Purification and testing of class-specific anti-chicken immunoglobulin antibodies.

The purification and testing of chicken immunoglobulins and of class-specific antichicken immunoglobulin antibodies are described. This work suggests the existence of as yet uncharacterized classes of chicken immunoglobulins. Their eventual determination in a species that has a B lymphopoietic organ may promote further progress in the understanding of B cell differentiation and function.     

Preparation of specific anti-thymocyte and anti-bursacyte sera in rabbits.

A method of producing anti-thymocyte and anti-bursacyte sera in rabbits is described. Chickens which served as donors of cells were thoroughly perfused with saline to remove plasma proteins and circulating blood elements. For immunization, thymocytes were obtained from neonatally bursectomized birds, and bursacytes from chickens thymectomized at hatching. The purification of rabbit anti-lymphocyte sera included absorptions with leucocyte-free suspension of chicken erythrocytes, chicken liver cell membranes, thymocytes from bursectomized-irradiated and bursacytes from thymectomized-irradiated chickens, and chicken IgM and IgG immunoadsorbents. Cytotoxicity and fluorescent-antibody assays revealed that anti-thymocyte and anti-bursacyte sera thus produced clearly distinguished the surface antigenic determinants of thymocytes from those of bursacytes.     

两株抗鸡CARP单克隆抗体的制备及其生物学特性鉴定

目的:制备抗鸡CARP单克隆抗体(McAb)。方法:克隆并原核表达鸡CARPN端110个氨基酸,纯化CARP蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合,采用间接ELISA方法筛选阳性克隆;并利用ELISA、Western blot方法测定其效价和特异性。结果:获得2株能够稳定分泌抗鸡CARP的McAb杂交瘤细胞株,命名为4A8和4E6,亚型都属于IgG1,轻链为κ链;抗体腹水效价分别为3.2×104、1.28×105。Western blot检测到这两株抗体能够识别鸡CARP蛋白。结论:成功获得了2株能识别鸡CARP的杂交瘤及其分泌的特异性McAb。     

A method of obtaining specific anticancer sera

Summary An attempt was made to apply the known phenomenon of inhibited antibody production associated with antigen overdosage as a method of increasing the specificity of anticancer sera. In order to depress the production of noncancer antibodies, and to raise the titer of antitumor anitbodies, rabbits were immunized with a mixture of aqueoussaline extracts obtained from the tissue of human stomach cancer and from normal stomach tissue, at first in the ratio of 1:5 and then 1:7. In the first case, the production of noncancer antibodies was greater. Although no complete depression of antibody production against the investigated normal human tissue was achieved in the second ratio, in the majority of cases such immunization did not lead to the rise of the titer of noncancer antibodies over that of the anticancer ones. In the author's opinion, further increase in the amount of extract or fractions from normal tissue used in the immunizing antigen may possibly effect a greater depression on the production of antibodies against normal tissues, and increase the anticancer sera specificity.(Presented by Active Member of the Akad. Med. Nauk SSSR N. N. Zhukov-Verezhnikov)_ Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 56, No. 9, pp. 95–98, September, 1963     

Distribution of immunoglobulin G subclasses in anti-A and anti-B sera

Sera from 117 immunologically normal subjects, who had been selected for the presence of high titre ABO system antibody on routine screening, were further evaluated for the presence of IgG and its subclasses IgG1, IgG2, IgG3, and IgG4 using an indirect antihuman globulin technique. Subjects of all ABO groups had the capacity to produce IgG antibodies within each subclass, but those of group O produced the broadest spectrum of IgG subclasses and greatest strength of reactions.     

The specific immunoglobulin in hydatid disease

The variation in the serum level of specific IgG, IgM and IgA antibodies during different stages of hydatid disease has been demonstrated by a technique of fluorescent microscopy that uses monospecific anti-human immunoglobulin conjugates and freeze-dried antigens. The technique is easy to perform and our results suggest that the test is sensitive and specific. Specific IgG antibodies are present in patients with either current or past infections. IgM antibodies, detected during periods of antigenic activity, disappear soon after removal of the cyst. In many cases IgA antibodies also disappear soon after removal of the cyst. Cross-reactions between the antigens and antibodies of hydatid disease and schistosomiasis are shown to be present mainly in the IgG immunoglobulin and only to a much smaller extent in the IgA.     

Measurement of T-cell-derived antigen binding molecules and immunoglobulin G specific to Candida albicans mannan in sera of patients with recurrent vulvovaginal candidiasis

Immunoglobulin G (IgG) and T-cell-derived antigen binding molecules (TABM) specific to whole Candida extract and to Candida-derived mannans prepared by both the cetryltrimethylammonium bromide (CTAB) and alkaline degradation (PEAT) methods were measured in the sera of women with vulvovaginal candidiasis and controls. In the patients there were significantly higher levels of IgG to both CTAB and PEAT mannans and of TABM to CTAB mannan. TABM specific to CTAB mannan was purified from the serum of a patient with a high titer of this TABM. The purified TABM bound specifically to CTAB mannan and to other yeast and mold extracts. This TABM preparation was associated with transforming growth factor beta2 (TGF-beta2), and on specific binding to mannan there was a marked increase in the level of detectable TGF-beta2. This increase in TGF-beta2 level was critically dependent on the relative concentrations of the purified TABM and mannan, being smallest when either was in excess. The TABM specific to CTAB mannan was also shown to inhibit Candida-stimulated gamma interferon production. The results suggest that CTAB mannan-specific TABM may increase susceptibility to vulvovaginal candidiasis in association with a shift in the immune response to the Th2 type.     

Human antibodies against bovine immunoglobulin M in IgA deficient sera

Analysis of the sera of seventeen patients with a selective deficiency of IgA revealed that 24% gave a precipitin reaction on gel diffusion with normal bovine serum and milk. None of 100 normal adults and only one of approximately 500 patients with various diseases without an IgA deficiency had precipitins when studied by this same technique. Analysis of the nature of this reaction revealed that an IgG type antibody present in the human sera was reacting with the Fc fragment of the IgM present in bovine milk and serum.

An immunologically identical reaction occurred with goat and sheep, and reactions of partial identity with horse and pig sera. No reactions were found with sera from other species including man. In two of the IgA deficient sera additional but much weaker precipitin lines were noted with cow milk. These data are consistent with the hypothesis that oral immunization with the highly antigenic macroglobulin present in cow milk was facilitated by the mucosal antibody defect accompanying the IgA deficiency.

     

Microtiter solid-phase radioimmunoassay for specific immunoglobulin E

A microtiter solid phase-radioimmunoassay (MSPRIA) for perennial rye grass-specific IgE is described. The assay is carried out in flexible polyvinyl chloride "u" microtiter plates by sequentially incubating antigen, albumin, test sera, and finally radiolabeled antihuman IgE. Wells are cut free of the plate with a hot wire-cutting apparatus and counted individually in a gamma counter. The amount of radioactivity bound is proportional to the amount of specific IgE in the serum. The MSPRIA is shown to be antigen and antibody specific, reproducible, and easily done. Rye-specific IgE levels assayed with the MSPRIA correlated with quantitative end point titration skin testing using perennial rye grass antigen performed on 31 volunteers with a maximum correlation coefficient of 0.92. The MSPRIA was compared with the radioallergosorbent test (RAST) method using sera from the same 31 volunteers. The rye-specific IgE levels assayed by MSPRIA correlated with those assayed by RAST with a correlation coefficient of 0.95. The MSPRIA is well suited for mass screening and represents a useful method for measuring specific IgE.     

Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay

We have evaluated the diagnostic utility of eleven Toxoplasma gondii recombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94. 5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.     

Granulocyte specific cytotoxic antibodies in pregnancy and multitransfused sera

The myeloid cell line K562 was used to screen for specific antibodies in sera obtained from pregnant women and multitransfused patients. The alloantisera thus procured reacted with random granulocytes in a pattern suggestive of a polymorphic system. Some antibodies were found to react only in the cold. A simple reproducible method for the simultaneous separation of granulocytes and lymphocytes for microcytotoxicity is described.     

Bluetongue virus hemagglutination and its inhibition by specific sera

Summary Bluetongue-virus (BTV) was found to agglutinate a variety of erythrocytes including sheep-, chicken-, guinea pig- and mouse-erythrocytes. Hemagglutination was inhibited specifically with type specific serum. A temperature dependence was only found for chicken erythrocytes, which showed a hemagglutination optimum at 37° C. The hemagglutination was lost upon treatment of the virus with 0.4 per cent trypsin as well as after treatment with 0.01M KJO₄. Heating of the virus preparation to 56° C resulted in the loss of the HA-activity. Gelchromatographic studies indicated that the hemagglutinating capacity is associated with the complete virion. Whereas virulent strains of BTV hemagglutinate a number of different erythrocytes the avirulent type tested produced only a slight hemaglutination with sheep red blood cells. However, specific antiserum produced with the avirulent strain yielded strong hemagglutination inhibition (HI) with the corresponding virulent strain. Treatment of sera prior to their use in the HI proved necessary to remove nonspecific inhibitors. The efficiency of KJO₄ in removing nonspecific inhibitors indicates that carbohydrates represent the major group of nonspecific inhibitors. The data represented recommend the hemagglutination inhibition test as a new method to identify the various BTV serotypes.With 1 Figure     

Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera

The use of protein A from S. aureus (SpA) as an anti-IgG reagent in immunological techniques has extended in recent years, together with knowledge about its interaction with immunoglobulins of different species. Current data with respect to the binding of protein A to immunoglobulins and to the levels of immunoglobulins in the sera of some mammalian species are reviewed.     

A new method, distribution-analyzing latex immunoassay (DALIA), to measure specific immunoglobulin G against mite and wheat allergen in human sera

A distribution-analyzing latex immunoassay (DALIA), based on the agglutination of latex particles coupled with mite or wheat allergen, was developed to determine allergen-specific IgG in human sera. The immune complex between chemically coupled-allergen latex and specific IgG was agglutinated specifically and efficiently by employing an IgM-type monoclonal second antibody with strong amplification activity. The extent of agglutination was evaluated by determining the relative ratio of volumes (RV) of agglutinates to residual nonagglutinating particles with a particle counter. This method exhibited a high sensitivity (detection limit ≤ 5 munits/ml) in the determination of allergen-specific IgG, and no influence of inhibitory factors such as competitive antibodies (specific-IgA, -IgM) and nonspecific IgG (≤320 mg/ml) was observed. The concentrations of specific IgG against mite allergen in the sera of 130 allergy patients with atopic dermatitis and 52 normal subjects were 22.3 ± 12.3 and 16.5 ±4.2 units/ml, respectively, and the concentrations of specific IgG against wheat allergen in the same two groups were 5.4 ± 4.2 and 2.1 ± 2.2 units/ml, respectively. The coefficients of variation of intra- and interassay ranged from 3.4% to 11.2% in both cases. The present method is an excellent homogeneous immunoassay which may be used as a routine assay that can measure 50 samples per hour without prior treatment.     

Toward molecular targeting with specific intravenous immunoglobulin preparation

Intravenous immunoglobulin (IVIg) is used successfully for therapy of inflammatory and autoimmune diseases, especially in cases of conventional therapy resistance. Within the broad spectrum of immunomoregulatory activities of IVIg in vitro and in vivo, the anti-idiotypic activity neutralizing the related idiotypes is one of the main mechanisms. Futhermore, IVIg addresses integrins associated with inflammation and immune response thrombosis, such as the RGD (Arg-Gly-Asp) motif, expressed on a large number of cell surface and matrix proteins. In addition, during the last years, anti-Fas activity of IVIg was reported. We fractionated IVIg specific preparation (sIVIg) based on the multispecificites of the IVIg compound. We have generated an IVIg fraction that will show specific activity for lupus idiotypes in vitro. In NZBxW.F1 mice, results showed 200 times more beneficial effect. Using a peptide phage display library technology, we have identified a panel of lupus-related synthetic idiotypes that are mimetics of the idiotypes presented in patients with systemic lupus erythematosus. A column composed of these synthetic lupus-related idiotypes was used to prepare a large amount lupus-specific IVIg. Using the same approach, we prepared anti-anti-β-2-glycoprotein-I (β2GPI) specific IVIg for antiphospholipid syndrome (APS). This APS-specific IVIg reduced the fetal loss induced by anti-β2GPI antibodies by improving the implantation process in a mouse model. Others prepared specific preparation of IVIg to RGD or for Fas. The molecular targeting with specific IVIg may be used for therapeutical purposes, using a smaller amount of IVIg, and targeting more specifically autoimmune diseases, thrombosis, or inflammatory condition.     

Selective depletion of immunoglobulin isotypes after decomplementation of mouse sera by heat treatment

Inactivation of mouse serum by heat treatment (30 min at 56 degrees C) decreased antibody reactivity as shown by haemagglutination assays. This appeared to be due to loss of antibody since Ouchterlony analysis showed disappearance of antibody after heat treatment. By both isotype-specific ELISA and Ouchterlony analysis it was shown that mouse IgM, IgG2b and IgG3 are sensitive to heat treatment whereas IgG2a is relatively resistant. IgG1 is not sensitive to heat treatment. To avoid complement-dependent lysis in haemagglutination assays EDTA should be added to the serum samples.     

Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking. OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy. METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy. RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins. CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.