Serological Distinction Research Articles

The Genetic Basis of Human Vh4 Gene Family‐Associated Cross‐Reactive Idiotype Expression in CD5+ and CD5‐ Cord Blood B‐Lymphocyte Clones

In a recent study we have observed a high frequency expression of cross-reactive idiotypes encoded by genes from the relatively small VH4 family of immunoglobulin heavy chain genes in cord blood B-lymphocyte lines. Furthermore, we have demonstrated a selective pattern of expression of two VH4-associated cross-reactive idiotype (CRI) in B-lymphocyte lines established from CD5+ and CD5- cord blood B-lymphocytes. There was a restricted expression of one CRI marker recognized by the 9G4 monoclonal antibody in lines established from CD5+ B-lymphocytes but not in those established from the CD5- population. In the current study we examine the molecular basis for the selective pattern of CRI expression. Nucleotide-sequence analysis of functional immunoglobulin heavy chain (IgH) gene rearrangements in three CD5+ lines expressing the CRI recognised by 9G4 reveal that all use a single gene from the VH4 family, the V4.21 gene. However, all three lines have distinct third complementarity determining regions (CDR3) implying different clonal origins. In contrast, four cord blood cell lines (two established from CD5+ B-lymphocytes) expressing the CRI recognized by MoAb Lc1 have functional IgH gene rearrangements involving two different genes from the VH4 family, the V71-4, and V2-1 genes. Antigen specificity analysis reveals that all three 9G4-reactive lines produce antibodies that react with the I and/or i red blood cell carbohydrate antigens. These data suggest that the distinction in VH4 gene use in CD5+ B-lymphocytes in cord blood results from a selection process in vivo that shapes the repertoire of CD5+ B-lymphocytes. This study extends recent observations that the monoclonal anti-CRI antibodies 9G4 and Lc1 are markers of two distinct subgroups of proteins encoded by two subsets of genes within the VH4 family. Furthermore, it appears that amino acid residues in framework region one and complementarity determining region two are critical for the expression of the cross reactive idiotypes and the serological distinction between the two subgroups of proteins.

Diagnosis of infection with human T-lymphotropic virus type II (HTLV-II) in Norwegian HIV-infected individuals

Sera from 298 HIV-infected individuals from Southern Norway were examined for antibodies against HTLV. 30 sera (10.1%) were HTLV-II positive and 1(0.3%) HTLV-I positive. 25 of the HTLV-II infected subjects were intravenous drug abusers (IVDAs), giving a prevalence of HTLV-II infection of 24.5% in this group. Examination of blood samples by polymerase chain reaction followed by restriction enzyme analysis or sequencing confirmed the serological diagnosis. To evaluate current screening and verification HTLV tests, 44 sera were examined using a gelatin particle agglutination test, 5 different enzyme-linked immunoassays (ELISA) and 4 Western blots (WB). While earlier ELISAs and WBs were inadequate, a recent ELISA and WB including recombinant envelope glycoproteins from both viruses permitted serological diagnosis and distinction between HTLV-I and HTLV-II. Thus, HTLV-II now spreads among IVDAs in a North-European country. Health authorities in other countries should estimate the magnitude of the problem to decide upon measures to avoid transmission through blood transfusion.

Prevalence of IgA receptors in clinical isolates of Streptococcus pyogenes and Streptococcus agalactiae; Serologic distinction between the receptors by blocking antibodies

Group A and B streptococci ( Streptococcus pyogenes and Streptococcus agalactiae) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding epitopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.

Prevalence of IgA receptors in clinical isolates of Streptococcus pyogenes and Streptococcus agalactiae: serologic distinction between the receptors by blocking antibodies.

Group A and B streptococci (Streptococcus pyogenes and Streptococcus agalactiae) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding eptiopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.

Serologic distinction between human T‐lymphotropic virus (HTLV) type I and HTLV type II

In November 1988, the Food and Drug Administration approved reagents for serologic screening for human T-lymphotropic virus type I (HTLV-I) infection in blood donors and patients suspected of having HTLV-I-related illnesses. These reagents are able to detect HTLV type II (HTLV-II), a close relative of HTLV-I with no known pathologic effect. The distinction between the two forms of HTLV is important to the donor and to any recipient of blood containing HTLV. The application to sera from 38 seropositive blood donors and 2 recipients (37 "confirmed" positive and 3 indeterminate by Western blot) of two methods (Western blot and peptide enzyme immunoassay) for serologic distinction between HTLV-I and -II is described. These results were compared to those from polymerase chain reaction (PCR) analysis of HTLV proviral DNA obtained from donor peripheral blood mononuclear cells. The peptide enzyme immunoassay was found to be less sensitive than the Western blot, but completely concordant with PCR results when differential reactivity could be established. The Western blot pattern showed complete diagnostic concordance with the samples with confirmed-positive serologic tests, but was incorrect in two HTLV-II-infected donors with indeterminate serologic tests. Thirty-three (89%) of the 37 individuals from this predominantly Native American and Hispanic group of blood donors were found to have HTLV-II. These findings confirm and extend previous reports that HTLV-I infection may be less common, and HTLV-II infection more common, than previously believed. The peptide enzyme immunoassay can provide most individuals who have positive results with the HTLV-I/II screening test with serologic distinction between HTLV-I and HTLV-II.

Recombinant peptides derived from the env-gene of HIV-2 in the serodiagnosis of HIV-2 infections

We produced recombinant envelope-derived peptides of HIV-2 for use in a diagnostic enzyme-linked immunosorbent assay (ELISA) or Western blot by expressing several restriction enzyme fragments from the env gene of HIV-2 in the bacterial fusion vector pEX-3. On Western blots, 17 out of 18 anti-HIV-2-positive sera available to us reacted strongly with those recombinant peptides which were derived from, or extended into, the transmembrane protein of HIV-2. In contrast, recombinant peptides derived from the external envelope glycoprotein were only weakly recognized by two sera. We observed a cross-reactivity of some human sera containing antibodies to HIV-1 with the HIV-2 peptides derived from the transmembrane protein of HIV-2. In spite of this cross-reactivity, a serological distinction between anti-sera to HIV-1 and HIV-2 can be attempted by simultaneous testing in ELISA on recombinant peptides derived from the transmembrane protein of HIV-1 and HIV-2.

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO‐associated antigens

SummaryA method is described for obtaining from plants partially purified preparations of mycoplasma‐like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC‐labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross‐reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.

Diagnosis of infection caused by Chlamydia pneumoniae (strain TWAR) in patients with "ornithosis" in southern Sweden 1981-1987.

Serological distinction between ornithosis and infection by Chlamydia pneumoniae (strain TWAR) was attempted by microimmunofluorescence (MIF) of 156 cases previously considered as ornithosis on the ground of a positive complement fixation test with a chlamydia group antigen. At least 23% of these cases were TWAR infections according to the results at retesting by MIF. The highest frequency of TWAR infections was demonstrated during 1981 with a decreasing number and proportion thereafter. We contend that the sudden increase in the reported number of ornithosis cases in 1981 may have been caused by TWAR infections which seem to vary in incidence over time. Family clusters of respiratory infections were much more common with TWAR infections than with ornithosis.

Structure and expression of the Lyt-3a gene of C.AKR mice.

The mouse Lyt-3a gene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5' flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3a gene and the cDNA sequences reported for Lyt-3b (Nakauchi et al. 1987. Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3a encodes serine and Lyt-3b encodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5' to the Lyt-3a gene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3' to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3a gene together with a cloned Lyt-2a gene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk- and BW5147 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Revised Group Classification of the Genus Spiroplasma (Class Mollicutes), with Proposed New Groups XII to XXIII

Fourteen spiroplasma strains, primarily of insect origin, were analyzed according to criteria previously proposed for description of new serogroups of the genus Spiroplasma. When tested by reciprocal metabolism inhibition, growth inhibition, and deformation serological procedures, 12 of the strains were serologically unrelated to each other and to representative strains previously assigned to groups I to XI and subgroups I-1 to I-8. Examination by dark-field and electron microscopy indicated that each of the 12 strains possessed morphological features typical of spiroplasmas (helicity, motility, lack of a cell wall, and absence of periplasmic fibrils). All strains were resistant to 500 U of penicillin per ml and catabolized glucose but were unable to hydrolyze urea. Ability to hydrolyze arginine varied among strains. The guanine-plus-cytosine contents of the deoxyribonucleic acid of the 12 strains varied from 24 to 29 mol%. Two other strains (MQ-6 and Ar-1357) shared only a partial serological relationship to strain CC-1 (group XVI), suggesting that this group may consist of an assemblage of heterogeneous serovars. On the basis of the unique serological distinctions and other properties reported herein, we propose that the 12 representative strains be assigned new consecutive group designations XII to XXIII.

Genomic Analysis of the Human B-Lymphotropic Virus (HBLV)

The human B-lymphotropic virus (HBLV) has a double-stranded DNA genome of greater than 110 kilobase pairs, which is consistent with its morphological classification as a herpesvirus. A 9000-base pair cloned probe of HBLV detected specific sequences in DNA and RNA of infected cells but did not hybridize to the genomic DNA of other human herpesviruses including the Epstein-Barr virus, human cytomegalovirus, herpes simplex type I, and varicella-zoster virus. Conversely, while probes obtained from each of the known human herpesvirus readily detected the homologous viral DNA, they did not hybridize to genomic HBLV DNA. This evidence, in addition to serological and morphological distinctions and the biological effects of this virus demonstrate that HBLV is a novel human herpesvirus.

Pathogenicity and antigenicity of eleven isolates of chicken anaemia agent (CAA)

Pathogenicity for chicks of 11 isolates of chicken anaemia agent (CAA) isolated in Japan was examined. Each strain produced aplastic anaemia in 100% of chicks when inoculated at 1 day of age with mortalities ranging from 20 to 90%. Incidence of anaemia in chicks inoculated at 7 days of age varied from 0 to 87.5% according to the isolates, but no chick died. The susceptibility of chicks at various ages against the G1 (Gifu-1) and A2 strains was compared. Both strains produced anaemia in 100% of the chicks when inoculated at 1, 2, 3 or 4 days of age. Half of the chicks inoculated with the A2 strain at 8 days of age showed anaemia, but none of those inoculated with the G1 strain did. No chick showed anaemia from either strain when inoculated at 14 days of age. The antigenicities of the isolates were compared by cross neutralisation and indirect immunofluorescent antibody methods. No serological distinction was recognised among the isolates.

Serological distinction of A antigen between red blood cells and saliva in blood grouping of blood and body fluid stains.

A antigens of red blood cells and body fluids such as saliva, semen and sweat could be serologically distinguished using rabbit or guinea pig immune anti-A. As for antisera specific for red blood cell A, A+ rabbits were intravenously immunized with A group red blood cells. The resulting antisera were absorbed with O and B red cells and with A. Se saliva. The absorbed anti-A reacted with A red cells (titer 1:32) and was not inhibited with A. Se saliva. Guinea pigs were intramuscularly injected with A. Se saliva. Crude antisera contained agglutinins to human red cells which were abolished by absorption with A red cells. After absorption with O. Se saliva, the antisera were proved to have agglutinin activity with A group saliva using latex coated with A. Se saliva. A antigens from blood or body fluid stains could be distinguished by the elution method with these anti-A sera.

1H NMR studies of the Fc region of human IgG1 and IgG3 immunoglobulins: Assignment of histidine resonances in the C H3 domain and identification of IgG3 protein carrying G3m(st) allotypes

A 1H NMR study of the Fc region of human IgG1 and IgG3 immunoglobulins is presented. 1H NMR data were collected for the Fc and pFc' fragments obtained from human monoclonal IgG1 and IgG3 and also from rabbit IgG. The C2-H proton signal of His-435 in the C H3 domain of IgG1 was assigned by comparing the spectra of the Fc fragment of IgG1 with that of IgG3[G3m(g)] where there is a substitution of histidine by arginine at position 435. Chemical shifts and linewidths of the His-435 signal are quite different for the Fc and pFc' fragments. We suggest that His-435 is involved in the interdomain C H2-C H3 contact. Assignments of the C2-H proton signals of His-429 and His-433 in the C H3 domain were made on the basis of our previous 1H NMR results on the human light chain. NMR measurements clearly show that IgG3 Jir, which was isolated from a Japanese patient with cryoglobulinemia, has histidine at position 435 as in the case of IgG1. We also confirmed that IgG3 Jir reacts strongly with protein A. In marked contrast, IgG3[G3m(g)] does not bind protein A. These results show that binding of protein A to the Fc region is not subclass-specific and the existence of His-435 is a necessary condition for the protein A binding. It has recently been demonstrated that IgG3 proteins carrying G3m(st) allotypes. which are relatively common in Mongoloid populations but quite rare in Caucasians, bind protein A strongly. We confirmed that, as expected, IgG3 Jir carries G3m(st) allotypes. The pH titration curve of His-435 observed for IgG3 Jir is quite different from that for IgG1. This result makes it possible to identify by 1H NMR IgG3 proteins carrying G3m(st) allotypes. In the case of the pFc' fragments, His-435 gives identical titration curves for IgG1 and IgG3[G3m(st)]. This is consistent with the fact that no serological distinction can be made between the pFc' fragments obtained from these two types of proteins. We suggest that G3m(st)-specific antiserum differentiates IgG1 and IgG3[G3m(st)] by recognizing the difference in the way in which the C H2 and C H3 domains make contact with each other.

Analysis of the HLA-DRw8 haplotype: recognition by HTC typing of three distinct antigen complexes in Caucasians, Native Americans, and Orientals.

We have used seven HLA-D homozygous typing cells (HTC) in a comparative study of the DRw8 antigen complex in three racial groups. Three distinct HLA-D specificities were recognized, each associated with HLA-DRw8. Four of the HTC defined a DRw8-associated HLA-D specificity designated 8.1, one defined a specificity designated 8.2, and two defined a specificity designated 8.3. Each of the three specificities showed an association with a distinct racial group: Dw"8.1" in Caucasians, Dw"8.2" in Pacific Northwest Indians, and Dw"8.3" in Orientals. An informative primed lymphocyte (PLT) cell generated against a Dw"8.1" haplotype was able to distinguish 8.1 from 8.2 and 8.3. Using selected anti-DRw8 sera, a serologic distinction between 8.1 and 8.3 could also be made. It was thus possible, by using both cellular and serologic techniques in a comparative population study, to recognize at least three HLA-D-defined splits of the DRw8 haplotype.

Serological distinction of H antigen between saliva and red cell in forensic practice.

Rabbit antisera were prepared by immunization with O red cells or O.Se saliva. Anti-O red cells (anti-Hr) were diluted with O.Se saliva and each of diluents was adopted for elution method on blood or body fluid stains. Anti-Hr gave positive reactions only with red cell stains. On the other hand, anti-O.Se (anti-Hs) absorbed with O red cells kept activity reacting with latex coated with O.Se saliva. Anti-Hs reacted with various body fluid stains but not with red cell stain. It is considered that anti-Hr and -Hs are useful tools for individualization of blood or body fluid stains in forensic practice.

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies. II. Distinction between HLA-Dw2 and HLA-Dw12.

Three cytotoxic monoclonal antibodies, HU-11, HU-32, and HU-33, specific for human Ia-like antigens were used to analyze the two HLA-DR2-associated HLA-D specificities, HLA-Dw2 and HLA-Dw12. In the HLA-Dw2, DR2, MB1 homozygous B-cell line EB-CMG, the binding of radiolabeled HU-32 and HU-33 was strongly inhibited by the addition of nonlabeled HU-11, whereas no inhibition occurred in the HLA-Dw12, DR2, MB1 homozygous B-cell line EB-KT. To confirm this differential inhibition pattern further, F(ab')2 fragments were prepared from HU-11, and their ability to inhibit complement-dependent lysis mediated by HU-32 and HU-33 was assessed against a total of five homozygous typing cell lines homozygous for HLA-Dw2, DR2, MB1 or HLA-Dw12, DR2, MB1, including EB-CMG and EB-KT. Here again, the same differential inhibition pattern as that observed in the radiobinding inhibition assays was obtained. Thus, the data suggest that the two kinds of HLA-DR2-positive homozygous typing cell lines with distinct HLA-D specificity can be distinguished from each other by using solely serologic methods. This is the first clear-cut serologic distinction made between homozygous typing cells defining HLA-Dw2 and those defining HLA-Dw12, since no serologic means that enables one to distinguish one from the other has been available.

Enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to cysticerci of Taenia solium.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to cysticerci of Taenia solium has been developed that employs a pork muscle antigen control for the cysticercus test antigen, somewhat improving the serological distinction between infected and uninfected subjects. Serum antibody to cysticercus was detected in 79% of classical neurocysticercosis patients from Mexico, and in 61% of a group of cysticercosis patients with an unusually rapid invasion of the central nervous system in an endemic focus of disease in Irian Jaya. Antibody was absent in a group of healthy American laboratory personnel, and in residents of a non-endemic region of Papua New Guinea. Additional test on sera from patients with other parasitic diseases showed that cross-reactivity may occur in some patients with schistosomiasis, echinococcosis, and possibly angiostrongyliasis; however, these parasites are not known to cause human infection in Irian Jaya.

Serological distinction between DR antigens and lymphocyte activating determinants.

The Burkitt lymphoma (BL)-derived, HLA-DR antigen positive B cell line, EB1, is a consistently low stimulator in MLC. A rabbit antiserum raised against the strongly stimulating BL line DAUDI, after appropriate absorption with EB1, inhibits MLC stimulation by both B cell lines and allogeneic lymphocytes, whilst lectin-induced proliferation is not significantly affected. Indirect immunofluorescence and 125I-staphylococcal protein A binding to cells pre-incubated with this antiserum suggest that the antigen is present on both peripheral B and T cells, as well as on B lymphoblastoid and myeloma lines. We suggest that this antiserum is directed against lymphocyte activating determinant(s) (LADs) and that these are distinct from the serologically defined DR antigens.

SEROLOGICAL DIFFERENTIATION OF CONGENITAL AND ACQUIRED CYTOMEGALOVIRUS INFECTIONS DETECTED IN INFANCY

Abstract. A serological investigation on infants with incidentally detected cytomegalovirus (CMV) excretion was made in an attempt to differentiate between congenital and acquired infections. Generally, each of the patients, 0–12 months old at the detection of CMV‐excretion, was studied by indirect immunofluorescence (IIF) test for IgM‐antibodies in cord serum and by complement fixation (CF) test performed on cord serum and a number of sera drawn after the detection of CMV. Two out of 4 patients with virologically confirmed congenital CMV‐infection, as shown by positive virus isolation within 1 week of age, had a positive CMV‐IgM‐test in cord serum. One of these two children also had a persistently high CMV‐CF‐titer from birth until 2 months of age, indicating congenital infection. In the remaining 46 cases, all with the CMV‐excretion detected after 3 weeks of age, in half of the cases after 5 months, no positive IgM‐reaction was recorded in cord serum. No persistently high CF‐titer could be demonstrated among 13 out of the 46 patients from whom sera were drawn at birth and at 1–4 months. Six out of these 13 patients had a CF‐titer rise after the period of 1–4 months, indicating acquired infection. However, also one of the congenitally infected children had a similar titer increase. Many patients lacked characteristic serological patterns, some of them in spite of access to sera drawn at birth, as well as at 1–4 months of age and later on. It could be concluded that the possibility of making a serological distinction of congenital and acquired infant CMV infection found, by chance, during infancy is limited.