radioimmunoassay (RIA) but gave far better results than counterimmunoelectrophoresis. Quantitative titration of HBsAg in serial dilutions of the samples appeared to be more reliably performed by TEHA than by RIA. TEHA is a simple and sensitive technique for the detection of HBsAg and may offer several practical advantages over RIA.     

Comparison of the RPHA and EIA techniques for the detection of HBs antigen among pregnant Thai women

Five hundred serum samples obtained from pregnant women attending an antenatal clinic in Bangkok were tested for HBsAg by reverse passive hemagglutination assay (RPHA) and enzyme immunoassay (EIA). It was found that 21 (4.2%) and 28 (5.6%) of the sera were positive by RPHA and EIA, respectively. The sensitivity and specificity of the RPHA were 75% and 100%, respectively, when using EIA as the standard method. The RPHA positive predictive value was 100% and the negative predictive value was 98.5%. Accuracy was 98.6%. This study showed that the RPHA was simple and required inexpensive equipment, making it suitable for mass screening. However, the possibility of false negative readings due to low levels of HBsAg should be kept in mind, especially in the blood transfusion practice.     

Detection and quantitation of hepatitis-B surface antigen immune complexes (HBsAg-ICs) by an antigen-specific method. I. Detection and quantitation of in vitro prepared HBsAg-ICs

An antigen-specific method has been developed for direct detection and quantitation of HBsAg-ICs. The method involves (1) precipitation of HBsAg-ICs with 3.5% PEG; (2) dissociation of the PEG precipitated ICs by treatment with NaSCN, NaI, KBr, low or high pH buffers, trypsin or papain; and (3) detection and titration of HBsAg and/or anti-HBs liberated from ICs. Treatment with 2 M and 3 M NaSCN, papain or trypsin liberates HBsAg, while following treatment with 3 M and NaI free anti-HBs is detectable. Trypsin digestion (2 mg/ml, 30 min at 37 degrees C) proved to be most effective for disrupting HBsAg-ICs formed at equivalence as well as in excess of antigen or antibody. After trypsin digestion of the sample RPHA, RIA and ELISA may be used as a third step. Th     

Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg

A second generation radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) was developed which utilizes recombinant DNA-derived HBsAg (rHBsAg) in place of human plasma derived HBsAg. In these sandwich assays, rHBsAg immobilized on a solid phase was used to capture anti-HBs from the specimen and rHBsAg conjugated to horseradish peroxidase or radiolabeled with 125I was used as a detecting reagent. These rHBsAg-based assays were compared to a commercial radioimmunoassay for anti-HBs detection (AUSAB RIA). For a population of 1711 sera and plasma specimens, 99.2% overall agreement was demonstrated between the recombinant RIA and EIA and 98.6% agreement was observed between the recombinant assays and AUSAB-RIA. The recombinant assays demonstrated equivalent sensitivity and detectability to AUSAB RIA. Most discrepant samples were low-level reactive by AUSAB-RIA, generally less than 10 mIU/ml, and likely represent nonspecific reactivity since no other marker for hepatitis B infection was detected in these samples.     

Improved economics of HBsAg screening with commercial radioimmunoassay reagents.

Commercial 125I anti-HBs was processed to yield a sixfold improvement in economy without significant loss of sensitivity or specificity. Additional polystyrene beads were coupled with commercially supplied anti-HBs. The modified assay (Mod-RIA) was compared with commercial RIA, EIA, and RPHA using established HBsAg panels. Mod-RIA was also compared with HEPATEST (RPHA) for screening 71 200 fresh blood donations during an 11.5-month period.     

Detection of HBV-DNA and HCV-RNA viral sequences by polymerase chain reaction in selected Kenyan samples and the relationship to HBV seromarkers

We undertook a study on selected samples from patients who had presented with viral hepatitis and conditions of the liver (liver cirrhosis, chronic hepatitis and hepatocellular carcinoma). Diagnosis, screening and confirmation for viral hepatitis was done using a battery of techniques: ultrasound, conventional serological methods (Hepatitis B surface Antigen [HBsAg] - Reverse Passive Haemagglutination [RPHA], Hepatitis B core Antibody [HBcAb] - Passive Haemagglutination [PHA], Alpha-feto Protein - RPHA), Hepatitis B e Antigen/Antibody [HBeAg/Ab] - Radioimmunoassay [RIA], Hepatitis C antibody [HCV-Ab] - Enzyme Immunosorbent Assay [EIA]. Due to the high specificity and sensitivity of the Polymerase Chain Reaction technique [PCR] in detecting the viral genomes, it was used to establish the presence of the HBV-DNA and HCV-RNA to correlate the serological diagnosis of their respective seromarkers. A total of 39 serum samples were tested comprising 11 blood donors, 8 chronic liver disease patients and 20 hepatocellular carcinoma cases. 4/19 (21%) HCV-antibody (C-l) reactive samples were found to be positive for HCV-RNA by PCR. 14 of the 19 (73.7%) including the 4 HCV-RNA positive cases tested positive for HBcAb. 6 of 11 (55%) HBsAg positive cases also tested positive for HBV-DNA by PCR, In 8 of 20 (40%) hepatocellular carcinoma cases, no aetiological role could be assigned to hepatitis B or C as only HBcAb was demonstrated in those cases.     

Serological detection of HBeAg and anti-HBe using automated microparticle enzyme immunoassays.

Fully automated microparticle enzyme immunoassays (EIA) were developed for the detection of HBeAg (IMx HBe) and antibodies against HBeAg (IMx anti-HBe), respectively. Specimens from blood donors, diagnostic and hospital patients and individuals with a variety of infectious and immune diseases were tested both in house and at four clinical sites. The overall agreement between IMx HBe and Abbott HBe RIA/EIA was 99.7% (2985 of 2994) and between IMx anti-HBe and anti-HBe RIA/EIA was 95.8% (2330 of 2432). Almost all anti-HBe discordant specimens (94.1%, 96 of 102) were reactive by IMx anti-HBe but negative by anti-HBe RIA/EIA. off anti-HBe discordant specimens were also reactive for anti-HBc. The IMx anti-HBe assay was 2- to 4-fold more sensitive than the current RIA as determined by serial dilution of anti-HBe reactive specimens. The ability of these IMx assays to detect HBeAg and anti-HBe in 199 HBsAg reactive specimens was also evaluated. 43.7% (87 of 199) and 66.3% (132 of 199) specimens were reactive for HBeAg and anti-HBe by IMx, respectively. Only one specimen was negative for both IMx assays compared to 14 (7.0%) non-reactive for both HBe and anti-HBe RIA. There were 24 specimens (12.1%) positive for both HBeAg and anti-HBe by IMx compared to 1 (0.5%) positive by the corresponding RIAs. This increased detectability of anti-HBe in HBsAg carriers using IMx anti-HBe may result from increased sensitivity for 'free' anti-HBe and/or increased ability to detect anti-HBe in immune complex. IMx anti-HBe also detected more reactives among volunteer blood donor specimens reactive for anti-HBc but negative for HBsAg (55.5%, 86 of 155), compared to RIA (38.7%, 60 of 155). IMx anti-HBe may be useful in confirming prior exposure to HBV in blood screened positive by Corzyme.     

Improved stool concentration procedure for detection of Cryptosporidium oocysts in fecal specimens.

Epidemiologic and laboratory data suggest that coprodiagnostic methods may fail to detect Cryptosporidium oocysts in stool specimens of infected patients. To improve the efficacy of stool concentration procedures, we modified different steps of the Formalin-ethyl acetate (FEA) stool concentration technique and evaluated these modifications by examining stool samples seeded with known numbers of Cryptosporidium oocysts. Because these modifications failed to improve oocyst detection, we developed a new stool concentration technique that includes FEA sedimentation followed by layering and flotation over hypertonic sodium chloride solution to separate parasites from stool debris. Compared with the standard FEA procedure, this technique improved Cryptosporidium oocyst detection. The sensitivities of the two concentration techniques were similar for diarrheal (watery) stool specimens (100% of watery stool specimens seeded with 5,000 oocysts per g of stool were identified as positive by the new technique, compared with 90% of stools processed by the standard FEA technique). However, the most significant improvement in diagnosis occurred with formed stool specimens that were not fatty; 70 to 90% of formed stool specimens seeded with 5,000 oocysts were identified as positive by the new technique, compared with 0% of specimens processed by the standard FEA technique. One hundred percent of formed specimens seeded with 10,000 oocysts were correctly diagnosed by using the new technique, while 0 to 60% of specimens processed by the standard FEA technique were found positive. Similarly, only 50 to 90% of stool specimens seeded with 50,000 oocysts were identified as positive by using the standard FEA technique, compared with a 100% positive rate by the new technique. The new stool concentration procedure provides enhanced detection of Cryptosporidium oocysts in all stool samples.     

A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections.

A simple, specific and economical dipstick immunobinding enzyme-linked immunosorbent assay (DIA) for detecting hepatitis B surface antigen (HBsAg) and antibodies to hepatitis delta virus (anti-HDV), utilizing cellulose nitrate membrane is described. Screening of 815 serum specimens for HBsAg by DIA and micro ELISA revealed a positivity of 22.69% and 22.94% respectively. In the detection of antibodies to delta antigen, DIA was compared with an indirect immunofluorescence technique using A3 cell line as antigen substrate and a commercial macro ELISA. Of the 143 HBsAg positive sera tested for anti-HDV, 59 (41.25%) were positive by both immunofluorescence and macro ELISA and 61 (42.65%) by DIA. While the positive and negative predictive values of DIA for HBsAg were 100% and 99.6%, for anti-HDV by DIA these were 96.7% and 100% respectively. Based on the simplicity of performance and the economical nature of the test system, DIA is recommended as a diagnostic tool for field surveys and small laboratories in developing countries.     

Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays

A solid-phase radioimmunoassay (RIA) for the detection of hepatitis B e antigen (HBeAg) and antibody (anti-HBe) was developed. The RIA was approximately 1,000-fold more sensitive than rheophoresis for HBeAg, and approximately 6,000-fold more sensitive than rheophoresis for anti-HBe. Generally, less than one-fifth of hepatitis B antigen (HBsAg)-positive sera from blood donors were positive for either HBeAg or anti-HBe by rheophoresis; in contrast, more than 90% of the samples were positive by the RIA method. The ratio of HBeAg to anti-HBe among HBsAg carriers varied in different geographic localities. Also, the presence of HBeAg correlated directly with the titer of HBsAg and the presence of Dane core particles. Anti-HBe was associated with lower titers of serum HBsAg.     

Detection of genital herpes simplex infections by a tissue culture-fluorescent-antibody technique with biotin-avidin.

Several cell lines were evaluated for their suitability for rapid detection of herpes simplex virus (HSV) from clinical genital specimens. Human foreskin fibroblast (Flow 7000) cells were found to be most suitable in terms of sensitivity and adherence characteristics. HSV in clinical specimens was isolated by a standard tissue culture method by monitoring the cytopathic effect, and the titers of the HSV-positive specimens were determined. More than 65% of the HSV-positive genital specimens showed titers of less than or equal to 10(4) 50% tissue culture infective doses per ml. The standard tissue culture-cytopathic effect method required 3 to 10 days for detection of HSV in clinical specimens of low infectivity. A more rapid technique was developed which involved a short-term tissue culture (24 h) on Lab-Tek chambers followed by staining with biotin-linked HSV antibody and avidin-fluorescein conjugate. Because of the high binding affinity of this system due to multiple binding of biotin to avidin and multiple attachment of biotin to the antibody molecule, the biotin-avidin fluorescent-antibody technique produced a quality of fluorescence far superior to that of the conventional fluorescent-antibody techniques. The tissue culture-biotin-avidin fluorescent-antibody method was as sensitive as the tissue culture-cytopathic effect test. This method provides an improved, more rapid test (26 h) for detecting HSV in clinical specimens.