Production of monospecific serum against secretory IgA (anti-SC) and a standard for quantitative analysis]

The authors describe a method of obtaining monospecific serum against secretory IgA and the corresponding standard. An immunochemically pure (11.6S) secretory human IgA was extracted from the colostrum by salt fractionation and gel-filtration through Sephadex G-200 and Sepharose 6B; this IgA was used as an antigen for the immunization and the standard for the quantitative determination of SIgA in the secretions. Monospecific anti-SC-serum was obtained by successive exhaustion of the antiserum against the S IgA immunosorbents prepared from normal human serum and the serum of a patient suffering from A myeloma containing polymeric IgA forms.     

Isolation of monospecific antisera to guinea pig immunoglobulins

The results of the production and analysis of monospecific rabbit antisera to guinea pig IgG1, IgG2, IgA and IgM are presented. Isolated immunoglobulins of different isotypes, as well as immune precipitates obtained by immunoelectrophoresis, were used for immunization. After adsorption antisera of each type there formed one precipitation line with guinea pig serum in immunoelectrophoresis, thus indicating that they contained antibodies to immunoglobulins of the definite isotype.     

Study of human secretory immunoglobulin A. I. Obtaining monospecific antiserum to human secretory immunoglobulin A]

A method of obtaining monospecific antiserum to the human secretory IgA is described. Immunochemically pure secretory IgA (isolated from human colostrum by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200) was used for immunization of rabbits or sheep. Heterologous antibodies were removed by adsorption with commercial gamma globulin, normal serum, the serum of a patient suffering from A-myeloma with the IgA polymere and purified lactoferrin. Monospecific antiserum to the secretory IgA gave a reaction of complete immunological identity with the secretory IgA and a free secretory component.     

Arylamidases of rat liver and chemically induced hepatomas. II. Preparation and characterization of monospecific antisera against two distinct arylamidase-active antigens.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity.The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.     

Sheep haemopexin: immunological cross-reactivity with other vertebrate sera and evidence for the absence of haemopexin in some mouflon

A monospecific antiserum to sheep haemopexin was produced in rabbits. By using this antiserum, as well as absorbed antisera to sheep and mouflon serum proteins, it was proved that some mouflon sera lack haemopexin. In immunodiffusion, when using the monospecific antiserum, immunoprecipitates were present only in species' belonging to suborder Ruminantia. None of the other mammalian, nor any of the avian, reptilian, amphibian and fish species tested, showed reaction.     

Isolation of antisera to different IgA determinants and an analysis of the antigenic structure of human monoclonal IgA

Different molecular forms and subclasses of monoclonal IgA, identified by the method of electrophoresis in acrylamide gel with sodium dodecyl sulfate, were used as antigens for immunization and as adsorbents for the production of antisera to various IgA subclasses and to Fc alpha, Fab alpha and P-determinant of IgA. The antisera thus prepared were used for the analysis of the antigenic structure of monoclonal IgA. True IgA polymers and monomers were detected in the precipitation test with antiserum to P-determinant; in some cases monoclonal IgA synthesized by a single clone of plasmatic cells consisted of IgA monomers and polymers, equally capable of binding free SC in vitro. The results of the determination of IgA sub-classes with the use of antisera to IgA1 and IgA2 coincided with the distribution of subclasses established by the study of these proteins in polyacrylamide gel with sodium dodecyl sulfate. The antisera obtained in these investigations made it possible to detect three cases of heavy alpha-chain disease and to characterize the antigenic structure of protein occurring in this disease.     

Isolation and identification of the third component of complement of Japanese quails

The identification of the third component of complement (C3) of Japanese quails was attempted by using rabbit antiserum prepared against quail serum-treated zymosan (ZX) as an initial reagent. This antiserum (anti-ZX) had agglutinating activity on rabbit erythrocytes reacted with quail antibody and quail complement (EACq) but not on EAq, and developed two precipitin lines against quail serum at beta- and gamma-regions in crossed immunoelectrophoresis. Subsequently, monospecific antisera to each of these precipitin lines were prepared in rabbits, and quail serum proteins reactive with these antisera were purified by salt precipitation followed by Sephadex gel filtration and DEAE cellulose column chromatography. One protein with a m.w. of 184,000 (184K) resembled mammalian C3 in that: 1) monospecific antiserum (anti-184K protein serum) agglutinated EACq but not EAq; 2) treatment of fresh quail serum with either inulin or zymosan resulted in the conversion of the precipitin line developed against 184K protein from gamma to beta in crossed immunoelectrophoresis; 3) the 184K protein was shown to consist of two polypeptide chains of 110K and 73K linked by disulfide bonds. Furthermore, the 184K protein in serum was cleaved through the incubation with inulin to 174K and 140K proteins that might correspond to C3b and C3c of human complement; 4) the 184K protein bound to zymosan was eluted with hydrazine or methylamine but not with Nonidet P-40, indicating that 184K protein binds to zymosan by a covalent bond but not by a hydrophobic one; and 5) by treatment of fresh quail serum with methylamine, complement reactivity was reduced, although its activity was restored by the addition of purified 184K protein. These results suggest the 184K protein is the quail's equivalent to mammalian C3. When quail serum was reacted with cells that had complement-activating capacity, quail C3 deposited on their membrane as in mammalians; however, no conversion of quail C3 was noted by the reaction with CVF. Antibody to quail C3 failed to cross-react with that in mammals.     

Quantitation of serum immunoglobulins G, M, and A in the rhesus monkey (M. mulatta) using human monospecific antisera in the enzyme-linked immunosorbent assay: developmental aspects

Using human monospecific antisera, several parameters have been optimized for the micro-ELISA "sandwich" technique used in the quantitative measurement of total serum IgG, IgM and IgA levels in rhesus monkeys. Representation of the optical density as a four-parameter logistic function provided excellent fits of the data over a wide choice of dilutions of human antisera used for coating the ELISA plates and for the peroxidase-conjugated antisera used in the system. The micro-ELISA "sandwich" technique was shown to be specific, reliable, sensitive, and economical for use in the routine measurement of total serum IgG, IgM and IgA levels in the rhesus monkey.     

Isotypic characteristics of serum and secretory O antibodies and local immunological memory in the parenteral immunization of guinea pigs with a ribosomal Shigella vaccine

Guinea pigs were immunized subcutaneously with ribosomal vaccine prepared from S. sonnei and their systemic and local humoral response was studied by means of ELISA techniques with the use of monospecific antisera to guinea pig IgA and IgG. Injection of the ribosomal vaccine leads to a significant rise in the levels of IgA O-antibodies in tears, IgG and IgA O-antibodies in the serum. The presence of IgA O-antibodies in tears was seemingly the result of their local synthesis rather than the seepage of serum IgA. The stimulation of the local and systemic anti-O response was more pronounced after parenteral immunization with the ribosomal vaccine than after immunization with the corresponding dose of lipopolysaccharide (LPS). Parenteral immunization with the ribosomal vaccine induced the development of both systemic and local memory. The priming effect produced by relatively small doses of this vaccine (40 micrograms), administered parenterally, was similar to the effect of prolonged and intensive stimulation ensured by 10-day feeding with LPS (the total dose being 5,000 micrograms).     

Quantification of human urinary free secretory component, secretory and nonsecretory IgA by ELISA

The specific quantification of human urinary free secretory component (FSC), secretory IgA (SIgA) and total IgA using ELISA has been hampered by mutual interferences of these three molecules. Using affinity chromatographically purified antisera an attempt was therefore made to reduce these interferences without necessitating further assay steps. FSC and total IgA were measured in unprocessed urine by means of anti-FSC and anti-IgA as well as alkaline phosphatase-coupled anti-FSC or anti-IgA antisera. SIgA was determined using anti-IgA as well as alkaline phosphatase-coupled anti-FSC. Nonsecretory urinary IgA was calculated from the measured SIgA and total IgA. The mutual interferences of FSC, SIgA or nonsecretory IgA in the three assay systems were low and not relevant for normal samples. Normal urinary concentrations were: FSC 344 +/- (SD) 208 ng/ml (n = 120), SIgA 1,874 +/- 1,133 ng/ml (n = 123) and nonsecretory IgA, depending on the way of standardization, 712 +/- 699 (n = 56) or 878 +/- 732 ng/ml (n = 51). SIgA excretion increased with age. Lower urinary SIgA as well as total and nonsecretory IgA levels were observed in males as compared to females. No correlation evolved between the hormonal status of women and the excretion of FSC, SIgA or IgA. In IgA-deficient patients virtually no nonsecretory IgA or SIgA was detected in the urine while the FSC concentration was in the normal range.     

Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells.

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera. Higher concentrations of glutaraldehyde lowered the sensitivity of detection. Purified SIN capsid protein specifically inhibited antibody-dependent, complement-mediated cytotoxicity by the monospecific anti-capsid protein serum on SIN- and Semliki Forest virus-infected target cells. That hyperimmune anti-SIN serum also cross-reacts with capsid protein on the surface of Semliki Forest virus-infected cells was suggested by the fact that capsid protein inhibited cross-cytolysis in the antibody-dependent, complement-mediated cytotoxicity assay. The latter antiserum was collected after repeated injections of purified virions over a 9-month period. The results suggest that hyperimmune monospecific antisera made to SIN capsid protein or hyperimmune antisera to SIN or Semliki Forest virions detect homologous and cross-reacting capsid protein determinants on the surface of infected cells.     

Method of preparation and evaluation of antisera for the differential determination of secretory immunoglobulin A and free secretory component in biological fluids]

One of the pressing tasks in the study of local nonsusceptibility to infectious diseases and immunochemical analysis of the external secretion is recording of the level of various forms of the secretory IgA (SIgA) and of the secretory component (SC) in various biological fluids. Indication and measurment of the concentrations of the mentioned proteins encounter serious difficulties caused by heterogeneity of their molecular forms. It was shown that the antisera to the whole molecule of SIgA and SC are of no use. On the basis of a new method of purification of free SC and technology of preparation of monospecific antisera capable of separation of SIgA and free SC there were obtained diagnostic antisera for the quantitative recording and differentiation of various forms of IgA and SC in biological fluids. A reliable measurement of the SIgA and SC concentration in some external secretion was carried out with the aid of the mentioned preparations without any complicated chromatographic experiments.     

Preliminary results concerning the presence of secretory immunoglobulin A (SIgA) in the serum of patients with IgA myeloma

Using the immunosorbents realized by binding either antisecretory immunoglobulin A antibodies or antisecretory component ones to beads of cross-linked polyvinyl alcohol matrix, secretory immunoglobulin A, from the serum of patients with IgA myeloma was investigated. The related protein isolated with the same method from colostrum was comparatively studied. Studies were done by polyacrylamyde gel electrophoresis (PAGE) and SDS-PAGE, as well as by immunochemical methods. The two proteins obtained presented the same characteristics: a single fraction in PAGE and four fractions in SDS-PAGE. The determinations were carried out by immunoelectrophoresis and double diffusion using monospecific antisera proved the identity of the isolated proteins with secretory IgA.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Immunochemical study in two cases of alpha chain disease

First two cases of alpha-CD recognized in the USSR are described. Their immunochemical patterns were typical of this disease. Alpha chain proteins in sera were identified with the help or antisera to IgA, containing antibodies to alpha-chains and to Fab-fragment of IgA. Not only IEP but also SRID were proved to be useful for detecting alpha-CP since double rings were formed by alpha-CP-containing sera: the external ring was formed by alpha CP and the internal ring by normal IgA. Alpha-chain proteins were found in all the patient's secretions (coprofiltrates, saliva, urine), but only in coprofiltrates alpha-CP was bound to SC; in urine and saliva free alpha-CP in these secretions to bind SC. With the help of antiserum to P-determinant alpha-CP was shown to exist in true polymeric (dimeric) form only in coprofiltrates, but not in urine or saliva. A marked shift of kappa/lambda ratio towards kappa chains was revealed in the whole serum and IgG-fraction of one patient; this can be considered either as a result of a peculiar immune response to various antigens because of the deficiency of local immune system, or as an initial phase of development of monoclonal IgGk gammopathy.     

Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion

Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct.     

Study of the subclasses of immunoglobulin G. II. Qualitative and quantitative characteristics of IgG subclasses using antisera systems]

Bispecific antisera, or "antisera-systems", containing class- and subclass-specific antibodies to IgG were obtained from rabbits, goats and guinea pigs after brief courses of immunization with purified G1, G2, G3 and G4 paraproteins. After the elimination of antibodies to light chains by adsorption these antisera were tested in immunoelectrophoresis and radial immunodiffusion in gel with sera containing G paraproteins of different subclasses. In immunoelectrophoresis double lines and in radial immunodiffusion with G paraproteins of heterologous subclasses double rings were obtained: the external lines (or the external rings) were formed as a result of interaction between G paraproteins and antibodies to class-specific IgG determinants, the inner lines (or the inner rings) were formed as a result of interaction between the corresponding subclass of normal IgG and subclass-specific antibodies. The identification of different G paraprotein subclasses gave similar results when carried out with "antisera-systems" and with monospecific antisera to the corresponding IgG subclasses. "Antisera-systems" proved to be suitable for use in the identification of G paraprotein subclasses, as well as in the quantitation of different subclasses in normal IgG.     

Purification of mouse alpha-foetoprotein by ampholyte displacement chromatography.

Mouse alpha-foetoprotein (alphaFP) was isolated from H4 hepatoma tissue using Con A-Sepharose salt gradient ion exchange chromatography and ampholyte displacement chromatography, the latter being a new method for a sharp separation of proteins based on their different isoelectric point. The purity of the alphaFP was demonstrated by (i) the absence of contaminant on sodium dodecyl sulphate polyacrylamide gel electrophetic gels, (ii) Ouchterlony's immunodiffusion against monospecific antimouse alphaFP and the absence of precipitation against a polyvalent antinormal mouse serum, (iii) the production of a monospecific antiserum in a rabbit after injection of the purified antigen, and (iv) immunological unreactivity of the produced antiserum against normal hepatic tissue.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines fromHLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelatedHLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.