Measuring IgG anti-A/B titres using dithiothreitol (DTT).

Knight, Rob

doi:10.1136/jcp.31.3.283

Cited by 14 publications

(7 citation statements)

References 8 publications

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“…It has been shown that anti-A agglutinins treated with sulphydryl compounds would inhibit agglutination although the treated agglutinin no longer agglutinates the cells. Similarly, they are shown to give a positive antiglobulin test with appropriate cells (Moore and Steane, 1976;Knight, 1978). It is interesting to note in our studies (Table 4) that no changes of scores (neither increase nor decrease) are observed in the antiglobulin phase of reaction after DTT treatment.…”

Section: Discussionsupporting

confidence: 62%

“…There appears to be disagreement regarding the optimal conditions, such as incubation period, incubation temperature, or pH, for the 2-mercaptoethanol method of inactivating IgM antibodies (Freedman et al, 1976), and only a few studies on dithiothreitol reagent are available in the literature (Pirofsky and Rosner, 1974;Knight, 1978). The procedure for DTT is generally the one described by Pirofsky and Rosner.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of dithiothreitol (DTT) for inactivation of IgM antibodies.

Okuno¹,

Kondelis²

1978

J Clin Pathol

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The identification of red cell antibodies as IgM or IgG immunoglobulins often provides useful information in blood banking (Hasekura, 1974; Moor and Steane, 1976). IgM immunoglobulin may be inactivated by treatment with sulphydryl compounds such as 2-mercaptoethanol (2-ME). The drawbacks are its obnoxious odour and the need for dialysis of the specimen in some cases (Pirofsky and Rosner, 1974; Moor and Steane, 1976 (Webb, 1972).DTT was obtained from Sigma (St. Louis, Missouri). The procedure used was essentially the same as that described by Pirofsky and Rosner. Briefly, 0O01 M DTT is prepared in pH 7-4 phosphate buffered saline (PBS). After addition of 025 ml DTT in PBS to 025 ml of test serum, resulting in a final concentration of 0O005 M DTT, the mixture is incubated at 370C for two hours. For evaluation of the optimal conditions of DTT inactivation of IgM antibodies, changes of molarity of DTT were made by appropriately diluting the stock solution in phosphate buffered saline. The pH of the solution was altered by changing appropriately the ratio of Na2HPO and KH2PO4.The titration of antibodies was scored according to the procedure set out by the American Association of Blood Banks 1974. ResultsThe effects of incubation time, incubation temperature, and concentrations and pH of DTT were evaluated by using high-titre group 0 sera in the saline phase. The pretreatment titration of the sera ranged from 32 to 128. The effects of incubation temperature and incubation time on the reduction of titration scores by DTT are shown in Figure 1. A marked reduction was seen after a 15-minute 1152 copyright.

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Evaluation of dithiothreitol (DTT) for inactivation of IgM antibodies.

Okuno¹,

Kondelis²

1978

J Clin Pathol

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“…DTT treatment is needed to measure the exact IgG titration. However, the process for DTT treatment is cumbersome, time consuming, and may destroy IgG [ 14 ]. According to the 'uniform procedure' for ABO Ab titration suggested by the College of American Pathologists, the tube test can be converted to the AHG phase after 30 min of RT incubation without DTT treatment [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

Comparison of ABO Antibody Titers on the Basis of the Antibody Detection Method Used

2014

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BackgroundDetection methods for ABO antibody (Ab) titers vary across laboratories, and the results are different depending on the method used. We aimed to compare titer values using different detection methods for the measurement of ABO Ab titers.MethodsFor ABO Ab detection, pooled group A or B red blood cells (RBCs) were reacted with each of 20 sera from blood groups A, B, or O without dithiothreitol treatment. The room-temperature (RT) incubation technique and the indirect antiglobulin test (IAT) were used in the tube test and gel card test. Flow cytometry (FCM) was performed by using anti-IgM and anti-IgG Abs.ResultsRegardless of the blood groups tested, the FCM assay with anti-IgM showed the highest titer compared to the tube test and gel card test with RT incubation in both. The tube test with IAT showed a higher titer than the gel card test with IAT (Gel-IAT) or FCM with anti-IgG in blood group A and B, while Gel-IAT showed the highest titer relative to the other tests, only for the anti-A Ab in blood group O.ConclusionsThere were significant differences in the titers depending on the detection method used, and each method showed a different detection capacity for each ABO Ab depending on the ABO blood group tested. Therefore, caution should be exercised in interpreting ABO Ab titer results, taking into consideration the detection method used and the blood group.

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“…The dilution process was same as that for IgM. After incubating for 30 min in a 37°C water bath , 100 µl of 2% type A1 blood cell suspension or type B blood suspension (Ortho‐Clinical Diagnostics, High, Wycombe, UK) was dispensed into each tube and incubated for another 30 min in a 37°C water bath. The mixture was processed with the UltraCW automatic cell washing system (Helmer, Noblesville, USA) four times, and two drops of anti‐globulin reagent (Millipore, Livingstone, UK) were added and the sample was centrifuged for 15 s at 1000 g and then interpreted (Tube IgG).…”

Section: Methodsmentioning

confidence: 99%

Comparison of ABO isoagglutinin titres by three different methods: tube haemagglutination, micro‐column agglutination and automated immunohematology analyzer based on erythrocyte‐magnetized technology

et al. 2019

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Background and Objectives ABO isoagglutinin titre is important for evaluating and monitoring ABO-incompatible (ABOi) stem cells or solid organ transplantations. There are several methods to measure the titre level, including the tube haemagglutination method, micro-column agglutination and erythrocyte-magnetized technology (EMT). However, few studies have reported isoagglutinin measured by EMT. Here, we compared the isoagglutinin titre of normal individuals obtained by an automated instrument with that obtained by conventional manual methods to evaluate the feasibility of replacing the manual method with the automated instrument. Materials and MethodsThe ABO isoagglutinin titre was measured on residual samples of healthy individuals who visited the health promotion centre of the National Cancer Center, Korea, from April to October 2015. Samples from 120 patients were collected, which included 20 males and 20 females for each blood group (A, B and O). IgM and IgG ABO isoagglutinin titres of each blood group were measured by the tube haemagglutination, micro-column agglutination and EMT techniques. The median (minimum-maximum) titres were compared, and the concordance between two methods was evaluated with the rate of results showing within one titre difference. ResultsThe median ABO IgM and IgG titres of all blood groups obtained by the EMT method were higher than that obtained by the conventional tube haemagglutination and micro-column agglutination. ConclusionThe agreement between the two methods was comparable in case of IgM but low in IgG.

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Measuring IgG anti-A/B titres using dithiothreitol (DTT).

Cited by 14 publications

References 8 publications

Evaluation of dithiothreitol (DTT) for inactivation of IgM antibodies.

Evaluation of dithiothreitol (DTT) for inactivation of IgM antibodies.

Comparison of ABO Antibody Titers on the Basis of the Antibody Detection Method Used

Comparison of ABO isoagglutinin titres by three different methods: tube haemagglutination, micro‐column agglutination and automated immunohematology analyzer based on erythrocyte‐magnetized technology

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