Specificity of rabbit antisera against mouse leukaemias in vitro

Rabbit antisera were prepared against mouse leukaemias from several inbred strains, and against a mixture of these leukaemias. The potencies of these antisera were tested against each of the different leukaemias, and against certain normal cells and tissues.

As determined by immune cytolysis, the potencies of antisera were strongest against the homologous leukaemias. The potencies against unrelated leukaemias were generally within 50 per cent of those against the homologous leukaemias, with some exceptions. Antisera prepared by injection of a mixture of the leukaemia cells showed remarkably high potencies against all leukaemias tested.

As measured by C fixation, the same antisera showed less specificity for the homologous leukaemias. The potencies of antisera for C fixation on a variety of normal tissues was quite strong, and decreased approximately in the following order: testis, kidney, liver, lung, brain, muscle, erythrocytes. For antiserum prepared against DBA/2 leukaemia L1210, the C fixation reaction against normal lymph node lymphocytes was about 20 per cent as strong as the reaction against the homologous leukaemia L1210. A hypothesis concerning the mechanism of immune cytolysis is presented to explain these results. Exhaustive absorption of anti-L1210 sera with normal DBA/2 tissues produced only a slight increase in antileukaemia specificity.

     

Proportional absorption. A method for determination of the relative specificity of antisera prepared against cells.

Antisera prepared against a complex of antigens such as a tissue cell may produce a mixture of antibodies of different specificities, affinities and types. Proportional absorption permits determination of the comparative specificity of such antisera. It is performed by absorbing the antisera with an amount of absorbent proportional to the initial content of (usually undesired) antibody to this absorbent; the potencies of desired and undesired antibodies are then separately determined. The method has been used to determine the specificity of a conventionally raised rabbit anti-mouse leukaemia serum, relative to one prepared with leukaemia cells admixed with rabbit antiserum against normal mouse lymphocytes (to block normal antigen sites on the leukaemia cell inoculum). The latter antiserum was cells as compared to normal splenic lymphocytes.     

Xenogeneic recognition of tumour specific plasma membrane antigens derived from mouse lymphoma cells

Rabbit antisera were prepared to two mouse ascitic lymphomas, one of them being highly immunogenic, the other one weakly immunogenic in syngeneic hosts. A comparison was made between antisera raised with intact cells and antisera raised with cell-free fractions composed of purified large plasma membranes. The antisera were tested in vitro for their lytic capacity by the ⁵¹Cr-release technique, and in vivo for their capacity to protect syngeneic mice against a tumour challenge. The antisera were used unabsorbed, absorbed with syngeneic mouse tissue, and also after passage through syngeneic animals. The antisera with the highest specificity for the respective tumour in mice were those raised by plasma membranes derived from the strongly immunogenic tumour. Lysis in the ⁵¹Cr test corresponded roughly with these findings. Xenogeneic recognition of tumour distinct antigens as well as the therapeutic value of xenogeneic antisera against established lymphomas growing in the ascitic form are discussed.     

Trypanosoma vivax : Soluble Antigens of • and of other Trypanosomes

Precipitins against trypanosomal antigens occurred in serum from Zebu cattle which had been infected for prolonged periods with Trypanosoma vivax transmitted by Glossina morsitans.

Precipitating antisera against Trypanosoma vivax were used to detect complexes of soluble trypanosomal antigens in sera from rats infected with blood-passaged strains of T. vivax, T. gambiense and T. brucei and in sera from goats infected with a cyclically transmitted strain of T. vivax.

The immunological relationships of antigens of these three species of trypanosomes were studied in terms of antisera against T. vivax.

It was found that the antisera contained antibodies which reacted with antigens common to the three species of trypanosomes. The antisera also contained antibodies which reacted with antigens which may be specific to T. vivax.

     

Reactions of purified hemagglutinating antigens of flaviviruses with 19S and 7S antibodies.

The rapidly sedimenting hemagglutinin (RHA) representing the purified virion and the slower-sedimenting hemagglutinin (SHA) of several flaviviruses were separated and used in hemagglutination inhibition tests with the 19S (immunoglobulin M) and 7S (immunoglobulin G) immunoglobulin fractions of rabbit antisera, prepared against purified viral antigens or against crude virus pools. The antibody specificity in tests with RHA was identical to the specificity in those employing SHA. 7S antibody cross-reacted broadly with all flavivirus antigens, whereas 19S antibodies were relatively specific in cross-reactions among flaviviruses (RHA or SHA). SHA was consistently inhibited by antibody to a greater extent than RHA. Anti-envelope protein, anti-RHA antibodies and anti-SHA antibodies were unable to discriminate between RHA and SHA. It was concluded that the relative amounts of RHA or SHA in crude hemagglutinin preparations have no influence on the result of hemagglutination inhibition tests with flaviviruses.     

Quantitative studies on antigenic recognition. 3. Nonspecific inhibition of the recognition process

Attempts were undertaken to evaluate the inhibitory effect of various agents on the process of recognition of transplantation antigens by lymphoid cells from normal mice, rats and Syrian hamsters. These effects were measured by estimating quantitatively a product of antigenic recognition (PAR) present in supernatants of mixed spleen cell cultures in which either the recognizing (parental strain aggressor) cell partner or the recognized ( F₁ hybrid target) cell partner was treated prior to cocultivation. The inhibitory agents used were heat, trypsin, ALS and rabbit anti-PAR antisera. Heat inactivation of cells resulted in inhibition of the process and affected both partners of cell mixtures. All other agents failed t o harm antigens on F₁ target cells but neutralized the recognizing potential of aggressor cells. In this respect, trypsin inhibited both mouse and rat aggressor cells to about equal extents. Anti-lymphocyte serum failed to show strain and even species specificity. Sera prepared against PAR also displayed no strain specificity but revealed species specificity.     

Leukaemia-associated antigens in man. Detection by antibodies in maternal sera

A membrane antigen on peripheral blood lymphocytes from cases of chronic lymphatic leukaemia (CLL) is described. The antigen was detected by complement-dependent cytotoxicity using serum from a healthy pregnant woman, and appeared to be absent from the normal lymphocyte population in these patients. The serum was also cytotoxic for some acute leukaemia blast cells and for cultured Burkitt lymphoma cells; absorption studies suggested that the CLL antigen is identical to the acute leukaemic antigen.

Similar antibody activity was also found in a number of HL-A typing antisera and could be separated from the HL-A specificity by absorption.

Although these antibodies are probably directed against foetal antigens, the leukaemic antigen could not be demonstrated on foetal tissues.

     

Immunosuppressive ALS. II. Antibody to Ia antigens in heterologous anti-lymphocyte serum.

The relationship between Ia alloantigens and xenoantigens detected by immunosuppressive heterologous anti-lymphocyte sera has been investigated. Three rabbit anti-rat lymphocyte sera were examined for the presence of antibodies to Ia antigens. Two of these sera, an anti-thymus membrane and anti-lymphocyte sera detected labelled cell-surface Ia antigens (mol. wt 35,000 and 27,000) present on rat spleen B cells. The third antiserum, prepared against fractionated soluble lymphocyte antigens, was essentially non-reactive with these antigens. Of these three heterologous antisera, the anti-membrane serum reacted with the same antigens detected by two alloantisera. It seemed possible that such an antiserum could modify a recipient animal's immune response in vivo in a fashion identical to alloantibody to Ia antigens. In fact, all three heterologous antisera, including one devoid of antibody to Ia proved immunosuppressive in vivo. These results suggest that antibodies to antigens other than Ia can induce prolonged allograft survival. Since heterologous sera bind Ia antigens, it remains to be determined whether monospecific heterologous antisera to Ia antigens can allograft survival. The results raise the prospect that more than one antibody specificity may contribute to the immunosuppression achieved with ALS.     

Preparation of anti-β1C antisera and their use in fluorescent antibody techniques

Antisera against components of guinea-pig complement were raised in rabbits by:

(1) Using as antigen a suspension of killed Proteus species bacteria which had been allowed to combine with their homologous antiserum in the presence of guinea-pig complement in optimum proportions.

(2) By injection of the β_1C component of guinea-pig complement adsorbed to zymosan particles. The antisera raised by the two methods contained antibodies mainly against the β_1C component of complement.

When coupled with FITC both antisera were found useful in detecting sites of complement fixation. The fluorescence anti-complement technique was found four times more sensitive than the indirect method for detecting antigen—antibody reactions in the presence of diminishing concentrations of antigen. It was only twice as sensitive for detecting antigen—antibody reactions in the presence of diminishing concentrations of antibody. The comparisons were based upon both visual assessment and photometric measurement.

Coupled antisera raised by the first method gave brighter specific fluorescence than antisera raised by the second method when used in the highest concentration which did not give non-specific staining.

The usefulness for detection of viral antigens of sera raised by both methods is discussed.

     

The immunology of fascioliasis: II. Qualitative studies on the precipitin reaction

Antigens prepared from Fasciola spp. precipitated in agar with antisera obtained from mice or rabbits infected with Fasciola hepatica, and with antisera from cattle infected with F. gigantica, or from rabbits immunized with preparations of adult F. hepatica. In the latter there were at least thirty-eight different antibodies, reacting with both protein and species specific non-protein antigens.

The reactions of the antisera from infected animals were less complex. Antisera from infected cattle or mice gave little or no reaction with protein antigens, whereas antisera from infected rabbits reacted predominantly with one of the protein antigens.

Certain non-protein components of the somata or metabolic products of the flukes also reacted with antisera from infected animals. These components were not species specific, and appeared to be haptens as they did not react with antisera from rabbits immunized with killed fluke antigen.

     

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an ¹²⁵I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (?1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an ¹²⁵I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).     

Role of Biological Mimicry in the Pathogenesis of Rat Arthritis Induced by Mycoplasma arthritidis

Complement fixation (CF), immunofluorescence, and agar gel double-diffusion tests were used to demonstrate an antigenic relationship between rat tissues and Mycoplasma arthritidis. Rabbit antisera against six strains of M. arthritidis exhibited positive reactions in the CF test with an ethyl alcohol-saline extract of rat muscle, whereas only 6 of 18 antisera against other Mycoplasma species were positive. With the use of gel diffusion techniques, absorption of various M. arthritidis antigens with antiserum against rat muscle removed at least one precipitin band when the absorbed mycoplasma antigens were reacted against homologous antisera. Rabbit antiserum against M. arthritidis was conjugated with fluorescein isothiocyanate and reacted against frozen sections of muscle tissues of various animals. As controls, unlabeled normal rabbit serum and rabbit anti-M. arthritidis serum were included to determine the specificity of the reaction. Rat, hamster, and mouse skeletal muscle exhibited specific fluorescence, whereas chicken, beef, frog, and turtle muscles exhibited no specific fluorescence. Mice injected at birth with rat lymphocytes were found to be more susceptible to subsequent infection by M. arthritidis than were normal mice or mice injected at birth with mouse lymphocytes. These results indicate the occurrence of a heterogenetic antigen(s) common to M. arthritidis and rat tissues. Preliminary evidence suggests that this heterogenetic antigen(s) may enable the mycoplasmas to become established in their host.     

Immunological Studies on Adrenal Glands: III. Interspecies Relations of Thermostable Adrenal-Specific Antigens

In continuation of the previous work on organ specificity of bovine adrenal, rabbit antisera against human, porcine and equine adrenal were obtained. It was demonstrated that, in addition to ox, man, pig and horse contain adrenal-specific antigens that can be associated with organ preparations obtained by extraction at 100° followed by precipitation at a 72 per cent ethanol concentration.

The present study allowed identification of two thermostable adrenal-specific antigens, one of them being species-restricted and the other crossing the species line. The former antigen was serologically dissimilar in man, ox, pig and horse. It could be detected only by homologous anti-adrenal sera and was represented by a thicker precipitation line. The latter antigen could be detected by both homologous and heterologous anti-adrenal sera and it was represented by a thinner precipitation line. This antigen was closely related, though possibly not identical, in man, ox, pig and horse.

     

Specificity of isoelectric focusing-purified antigens in the diagnosis of human cysticercosis

Specific antigens were isolated from the cystic fluid of larval Taenia solium by preparative isoelectric focusing (PIEF). A total of 20 fractions were produced by a rotating ampholine column with pI 3–10 ampholytes. The specificity of each fraction (F) was tested by double-antibody enzyme-linked immunosorbent assay (ELISA) using antisera from patients suffering from cysticercosis or one of six other parasitic diseases. F8–F15 cross-reacted strongly with sera from patients with hydatidosis. F9 and F10 also cross-reacted with the antisera against ascariasis and F15, with antisera against angiostrongylosis. However, F16 and F17 were highly specific as they yielded no cross-reaction with any of the heterologous antisera. PIEF is a good method for the production of specific antigens from larval T. solium because it is easy to perform and relatively inexpensive to run. Received: 29 November 1997 / Accepted: 10 February 1998     

Detection of T and B cell-specific heteroantigens on electrophoretically separated lymphocytes of the mouse

Mouse spleen lymphocytes were electrophoretically separated into pools of T and B lymphocytes. Heteroantisera were raised in rabbits against these cell pools, the IgG fractions were isolated and their lymphocytotoxicity tested. After appropriate absorption, the antisera were specifically directed against lymphocyte antigens. Further cross-absorption with T and B lymphocytes made the antisera specific for antigens which were mutually exclusively present on T and B cells and were related to neither alloantigens nor immuno-globulins. These anti-B and anti-T cell antisera killed antibody-forming cells and graft-versus-host reactive cells, respectively, in vitro. Furthermore, anti-B cell antiserum was cytotoxic to lymphocytes of low electrophoretic mobility in bone marrow and peripheral organs, except in the thymus, whereas anti-T cell antiserum was cytotoxic to lymphocytes of high electrophoretic mobility in all organs and, in addition, killed all thymocytes of low electrophoretic mobility, which had not been affected by anti-B cell antiserum. This distribution of lymphocytes in the electrophoretic distribution profiles, together with the described properties, gives evidence that heteroantigens were found to be exclusively present on T and B cells. They were designated “mouse B cell-specific” and “mouse T cell-specific” antigens and are part of mouse lymphocyte-specific antigen.     

The in vitro determination of the D antigens of poliomyelitis viruses: I. The gel diffusion test

1. A technique for the measurement of poliomyelitis virus D antigens by means of gel diffusion tests against antisera obtained from hyperimmunized calves is described in detail.

2. The precision of the method was estimated by performing several replicate tests with three or four dilutions of concentrated antigen of each of the three types. The D antigen content, if measured by means of twelve precipitation hexagons in agar can be calculated with a precision of about 10 per cent (95 per cent confidence limits).

3. The calf sera gave no precipitation with C antigens in the concentrations suitable for measuring D antigens.

4. The local reference antigens, the D antigen concentrations of which were adjusted to equal that of the reference antigens used in the Glaxo Laboratories, contained C as well as D antigens. This was observed in gel diffusion tests with specific anti-C sera from guinea-pigs.

5. The guinea-pig anti-C sera showed a precipitation with the immune calf sera as well as with normal calf serum. This indicated the presence of anti-calf antibodies in the guinea-pig sera, presumably elicited by traces of calf proteins in the vaccines. These antibodies might give rise to non-specific precipitation if antisera used in the test for D antigen were obtained from animal species other than those used to provide components in the tissue culture medium from which the antigens were derived.

     

Function and distribution pattern of human T lymphocytes. I. Production of anti-T lymphocyte specific sera as estimated by cytotoxicity and elimination of function lymphocytes

Antisera were prepared in rabbits against lymphoid cells from peripheral blood of a patient with Bruton-type agammaglobulinaemia. Such antisera could be shown to display a two plateau level of cytotoxicity against human peripheral lymphocytes in the presence of complement. This suggested the presence of species- as well as subgroup-specific antibodies in these antisera.

The subgroup-activity of the antisera could be shown to be directed against human T lymphocytes on the basis of the following results. When lymphocytes are filtered through columns coated with anti-immunoglobulin antibodies lymphocytes with high surface concentrations of immunoglobulin are retained. The filtered cells are highly enriched in cells sensitive to the subgroup-specific antibodies. A close to complete inactivation of mixed leucocyte reactivity or stimulability with soluble PHA was induced by preincubating with the antisera and complement. Using identical conditions only marginal inhibition of immunoglobulin production of peripheral lymphocytes in vitro was induced.

In conclusion, we believe these antisera to selectively kill human T lymphocytes at serum concentrations, which will not kill or inhibit the function of human B lymphocytes.

     

Serological relatedness of herpes simplex viruses. Type-specificity of antibody response.

The serological relatedness of forty-seven strains of type 1 and type 2 herpes simplex virus was investigated by reciprocal and non-reciprocal neutralization kinetics. Early rabbit antisera divided the virus strains into two distinct groups where confident indentification of virus type was possible. Hyperimmune mouse and rabbit antisera did not divide the two virus types into two distinct non-over-lapping groups. The extent of overlap varied with the particular attribute of the virus being studied. The virus types were best discriminated by their neutralizability by type 1 antisera and least well by their neutralizability by type 2 antisera. The results of reciprocal kinetic neutralization test with hyperimmune mouse antisera were analysed by multi-dimensional cluster analysis. Hyperimmune mouse or rabbit antisera could not be discriminated with respect to their immunogenic type by their absolute neutralization rate constants against either type 1 or type 2 virus, but could be distinguished on a group basis by their relative neutralizability against both virus types (antiserum specificity attribute); however, using this latter criterion, the type of immunogen could only be predicted in seven of the forty antisera under test. 'Early' mouse antisera could also be distinguished as groups by their absolute k-values against type 1 herpes virus. Thus, immunogenic identification, on other than a group basis, was unreliable. The specificity of a given serum was inversely related to its titre. There was a positive correlation between the specificity of a given virus strain and of its corresponding antiserum.     

A comparison of passive protection tests against intranasal and intracerebral challenges with Bordetella pertussis

A survey of a number of rabbit antisera to Bordetella pertussis revealed the existence of two distinct antibodies, one passively protecting mice against lethal infection by the intracerebral route, the other passively protecting mice against lethal infection by the intranasal route. Neither is the antitoxin. Antisera against most, if not all, S forms of B. pertussis contain both types of protective antibody, and so to a lesser extent do B. parapertussis and B. bronchisepticus. Neither of the protective antigens is an agglutinogen, or the haemagglutinin. The two antigens can also be distinguished by active protection tests. Extensive investigations, however, had not led to an in vitro test for either of the protective antigens or their antibodies that would replace the mouse test.

The practical importance of the two distinct antigens and their antibodies is shown by the fact that assay by the intranasal and intracerebral routes of challenge will not arrange vaccines in the same order of potency either in active or in passive protection tests.

     

Microfluorometric evaluation of the specificity of fluorescent antisera against mouse immunoglobulins with the defined antigen substrate spheres (DASS) system

Highly purified MOPC-21 IgG₁, MOPC-173 IgG_2a, MOPC-195 IgG_2b, MOPC-104 E IgM, and MOPC-315 IgA paraproteins, heterogeneous mouse IgG, Fab and Fc fragments of heterogeneous IgG were prepared and coupled to Sepharose beads. These beads were then used as artificial substrates to test the specificity of fluorescent antisera against mouse immunoglobulins by microfluorometry. By comparing the visual evaluation of stained plasma cells and measurements on beads, the highest permissible percentage impurity in a conjugate was determined. It was 14% for a fluorescein iso thiocyanate (FITC) and 6% for a tetramethyl rhodamine iso thiocyanate (TRITC) conjugate. By application of these criteria, 1 out of 7 tested commercial antisera and 6 out of 8 conjugates prepared in this laboratory proved to be satisfactory. The most common impurities were anti-light chain antibodies, as revealed by their reaction with Fab. With the bead system, a good impression of the specificity of an antiserum can be obtained. It gives, however, only approximate information on whether the conjugate will cause a high background staining in the biological specimen.