Detection of anti-HLA antibodies by enzyme-linked immunosorbent assay, fluorescence activated cell sorter and microlymphocytotoxicity testing: a comparison of sensitivities and suggestions for standardization of ELISA

Loewenthal, Ron; Shmuelian, I; Efter, T.; Avishai, Ophelia; Kalt, Rivka; Moskovich, Y; Gazit, Ephraim

doi:10.1016/s0041-1345(99)00155-4

Cited by 3 publications

(5 citation statements)

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“…When titrating the mab BRO11F6, the CDC was slightly superior to the other methods, while the PRA-STAT exhibited the lowest antibody titer. These results contradict those of another study comparing the sensitivity of ELISA, CDC, and flow cytometry (35). In that study, they found that their HLA antigen screening ELISA, which is based on bound HLA antigen preparations, was generally more sensitive than the CDC, with an advantage of some serial dilution steps.…”

Section: Discussionmentioning

confidence: 62%

“…the assays, but antibody specificities and test sensitivities vary in detail due to test-specific characteristics (19,29,34,35). The IPIm detects a higher percentage of panel reactivity in sera from high-risk patients awaiting a kidney than the CDC, and this is in good agreement with graft failure (14).…”

Section: Discussionmentioning

confidence: 94%

“…But the problem of IgM reactivity of the CDC and of nonreactivity with non-complement-fixing antibodies remains. Many studies comparing different assays for the detection of HLA antibodies determine the percentage of PRA in pre-or posttransplant sera (32,35,36). These PRA values then may be correlated with the clinical outcome of the transplantation.…”

Section: Discussionmentioning

confidence: 99%

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HLA‐antibody testing: The immune phagocytosis inhibition test is superior to the PRA‐STAT and NIH lymphocytotoxic test with respect to specificity

Flesch¹,

Philipp²,

Cassens

et al. 2001

Clinical Laboratory Analysis

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We compared the specificity and sensitivity of four different methods for the detection of antibodies specific for HLA antigens. The NIH version of the complement-dependent cytotoxic test (CDC) was used as the gold standard to which we compared two Fcgamma receptor (FcgammaR)-dependent immune phagocytosis inhibition tests (IPI) and one commercial enzyme-labelled immunosorbent assay (ELISA) with soluble HLA class I-antigen preparations bound to the plate (PRA-STAT). Both IPI tests are based on the fact that HLA-antibodies specifically bind to antigens on the monocyte surface via their Fab portion, and in so doing block a neighbouring FcgammaR with their Fc region. This blockade prevents phagocytosis of IgG-coated red blood cells (RBCs), which can be measured either microscopically (IPIm) or photometrically (IPIp). The four assays were used in blind tests on 20 human alloantisera or monoclonal antibodies with known HLA-antigen reactivities. Additionally, two monoclonal antibodies and one human serum were titrated to elucidate the sensitivity of each test. After all tests were completed, the identities of the samples were disclosed. Both IPI methods detected and identified all clinically relevant HLA class I and class II specific antibodies. In contrast, the CDC was not able to detect noncytotoxic HLA-antibodies and HLA class II specific antibodies; however, it detected clinically insignificant IgM lymphocytotoxins. The PRA-STAT assay enabled identification of all cytotoxic and noncytotoxic IgG antibodies with specificity for HLA-class I antigens. With respect to sensitivity, the CDC and the IPI methods were superior to the PRA-STAT. These facts demonstrate the advantage of IPI methods in the detection of clinically relevant HLA-antibodies.

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Section: Discussionmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

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HLA‐antibody testing: The immune phagocytosis inhibition test is superior to the PRA‐STAT and NIH lymphocytotoxic test with respect to specificity

Flesch¹,

Philipp²,

Cassens

et al. 2001

Clinical Laboratory Analysis

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“…The ELISA technique is more sensitive than CDC in detecting HLA antibodies (21, 22) but has the potential drawback of not distinguishing between complement-fixing and non-complement-fixing antibodies. This assay however has been used as a very effective method for detecting pre- and postsensitization in solid organ transplants (23–25) but has been somewhat superseded by the introduction of fluorescently labeled beads to which HLA molecules have been attached.…”

Section: Hla Antibody Detection Assaysmentioning

confidence: 99%

Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

Tait

2016

Front. Immunol.

113

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This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques.

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“…The main conclusion from other studies is that ELISA methods (most studies used the PRA-STAT assay) serve as a good alternative to the CDC, with an approximate agreement of 80 % between both assays [26,29,41,42]. Some laboratories clearly prefer the ELISA method [15,38,43,44] while some recently published papers describe the flow cytometric analysis [27,45] as more sensitive. A recent study described flow cytometry as the most sensitive technique for detecting HLA class I antibodies, while the ELISA was superior in HLA class II antibody detection [46].…”

Section: Test Specificity and Test Sensitivitymentioning

confidence: 99%

Laboratory Diagnostics of HLA Antibodies/Labordiagnostik von HLA-Antikörpern

Flesch¹

2003

LaboratoriumsMedizin

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Detection of anti-HLA antibodies by enzyme-linked immunosorbent assay, fluorescence activated cell sorter and microlymphocytotoxicity testing: a comparison of sensitivities and suggestions for standardization of ELISA

Cited by 3 publications

References 0 publications

HLA‐antibody testing: The immune phagocytosis inhibition test is superior to the PRA‐STAT and NIH lymphocytotoxic test with respect to specificity

HLA‐antibody testing: The immune phagocytosis inhibition test is superior to the PRA‐STAT and NIH lymphocytotoxic test with respect to specificity

Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

Laboratory Diagnostics of HLA Antibodies/Labordiagnostik von HLA-Antikörpern

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