Identification of Group a Streptococci by Reverse Passive Hemagglutination

A method for preparing sensitized human 0 erythrocytes with specific antibody is reported. Whole sera or 40% ammonium sulfate insoluble antibody globulin fractions did not satisfactorily sensitize erythrocytes to agglutinate in the presence of either group specific polysaccharide of group A beta-hemolytic streptococci or antigen produced during colony formation. Antibody purified-by affinity chromatography sensitized the erythrocytes to rapidly agglutinate in the presence of either antigen. No spontaneous or “pseudo-immune” agglutination occurred when the sensitized erythrocytes were suspended in human serum. Such sensitized human cells, while more difficult to prepare than sensitized staphylococci, should be suitable for detecting bacterial or viral antigens in vivo.     

A method for measuring different classes of human immunoglobulins specific for the penicilloyl group

A method is described for the detection of human immunoglobulins of the four main classes specific for the penicilloyl group. The technique is an adaptation of the red cell linked antigen antiglobulin reaction based on the finding that benzyl penicilloylated rabbit γ-globulin, specific for human erythrocytes, reacted specifically with erythrocytes but did not agglutinate them. In turn this complex reacted specifically with human penicilloyl antibody and it was then possible to titrate each immunoglobulin class by the addition of anti-immunoglobulin sera.The method described here was used to compare titres of penicilloyl specific immunoglobulins of the same class between different sera.The test was found to be less sensitive than the hapten modified bacteriophage reduction test but had the advantage that individual immunoglobulin classes could be compared. In the absence of a reliable method for the diagnosis of pencillin allergy, it is hoped that the technique described will be a useful addition to existing in vivo and in vitro methods of determining the antibody response of the patient to the penicilloyl group.     

Application to immunoglobulin M capture hemadherence assays of hemagglutination of monkey erythrocytes by native and recombinant human parvovirus B19 antigens.

Human parvovirus B19 recently was shown to agglutinate baboon and human erythrocytes. We have now demonstrated that both recombinant and native B19 antigens agglutinate rhesus, cynomolgus, and Saimiri monkey erythrocytes. Using cynomolgus erythrocytes and the recombinant antigen, we developed an immunoglobulin M (IgM) antibody capture hemadherence test (MACHAT) for the detection of specific B19 IgM antibodies in human sera. The results obtained with MACHAT were compared with those obtained with an IgM capture enzyme immunoassay (MACEIA) employing the native antigen routinely used in our laboratory. For 229 patient serum samples, we found 96% agreement between the results of the two assays. There was some evidence that MACHAT was slightly more sensitive than MACEIA. Our results add to the range of erythrocytes that can be agglutinated by B19 virus and show that native as well as recombinant antigens may be used in MACHAT.     

Detection of Salmonella in foods using a novel coloured latex test

A novel coloured latex test for the detection of Salmonella in naturally contaminated foods is described. The test consists of two reagents which are mixtures of red, blue and green latex suspensions; each colour of latex is sensitized with specific antibodies to different groups of Salmonella. In the presence of an individual group of Salmonella organisms, the latex sensitized with the antibody specific to that grouping will agglutinate to produce a crescent of single colour latex particles and a change of background colour. Seven trial centres, within the UK, evaluated the performance of the coloured latex test for the detection of Salmonella in natural and artificially contaminated foods. The results obtained by the trial laboratories are presented and show that it is possible to combine the coloured latex test with traditional or electrial methods to give detection and/or confirmation of Salmonella at least 24 hours in advance of conventional cultural methods.     

Relationship of hemagglutination to other biological properties of serologically classified isolates of Escherichia coli.

The ability of 170 serologically classified strains of Escherichia coli to agglutinate human erythrocytes was examined. Erythrocytes of blood group A were more sensitive indicators of this property than were those of groups B or O. The predominant receptor was shown to be mannose containing; however, an additional receptor was found in two of nine strains studied. Natural mannose-like inhibitors were not found in unconcentrated urine obtained from 12 humans. Isolates from the urine or blood of patients with infections agglutinated erythrocytes significantly more frequently than did isolates from feces. Urine isolates of 10 common serogroups and isolates of less common serogroups did not differ in their ability to agglutinate erythrocytes. Among isolates from the urine of patients with infections, the ability to agglutinate erythrocytes did not correlate with either the serogroup of the strain or the clinical syndrome of the patient. Of the several other biological properties that were examined, only the production of colicins showed a significant association with the ability to agglutinate human erythrocytes.     

Monoclonal antibodies against glycophorin A

Two mouse IgM monoclonal antibodies, 177.1 and 179.3, are directed against glycophorin A, the major sialoglycoprotein of human erythrocytes. Both antibodies agglutinate blood group M and N erythrocytes equally well, both before and after treatment with neuraminidase or trypsin, but fail to agglutinate erythrocytes treated with papain. Antibody 179.3 agglutinates MiVII(K.T.) cells, whose glycophorin A probably contains some alterations in amino acid sequence between residues 46-56, but antibody 177.1 does not agglutinate these cells. Neither antibody agglutinates En(a-)G.W. cells, which lack glycophorin A completely. The hemagglutinating activity of antibody 177.1 is inhibited by purified glycophorin A and its chymotryptic glycopeptides CH1 (amino acid residues 1-64) and CH3 (amino acid residues 35-64), whereas the hemagglutinating activity of 179.3 is inhibited weakly by glycophorin A but not by chymotryptic peptides. These antibodies both are classified as anti-En(a-)FS but apparently bind different epitopes.     

A mouse monoclonal antibody against glycophorin A produced by in vitro stimulation with human red cell membranes

Human erythrocyte membranes were used as antigen for production of mouse monoclonal antibodies against blood group related structures by in vitro immunization. Culture medium supernatant of PHA and PMA stimulated mouse thymus cells was used as source of cytokines. The selected antibody designated 124,D-7 (isotype IgM) was found to directly agglutinate all human red cells, except the rare erythrocytes En(a-) which lack glycophorin A. Immunoblotting showed faint bands in the positions of glycophorin A, whereas no binding occurred to glycophorin B. Inhibition of agglutination with purified glycophorin A and peptides suggests that the epitope is located within the amino acid residues 35-40. Rat and chicken erythrocytes also reacted with the antibody, whereas mouse erythrocytes were only agglutinated at very low dilutions of ascitic fluid.     

Further evidence that carbohydrates are the immunodeterminant structures of blood group M and N specificities

Terminal beta-D-galactopyranosyl (Gal) groups are implied in blood group N but not M specificity by the following findings: (a) Rabbit anti-asialoganglioside sera specific for terminal beta-Gal-(1 leads to 3)-GalNac agglutinate human group 0 M and N erythrocytes, the latter to a significantly higher titer, while rabbit anti-ganglioside GMl sera, whereas sialic acid modifies the antibody specificity, do not. The agglutination score with N erythrocytes was about twice that with M red blood cells. The asialoganglioside antibodies were readily absorbed by group 0 N erythrocytes, which were up to 10 times more efficient than group 0 M erythrocytes. Erythrocyte agglutination by the anti-asialoganglioside sera was inhibited by M and N antigen preparations isolated from group 0 red cells ghosts. N antigen was a better inhibitor. Asialoganglioside effectively inhibited the red cell agglutinations by anti-asialoganglioside serum. Ganglioside GMl did not inhibit. (b) Horse anti-pneumococcus Type XIV serum, which has anti-beta-Gal specificity, precipitated highly active N but not M substances. This precipitation was specifically inhibitable by oligosaccharides with terminal beta-Gal. (c) Beta galactosidase specifically inactivated native N and "acid-produced' N substances with the release of about three moles Gal per subunit of N antigen. It did not affect M antigen.     

Further Evidence that Carbohydrates are the Immunodeterminant Structures of Blood Group M and N Specificities

Terminal β-δ-galactopyranosyl (Gal) groups are implied in blood group N but not M specificity by the following findings: (a) Rabbit anti-asialoganglioside sera specific for terminal β-Gal-(1→3)-GalNac agglutinate human group 0 M and N erythrocytes, the latter to a significantly higher titer, while rabbit anti-ganglioside G_MI sera, where sialic acid modifies the antibody specificity, do not. The agglutination score with N erythrocytes was about twice that with M red blood cells. The asialoganglioside antibodies were readily absorbed by group 0 N erythrocytes, which were up to 10 times more efficient than group 0 M erythrocytes. Erythrocyte agglutination by the anti-asialoganglioside sera was inhibited by M and N antigen preparations isolated from group 0 red cell ghosts. N antigen was a better inhibitor. Asialoganglioside effectively inhibited the red cell agglutinations by anti-asialoganglioside serum. Ganglioside G_MI did not inhibit, (b) Horse anti-pneumococcus Type XIV serum, which has anti-β-Gal specificity, precipitated highly active N but not M substances. This precipitation was specifically inhibitable by oligosaccharides with terminal β-Gal. (c) Beta galactosidase specifically inactivated native N and 'acid-produced' N substances with the release of about three moles Gal per subunit of N antigen. It did not affect M antigen.     

Immunity and paralysis in mice. Serological and biological properties of two distinct antibodies to type III pneumococcal polysaccharide

The nature of two distinct anti-pneumococcal antibodies of mice has been further investigated. These antibodies, produced after the injection of purified SIII (the capsular polysaccharide of Diplococcus pneumoniae Type III), were examined for their serological properties in vitro and for their protective potency in vivo.The first anti-SIII antibody, distinguished by its ability to agglutinate antigen-coated erythrocytes, was not only unable to lyse these erythrocytes (with complement), but it protected them against lysis by the other antibody, because of its high affinity for the antigen. This haemagglutinating antibody did not precipitate soluble antigen, was of the IgA class, and could protect mice against challenge with virulent pneumococci. It was produced by mice given small doses (0.1 μg) of SIII, but not by mice given 50 μg. These latter were susceptible to pneumococcal challenge.The second antibody, which was haemolytic and precipitating but not haemagglutinating, had a comparatively low affinity for the antigen: thus it was not detectable when mixed with the first antibody, especially if the test erythrocytes were inadequately sensitized. This haemolytic antibody was of the IgM class. It was found in high titre in mice given 50 μg of SIII, but did not protect them against challenge.     

A simple procedure to use whole serum as a source of either IgG- or IgM-specific antibody

Absorption of rabbit antiserum to Forssman antigen with immobilized staphylococcal Protein A or concanavalin A selectively removed IgG or IgM antibodies, respectively. This absorption procedure was more rapid than ion exchange chromatography on DEAE cellulose or molecular sieving on Sephadex G-200 and gave a better yield of functionally purer antibody. This absorption method gave antiserum suitable for the preparation of either IgM or IgG sensitized sheep erythrocytes and should be of value for the large-scale preparation of indicator cells required for the study of complement action and for detection of specific receptors on cell surfaces.     

Characterization of functional Fc receptor material from human lymphoblastoid cell lines—I. Large scale purification and biochemical analysis

A receptor, specific for the Fc portion of IgG, was purified from several lymphoblastoid cell lines using immunoadsorbents prepared from either immobilized antigen-antibody or heat aggregated immunoglobulin columns. Biochemical analysis revealed that the receptor is a multimeric glycoprotein with a subunit polypeptide mass of 46,000 daltons, held together by disulfide bonds. Integrity of these disulfide bonds is essential for successful binding of the receptor complexes to the Fc portion of complexed Ig. The association of receptor with immunoglobulin, once formed, was found to be highly stable even in the presence of low pH or high concentrations of chaotropic agents. The receptor, present on the surface of several human lymphoblastoid cell lines, was found to represent as much as 4–5% of the total cytoplasmic membrane proteins. The receptor showed no affinity to staphylococcal Protein A, but did bind to Protein A-immunoglobulin complexes, indicating separate binding sites for these proteins on the Fc portion of IgG. The purified receptor was found to be insoluble in aqueous buffers after detergent removal. Nevertheless, in functional assays, the precipitated receptor was able to agglutinate sheep erythrocytes sensitized with rabbit anti-SRBC IgG, but not unsensitized SRBC or SRBC sensitized with IgM. Antiserum against the receptor was found to react with a specific subclass of normal human or mouse peripheral blood lymphocyte, and with all Fc receptor positive but not with Fc receptor negative cell lines tested.     

Morphological differences in Neisseria meningitidis pili.

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.     

Antibody-induced destruction of colon antigen-coated chicken erythrocytes by normal human lymphocytes

⁵¹Cr-labeled chicken erythrocytes were sensitized with a polysaccharide antigen from colon of germ-free rats. When exposed to highly purified human blood lymphocytes in excess (25 lymphocytes: 1 erythrocyte) in the presence of heat inactivated rabbit antiserum to rat colon antigen, the modified red cells were lysed within 16–20 h. Lysis was specific and could be inhibited by addition of increasing amounts of soluble colon antigen. Maximal lysis was induced by antibodies from hyperimmune rabbits. While antibodies of the IgG class were active in this test, IgM antibodies lacked the capacity to induce lymphocyte mediated lysis. Under the experimental conditions applied, as little as 1 ng IgG antibody was required for 50 % lysis. This corresponded to ～ 80 000 antibody molecules for lysis of one erythrocyte. The model system described should provide a basis for further studies of the cytotoxic potential of lymphocytes and/or antibodies to colon from patients with ulcerative colitis.     

From IgG monoclonals to IgM-like molecules

One problem in blood group testing is that IgG monoclonal antibodies, in contrast to IgM, do not usually agglutinate erythrocytes. One of the reasons is the high zeta potential induced by the negative charge of the cell surface. During the last few years, we have produced a series of human monoclonal antibodies by the conventional fusion technique directed against antigens of the Rh blood group system. Some of these monoclonals, especially those directed against Rh-subgroups such as the c-antigen, were mainly of the IgG-subtype and unsuitable for agglutination tests. We have therefore tried to establish a molecular biological method to make IgM-like molecules from IgG monoclonals. From the c-antigen specific human hybridoma BS 240 (IgG subtype), we isolated mRNA that was transcribed into cDNA and then amplified by PCR using family specific primers. The heavy and light chain products were cloned into the pHen vector containing a DNA linker fragment, a myc-tag for identification and a His-tag for purification. After transformation in E.coli and phage rescue with helper phage, the culture supernatant was screened for antigen positive recombinant phage antibodies as a first control for specificity using c-antigen positive erythrocytes and anti-M13 antibodies as bridging antibodies (Coombs technique). Erythrocytes being negative for the c-antigen served as a negative control. After changing the culture conditions, soluble single chain fragments (scFv) were obtained from the periplasmatic extract. Specificity was shown using the c-antigen positive and negative erythrocytes and the 9E10 antibody (anti-myc) as a bridging antibody. To obtain IgM-like molecules, DNA coding for the specific scFv was cloned into the vector pSTE containing DNA coding for the monomer of core streptavidin. After expression, purification and refolding of the monomer, the core streptavidin combines to form tetrameric structures, termed scFv::strep, that are able to bind biotin as shown using ELISA plates coated with biotinylated BSA. Binding was detected with 9E10 and a peroxidase conjugated secondary antibody. In the agglutination assay, the construct was able to agglutinate c-antigen positive erythrocytes but not the negative erythrocytes. These experiments show that it is possible to construct IgM-like agglutinating molecules from cells containing secreting IgG antibodies. Experiments employing human antibody libraries instead of hybridoma cell lines are now in progress.     

Analysis of the fine specificities of 11 mouse monoclonal antibodies reactive with type 2 blood group determinants.

The specificity of 11 mouse monoclonal antibodies reacting selectively with type 2 blood group structures was analyzed in detail by studying their reactivities with a panel of standard glycolipids, glycolipids from erythrocytes and blood group glycoproteins. The antibodies reacted with monofucosyl type 2 H, difucosyl type 2 structures (Le gamma) or both; none of the antibodies reacted with type 1 (H, Lea, or Leb) structures. Only a small proportion of the antibodies were completely specific for either type 2H or Le gamma structures. None of the antibodies had identical patterns of reactivity and their specificities were individually distinct. Seven antibodies preferentially agglutinated O and A2 erythrocytes. Anti-Le gamma-specific antibodies, except mAb101, did not agglutinate erythrocytes or react with glycolipids from erythrocytes, indicating the absence of Le gamma structures in erythrocyte glycolipids. The ability of some antibodies to react with A erythrocytes was shown to be due to cross-reactivity of the antibodies with type 3 (repetitive) A structures. The study demonstrates that monoclonal anti-carbohydrate antibodies tend to react with a range of related, and even distantly related, structure in a pattern characteristic of each antibody and that very few antibodies have extremely restricted specificities.     

A functional role for Fcμ receptors on human lymphocytes

The essential findings in this paper are that ox erythrocytes (ORBC) sensitized with human heterophile IgM antibody failed to detect Fcμ receptors on human lymphocytes. These results contrast to those obtained when the ORBC were sensitized with mouse hybridoma IgM antibody (SCC I). It is therefore suggested that the functional role of Fcμ receptors on human lymphocytes is not to bind IgM/antigen complexes.     

Indirect hemagglutination test for detection of antibody to Rickettsia rickettsii in sera from humans and common laboratory animals.

Antibody production in humans and three species of laboratory animals infected with Rickettsia rickettsii was determined with the indirect hemagglutination test. Rabbits, guinea pigs, and mice were inoculated with R. rickettsii and bled at intervals. Antibody which agglutinated both fresh and glutaraldehyde-fixed sheep erythrocytes sensitized with antigen prepared either from purified rickettsiae or from infected yolk sacs was found in rabbit sera at all intervals tested (10 to 59 days postinfection). Antibody which agglutinated fresh but not glutaraldehyde-fixed erythrocytes sensitized with either of the above antigens was detected in guinea pig sera obtained 7, 14, and 28 days postinfection. Antibody was found in mice inoculated with 5.6 x 10(6) plaque-forming units of R. rickettsii but not in mice given 5.6 x 10(2) plaque-forming units. Peak indirect hemagglutination titers occurred in nonvaccinated human Rocky Mountain spotted fever patients about 3 weeks after onset of illness, and antibody was still detectable after 1 year. Both human immunoglobulin G and human immunoglobulin M antibodies agglutinated sensitized cells, but immunoglobulin M antibodies apparently were more efficient. The indirect hemagglutination test is useful for the titration of human, rabbit, guinea pig, and mouse antibodies when the appropriate erythrocytes are used.     

Requirement for continuous antigenic stimulation in the development and differentiation of antibody-forming cells. Effect of antigen dose

The concept that antigen has a continuous role in the recruitment and differentiation of immune progenitor cells was tested with optimum and suboptimum doses of heterologous erythrocytes in mice. These studies further evaluated an immune cell maturation scheme in which continuous antigenic stimulation is required for both the recruitment of `antigen-sensitive units' and the expansive proliferation of a distinct sensitized cell compartment, which undergoes irreversible differentiation to functional antibody-forming cells. Haemolytic plaque-forming cell capacity during both the primary and secondary immune reactions were studied, both in the intact animal and with the spleen cell transfer technique. This in vivo culture technique was used to measure the sensitized cell compartment in the absence of existing antibody regulatory mechanisms. The results clearly demonstrate a higher detectable secondary immune capacity in the suboptimum antigen dose group than in the optimum antigen dose group. This was demonstrated for both the 19S and 7S cellular responses, as well as with humoral antibody levels measured in the spleen cell recipient mice. It can be concluded that in the presence of a suboptimum dose of antigen, which rapidly diminishes during the early intervals of the primary response, there is adequate recruitment with subsequent preservation or rescue from antigen-mediated depletion of the sensitized cell compartment, at the expense of the detectable primary response.     

Human antibody response to three meningococcal outer membrane antigens: comparison by specific hemagglutination assays

Three cell surface antigens, protein, lipopolysaccharide, and polysaccharide, were purified from group B and group C strains of Neisseria meningitidis representing a variety of serotypes. Chemical analysis indicated that cross-contamination was on the order of 1%. Sensitization of sheep erythrocytes with these antigens resulted in highly specific passive hemagglutination assays for the three kinds of antigens. Paired human sera from several groups of individuals were tested by hemagglutination for antibody against each of the antigens. Patients with group B or C systemic meningococcal disease showed increases in antibody titer against all three kinds of antigens, but the antibody response to B polysaccharide was low compared with the response to C polysaccharide. Nasopharyngeal carriers of group B meningococci showed significant increases in titer only against the protein antigens, and noncarriers who received a C-polysaccharide vaccine had a specific response to the C polysaccharide. A given protein or lipopolysaccharide antigen reacted on the average equally well with either group B or C convalescent sera. These results suggest that all three antigens may play a role in the broad human immunity following natural infection.