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Three previously defined antigenic subsets of C3 (C3(S), C3(D) alpha and C3(D)beta) clearly distinguish native C3 from C3 that has been denatured in the presence of sodium dodecyl sulphate (C3-SDS): C3-SDS expresses antigens of all 3 subsets, while native C3 only expressed C3(S) antigens. The radioimmunometric assessment performed in this study revealed that 90% of the C3(S) and C3(D)alpha and 70% of the C3(D)beta antigens demonstrable in C3-SDS also was expressed by physiologically bound C3b on sheep erythrocytes. The antigenic properties of bound C3b were not shared by soluble C3b, which (like soluble C3) only expressed C3(S) antigens, nor did soluble C3b 'spontaneously' released from complement-treated target cells express antigens other than C3(S). We therefore conclude that the expression of C3(D) antigens, under physiological conditions of activation, reflects a conformational modification unique for bound C3b and occurring to an extent comparable to that of C3 in the presence of a strong denaturant. Additional studies further revealed that the antigenic profile of bound C3b is determined by a heterogeneous population of molecules differing in their relative expression of the C3(S), C3(D)alpha and C3(D)beta subsets.

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis immunoblotting.

A mixed culture in which cellulose is capable of being converted to methane and carbon dioxide was obtained from an inoculum procured from a sewage-treatment plant and maintained in a synthetic medium containing tissue paper and an inorganic salt and vitamin mixture. The culture was tested for its ability to degrade 12 different paper and cotton products under batch conditions in 3-ℓ anaerobic fermenters. This culture degraded 6–8 mmol/ℓ per week of cellulose, expressed as glucose equivalents, with total gas yields of 0.3 m3/kg of cellulose degraded. The gas produced contained between 56 and 59% of methane. Maximum cellulose degradation occurred at chemical oxygen demand: nitrogen: phosphorus level of 80:5:1 and was adversely affected by high stirring rate. Also the presence of higher proportions of lignin in cellulose products adversely affected the ability of this culture to degrade cellulose.

Antigenic competition is known to be a widespread phenomenon when using crude extracts of antigens (e.g., Escherichia coli cytoplasmic proteins) for immunization. This non-specific form of immune suppression can be partially overcome by passive immunization with antibodies against dominant antigens (which are the suppressive molecules) before injection of the antigenic mixture. Blocking these immunodominant antigens or antigenic determinants by a passively administered antibody permits antibody responses against hitherto weakly or non-immunogenic molecules.

SUMMARYTo assess the potential of fomites and environmental surfaces as vehicles in the transmission of rotaviral diarrhoea, disks (1 cm diameter) of various porous and non-porous materials were contaminated with about 105plaque-forming units of the Wa strain of human rotavirus (HRV) suspended in faecal matter. The contaminated disks were then held for 10 days at either room temperature (22±2 °C) or 4°C with the relative humidity (RH) at the high (85±5%), medium (50±5%) or low (25±5%) level. Survival was longer on non-porous surfaces at the lower temperature and at lower humidity. In contrast, survival on porous surfaces was very variable; better on cotton-polyester than on poster card or paper currency on which HRV survived very poorly. These results suggest that under the right environmental conditions, HRV-contaminated objects could play a role in the transmission of rotavirus infections in hospitals, nursing homes and day-care centres.

Conventional cleavage of linked polypeptide chains by heating in SDS can so alter molecular structure as to interfere with antibody binding, on which both immunoprecipitation and 'western blotting' depend. As an alternative, gentle treatment with acid at room temperature or at 0 degrees C was effective in separating the alpha and beta chains of human MHC Class II glycoprotein dimers and proved superior in terms of preservation of at least one labile epitope on the beta chain.

LAV/HTLV-III has been closely linked to the acquired immunodeficiency syndrome (AIDS). We have studied and correlated the prevalence of AIDS-associated retrovirus and retroviral antibodies in several groups of male homosexuals from Greenwich Village. Retrovirus was detected in cultured peripheral blood lymphocytes by testing for reverse transcriptase (RT) and confirmed by establishment of virus-producer cell lines, and electron microscopy. Seventy-six percent of patients with AIDS, 93% with AIDS-related complex (ARC), 69% with generalized lymphadenopathy (LAS), and 35% of asymptomatic homosexuals were positive for virus in the RT assay. Transmission of the virus from RT-positive lymphocytes into the CEM cell line was successful in 10 of 11 randomly chosen cases. No virus isolates were obtained from lymphocytes of 8 heterosexual individuals. Serum antibodies against AIDS-associated virus were detected by indirect immunofluorescence assay and confirmed by Western blotting, using an LAV/HTLV-III-producer cell line, LAV-N1, which we established. LAV/N1 virus was purified by ultracentrifugation through sucrose gradient and the pattern of its proteins was determined by SDS-gel electrophoresis and Western blotting using sera from an AIDS patient. The major polypeptides of LAV/HTLV-III (19, 25-27, 32, 42 and 54 kilodalton) were present. These proteins did not react in Western blots with sera positive for Adult T cell leukemia virus (ATLV). thus, LAV-N1 and ATLV were not antigenically related. In our assay for LAV/HTLV-III antibodies, 18 (100%) of patients with AIDS, 13 (100%) of patients with ARC, 24 (69%) of 35 patients with LAS and 9 (39%) of 23 asymptomatic homosexuals were sero-positive. Heterosexual controls were negative. All IF-positive sera tested by Western blot contained antibodies against specific viral proteins. High titers (greater than or equal to 1:1280) of serum antibodies against LAV/HTLV-III virus were detected in 71% of AIDS patients, 62% with ARC, 38% LAS and 13% among asymptomatic homosexuals. Our data show that the presence of LAV/HTLV-III antibodies correlates with the presence of infectious virus. Antibody titers may also correlate with progression toward AIDS.

Previous studies have shown that in sickle trait (AS) donor blood very little sickling is visible. AS donor blood was evaluated by a method of blood filtration, which would be sensitive to intracellular gellation of S hemoglobin which might not be evident morphologically. Blood was collected from normal and sickle trait subjects into CPD solution and stored at 4 C. Blood was removed over a 21-day period and filtered through paper filters at unit gravity and also examined microscopically. Sickling was not evident in saline wet preparations, but was seen in each AS sample in 10 per cent formalin-saline. At 4, 25, and 37 C, AS blood filtered less rapidly than did normal (P < 0.01). AS blood filterability became progressively prolonged as temperature rose; conversely normal blood filterability shortened with increasing temperature. Over 21 days, AS blood remained less filterable than normals. AS samples with high hemoglobin S percentages filtered significantly more slowly than did those with less S hemoglobin. Recognizing that these studies refer only to blood in vitro, no conclusions are drawn with respect to reversibility of the altered filterability or any possible clinical implications.

The health effects of both indoor and outdoor air pollutants are of increasing concern. The health effects of outdoor air pollutants traditionally have been assessed through measurements of lower respiratory tract changes. However, it has been shown that one outdoor air pollutant, sulfur dioxide, decreases nasal mucus flow and increases nasal airway resistance. Along with cigarette smoke, indoor air pollutants such as formaldehyde, cadmium, and ammonium or sulfate ions have been shown to alter upper airway mucociliary function. Emissions from wood stoves are known to irritate the upper airways. Measurement of nasal airway resistance using posterior rhinomanometry allows quantification of nasal function. This technique recently has been used to demonstrate that adolescents with allergic asthma have increased work of breathing after inhalation of 0.5 ppm sulfur dioxide. Another study using posterior rhinomanometry showed that clerical workers had increased work of breathing after exposure to carbonless copy paper as compared with bond paper. This brief review of upper respiratory tract changes after pollutant exposure should serve as a reminder that a complete clinical history must include questions designed to ascertain the patient's exposure history to both outdoor and indoor air pollutants. These exposures can have a major impact on the health of the upper respiratory system.

The antigenicity of human syncytiotrophoblast membrane (TM) was analyzed using rabbit antiserum raised against TM. From immunoblot analysis, about ten protein bands in TM were recognized by the anti-TM. These included placental alkaline phosphatase and TM-bound albumin. From limited antigenic specificity studies, these antigens, with the exception of albumin, were not detectable in membranes of liver, kidney, heart, and erythrocyte, or in human normal serum. Therefore, these antigens appear to be placental membrane specific. Analysis of lymphocyte membrane by Immunoelectrophoresis, crossed Immunoelectrophoresis, and immunoblot techniques revealed that a single protein band, designated “40 kDa,” was cross-reactive with the anti-TM antiserum, indicating that this antigen is shared commonly between placenta and lymphocyte membranes. Experimental evidence suggesting that the 40-kDa membrane antigen is probably a unique trophoblast-Iymphocyte cross-reactive antigen is based on the following observations: 1) the 40-kDa antigen was not detectable in liver, heart, and kidney membrane; 2) γ2-microglobulin (12,000 daltons) was not detectable in our TM preparation; and 3) the lymphocyte membrane showed only a single protein band at 40,000 daltons, not at 12,000 for γ-microglobulin.

The allergen composition of moulds (Alternaria alternata and Aspergillus fumigatus) and mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) was studied by electroblotting. The allergens were separated by SDS-gPAGE and transferred to a nitrocellulose membrane by electroblotting. Specific IgE antibodies directed against mould and mite allergens were utilized as probes to detect the allergens. The bands were localized on the nitrocellulose membrane by means of enzyme- or isotope-labelled antibody. Circulating specific IgE antibodies in mould and mite sensitive patient sera were also analyzed and characterized with respect to their IgE specificity. Under appropriate experimental conditions this electroblotting technique could also be used for quantitation of individual allergens by scanning the isotope- or enzyme-stained nitrocellulose strips.

International audience; A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.

The karyotypic abnormality t(14;18)(q32;q21) is reported to occur in 75% of follicular lymphomas. This translocation results in the rearrangement of a putative oncogene bcl-2, which resides at chromosome 18 band q21 (the 18q21 gene). Using two human genomic DNA fragments cloned from the chromosome 18 band q21 as probes, we analyzed 65 uncultured human lymphoma samples by the Southern blot technique. The 18q21 gene was rearranged in 18 of 26 (69%) follicular lymphomas, 3 of 5 (60%) follicular lymphomas transformed to large cell lymphomas, 8 of 20 (40%) diffuse large cell lymphomas (DLCLs), and 2 of 7 (29%) small noncleaved cell lymphomas (SNCs). Our analysis detected rearrangement of the 18q21 gene in 10 of 13 (77%) cases in which the t(14;18)(q32;q21) translocation was found by cytogenetic techniques. Our analysis also proved helpful in difficult karyotyping situations: (a) identifying the donor chromosome fragment as chromosome 18 band q21 in 4 of 9 (44%) cases that cytogenetically displayed a 14q+ chromosome of unknown origin, and (b) identifying a rearrangement of chromosome 18 band q21 in 12 of 18 (67%) cases that cytogenetically yielded no cells in metaphase. We also demonstrated three cases of submicroscopic rearrangement of the 18q21 gene. In our studies, patients with DLCLs and rearrangement of the 18q21 gene had a significantly higher incidence of extranodal involvement when compared with patients with DLCLs and no 18q21 gene rearrangement (P = 0.03).

The coliform flora of a pulp and cardboard mill that uses birch as the raw material and ammonium sulphate as the process chemical was studied. Escherichia coli was observed to multiply in the mill. It persisted as the dominant thermotolerant coliform in the effluent. Klebsiellae were encountered among total coliforms only. The E. coli strains isolated had the biochemical characteristics and maximum growth temperatures typical to the species. However, serotyping and hemolysin test differentiated these strains from pathogenic and fecal E. coli.

All AIDS retroviruses isolated from different patients have shown degrees of heterogeneity as defined by restriction fragment polymorphisms. Despite this variability, all these virus isolates share a number of structural features, including immunological cross-reactivity of virally encoded proteins. In this paper, we compare restriction patterns of integrated proviral DNA from viral isolates of patients belonging to different geographical groups, at risk or not for the disease. We confirm the existence of different clones in the same isolate of the Lymphadenopathy-associated virus (LAV) and of Human T lymphotropic virus (HTLV-III), which are identical. One of these forms is very similar to a Haitian isolate, as well as to an isolate from an early recognized New York case, suggesting a common origin for these viruses. More variation is apparent with Zairian viral isolates, and one of two clones found in a virus from a child who received an allogeneic bone marrow transplant. Greater disparity was also found in the restriction pattern of an isolate from an individual belonging to none of the so-called high-risk groups. We also show that this variation occurs mainly but not only in the env region of the genome, as previously described.

An immunobinding assay is described that tests antigens attached to nitrocellulose paper against antisera contained in microtiter plates. In comparison to a conventional microtiter plate radioimmunoassay, the nitrocellulose paper radioimmunoassay is clearly superior in both antigen attachment and antibody binding. Studies using bovine serum albumin and human IgG demonstrated superior antigen attachment extending from 5-fold in a physiological solution, to 50-fold in 50% fetal calf serum, to over 1000-fold in detergent solutions. With titrations using a rabbit anti-human IgG serum, the antibody binding in the nitrocellulose paper radioimmunoassay averaged over 5 times the binding in the microtiter plate radioimmunoassay. The nitrocellulose paper radioimmunoassay was also modified to quantitate human IgG. With this assay, 15 pg of human IgG inhibited the antibody binding by 50%. The nitrocellulose paper radioimmunoassay is easy to perform, and, since it combines the antigen-binding properties of the nitrocellulose paper with the convenience of assaying samples in microtiter plates, this assay should prove useful for investigating the many antigens that attach to nitrocellulose paper.

In order to examine how rat erythrocytes stimulate erythrocyte autoantibody production at the molecular level, we have identified rat erythrocyte antigens by immunoprecipitation and western blotting using monoclonal antibodies and antisera. A novel non-radioactive immunoprecipitation technique was used, which employed biotin as a label and a luminescent detection system. The new method was validated by comparison with conventional immunoprecipitation using 125I. Glycophorins of relative molecular mass (Mr) 81,000 and 38,000 were found to be the major antigenic components of rat erythrocytes, while band 3 (the most abundant erythrocyte membrane protein) was not recognized by rat-specific antibodies. The same surface antigens were recognized by sera from mice producing erythrocyte autoantibodies and by sera from mice in which autoantibody production was suppressed. Nine other minor rat-specific antigens were identified by blotting, ranging in Mr from 23,000 to 147,000. Analysis of the integral membrane proteins of rat and mouse erythrocytes by sodium dodecyl sulphate (SDS) electrophoresis followed by silver or periodic acid-Schiff (PAS) stains revealed differences between the glycophorins, but not between rat and mouse band 3. Thus, the major antigenic differences correspond to discernible biochemical differences between rat and mouse erythrocyte sialoglycoproteins.

Using the protein immunoblot technique, antibodies to an Epstein-Barr virus-induced 92 kD polypeptide (EBNA-2) were more frequently present in the sera of patients with rheumatoid arthritis and their consanguineous relatives when compared with a control group. No association of anti-EBNA-2 antibody with the HLA-DR antigens was observed.

Horse anti-alpha-fetoprotein was coupled to CM-cellulose discs by a modified carbodiimide reaction. The resulting coupling CM-discs were used in solid-phase radioimmunoassay of human alpha-fetoprotein. The sensitivity of these discs and conventional BrCN activated filter paper discs coupled anti-alpha-fetoprotein was approximately the same. A fair correlation between the alpha-fetoprotein levels determined by both methods was observed. The coupling procedure with carbodiimide is simple and the use of hazardous BrCN is eliminated.

Sera of 60 allergic persons and 20 healthy RAST-negative control persons were examined using immunoblotting to establish the presence of grass pollen-specific antibodies. These antibodies were depicted by an enzymatic, immunoglobulin class-specific indicator system. Seven bands of the timothy pollen extract proved to be major allergens. One of the 4 grass pollen-specific monoclonal antibodies tested (Bo 1) recognized the 7 major allergen bands. The possibility of employing monoclonal antibodies for the isolation of allergens is discussed.

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

A miniaturization of the immunoblot technique permitting the use of as little as 50 μl of antibody is described. This method does not require any special equipment and the whole experimental procedure (incubation, washing, detection) can be performed in the same chamber, with economy of reagents at all steps and no prenumbering of blotted strips. This method has been applied for screening the presence of antibodies in hybridoma culture supernatants and is of special interest for testing any scarce material (immune sera or antigens).

Monoclonal and polyclonal antibodies to the ABH blood group antigens were tested for their specificity to glycoproteins with ABH activity on immunoblots of solubilized erythrocyte membranes. Immunoblots were stained with monoclonal antibody G10 to the H type-2 carbohydrate structure or with commercially prepared monoclonal and polyclonal antibodies to A, B, and H blood group antigens. G10 antibody specifically stained antigens in the regions that contain the erythrocyte membrane bands 3 and 4.5; the staining was proportional to the expected H content of the erythrocytes (O greater than A2 greater than B greater than A2B greater than A1 greater than A1B). No specific staining was observed with membranes derived from Oh (Bombay) erythrocytes which lack the H type-2 structure. A commercially prepared monoclonal anti-H did not specifically stain erythrocyte membrane antigens. Monoclonal and polyclonal anti-A specifically stained bands from A and AB but not O, B, or Oh erythrocytes (A1 greater than A1B greater than A2 greater than A2B). Polyclonal anti-B serum specifically stained bands from B and AB but not O, A, or Oh erythrocytes (B greater than A2B greater than A1B). However, no specific staining was observed in tests with monoclonal anti-B. Monoclonal antibodies G10 and anti-A and polyclonal anti-A and -B blood typing sera will be useful in the further characterization of the molecular nature of the ABH antigens.

Two DNA probes isolated from the region 5′ to the HLA-B gene are described. One of these, pCH6, detects a TaqI polymorphism which is correlated with serological alleles of HLA-B. Both probes are used to show a high level of DNA sequence homology between the 5′ flanking region of HLA-B and HLA-C.

A sensitive immunoblot technique for platelet glycoprotein IIIa (GPIIIa) was used to analyze the platelets of patients living in Israel who meet the diagnostic criteria for type I Glanzmann thrombasthenia. When reacted with solubilized normal platelets, a rabbit antiserum to GPIIIa identified a major band at molecular weight (mol wt) 90,000 and three additional minor bands at Mr 110,000, 81,000, and 64,000. The major band could not be detected, and the minor bands were either markedly reduced or absent in the platelet samples from 14 of the 15 patients from the Iraqi-Jewish population. In contrast, in all four Arab patients tested, the major band was detectable, although at markedly reduced levels, and the minor bands were either markedly reduced or absent; an additional minor band at mol wt 47,000 was also present in the platelets from these patients. One Iraqi-Jewish patient had a unique pattern in which two of the bands were present but reduced and two were undetectable. We conclude that the protein defect, and thus presumably the genetic defect, causing Glanzmann thrombasthenia in the majority of patients in the Iraqi-Jewish population differs from that in the Arab population, and we confirm that there is considerable biochemical heterogeneity among the patients who meet the criteria for type I Glanzmann thrombasthenia.

The survival of gonococci on various materials contaminated with gonococcal pus and stored at room temperature was studied. Gonococci were recovered for up to 3 days from a wide variety of hard and soft materials. It is possible that gonorrhoea is transmitted nonvenereally more often than is usually acknowledged, and these results may have medicolegal and social significance.

When pilus+ Gc were introduced into a male subject's urethra, they gave rise to pilus+ variants whose pilin mRNAs differed from that of input Gc. The differences stemmed from the Gc genome's single complete pilin gene having undergone gene conversion by different partial pilin genes' sequences and by different length stretches of a single partial pilin gene. In some instances, the variant's pilin mRNA appeared to reflect two independent gene-conversion events that used sequences from two different partial pilin genes. The resulting variants' pilins exhibited antigenic differences compared with the pilin polypeptide of input Gc; these differences were discernible by immunoblotting with mAbs. Amino acid and antigenic changes occurred in a segment of the variants' pilin polypeptides that previously was thought to be conserved or constant in sequence.

Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.

The mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, previously reported to exhibit greatly reduced expression of human HLA class I and II antigens (DeMars et al., Hum Immunol 11:77, 1984), were analyzed by Southern blotting using class II cDNA and genomic clones as hybridization probes. All genomic sequences complementary to DR alpha, DR beta, DQ alpha, and DQ beta probes were absent from these mutants. DZ alpha genomic sequences were deleted as were the DP alpha 1 and DP beta 1 loci but the DP beta 2 and most, if not all, of the DP alpha 2 locus were retained. However, no RNA transcripts for either DP alpha 2 or DP beta 2 could be detected. The mapping of the deletion breakpoint within the DP cluster allows the orientation of the loci in the DP region with respect to the centromere as follows: centromere, DP beta 2, DP beta 1, DP alpha 1, (DQ, DR). In addition, the analysis of a set of DR-, DQ-, DP+ homozygous deletion mutants (721.82, 721.84, and 721.101) reveals a deletion breakpoint between the DQ alpha 1/DQ beta 1 loci and the DQ alpha 2/DQ beta 2 loci. These mutants retain DZ alpha genomic sequences, tentatively mapping the DZ alpha locus between the DQ and the DP region. The residual ability of the DR-, DQ-, DP- mutants (174 and 180)* to stimulate allogeneic and autologous lymphoproliferative responses must be attributed to expression of as yet unidentified class II antigens, or to non-class II antigens.

With a view to specifically suppressing the IgE isotype, rats of high (BN) and low (PVG.RT1u) IgE-responding phenotypes were immunized with a highly purified rat IgE myeloma (IR2) in an attempt to induce an anto-anti-IgE response. Rat IgE antibodies against epsilon determinants were detected in the serum of IR2-immunized animals using a solid-phase (plate) radioimmunoassay. The auto-anti-IgE antibodies detected were found to bind to IR2, to a second rat IgE myeloma (IR162) and to mouse monoclonal IgE but not rat IgG. The specificity of the anti-epsilon binding was shown by inhibition studies. The raising of an auto-anti-IgE response in PVG.RT1u rats severely depleted the serum level of circulating IgE for at least 8 weeks. In BN rats, immunization with IR2 caused marked fluctuations in serum IgE levels. The rats in both strains remained healthy throughout the experiment. The rate and route of IgE break down was not altered in anti-IgE-producing rats. The relevance of the present model in understanding and possibly controlling allergic disorders is considered.

We examined the interaction of complement component C3 with surface molecules on Trypanosoma cruzi. Five- to six-fold more C3 was bound to epimastigotes (Epi) than to metacyclic trypomastigotes (CMT) of strain M88. Epi and CMT were surface iodinated, then incubated in C8-deficient serum, and detergent lysates were applied to anti-C3 antibody that had been coupled to Sepharose. We found that 9.20-10.24% of applied 125I-Epi protein bound to anti-C3-sepharose, compared to 2.64% binding of 125I-CMT protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that C3 was attached to 125I-Epi protein by a covalent bond. Samples eluted from anti-C3-sepharose with hydroxylamine revealed a single, major, 72 kD band, suggesting that C3b attaches almost exclusively to the 72 kD glycoprotein of Epi by a hydroxylamine-susceptible ester bond. An antiserum was prepared from lysates of serum-treated Epi that had been affinity-purified on anti-C3-sepharose. This antiserum immunoprecipitated a single 72 kD component (gp72) from surface-iodinated Epi, and specifically recognized only gp72 from Epi in immunoblots. In contrast to the results with Epi, gp72 on CMT was not found to be an efficient acceptor molecule for C3 deposition. The results are the first to evaluate the acceptor site for C3 deposition on a parasite, and they show that gp72 on Epi, but not gp72 on CMT, serves as the preferential acceptor for C3 during antibody-independent alternative complement pathway activation.

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that posses phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.

Immunochemical studies (sequential immunoprecipitation followed by SDS-PAGE and two-dimensional gel electrophoresis) have shown that the monoclonal antibody (MoAb) 26.163 reacts with HLA-DR, DQ, and DP antigens. Testing with isolated alpha and beta chains of HLA class II antigens and immunoblot analysis also demonstrated that the determinant defined by the MoAb 26.163 is localized on beta chains and does not require their association with alpha chains for its expression. The MoAb 26.163 appears to be the first example of a monoclonal antibody with specificity for the gene products of HLA-DR, DQ, and DP loci.

Monoclonal antibodies against major allergens of the short ragweed pollen were produced by fusion of NS-1 cells with splenic cells from BaLB/C mice that had been immunized separately with the major allergens, AgE-B+E-C and AgK, of the short ragweed pollen. These monoclonal antibodies were detected by enzyme-linked immunosorbent assay and further characterized by immunoblot analysis using the crude extract and highly purified allergens of the ragweed pollen. Three monoclonal antibodies obtained by immunization with AgE-B+E-C, designated as 36-6, 27-2 and 48-5, reacted mainly with the beta (36-6) and alpha (27-2) subunit of AgE and both AgE and AgK (48-5), respectively. Two monoclonal antibodies obtained by immunization with AgK, 4-7 and 8-5, had a similar reactivity with AgE-B+E-C, AgK, AgK-A and AgK-B. In addition, however, antibody 4-7 also reacted with AgE-B2 as well as the 36- and the 24- to 22-kilodalton antigens of the crude extract. All 5 monoclonal antibodies were characterized as IgG1 subclass. Besides, monoclonal antibody 48-5 also showed cross-reactivity to components of sage pollen. Beyond academic interests, the monoclonal antibodies described here may also be useful in clinical allergy.

C5-deficient mice differed from C5-sufficient mice both quantitatively and qualitatively in C5 protein, C5 mRNA, and the C5 gene. C5-deficient protein was present as decreased amounts of an unprocessed, single-chain precursor. C5-deficient mRNA was decreased in amount and present in two forms, the smaller of which was the same as the single form in normal cells. Nuclei from both normal and deficient cells contained the larger form of C5 mRNA, and C5-deficient DNA demonstrated differences from the normal pattern on Southern analysis for two restriction enzymes. These data suggest that the primary transcript of the C5-deficient gene is abnormal, retarding the processing of the C5 mRNA, and that the C5-deficient mRNA codes for an abnormal protein.

Allergenic proteins were extracted from one silk batch that was imported to be used as filling material for bed mattresses and rugs. IgE and IgG antibodies to the extracted silk proteins were measured by RAST in sera of nine silk-sensitive persons as well as in sera of healthy control donors. Silk proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 polypeptides of molecular weights between 14 and 70 kilodaltons. By means of the immunoblot technique, IgE and IgG antibodies to the individual silk polypeptides could be detected. Sera of silk-sensitive persons contained high titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Sera of control donors contained low IgG antibody titers to a limited number of these polypeptides.

To study the possible involvement of T lymphocytes in Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) we analyzed the arrangement of the bcr gene in T cell and non-T cell samples of 12 CML patients. Although all the patients showed bcr rearrangements in non-T cell fractions, T cell populations lacked respective gene recombinations. Moreover, by Southern blot analyses using T cell receptor beta chain sequences our data indicate polyclonality of T cell samples from 11 of 12 cases; in one patient a clonal T cell population could be identified. These results suggest that T lineages of most Ph- positive CML patients are not derived from pluripotent stem cells involved in leukemogenesis and thus confirm previous investigations based on cytogenetic or glucose-6-phosphate dehydrogenase analyses. The demonstration of polyclonal T cell populations may reflect persistence of stem cells committed to differentiate only into T cells.

Human urinary erythropoietin has been highly purified by a combination of conventional purification methods and immunoadsorbent columns packed with hybridoma-produced antibodies against contaminants that seemed difficult to separate from erythropoietin by the usual means. By using the partially purified erythropoietin as an antigen, three hybridoma clones have been obtained that secrete monoclonal antibodies against erythropoietin. One of the clones has been quite stable, with a rapid growth rate and high production of antibody. Western blotting technique with monoclonal antibodies revealed occurrence of two species of erythropoietin. The monoclonal antibody will be useful as a probe for the purification of erythropoietin and for further studies of the hormone and its mechanism of action.

During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that (1) phosphorylated neurofilaments are protected against proteolysis, and (2) dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.

Autoimmune thyroid disease in man is commonly associated with autoantibodies against thyroglobulin, microsomes, and the TSH receptor, and the character and specificity of these antithyroid antibodies have been extensively utilized in investigating these conditions. In the present study we have asked whether other thyroid-related antigens exist, against which autoantibodies may be directed. A crude thyroid extract was separated by polyacrylamide gel electrophoresis followed by immunoblotting with serum obtained from patients with Graves' disease or Hashimoto's thyroiditis. Antibodies in sera from patients with Graves' disease and Hashimoto's thyroiditis reacted with many antigenic determinants in immunoblots of the thyroid membrane preparation (2000g supernatant). These determinants were disease specific in that sera from normals and patients with Addison's disease and rheumatoid arthritis did not react, but there was no difference between the patterns of reactivity with Graves' disease or Hashimoto's thyroiditis sera. Thyroglobulin produced two predominant bands of reactivity at 320 and 200 kDa, whereas purified microsomal antigen produced a triplet of bands around 105 kDa, when these preparations were reacted with appropriate autoimmune sera. Nonetheless, some sera produced additional bands with the microsomal antigen blots, indicating that some of the antigens which were detected using crude thyroid membrane remained in the microsome preparation to produce multiple antibody binding reactivities. We were unable to inhibit any of the antibody binding with TSH. Purification of individual thyroid antigens on the basis of their molecular weights should standardize current antibody assays and permit more detailed evaluation of the cellular immune responses in Graves' disease and Hashimoto's thyroiditis.

This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established double diffusion in gel for detection of anti-Scl-70 antibodies this ELISA has advantages.

Utilizing the principles of counterimmunoelectrophoresis, a technique was devised to immunologically identify non-denatured protein antigens resolved by polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred from polyacrylamide gel to antibody-containing agarose gel in a commercially available blotting apparatus. Detection of the antigen/antibody complex by Coomassie blue staining (in the ng range for unlabeled antigen) or autoradiography (in the pg range for radiolabeled antigen) established the sensitivity and specificity of this method. Similar sensitivity could be obtained following conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis after partial removal of the detergent and possible renaturation of proteins. Placement of a nitrocellulose sheet on the anodal side of the agarose gel produced a ‘negative replica’ (proteins/polypeptides not reacting with specific antibodies in the agarose gel). Counterimmunoblotting provides an additional method for molecular weight determination of protein antigens with sensitivity and specificity comparable to established nitrocellulose blotting (unlabeled proteins) or immunoprecipitation (labeled proteins) procedures.

A method for the clonal analysis of antibodies against high M.Wt. antigens is described. It involves isoelectric focusing, electrophoretic transfer of the focused antibodies to a nitrocellulose membrane and detection of the membrane-bound antibodies by overlay with radiolabelled antigen (reverse immunoblotting). The application of this technique to the clonal analysis of autoantibodies against thyroglobulin and DNA is described.

Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.

The possible risks of infection associated with the practice of licking envelopes, stamps and labels were investigated. Although pathogenic bacteria and viruses were not isolated from sample envelopes obtained from various sources, the gums used in manufacture were found to exert a protective effect against death from desiccation on the bacteria and viruses which had been introduced into them.Staph. aureus and Salm. paratyphi Bremained viable for several months in dried films of two out of four gums tested. An echovirus could be recovered from similar films for up to 30 days, and an adenovirus for up to 10 days.Bacterial multiplication occurred in the gum used for the manufacture of postage stamps. The comparison is drawn between the possible consequences of minimal contamination of this gum and of cans of corned beef in the light of recent studies.

A method has been developed for the assay of allergens and may be summarized as follows: 1. Filter paper discs are activated with BrCN. 2. The allergens are coupled to the reactive groups on the paper disc carrier. 3. IgE immunoglobulin, present in the sera of allergic patients, then attaches itself to the allergen coupled to the paper discs. 4. 125 I-labeled anti-IgE antibodies thereafter interact with the IgE molecules attached via the allergens to the paper carrier. Examples of the assay with the use of dog, cat, horse, cow, and timothy allergens are presented. The allergen assay method can conveniently be used to compare the "potency" of different allergen extracts and to check the extraction procedure, storage, and further treatment of allergens.