Immune Adherence Hemagglutination Research Articles

Correlative expression of C3b receptors in the glomerulus and on erythrocytes

The reactivity of glomerular C3b receptors (CR1) measured by the number of sheep erythrocytes bearing C3b (EAC) that adhered to glomeruli in frozen sections, was compared with the reactivity of erythrocyte CR1, which was determined by immune adherence hemagglutination (IAHA), in 22 patients with renal and non-renal diseases. Among seven patients with primary glomerulonephritis whose erythrocytes were positive for IAHA, the reactivity of glomerular CR1 was high in five. In the remaining two, the reactivity of glomerular CR1 was low, accompanied by severe sclerotic glomerular changes. Erythrocytes from five of six patients with systemic lupus erythematosus were IAHA negative, and their glomeruli failed to produce adherence of EAC, even in three cases in which there were no detectable C3 deposits or histopathological changes. In the other nine patients without appreciable glomerular changes, the reactivity of glomerular CR1 was low in three along with negative erythrocyte IAHA, whereas the remaining six exhibited high CR1 reactivity both in glomeruli and on erythrocytes. The results indicate a close correlation between the expression of CR1 in the glomerulus and on erythrocytes.

Two cases of Mesangiocapillary Glomerulonephritis that show defective C3b receptor on erythrocyte

We examined CR 1 on the red blood cell in the patients with primary glomerulonephritis using immune adherence haemagglutination (IAHA) method, and we found 2 patients of Mesangiocapillary Glomerulonephritis (MCGN) with CR 1 deficiency on the red blood cell. The two patients were Nephrotic syndrome. One patient's serum was found to contain anti Nuclear antibody (ANA), and C3 Nephritic factor (C3NeF) causing hypocomplementaemia was detected in the other patient. The deficiency of CR 1 was found in one of the parents of both these MCGN patients. It is thought that the cause of CR 1 deficiency on the red blood cell is hereditary and that it might be influencing the pathogenesis of these MCGN patients.

Freezing and rejuvenation of human O erythrocytes for use in the immune adherence hemagglutination test.

A method was established to freeze selected human O erythrocytes and to thaw them as necessary for use in the immune adherence hemagglutination test. This method ensured interrun reproducibility and eliminated the necessity to screen blood donors (fresh cells) for acceptable C3b receptor site sensitivity.

1074 SAFETY AND IMMUNOGENICITY OF A LIVE ATTENUATED RHESUS MONKEY ROTAVIRUS VACCINE

Rotavirus is a major worldwide cause of acute gastroenteritis in children. We recently evaluated a rhesus monkey rotavirus (RRV-1) which shares neutralization specificity with human sero-type 3, as a potential vaccine candidate. We administered RRV-1 (106 TCID50) or placebo orally to 40 adult volunteers who had low or undetectable levels of serum neutralizing antibody under double-blind conditions. Volunteers were evaluated daily for signs and symptoms of gastroenteritis, and stools were examined for RRV-1 by ELISA and tissue culture. Administration of RRV-1 did not induce illness. RRV-1 was shed in the stools of 11/21 (53%) vaccinees for 1 to 6 days without transmission of RRV-1 to other subjects, although one placebo recipient shed wild-type rotavirus from a community-acquired infection. Fourfold or greater serum neutralizing antibody rises to RRV-1 were noted in all vaccinees (21/21) and in 1/18 placebo recipients. Serum antibody rises were also demonstrated by immune adherence hemagglutination in 20/21 volunteers who received RRV-1 and in 1/18 who received placebo, and by complement fixation in 17/21 who received RRV-1 and 0/17 who received placebo. Transient asymptomatic serum ALT and AST rises were seen with similar frequencies in RRV-1 (3/21) and placebo (5/19) recipients. Thus, RRV-1 appears to be attenuated and highly immunogenic in young adults, and warrants additional study in children.

Polymorphonuclear leukocyte C3b receptors (CRl) and its relationship to the expressin of erythrocyte CRl in patients with systemic lupus erythematosus

The defective C3b receptor (CRl) activity on erythrocytes is frequently seen in systemic lupus erythematosus (SLE) and leukemia. Defective CRl activity on erythrocytes in SLE is thought to be inherited as an autosomal codominant manner. CRl exsists on cell types other than erythrocytes, including neutrophils, eosionphils, monocytes, macrophages, B and some T lymphocytes, mast cells, and glomerular podocytes. The relationship of the expression of erythrocytes with other cell types is unclear. In this study we investigated the relationship of CRl activity between erythrocytes and polymorphonuclear leukocytes (PMN).The CRl activity on erythrocytes was measured by immune adherence hemagglutination (IAHA) using aggregated human IgG sensitized erythrocytes and guinea pig complement. Patients with SLE were grouped into two, group 1, those who possess CRI activity on erythrocytes, and group 2, those who showed defective CRl activity on erythrocytes.The activity of CRl on PMN was studied by; 1) rosette formation of PMN by EA (IgM) C3b, and 2) inhibition test of IAHA using EA (IgM) C3b lysate by PMN.Compared with healthy controls, SLE patients revealed significantly decreased rosette formation.

Low C3b receptor reactivity on erythrocytes from patients with systemic lupus erythematosus detected by immune adherence hemagglutination and radioimmunoassays with monoclonal antibody.

C3b receptor (CR1) on erythrocytes from 23 patients with systemic lupus erythematosus (SLE) and 124 normal controls was determined by immune adherence hemagglutination (IAHA) and radioimmunoassay. The binding of radiolabeled monoclonal anti-CR1 to erythrocytes and their lysate was distributed continuously in a wide range. The majority of SLE patients showed low binding by both assays. CR1 sites on erythrocytes were determined also by Scatchard plot analysis and standardized by the number of similarly determined lectin-binding sites that served as a measure of erythrocyte surface. The numbers of standardized CR1 sites were classified as high, intermediate, and low. Thirty-six percent of control subjects had high numbers of CR1 sites, 53% had intermediate numbers, and 11% had low numbers. Of SLE patients, the numbers of CR1 sites were high in 0%, medium in 52%, and low in 48%. Negative IAHA was found in 10 controls (8%), all of whom had low numbers of standardized CR1 sites. Among 13 SLE patients with negative IAHA, 11 had low numbers of CR1 sites and the remaining 2 had low intermediate numbers. IAHA, therefore, was particularly efficient in detecting the low numbers of CR1 sites in SLE, which would impair the disposal of circulating immune complexes and accelerate the development of tissue injuries.

Infectivity titration of hemorrhagic fever with renal syndrome virus: use of immune adherence hemagglutination for detection of virus growth.

Serial dilutions of hemorrhagic fever with renal syndrome viruses were inoculated into Vero-E6 cells in microplates. After 2 weeks of incubation, infected cells were disrupted by freezing and thawing, and virus antigens were detected by immune adherence hemagglutination. The infectivity titers of the virus as determined by this method were in close agreement with those obtained by the immunofluorescent antigen endpoint method. Then, a neutralization method was established. Japanese hemorrhagic fever with renal syndrome isolates, strains SR-11 and TR-352, were found to be distinct from Hantaan virus, strain 76-118, by the neutralization test.

Immunoglobulin-mediated shifting of immune complexes to increased complement-dependent solubility and immunoadherence

The effect of a monomeric polyclonal polyspecific IgG preparation (mIgG) for i.v. use on the capacity of fresh normal human serum (NHS) to inhibit precipitation of immune complexes of tracer-labeled bovine serum albumin (BSA) and anti-BSA (aBSA) rabbit antibody was investigated. Relative to heat-inactivated serum which showed no capacity to inhibit immune complex precipitation (CIICP), fresh NHS at a final dilution of 1:3 or 1:2 showed a 12 and 46% CIICP, respectively, with a BSA:aBSA ratio at equivalence. Addition of incremental amounts of mIgG dose-dependently enhanced CIICP of NHS to reach 63 and 90%, respectively, at a concn of 13.0 mg/ml mIgG. Human serum albumin instead of mIgG had no influence on CIICP of NHS indicating that the phenomenon was not dependent on non-specific protein-protein interactions. Although minimal amounts of BSA cross-reactivity could be demonstrated in mIgG, the extent of this activity was too small to explain the CIICP-supporting effect of mIgG as determined by comparing the effect of mIgG and aBSA serum on CIICP of fresh serum. Furthermore, absorption ofmIgG on BSA-Sephrose did not lead to impairment of its CIICP-supporting effect. A direct binding of tritium-labeled mIgG ( 3H-mIgG) to either insoluble BSA:aBSA complexes or latex-bound IgG (IgG-latex) was found. Incubation of 2.7 μg 3H-mIgG with 90 μg IgG-latex resulted in a specific binding of 5.2%. of the labeled compound. Such binding could be inhibited by unlabeled mIgG. Binding of mIgG was mediated through the Fab as well as the Fc part of the molecules: the 3H-Fc as well as the 3H-Fab fragment of mIgG bound to IgG-latex. The extent of binding of Fc was more than twice that of Fab and amounted to 70–90% of the binding found with intact mIgG. In an immune adherence hemagglutination system that depends on the interaction of complement-reacted immune complexes with C3b receptor-bearing erythrocytes, evidence was obtained that the mIgG preparation facilitated fixation of C3b to preformed BSA:aBSA complexes. Addition of 1.45mg/ml mIgG reduced the quantity of antigen-complexed C3b-bearing aBSA antibodies required to show a given agglutination by a factor of 3–4. We conclude that the facilitating effect of high doses of mIgG on complement-dependent CIICP of NHS and on immune adherence hemagglutination is the amplifying effect of complement after an initial interaction of antigen-nonspecific polyclonal polyspecific IgG with antigenreacted antibody.

Relative frequency of rotavirus subgroups 1 and 2 in Venezuelan children with gastroenteritis as assayed with monoclonal antibodies.

Monoclonal antibodies recently developed against the 42,000-dalton protein of two rotavirus strains were used in an enzyme-linked immunosorbent assay to determine the subgroup specificity of 252 specimens collected during a 45-month period from Venezuelan children with rotavirus gastroenteritis. Subgroup 2 rotavirus was shed by 85% of the children, whereas only 14% shed subgroup 1 rotavirus (one-half of them in a 3-month period). No differences were found in the occurrence of fever and vomiting between children shedding either rotavirus subgroup, but it appeared that the syndrome tended to last longer in children shedding subgroup 2 rotavirus. The monoclonal subgrouping enzyme-linked immunosorbent assay seemed to be more sensitive than an immune adherence hemagglutination assay, an enzyme-linked immunosorbent assay with polyclonal antibodies, or the electrophoretic analysis of RNA extracted from the virus. Overall, 99% of the specimens could be subgrouped by this assay.

Immune adherence hemagglutination test for detection of antibody to respiratory syncytial virus. Brief report.

An immune adherence hemagglutination test was established for detection of antibodies to respiratory syncytial virus. The test was shown to be as specific as and much more sensitive than the neutralization and complement fixation tests.

An Immune Adherence Assay for Discrimination Between Etiologic Agents of Hemorrhagic Fever with Renal Syndrome

Antigens of the viruses that cause hemorrhagic fever with renal syndrome (HFRS) and antibodies to these antigens were titrated in an immune adherence hemagglutination (IAHA) test and a CF test. Vero-E6 cells infected with the SR-11 strain of HFRS virus, an isolate from rats associated with a laboratory outbreak in Japan, and the 76-118 strain of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, were used as antigens in the IAHA, CF, and indirect immunofluorescent antibody (IFA) tests. Sera from patients with different types of HFRS were tested against both strains of virus. Titers of antibody detected by the IFA test were similar for the two strains, but the IAHA test discriminated between antibodies to the two types of HFRS virus. Antigenic differences between HFRS viruses were suggested by the results of the IAHA test.

C3b receptor (CR1) on erythrocytes in various diseases

Complement receptor for C3b (CR1) on erythrocytes was investigated in various diseases by immune adherence hemagglutination (IAHA) using aggregated human IgG. In normal controls, 21 out of 312 (6%) revealed defective CR1 reactivity, and there was no difference in the prevalence of defective CR1 reactivity between female ( 11 157 , 7%) and male ( 10 155 , 6%). Among various diseases examined significantly high prevalence of defective reactivity of CR1 on erythrocytes was seen in systemic lupus erythematosus (SLE) ( 22 30 , 73%) and malignancy of hematopoietic system, especially in acute myelogenous leukemia (AML) ( 6 11 , 55%).

A serological survey on rotavirus infection in dogs by immune adherence hemagglutination test.

A serological survey on rotavirus infection in dogs by immune adherence hemagglutination test.

Control of the immune complex-complement interaction by protein H of the alternative complement pathway and the natural inhibitor heparin.

The potential of the negative regulatory protein H of the alternative complement pathway convertase and of heparin in modulating the complement-dependent capacity of fresh serum to inhibit immune complex precipitation (CIICP) between bovine serum albumin (BSA) and rabbit anti-BSA as well as tetanus toxoid (TT) and human anti-TT was assessed. Additions of purified H to serum to increase the intrinsic concentration of this protein by 80% (BSA-anti-BSA system) and 190% (TT-anti-TT system) resulted in an inhibition of CIICP by 50% and 60%, respectively, whereas further increase of the amount of H lead to a decrease of its inhibitory activity. A similar effect was observed with heparin: at a concentration of 400 U/ml a 90% inhibition of CIICP in the TT-anti-TT system was obtained which diminished at higher heparin concentrations. The effect of H on C3 deposition to immune aggregates was assessed through its influence on C3b-mediated immune adherence hemagglutination; factor H dose-dependently suppressed such hemagglutination induced by aggregated human IgG or preformed TT-anti-TT complexes when added to the immune complex-fresh serum mixture at the outset but not after 45 min of the 37 degrees C incubation period which means that H inhibited more likely decoration of immune complexes with C3b than it did inhibit the interaction of C3b-coated immune complexes with erythrocytes. This suppressive effect of H was reversed by the simultaneous addition of the activating protein B. Complement-mediated binding of tritiated C3 to latex-bound human IgG was assessed and H was found to dose-dependently inhibit such binding with a maximal inhibition of 37% at a H concentration of 7 micrograms/ml.

Antibody assay for measles virus: Comparisons of immuneadherence haemagglutination, single radial haemolysis, enzyme immunoassay and haemagglutination inhibition

Immune-adherence haemagglutination (IAHA); single radial haemolysis and enzyme immunoassay (ELISA) versus haemagglutination inhibition (HAI) for detecting antibodies to measles virus were evaluated. The rank correlation computed according to Spearman for the sera of healthy individuals gave values of 0.66, 0.73, 0.72, respectively, for HAI-IAHA, HAI-single radial haemolysis (SRH) and HAI-ELISA. High percentages of accordance were observed as regards sera from both healthy and vaccinated individuals. In the case of vaccinated individuals the discordance analysis showed significant differences between HAI and IAHA, HAI and SRH, SRH and ELISA. The discordances indicate higher sensitivity of HAI and ELISA for the detection of seroconversion.

Live Attenuated Varicella Vaccine: The KMcC Strain in Healthy Children

The KMcC strain of live, attenuated varicella-zoster virus vaccine was studied in healthy children as a preliminary step toward varicella vaccine studies with this strain in children with leukemia. Forty-three children were immunized: 26 with the 40th passage vaccine and 17 with the 50th passage. Studies included surveillance for clinical reactivity, oropharyngeal excretion of vaccine virus, viruria, and viremia. Antibody responses were assayed by fluorescent antibody to membrane antigens and immune adherence hemagglutination. Cell-mediated immune responses were assayed by lymphocyte proliferation to varicella-zoster virus specific antigens. There was 100% seroconversion to the KMcC passage 40 and 50 vaccines (by fluorescent antibody to membrane antigen assay). Every child studied developed in vitro lymphocyte proliferation to varicella-zoster virus antigens. Papular skin lesions, probably vaccine related, occurred in 31% of the 40th passage vaccinees but in only 6% of the 50th passage vaccinees. The 50th passage KMcC strain vaccine is sufficiently immunogenic and safe to initiate clinical studies with leukemia patients.

Tumor-associated antigen from a human breast tumor cell line

Membrane-bound antigens were solubilized and purified from the continuously growing human mammary adenocarinoma cell line SW527. Two distinct breast tumor-associated antigens (BTA-1 and BTA-2) were obtained, as well as purified HLA antigens. The molecular weights of BTA-1 and BTA-2 were estimated at 112,000 and 65,000, respectively, by analytical polyacrylamide gel electrophoresis. The two mitogens also differed in carbohydrate content and hexose/pentose ratio. Antibodies to these antigens, raised in rabbits, were shown to be directed against SW527 surface markers by an immune adherence hemagglutination assay. Purified BTA-1 and BTA-2 reacted with rabbit antibodies to SW527 and with antibodies to a tumor-associated antigen previously isolated from tumor tissue freshly obtained from a patient with mammary adenocarcinoma, but did not react with anti- β 2-microglobulin. By indirect immunofluorescence assay, SW527 surface markers were shown to be recognized by antibodies in the serum of a patient with mammary adenocarcinoma; this reactivity was blocked by treatment of the serum with the tumor-associated antigen purified from fresh tissue. These results indicate that the antigens isolated from tumor cells in tissue culture and directly from human tumor tissue have common immunological properties.

Detection of Human Rotavirus by Reversed Passive Hemagglutination (RPHA) Using Antibody against a Cultivable Human Rotavirus as Compared with Electron Microscopy (EM) and Enzyme‐Linked Immunosorbent Assay (ELISA)

Detection of Human Rotavirus by Reversed Passive Hemagglutination (RPHA) Using Antibody against a Cultivable Human Rotavirus as Compared with Electron Microscopy (EM) and Enzyme‐Linked Immunosorbent Assay (ELISA)

EBV-IgA and new heterophile antibody tests in diagnosis of infectious mononucleosis.

An evaluation has been made of the EBV-IgA tests and the immune adherence hemagglutination assay (IAHA) as compared with standard diagnostic procedures in 119 serial sera from 22 clinical and 113 sera from 42 subclinical cases of EBV infectious mononucleosis. EBV-IgA antibody was demonstrable in 86.4 per cent of patients using EB3 cells as antigen and in 68.2 per cent with P3/HRIK cells. For heterophile antibody the IAHA test was more sensitive, gave higher titers, and was positive longer than the standard absorbed horse or sheep RBC tests in both clinical and subclinical EBV infections.

Natural and experimental infection of woodchucks with woodchuck hepatitis virus, as measured by new, specific assays for woodchuck surface antigen and antibody.

Solid-phase radioimmunoassays for woodchuck hepatitis virus (WHV) surface antigen (WHsAg) and antibody to it (anti-WHs) were developed. The test for WHsAg could detect as little as 10 ng/ml. In both tests it was necessary to employ radiolabeled WHsAg instead of anti-WHs as the probe because the latter appeared to be labile to the conditions of labeling. The tests were used to characterize naturally acquired and experimental WHV infections of woodchucks. Forty-three of 72 wild-caught woodchucks had serological evidence of WHV infections; 16 of these resulted in chronic infection, and the remainder were self-limiting. All chronically infected animals were positive for WHsAg and DNA polymerase activity. During 3 years of observation, 11 of the 16 WHsAg-positive animals and 3 of the 27 anti-WHs-positive animals, but none of the 21 uninfected animals developed hepatocellular carcinoma. Seroconversion, possibly resulting from infection with WHV, was documented in a chimpanzee inoculated with WHV. An immune adherence hemagglutination test for WHsAg was also developed by using anti-WHs of chimpanzee origin as a reagent, but the test was not useful for detecting anti-WHs of woodchuck origin because of the lability of the latter.