A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic fever and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten 125I-L-Ala-gamma-d-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis.

Acute Disease↗

PubMed1975

Primary structure of the L chain from a rabbit homogeneous antibody to streptococcal carbohydrate. II. Sequence determination of peptides from tryptic and peptic digests.

A complete amino acid sequence containing 210 residues of a rabbit light chain of a homogeneous Group C streptococcal antibody has been determined as: See journal for formula. This light chain is allotype b4 and is of the V-K1 subgroup.

Amino Acid Sequence↗

PubMed1975

Primary structure of the L chain from a rabbit homogeneous antibody to streptococcal carbohydrate. I. Purification of antibody and sequence determination of peptides from alpha-chymo-tryptic and thermolytic digests.

Primary structural studies have been carried out on the light chain of a homogeneous rabbit antibody to Group C streptococcal carbohydrate. Approximately 20 g of this antibody were obtained by multiple exchange transfusions at the peak of the antibody response. The isolated antibody was homogeneous by several antigenic and chemical criteria. The light chain was isolated and modified, and then digested with alpha-chymotrypsin or thermolysin. The resulting peptides were isolated by gel filtration, paper electrophoresis, and paper chromatography. The amino acid sequences of these peptides were determined by Edman degradation plus dansylation. This supplied sufficient information to assign approximately 90 percent of the residues in the chain. The destruction of tyrosine during acid hydrolysis of peptides which had been eluted from a paper chromatogram was investigated. This destruction is due to inpurities in the paper which contaminate the peptides. Prevention of such destruction can be achieved by predevelopment of the paper with 1 N NH4OH prior to paper chromatography.

Amino Acid Sequence↗

PubMed1975

A peptide mapping technique--a three map system.

Explore the source record for details and available documents.

Chromatography, Paper↗

PubMed1975

Prevention of streptococcal sequelae by penicillin prophylaxis: a reassessment.

In conclusion, the diagnosis of streptococcal infection lacks precision, and as long as this is so, reliance must be placed on trained clinical judgement together with bacteriological studies. There is some evidence for an evolutionary drift in the relationship between the streptococcus and man, a drift that is compatible with the natural history of other diseases and with the accomodations observed in nature among other species during the course of evolutionary events. If, as a result of recent accomodations between man and the group A streptococcus, the risk of rheumatic fever is less, an undefinable risk nevertheless remains. There was even a military outbreak of rheumatic fever in England several years ago, and on that island--we are told--rheumatic fever has been banned for years. The current recommendations of the American Heart Association for the secondary prevention of rheumatic fever I judge acceptable for use in the United States. I have, however, raised the possibility that they may be inadequate for the prevention of second attacks in other populations where the environmental, socioeconomic, bacteriological, and nutritional circumstances are different from our own. Studies designed to reexamine these questions may, in the end, refocus our attention on the importance of host factors in the occurrence and pathogenesis of rheumatic fever.

Developing Countries↗

PubMed1975

Newly recognized distinctive M protein associated with certain M type 1 group A streptococci.

Immunological and epidemiological characteristics of M1T1 and M1T8 (T8, 25, Imp.19) group A streptococci isolated in Egypt have been described. Both strains possess a common M1 antigen. The M1T8 strains possess a second antigen which has the immunological and biological characteristics of M protein. The M1T1 strains do not possess this second M antigen. Absorbed rabbit antisera which are specific for this second antigen do not react with extracts of any of the known M protein serotypes. This second antigen is therefore a newly recognized M protein. The M1T1 and M1T8 strains had mutually exclusive geographical and temporal distributions. This finding may have been a consequence of the immunity which occurred after the initial occurrence of the M1T8 strain.

Absorption↗

PubMed1975

Use of radioimmunoassays to determine the concentration of streptococcal group-specific antibodies in rabbit antisera.

A radioimmunoassay was developed for the quantitation of antibodies to the Group A, A-variant, and C carbohydrates in rabbit streptococcal antisera. The assay employs radiolabeled purified Group carbohydrate which has been tyrosylated to allow for incorporation of 125-I. This assay has an advantage over the intitative precipitin test because it measures non-precipitating antibody in addition to precipitating antibody. Furthermore, 7S anti-IgG in rabbit antisera give a falsely elevated value for the antibody concentration in the quantitative precipitin test. This did not occur with the radioimmunoassay. The assay described is reproducible, sensitive, and uses little antisera.

Adsorption↗

PubMed1975

A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan.

A major portion of the humoral immune response to peptidoglycans is directed against the non-cross-linked pentapeptide side chains of these ubiquitous bacterial antigens. At present, no specific and sensitive assay for pentapeptide antibody determination is available. Therefore, a radioimmunoassay has been developed which employs the synthetic pentapeptide hapten L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, labeled by the active ester method of Bolton and Hunter to high specific activities (6.74 to 18.18 muCi/mug) with 125I, and used as a reagent for measuring pentapeptide antibody. A-variant streptococcal antisera, known to contain pentapeptide antibodies as shown by quantitative precipitation, would bind more than 95% of the radiolabeled hapten in contrast to 2 to 3% by preimmune rabbit sera. Specificity of the binding reaction was demonstrated by inhibition experiments imploying various synthetic oligopeptides related or unrelated to the pentapeptide in the radioimmunoassay. Binding curves established with serial dilutions of peptidoglycan antiserum were linear from 15 to 500 mug/ml of antibody permitting pentapeptide antibody measurement within this range. Comparative data on pentapeptide antibody determinations by quantitative precipitation and radioimmunoassay are given and the time course of the production of this antibody in 14 rabbits hyperimmunized with A-variant streptococcal vaccine is reported.

Ammonium Sulfate↗

PubMed1975

Immunological activity of the peptidoglycan.

Studies on the immunology of peptidoglycan received impetus from the initial observation that the rabbit Group A-variant streptococcal antisera were a rich source of antibodies to peptidoglycan. Indeed, quantitative precipitin studies revealed concentrations as high as 7-10 mg/ml of antiserum. The amount of antibody after Group A-variant streptococcal immunization is much greater than the amount in the sera following immunization of rabbits with the Group A or C streptococci. Furthermore, earlier studies had shown that the purified peptidoglycan obtained as a residue following extraction of streptococcal cell walls with hot formamide was a poor antigen. Both the hexosamine polymer and the peptide moiety are antigenic. Use of the solid phase synthesized pentapeptide L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala and related similar peptides facilitated the determination of the fine structure of the immunodominant site of pentapeptide. The evidence points to the C-terminal D-Ala-D-Ala as the immunodominant determinant. Because of the similarity of the peptidoglycans of a number of different bacteria, it would be anticipated that they would cross-react immunologically, and this has been shown to be the case. The biological and medical significance of antibodies to peptidoglycan has yet to be determined. Certainly the exposure to this ubiquitous substance which occurs in all the indigenous bacteria of the respiratory and the gastrointestinal tract must mean that from an early age and through advancing years there is a constant stimulation of the immune response to peptidoglycan. Because the immunochemistry of these substances is now firmly established, there is a scientific basis for proceeding with the medical and biological implications of peptidoglycan immunity.

Antibodies, Bacterial↗

PubMed1975

Measurement of peptidoglycan antibodies by a radioimmunoassay.

Staphylococcus epidermidis peptidoglycans solubilized by sonication or lysozyme digestion, and synthetic peptidoglycan analogs such as HSA-carboxymethyl-Gly-L-Ala-L-Ala-D-Ala-D-Ala (HSA-pentapeptide) or L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) have been labeled with 125I and tested for their applicability in the radioactive antigen binding assay. Use of radioiodinated Staph. epidermidis peptidoglycans was found to be considerably impeded by the presence of at least 2 different antigenic sites on such molecules, the pentapeptide and the glycan determinant. Application of labeled HSA-pentapeptide was limited by the necessity to use PEG for precipitation of Ag-Ab-complexes and by short linear potions of binding curves. However, the synthetic pentapeptide hapten, radioiodinated by the active ester method of BOLTON and HUNTER, proved to be a most useful regent for the selective measurement of pentapeptide antibody. Inhibition studies indicated that the immunological specificity of the labeled hapten was retained. Pentapeptide binding curves were linear from 15-500 g/ml of antibody. Generally, there was good agreement between pentapeptide antibody concentrations measured by radioimmunoassay and quantitative precipitation.

Antibodies↗

PubMed1974

Amino-acid sequence of an allotype b4 light chain from a rabbit antibody to streptococcal carbohydrate.

The complete amino-acid sequence was determined for a rabbit-antibody light chain of allotype b4. The light chain was recovered from approximately 20 g of homogeneous antibody to group C streptococcal carbohydrate, which was obtained from an immunized rabbit. Several enzymic digestions were used to obtain the overlapping peptides necessary for the sequence determination. The L chain has 210 amino-acid residues, as determined by sequence analysis, and a molecular mass of 23,060 daltons. This antibody light chain contains seven half-cystine residues characteristic of the (kappa)B light-chain class. The amino-terminal sequence was determined to be Ala-Asp-Ile-Val-Met, indicating that the chain belongs to the V(kappa)I subgroup.

Amino Acid Sequence↗

PubMed1974

Specificity and sensitivity of the streptozyme test for the detection of streptococcal antibodies.

A comparison between the results of the streptozyme hemagglutination test and serological titers for anti-streptolysin O (ASO), anti-hyaluronidase (AH), anti-deoxyribonuclease B (ADN-B), and anti-nicotinamide adenine dinucleotidase (ANAD) was made in two groups of human sera. In one group, serological titers for all the four antibodies were lower than the threshold of sensitization reported by the producing firm. In the second group, the titer of at least one of the four antibodies was equal to or higher than the threshold. False-positive and false-negative reactions occur with those sera when one or more antibody titer is at or near the threshold of the test as described by the manufacturer. The test was positive for all sera where either the ASO was greater than 166 or the ANAD was greater than 270, and for 98% of the sera with ADN-B greater than 360. It is, therefore, concluded that the streptozyme test can be used as an adjunct to the clinical diagnosis of streptococcal infections and their nonsuppurative sequelae. It is less useful to assess the levels of antibodies in sera from general population surveys. For such sera, the relative specificity and sensitivity of the test might yield misleading results. Until more experience is gained with the test, caution should be used in its application to infant and older adult age groups, where significant streptococcal antibody titers are frequently near the threshold of the test.

Antibodies, Bacterial↗

PubMed1974

Simplified extraction procedure for serological grouping of beta-hemolytic streptococci.

An adaptation of the nitrous acid extraction of streptococci proved to be a reliable and practical method for the preparation of extracts for routine serological group identification. The extracts of all groups tested gave strong capillary precipitin reactions as well as reactions of double diffusion in gel. For routine grouping, extracts were prepared from the first one-half-plate subculture of the initial throat culture. The technique is simple and reliable, and it requires a minimum of technical skill, reagents, and equipment. Its use would facilitate epidemiological surveillance of group A streptococci and rapid diagnosis of streptococcal infections at a low cost.

Antigens, Bacterial↗