Report of the second international soluble HLA (sHLA) workshop. Phoenix, Arizona, October 2, 1993.
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Antibodies, Monoclonal↗
Biomedical subjects
P Pouletty
Publications and source records attributed to P Pouletty.
Typing of a panel of soluble HLA class I antigens by enzyme-linked immunosorbent assay.
Two ELISA assays were developed to test the reactivity of soluble sHLAs with anti-HLA class I mAbs of IgG or IgM isotype. A panel of 40 different alleles of sHLA antigens was produced using 57 lymphoblastoid B-cell lines, which had been generated from class-I-phenotyped PBLs. Using 14 mAbs, the expression of 13 different sHLA antigens (sHLA-A2, -A3, -A11, -A24, -A29, -B7, -B8, -B13, -B14, -B27, -B44, -B57, and -B58) by 43 different cell lines was confirmed. In addition, the expected absence of these alleles from the culture supernatant of 11 cell lines was confirmed. Cross-reactivities of mAbs observed by microlymphocytotoxicity assays were also detected by ELISA. The results of this extensive analysis confirmed previous results demonstrating that sHLA class I typing by ELISA correlated with HLA class I cell typing by microlymphocytotoxicity [1-3]. Furthermore, additional information about the fine specificities of two mAbs was obtained. An anti-B27/44 IgM mAb appeared to react only with sHLA-B44 but not with sHLA-B27; mAb CR11-351, previously reported to react with HLA-A2, 28, bound also to sHLA-A1, -3, -11, and -A24.
Alleles↗
Rapid detection of anti-OKT3 antibodies with the Transtat assay.
The ability to successfully reuse OKT3, a mouse monoclonal antibody, is dependent upon the host's response to the antibody during and following the first treatment course. Antiidiotypic and/or antiisotypic antibodies may develop after exposure to OKT3. Antiidiotypic antibodies will bind OKT3, rendering it ineffective, while antiisotypic antibodies do not influence the efficacy of OKT3. A new membrane-based immunoassay, Transtat OKT3 (Sangstat Medical Corp, Menlo Park, CA) detects anti-OKT3 antibodies in less than 15 min. It allows simultaneous detection of antiidiotype and antiisotype antibodies. A total of 180 serum samples were initially analyzed by ELISA; results were negative, low-titer (1:100), or high-titer (> or = 1:1000). Retrospectively, these same samples were analyzed by Transtat for both anti-OKT3 (idiotype) and IgG2a (isotype). A total of 109 samples of 180 (60.6%) tested negative by ELISA and Transtat, while 71 (39.4%) tested positive. Of the negative samples by ELISA, 98 of 109 (89.9%) also tested anti-OKT3-negative by Transtat. Of the 109 specimens that were anti-OKT3 negative by Transtat, 98 (89.9%) tested negative by ELISA. There were 22 discrepant samples between the two methods; all were low-titer-positive (ELISA and Transtat). The 71 positive ELISA samples consisted of 53 low-titer (1:100) and 18 high-titer (> or = 1:1000), while the 71 anti-OKT3 positive Transtat samples consisted of 44 low-titer (1:10) and 27 high-titer (1:50). Sixty of 71 (84.5%) ELISA-positive samples were also positive by Transtat. Similarly, 60 of 71 (84.5%) Transtat-positive samples were also positive by ELISA. Of 71 patient samples positive for anti-OKT3 antibodies, 63 had an antiisotypic component present by Transtat. In conclusion, the Transtat OKT3 assay for measuring OKT3 and IgG2a antibodies offers a rapid and accurate assay for OKT3 monitoring.
Animals↗
Typing of serum-soluble HLA-B27 antigen by ELISA.
An ELISA using serum as soluble HLA antigen source was developed for HLA-B27 typing. Two sandwich assays were run in parallel. The first assay utilized a monoclonal antibody (mAb) reacting with a determinant expressed by both HLA-B7 and B27 antigens; the other assay utilized a mAb reactive with HLA-B7 antigens but not with HLA-B27 antigens. After incubation with serum samples, bound HLA antigen was detected using an anti-beta 2m antibody conjugated to peroxidase and a chromogenic substrate. Absorbance of each well was measured at 490 nm. Based on analysis of absorbances obtained with panels of specimens of known HLA phenotypes, a mathematical algorithm was developed to derive the specimen HLA-B27 phenotype from its ELISA absorbance values. Despite the lack of monospecific mAb, an accurate HLA-B27 typing was possible. 362 specimens (including 151 HLA-B27-positive) were tested. Agreement between microlymphocytotoxicity and ELISA was 99.2%. No correlation between the level of HLA-B27 antigen reactivity and the amount of total HLA class I antigen in serum was observed. This report demonstrates the possibility of using serum-soluble HLA antigen and ELISA technology for histocompatibility testing. The assay offers several significant advantages over microlymphocytotoxicity: no need for cell preparation, batch testing capabilities and objective, reproducible interpretation of results.
Algorithms↗
Summary report from the first international workshop on soluble HLA antigens. Paris, August 1992.
The First International Workshop on Soluble HLA antigens focused on the comparison of immunoassay procedures for quantitation of soluble HLA (sHLA) class I antigens and the selection of a sHLA class I antigen international standard. Several sets of serum, plasma, and cell culture supernatant specimens were assayed blindly for levels of sHLA class I antigens by 15 participating laboratories using different immunoassay formats. The sandwich ELISA using (i) for antigen capture: an anti-HLA class I heavy chain monoclonal antibody (mAb) specific for a monomorphic epitope, and (ii) for antigen detection: an anti-beta 2 microglobulin antibody-enzyme conjugate, was the assay format of choice. There was a high inter-laboratory correlation among the majority of laboratories. All serum and plasma specimens from normal donors, and from a single transplant patient, had detectable levels of sHLA class I antigens. Paired serum and plasma specimens had similar levels of sHLA class I antigens, although plasma sHLA antigens seemed more stable than serum sHLA antigens. sHLA-A2 and sHLA-B7 antigens were detected in all specimens from HLA-A2 and HLA-B7 donors, respectively, using allele-specific ELISAs. No difference in reactivity was observed for quantitation of native sHLA class I antigens whether the capture mAb was TP25.99 (alpha 3 domain-specific) or W6/32 (alpha 2 + alpha 3-specific). However, a human-mouse chimeric sHLA class I antigen reacted weakly in assays which used TP25.99 mAb. The wide variation among laboratories in their reporting of micrograms/ml units pointed to the need for an inter-laboratory standardization based on a calibrated sHLA antigen preparation. T.sB7, an sHLA-B7 antigen derived from a cell line transfected within human beta 2 microglobulin and HLA-B7 genes, was accepted as the First sHLA class I Antigen International Standard at the workshop meeting.
Alleles↗
Peptides corresponding to T-cell receptor-HLA contact regions inhibit class I-restricted immune responses.
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Amino Acid Sequence↗
Peptides corresponding to the CD8 binding region of HLA class I block the differentiation of cytotoxic T lymphocyte precursors.
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Animals↗
Human cytomegalovirus structural proteins: immune reaction against pp150 synthetic peptides.
In the present study, several peptides of the major structural antigen (pp150) of human cytomegalovirus (CMV) have been chemically synthesized and tested by a modified slot blotting procedure for their ability to bind CMV-specific immunoglobulin G (IgG) and IgM present in human sera. The sequences of the peptides were deduced on the basis of either (i) their presence in a fusion protein already known to be frequently recognized by human antibody or (ii) their high content of hydrophilic amino acids as deduced from the published nucleotide sequence. An important IgM-binding epitope was found to be located in the last 38 amino acids at the carboxy terminus of the molecule. This region reacts with anti-CMV IgM present in the great majority (83.3%) of IgM-positive human sera, and adsorption experiments have shown that IgM titers to the entire pp150 decrease 25 to 50% in most sera previously absorbed with this region. The overall results obtained endorse the continued synthesis of other sequences in order to define a group of peptides sensitive and specific enough to replace the virus and infected cells as an antigenic substrate in the serological evaluation of anti-CMV antibody.
Adult↗
Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.
We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.
Animals↗
Purification of murine IgG3 and IgM monoclonal antibodies by euglobulin precipitation.
We describe a simple and efficient non-chromatographic method for the purification of murine IgG3 and IgM monoclonal antibodies (MAbs) which takes advantage of their euglobulin properties. Following filtration, ascitic fluid is dialysed against demineralized water and centrifuged at 22,000 X g for 30 min. The resulting precipitate is dissolved in a high salt buffer (0.1 M Tris-HCl, 1 M NaCl, pH 8). A second cycle of dialysis and centrifugation yields a product of high purity. Nine IgG3 MAbs and eight IgM MAbs were purified by this procedure. Recovery was greater than 90% for seven of nine IgG3 MAbs. It was less reproducible for IgM MAbs and ranged from 40% to greater than 90% depending on antibody and batch. Purity was assessed by SDS-polyacrylamide gel electrophoresis. The purified immunoglobulin was essentially free of albumin, transferrin, and other mouse ascites proteins. No loss of antibody function was observed.
Animals↗
A new sensitive non-isotopic method using sulfonated probes to detect picogram quantities of specific DNA sequences on blot hybridization.
The use of non-radioactive systems to detect target DNA or RNA displays many advantages such as safe manipulation, potential use in non-specialized scientific area and prolonged lifetime of the probes (one year or more). We here describe a method we have improved and optimized using sulfonated DNA probes for hybridization on dot and Southern blots. Sulfonation is an easy chemical modification procedure which does not require enzymatic coctail as does nick-translation. Sensitivity of this method has been particularly improved by using a new blocking solution, containing heparin, which allows easy and fast detection of picogram quantities of DNA. This method allows the use of nitrocellulose as well as nylon membranes with very low background. Equal resolution is obtained in comparative experiments involving both sulfonated and 32P-radiolabelled probes. Single copy gene sequences are readily detected in nuclear DNA. These results allow the use of this procedure for restriction fragment length polymorphism (RFLP) studies.
Base Sequence↗
Optimization of a rapid test by using fluorescein-conjugated monoclonal antibodies for detection of Chlamydia trachomatis in clinical specimens.
A mixture of two fluorescein isothiocyanate-conjugated monoclonal antibodies (MAbs) was used to optimize a direct specimen test (Chlamydia Direct Specimen Test IF; Clonatec, Paris, France) for detection of chlamydial elementary bodies in clinical specimens. One MAb reacted with a subspecies-specific epitope of the major outer membrane protein (molecular weight 43,000) of Chlamydia trachomatis, whereas the other reacted with the periodate-sensitive genus-specific antigen (molecular weight 11,000) of Chlamydia spp. Nonfat dry milk was the most efficient additive at suppressing the fluorescent background and was included in the antibody preparation. Fc-dependent binding of fluorescein-conjugated MAbs to protein A-containing Staphylococcus aureus was inhibited by addition of purified rabbit immunoglobulin. The Chlamydia Direct Specimen Test IF was compared with tissue culture isolation by using 309 genital specimens. The sensitivity and specificity were 77.4 and 98%, respectively.
Animals↗
[Comparison of ELIFA and IGM-immunocapture technics in the diagnosis of 50 cases of congenital toxoplasmosis].
The authors have compared the diagnostic value in congenital toxoplasmosis of two recently developed immunological techniques, Enzyme Linked immunofiltration Assay (ELIFA) and IgM-immunocapture (or immunosorbent agglutination assay). The study involved 50 children suffering from congenital toxoplasmosis and 300 unscathed control children, whose mothers suffered from toxoplasmosis during their pregnancies. The ELIFA technique showed a sensitivity of 74 p. cent at the end of the first month after birth (day 30) and 84 p. cent after two months (day 60), whereas the IgM-immunocapture technique gave a sensitivity of 64 p. cent at day 30 and 70 p. cent at day 60. Both methods were at least 95 p. cent specific. The two techniques seem to be complementary to each other, and their association allowed the diagnosis to be confirmed in 80 p. cent of cases at day 30 and in 90 p. cent of cases at day 60. A serum sample taken on the tenth day after birth (day 10) was important both to eliminate the rare cases (5 p. cent) of transplacental transmission of maternal IgM antibodies and to detect transient neonatal synthesis of IgG or IgM toxoplasmic antibodies.
Age Factors↗
Detection of herpes simplex virus in direct specimens by immunofluorescence assay using a monoclonal antibody.
A monoclonal antibody (MAb), designated CHA 437, was developed against herpes simplex virus (HSV). This MAb (isotype, immunoglobulin G2b K) reacted with HSV type 1 and HSV type 2. It showed no cross-reactivity with varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. Direct detection of HSV antigen in clinical specimens using indirect immunofluorescence with this MAb was compared with tissue culture isolation. For the 682 specimens tested, the direct specimen test gave a sensitivity of 84.6% and a specificity of 95.7%.
Animals↗
IgM and IgG antibodies to HTLV-III in the lymphadenopathy syndrome and subjects at risk for AIDS.
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Acquired Immunodeficiency Syndrome↗
An anti-human mu chain monoclonal antibody: use for detection of IgM antibodies to Toxoplasma gondii by reverse immunosorbent assay.
A precipitating anti-human mu chain monoclonal antibody (designated Tibi 82 McAb) was produced by the cell fusion technique. This McAb (isotype: IgG1 kappa) reacted by radioimmunoassay with all 10 human IgM proteins tested. In contrast, no reactivity was observed with IgG, IgA, IgE, lambda and kappa chains. 19 S IgM proteins were precipitated by Tibi 82 McAb using the Ouchterlony method under standard conditions. Hence specificity of this McAb for the C mu 2 domain was characterized by inhibition of precipitin reactions using human IgM fragments. Despite its narrow specificity for the C mu 2 domain, such a McAb could be used for IgM capture in the detection of specific IgM to Toxoplasma gondii employing the IgM immunosorbent agglutination assay (IgM-ISAGA). Tibi 82 McAb was compared with 3 anti-human IgM polyclonal reagents in the routine analysis of 117 sera. With 2 of them, a correlation coefficient of 0.976 was obtained and Tibi 82 McAb was more sensitive than the third polyclonal reagent tested. The IgM-ISAGA technique was shown to be reproducible using Tibi 82 McAb and similar anti-human mu chain McAbs could permit the wider development of reverse immunosorbent methods for the detection of specific IgM in various infectious diseases.
Antibodies, Anti-Idiotypic↗
Lymphadenopathy in patients at risk for acquired immunodeficiency syndrome. Histopathology and histochemistry.
Lymph node biopsies of 12 patients at high risk for acquired immunodeficiency syndrome (AIDS) with generalized lymphadenopathy (AIDS-related complex [ARC]) and seven controls with conventional lymph node hyperplasia were examined by light microscopy and immunohistochemical staining of frozen tissue. The immunohistochemical results were quantified by planimetric means. Our findings show that the T helper/T suppressor-cytotoxic (Th/Tsc) ratio in lymph nodes from ARC patients is significantly decreased with respect to controls and that this decrease precedes the change in the Th/Tsc ratio in peripheral blood. Our findings distinguish between lymphadenopathy from patients with ARC and other forms of hyperplasia; the relation to AIDS is discussed.
Acquired Immunodeficiency Syndrome↗
An anti-human immunoglobulin M monoclonal antibody for detection of antibodies to Toxoplasma gondii.
An anti-human mu-chain monoclonal antibody, Tibi 82, was produced and tested for specificity by radioimmunoassay. Its reliability in detecting IgM antibodies to Toxoplasma gondii was tested by two reverse immunosorbent methods (IgM-ISAGA and IgM-SPIHA) and the IgM fluorescent antibody test (IgM-IFA) on 400 sera. Whereas the results obtained with Tibi 82 and with two polyclonal reagents were highly correlated, the third commercial polyclonal reagent provided many false negative results. By standardizing IgM binding, Tibi 82 allowed the comparison of IgM-ISAGA with IgM-SPIHA on 100 sera: 17% of the sera tested showed discrepancies due to the different toxoplasma antigens used. Although Tibi 82 facilitated the reading of results and enhanced sensitivity and specificity of the double-sandwich IgM-IFA method, the latter was still less sensitive than IgM-ISAGA with Tibi 82. Tests with the monoclonal antibody were consistently superior to tests with polyclonal antibodies.
Antibodies, Anti-Idiotypic↗