Immediate hypersensitivity to cockroach. Isolation and purification of the major antigens.

Crude cockroach extract elicited positive skin tests in 50% of patients with positive and in 4% with negative environmental history for cockroach exposure, suggesting a possible role of cockroach in perennial atopic disease. Three major allergens in crude American and German cockroach extracts have been identified using sequential purification steps on Sephadex G-75, diethylaminoethyl (DEAE) cellulose, and agarose gel electrophoresis. Cr-I elicits positive skin tests in 70% of patients sensitive to the crude extracts. It has a molecular weight of approximately 25,500 daltons, is highly acidic, and resists boiling for four hours. Boiling in 4 N acetic acid completely abolishes its allergenicity. The purified allergen elicits positive skin tests at a concentration of 3 mug/ml and is capable of inducing greater than 50% histamine release from sensitive leukocytes at 0.05 ng/ml. A second antigen, Cr-II, elicits positive skin tests also in approximately 70% of cockroach-sensitive individuals, has a molecular weight of approximately 63,000 to 65,000 daltons, and has similar heat stability and acid hydrolysis characteristics to Cr-I. A third, less well-characterized antigen, Cr-III, has a molecular weight less than 10,000 daltons and elicits positive skin tests in 30% of individuals sensitive to the crude extract.     

Rat transplantation antigens: I. Extraction and partial purification of a soluble antigen

A soluble histocompatibility antigen was extracted from normal rat lymphoid cells by exposure to an hypertonic KCl solution. The antigen was assayed by inhibition of monospecific cytotoxic (CT) alloantiserum and shown to have the specificity AgB 4 or Rt H1.1, the major locus private antigen of the dark agouti strain. Yields were ~2000 CT inhibitory units (ID₅₀) per 10⁹ cells. Gel filtration of the extract over Sephadex G-200 resulted in an approximate four-fold purification with a final concentration of 530 ID₅₀ units per mg protein. The major portion of the antigen appeared at the front of the inner bed volume but a minor included peak at about 65,000 mol wt. was also seen. Yields and specificity were greatly increased by (a) not washing the cell suspension before extraction (b) removing excess salt from the extract by dialysis rather than gel filtration (c) removing insoluble nucleic acids from the dialysed extract by ultracentrifugation.     

House dust, mite (D. farinae) and cockroach allergy in a midwestern population.

By using three different skin testing techniques and RAST, this study explores the relationship among house dust, mite (Dermatophagoides farinae) and cockroach allergy in atopic patients in the Kansas City area. Results suggest that the cockroach antigen may be as important as the mite in the etiology of house dust allergy in this population.     

Use of LOR-15C9 monoclonal anti-D to differentiate erythrocytes with the partial DvI antigen from those with either partial D antigens or weak D antigens

Historically, red blood cells (RBCs) with partial D antigens have been defined serologically by their pattern of reactivity with polyclonal and monoclonal anti-D. Although numerous variants have been described in tests with well-characterized monoclonal anti-D, definition remains difficult to ascertain serologically. RBCs of known partial D type were tested with LOR-15C9 (a monoclonal anti-D) and commercial anti-D by the tube indirect antiglobulin test (IAT), by micro typing system IgG gel cards, and by immunoblotting. By IAT, LOR-15C9 reacted strongly with DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs in addition to RBCs with common D antigens; weakly with DII, DNU, and DIIIb RBCs; and not at all with DIVa, DIVb, DBT, or R0 Har RBCs. Reactivity was variable (1+ to 4+), with RBCs classified as weak D (Du). As expected, the commercial anti-D agglutinated all D variants and weak D RBC samples by the IAT and by using IgG gel cards; however, the reactivity with DVI RBCs was weaker than with LOR- 15C9. By immunoblotting, LOR-15C9 detected a band with an apparent molecular mass of approximate Mr 30,000-34,000 in membranes prepared from D-positive, DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs and an additional band of Mr 20,000-22,000 in membranes prepared from DVI RBCs. No band(s) was detected in membranes from DII, DNU, DIIIb, DIVa, DIVb, DBT, R0 Har, weak D, or D-negative samples. LOR-15C9 provides a useful tool to identify positively DVI samples and thereby differentiate this partial D from other D variants and from weak D samples.     

IgE antibody response to mite antigens in mite infested mice.

Mice infested at birth with the mouse mite Myocoptes musculinus developed positive skin tests to mite antigens at the age of 5 weeks. Serum IgE antibodies directed against mite antigens were first detected at 6 weeks of age and high levels of IgE were present as long as 1 year later. Similar kinetics of IgE formation were observed in mice infected as adults. Mast cell degranulation by mite extract was demonstrated in connective tissue obtained from the skin of mite infested mice.     

Solubilization and partial purification of lymphocyte specific antigens in the chicken

Four different lymphocyte antigens were solubilized from chickens homozygous at the B locus. The antigens were identified by immunoelectrophoresis with rabbit anti-lymphocyte sera. 1. A bursa-specific cell surface antigen was extracted with 3 M potassium chloride and partially purified. The antigen was excluded from Sephadex G-200 gels, was heat labile and was a potent immunogen. The antigen could be detected on all bursa cells by immunofluorescence but not on thymus, spleen or blood lymphocytes. The findings do not exclude, however, the possibility that a small number of lymphocytes (e.g. plasma cells) possess the antigen. 2. A thymus-specific antigen was obtained by papain treatment of thymus cells. It migrated cathodically in immunoelectrophoresis. 3. A surface antigen which was specific for lymphocytes from both central lymphoid organs (thymus and bursa) was solubilized by pestle homogenization. 4. A fourth lymphocyte surface antigen was present on lymphocytes from blood, spleen, bursa and thymus. It was best solubilized by 3 M potassium chloride extraction and did not migrate under the conditions of immunoelectrophoresis. Studies did not reveal a stable antigenic marker with specificity for thymus or bursa cells and their progeny in the peripheral lymphocyte pool.     

Demonstration of close physicochemical similarity and partial immunochemical identity between the major allergen, Dp42, of the house dust mite, D. pteronyssinus and corresponding antigens of D. farinae (Df6) and D. microceras (Dm6)

House dust mite antigens Df6 and Dm6, corresponding to Dermatophagoides pteronyssinus (Dp) antigen 42 (= P1), were purified from cultures of Dermatophagoides farinae (Df) and Dermatophagoides microceras (Dm) using hydrophobic interaction chromatography in combination with gel filtration or acetone precipitation. Purified antigens from the three mite species (Dp42, Df6 and Dm6) were similar regarding molecular weight by gel filtration (D. pteronyssinus: 17,000, D. farinae: 17,000 and D. microceras: 18,500) and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (D. pteronyssinus: 30,000, D. farinae: 28,000 and D. microceras: 29,000). Widely spaced isoelectric points were observed for the antigens of all three species by sucrose gradient isoelectric focusing: (D. pteronyssinus: 4.5 and 6.3, D. farinae: 4.9 and 6.9 and D. microceras: 4.9 and 6.5). Species-specific rabbit antibodies were easily detected for all three species in crossed-line immunoelectrophoresis using mixtures of the two heterologous antigens in the intermediate gel. Antibody specificity for an epitope, common to D. farinae and D. microceras, but not shared by D. pteronyssinus, was further demonstrated. Potent rabbit antibody reagents with species specificity were produced by column immunoabsorption. Despite a probable common molecular structure and origin, the antigens of the three species exhibited gross differences in their immunochemical reactions with rabbit antibodies.     

K99 surface antigen of Escherichia coli: purification and partial characterization.

K99, a presumed colonizing factor of enterotoxigenic Escherichia coli of calf origin, has been purified. K99 was removed from K99+ bacteria by salt extraction and subsequently purified by ammonium sulfate precipitation and column chromatography on diethylaminoethyl-Sephadex. The purified material was homogenous in size, having an s20,w of 13 to 15 S. It was composed of two subunits: a major component with a molecular weight of 22,500 and a minor component of 29,500. When observed in the electron microscope, K99 appeared to be rod-shaped, with a strong tendency for self-aggregation. At concentrations where aggregation was minimized, individual rods were observed with diameters of 8.4 nm and mean lengths of 130 nm. Based on the subunit structure, exterior location, and rod-like shape of K99, it is concluded that K99 is a pilus or pilus-like structure. Chemically, K99 is composed primarily of protein and has an isoelectric point of greater than 10. Purified K99 did not hemagglutinate guinea pig erythrocytes.     

Identification and purification of IgE-reactive proteins in German cockroach extract.

Cockroaches have been implicated as a cause of respiratory allergy in urban areas worldwide. IgE-reactive German cockroach proteins were identified with molecular weights (MWs) of 90, 66, 50, 43 and 36 KD by immunoblot analysis in both immune BALB/c mice and sensitized humans. Prominent IgE-reactive proteins were purified using FPLC by ion-exchange chromatography, gel filtration and hydrophobic chromatography. The N-terminal amino acid sequence of a purified protein with a MW of 66 KD on SDS-PAGE was Val-Thr-Leu-Lys-Lys(Val)-Met-Ile-Lys-Thr-Phe-Tyr. No homologous protein was found through a search of GenBank that indicated a novel IgE-reactive protein in German cockroach extract. Another purified protein with a MW of 36 KD reacted strongly with a monoclonal antibody against Bla g 2.     

The levels of cockroach allergen in relation to cockroach species and allergic diseases in Thai patients

Recently, cockroaches have been established as the second most Important allergen, producing allergic diseases, especially in low socioeconomic populations. In Thailand, about 44-61% of atopic patients were positive to cockroach extract by a skin-prick test. This study examined cockroach allergen levels in relation to cockroach species and allergic diseases in the houses of cockroach-sensitive patients. Sixty households of allergic patients in the Bangkok metropolitan area were surveyed using open- and closed-ended questionnaires. Cockroaches were collected using commercial cockroach traps, while dust samples were obtained from the bedrooms, kitchens and living rooms of the houses using a vacuum cleaner. The cockroaches were counted and their species Identified. The levels of cockroach allergens were determined by specific monoclonal antibodies using a monoclonal antibody-polyclonal antibody based sandwich ELISA kit. Six cockroach species were Identified: Periplaneta americana (American cockroach, 72.15%), Supella longlpalpa (2.75%, found in only one house), Periplaneta brunnea (0.78%), Periplaneta australaslae (0.78%), Neostylopyga rhombifolla (0.78%), Blattella germanica (German cockroach, 0.39%) and nymphs (22.35%). Allergens of the predominant species, P. americana, were detectable in all homes studied, with the highest levels in the kitchen areas. The range of allergen levels in house dust varied from 0.40-162.00 microg per g of dust. The median and mean allergen levels in kitchen dust were 59.16 microg and 62.80 microg per g of dust, respectively, while the median allergen level in bedroom dust was only 15.90 microg per g of dust. The German cockroach allergen (Bla g 2) was undetectable in any of the houses. IN CONCLUSION: P. americana was the most common cockroach and may be the species causing allergic diseases, especially asthma, in Thailand, which differs from the USA and Europe     

Domestic allergens in public places II: dog (Can f 1) and cockroach (Bla g 2) allergens in dust and mite, cat, dog and cockroach allergens in the air in public buildings

Background Sensitization and exposure to indoor allergens are the major risk factors for asthma. It is possible that significant exposure to domestic allergens occurs outside the home. Objectives To investigate the levels of Can f 1 and Bla g 2 in the dust from carpeted floors and upholstered seats in public buildings and public transport and the airborne concentrations of Der p 1, Fel d 1, Can f 1 and Bla g 2 in schools and offices. Methods Can f 1 and Bla g 2 were measured in the dust collected by vacuuming a I m² area of carpet, as well as upholstered seats in five schools, six hotels, four cinemas, six pubs, three buses and two trains. Dust was also collected from the bedroom carpet, living room carpet, mattress and sofa in 20 homes with and 20 homes without a dog in the same area. Personal airborne sampling (2 L/min) was conducted for 8 h in offices (n= 16) and classrooms (n= 9). In addition, airborne samples in schools were collected using a high volume pump (60 L/min) for 1 h in three classrooms immediately after the children vacated the school. Can f 1, Bla g 2, Der p 1 and Fel d 1 were assayed using a two–site monoclonal antibody–based ELISA. Results Can f 1 was detected in all dust samples from public places, ranging from 0.2 to 52.5 μg/g, Significantly higher levels were found in upholstered scats (geometric mean – GM 9.4 μg/g) than in carpets (GM 1.5 μg/g; P < 0.001), and levels of Can f 1 > 10 μg/g were found in 40% of upholstered seats in public places. Can f 1 was significantly higher in upholstered seats in public places than in sofas in homes without a dog (GM 1.8 μg/g; P < 0.001). Detectable levels of Bla g 2 were found in all of the schools (GM 2.4 U/g, range 0.8–4.4 U/g). Bla g 2 concentration greater than 2U/g (provisional threshold level representing risk of sensitization) was measured in 65% of the classrooms sampled. Der p 1 and Bla g 2 were below the detection limit in all airborne samples. However, airborne Fel d 1 and Can f 1 were detected in schools and offices, albeit in low concentrations. Conclusions Upholstered seats from public places constitute a reservoir for the accumulation of dog allergen, and a source of exposure to Can f 1 inside public buildings or on public transport. Exposure to cockroach allergens in schools may be important for cockroach sensitized asthmatic children.     

Monoclonal antibodies to tissue-specific cell surface antigens. I. Characterization of an antibody to a prostate tissue antigen

Monoclonal antibodies were raised to PC-3 human prostate adenocarcinoma cells, and one hybridoma, designated F77-129, was extensively purified and used to characterize a PC-3 antigen. The F77-129 antibody also showed serological reactivity with the Du-145 prostate cancer line and with three of four breast carcinoma lines tested; it showed variable binding to a colon carcinoma line. Several other lines tested, including melanomas, fibrosarcomas, and leukemias, were completely negative. Immunoperoxidase staining of frozen surgical specimens showed binding to both normal and malignant prostate and breast tissue. Injection of radioiodinated F77-129 into tumor-bearing nude mice showed specific in vivo targeting to prostatic cancer implants. The antigen also showed surface modulation by bound antibody, suggesting possible clinical utility of this antibody in delivering immunotoxins to tumors.     

The detection of precipitating antibodies in equine infectious anemia and partial purification of the antigen

Summary Spleens from 12 ponies experimentally infected with the virus of equine infectious anemia (EIA) were evaluated as antigen sources against sera from known infected and normal horses using the Ouchterlony double diffusion technique. Spleens from 4 normal horses served as controls. Two of the 12 infected spleens could be used as antigen for detecting precipitating antibodies when the spleens were frozen and thawed, minced and placed in the antigen wells. When 9 of the infected spleens were treated to partially purify and concentrate the antigen, all 9 could be used as antigen. Using partially purified antigen, precipitating antibodies could not be detected in any of 107 non-EIA infected sera while antibodies could be demonstrated in 71 of 83 sera from infected horses. The 12 non-reacting sera were from 10 horses infected 14 days or less, from one horse infected 15 days, and from one horse infected 25 days. Eighty-two of 83 horses infected from 18 days to 5 1/2 years had precipitating antibodies detectable by this technique. A precipitinogen can be partially purified from EIA infected spleens and used to detect precipitating antibodies in EIA infected horses.     

Characterization and partial purification of a macrophage-stimulating factor from Mycoplasma hominis

PROBLEM: Mycoplasma hominis is one of the most common pathogens of the genital tract and is associated with increased production of proinflammatory cytokines in reproductive tissues during preterm labor. The mechanism by which M. hominis, an organism lacking cell walls, increases the production of proinflammatory cytokines is unknown. METHOD OF STUDY: We characterized and purified a macrophage-activating factor from this organism. RESULTS: Extraction of whole organisms with Triton-X-114 demonstrated that the activity was primarily associated with the detergent phase. Macrophage-stimulating activity (MSA) of detergent extracts of M. hominis was not inhibited by polymyxin B or heating but was completely abrogated by alkaline hydrolysis and partially reduced by proteinase K digestion. Further experiments that utilized Toll-like receptor (TLR)-2- and TLR-4-transfected cells, revealed that the detergent extracts activate TLR-2 but not TLR-4 signal transduction. Purification of the activity using preparative SDS-PAGE and reverse phase chromatography experiments led to the isolation of a 29-kDa protein. CONCLUSIONS: These experiments suggest that the MSA of M. hominis is due to a lipophillic factor that interacts with TLR-2 rather than TLR-4 (as does lipopolysaccharide), to increase tumor necrosis factor (TNF)-alpha by macrophages. It is known that TNF-alpha can cause preterm labor and intrauterine fetal death and that it is upregulated in amniotic fluid samples infected with M. hominis.     

Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract

Lyophilized Dermatophagoides pteronyssinus ( Der p ) allergen extract (AE) and partially purified Der p extract (PAE) Were Prepared and characterized. Partial purification of AE was performed by gel filtration on Sephadex G-1OO and Sephacryl S-300. Crossed immunoelectrophoresis (CIE) disclosed the same precipitating lines in AE and PAE preparations. The relative potencies of AE and PAE were determined and compared with the WHO International Standard for Der p by the RAST inhibition method. The potencies were 6.5 × 10⁵ IU and 1.5 × 10⁶ IU, respectively. Biologic standardization by quantitative skin testing was performed with AE (20 selected patients) and PAE (12 patients). Median C_h was calculated by linear regression analysis (log-log model). One ampoule of AE contained 65 300 BU and 1 ml (vial) of PAE contained 166000 BU. Der p AE could serve as a Croatian national standard for further production of Der p allergenic extracts.