Legal Medicine 16 (2014) 139–145

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Legal Medicine journal homepage: www.elsevier.com/locate/legalmed

Development of an indirect competitive ELISA for the detection of ABO blood group antigens Naoki Takada a,b,⇑, Chikahiro Mori b, Mizuho Iida b, Rie Takai b, Tomohiro Takayama b, Yoshihisa Watanabe b, Kohei Nakamura c, Kazuhiro Takamizawa c a b c

Science of Biological Resources, United Graduate School of Agricultural Sciences, Gifu University, Gifu 501-1193, Japan Forensic Science Laboratory, Gifu Prefectural Police Headquarters, Gifu 500-8501, Japan Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan

a r t i c l e

i n f o

Article history: Received 26 January 2014 Received in revised form 20 February 2014 Accepted 22 February 2014 Available online 3 March 2014 Keywords: ABO blood grouping Indirect competitive ELISA Monoclonal antibody Blood group antigen

a b s t r a c t We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in particular for blood stains. ABO blood group antigens conjugated to polyacrylamide were used for immobilized antigen. ABO blood group antigens were extracted from blood stains using a novel method involving pre-incubation with proteinase K (PK), followed by heat treatment. The extracts (analytes) were combined with either anti-A or -B monoclonal antibodies (mAbs), and added directly to the antigen-coated wells. The anti-A and -B mAbs were captured by either ABO blood group antigens present in the analyte or by the immobilized blood group antigens. Peroxidase-conjugated anti-mouse IgM antibody was used to detect anti-A and -B mAbs complexed with immobilized blood group antigens, and a colorimetric reaction using o-phenylenediamine/H2O2 used for its measurement. The ELISA developed in this study was able to detect blood group antigens in blood, saliva and blood stains. The detection limit for unknown blood, saliva and blood stain were determined as 1:200, 1:32 and 1:16. Overall the ABO blood grouping ELISA can be used with relative ease for the high throughput screening of biological samples for the detection of ABO blood group antigens. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The ABO blood group system has been used for many years as a general tool for the identification of human samples at crime scenes and for large-scale disasters. Conventional serological methods for detecting ABO blood group antigens such as the absorption–elution assay [1–3], absorption–inhibition assay [1] and mixed cell agglutination reaction (MCAR) [4] are all simple and inexpensive. However, these methods require the determination of the presence or absence of ABO antigens based on hemagglutination; making it difficult to determinate the ABO blood group objectively. Moreover, polyclonal antibodies required for the determination of ABO blood group antigens are hard to obtain for ethical reasons. Enzyme-linked immunosorbent assay (ELISA) is an immunological technique that proffers superior specificity, sensitivity and simplicity in the detection of antigens in complex samples using mAbs, and is able to provide the objective determination. ⇑ Corresponding author at: Forensic Science Laboratory, Gifu Prefectural Police Headquarters, Gifu 500-8501, Japan. Tel.: +81 58 271 2424; fax: +81 58 275 6041. E-mail address: [email protected] (N. Takada). http://dx.doi.org/10.1016/j.legalmed.2014.02.006 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

ELISAs have been used to determine ABO [5–12], Lewis [13,14] and Rh blood group phenotypes [15]. However, it is difficult to detect ABO antigens from dried stains such as blood stains, because the techniques used require the immobilization of analyte ABO blood group antigens onto the surface of microplate wells. Erythrocyte ABO antigens such as glycolipids and glycoproteins are located on the membrane surface [16], are difficult to extract with aqueous solutions and therefore require solubilization with detergents [17,18] or organic solvents [19]. Moreover, ABO blood group antigens cannot immobilize completely [12], forcing many forensic scientists to use other serological methods to detect ABO antigens from blood stains. Competitive ELISAs do not require the direct immobilization of analyte antigens and have been used extensively in the detection of low-molecular weight compounds such as pesticide residues in soil [20,21], dioxins [22], endocrine disruptors [23,24] and abuse drugs [25]. The competitive ELISA can be also applied to the detection of ABO antigens in human samples. We developed a simple technique for extracting ABO antigens from blood stains using proteinase K and heat treatment. The samples were then used in an indirect competitive ELISA developed and optimized in the current study. Our results demonstrated that

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the ABO blood grouping ELISA can be used to detect ABO antigens in saliva and blood, in particular for blood stains. 2. Materials and methods 2.1. Materials Polyacrylamide-conjugated blood group antigen type A (tri)PAA and type B (tri)-PAA were obtained from GlycoTech (Gaithersburg, MD, USA) and dissolved in phosphate buffered saline (PBS, pH 7.5). Proteinase K was purchased from Qiagen (Hildan, Germany). Microtiter-plates (F96 MaxiSorp Immunoplate) were obtained from Nunc (Roskilde, Denmark). Bovine serum albumin (BSA) and polyoxyethylene (20) sorbitanmonolaurate (Tween 20) were obtained from Wako (Tokyo, Japan). The washing solution was PBS and 0.05% Tween 20 (PBST). Blocking solution was PBST containing 1% BSA (PBST–BSA). The following six commercial anti-A and B mAbs were analyzed; Gamma clone (Gamma Biologicals, Houston, TX, USA), Novaclone (Dominion Biologicals, Dartmouth, Canada), Wako (Wako), Seraclone (Biotest AG, Dreieich, Germany), Immucor (Immucor, Norcross, GA, USA) and Ortho Bioclone, (Ortho Clinical Diagnostic, Tokyo, Japan). Horse-radish peroxidase-conjugated goat anti-mouse IgM mAb (anti-mouse IgM–HRP) was obtained from American Qualex (San Clemente, CA, USA). All mAbs were diluted with PBST–BSA. o-Phenylenediamine (OPD) was obtained from Sigma (St. Louis, MO, USA). Ten milligrams of OPD was dissolved in 20 ml McIlvaine buffer (0.2 M Na2HPO4 10.3 ml and 0.1 M citrate 9.7 ml, pH 5.0). 2.2. Samples Saliva stains were collected via the placement of a piece of filter paper in the mouths of healthy adult volunteers for 1 min. The saliva stain was then air-dried at room temperature. The ABO blood group and secretory (Se) or nonsecretory (se) status were determined by absorption–elution assays and absorption–inhibition assays, and the saliva samples categorized as type A Se (n = 5), type A se (n = 5), type B Se (n = 5), type B se (n = 5), type AB Se (n = 5), type AB se (n = 3), type O Se (n = 5), and type O se (n = 5). Bloods that could no longer be used for transfusions because of their expiration date were kindly provided by the Japanese Red Cross Society. The blood was categorized according to ABO blood type: type A (n = 5), type B (n = 3), type AB (n = 5), type O (n = 4). The blood samples were stored at 80 °C until required. Blood stains which prepared on bleached cotton cloth with human blood, air-dried and stored at room temperature and passed 5–10 years were used as samples. In addition, this study protocol was approved by the Ethical Committee of Gifu University. 2.3. Determination of optimal concentrations of immobilized antigens and mAbs by indirect ELISA Fifty microliters of serially twofold dilution blood type A (tri)PAA or blood type B (tri)-PAA (2.0 lg/ml) were added to the wells of a microtiter plate and incubated at 4 °C for 12 h. Unbound antigen was removed by washing three times with 250 lL PBST with a plate washer (MW-96CR, BioTec, Tokyo, Japan). This washing procedure was repeated for all other washing steps. Free binding sites on the well surface were blocked with PBST–BSA for 1 h at 37 °C. Unbound BSA was removed by washing, 50 lL diluted anti-A mAb or anti-B mAb with PBS was added to each well, and the plates incubated at 37 °C for 1 h. A 50-lL aliquot of anti-mouse IgM–HRP secondary antibody, diluted 1:1200 with PBST–BSA, was added to each well and incubated at 37 °C for 1 h. At the

completion of the incubation, the wells were washed and 50 lL OPD solutions added. The plates were incubated in dark place for 10 min at room temperature and 100 lL 1 M sulfuric acid added to each well to stop the reaction. The optical density (OD) at 490 nm was measured using a spectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA, USA). BSA–PBST without anti-A or -B mAb were included as blanks. The OD was corrected using the blanks. 2.4. Indirect competitive ELISA Saliva stains were cut into 1  2 cm2 pieces and the saliva extracted with 250 lL PBS. Blood was diluted 1:50 with PBS. The blood stain samples were cut into 0.25  0.5 cm2 pieces and the blood extracted with a solution of PBS (135 lL) and PK (15 lL) at 56 °C for 2 h. Blood was extracted from an equivalent sized sample of blood stain with PBS (150 lL) at 56 °C for 2 h and included as a control. Each extract was heat treated at 95 °C for 15 min, centrifuged at 25,000g for 10 min, and the supernatant recovered and aliquoted into two equal portions. Equal volumes (30 lL) of the supernatant (analyte) and either anti-A or anti-B mAbs were combined and incubated at 4 °C for 12 h. Wells of the microtiter plate were coated with the optimum concentration of blood group antigen (1.0 lg/mL) in 50 lL PBS at 4 °C for 12 h. The wells were washed three times with 250 lL PBST as described previously. Unbound binding sites were blocked with BSA–PBS (100 lL/well) at 37 °C for 1 h. The wells were washed and 50 lL of the analyte/ anti-A or anti-B mAb complex solution added to each well. The plates were incubated at 37 °C for 1 h, washed, and the colorimetric reaction performed as detailed above for the indirect ELISA. BSA–PBST without anti-A or -B mAb was included as the blank, and BSA–PBST without sample included as the control. The OD of the blank was subtracted from the gross OD of the sample and expressed as net OD of the sample. The results were expressed as B/B0(%): B/B0(%) = (ODsample)/(ODcontrol)  100%, where ODsample is the optical density of the well with sample, ODcontrol is the optical density of the well without sample. All procedural steps were summarized in Fig. 1. Samples for which B/B0(%) values were less than 90% were considered positive. 3. Results 3.1. Determination of optimum coating antigen concentration and mAb selection The optimum concentrations for the coating antigens, blood group type A and B (tri)-PAA, were determined by checkerboard titrations using an indirect ELISA. Blood group antigen probes were diluted 2.0  2 6–2.0  20 lg/mL with PBS and assayed against six commercial anti-A and B mAbs diluted 1:400. The standard curves for blood type A and B (tri)-PAA antigens for each of the commercial mAbs are presented (Fig. 2a and b, respectively). These results demonstrated that Seraclone (anti-A mAb) and Immucor (anti-B mAb) were highly reactive against blood type A and B (tri)-PAA, respectively. The optimum (saturation) concentration for type A and B (tri)-PAA antigen were established as 1.0 lg/mL (Fig. 2a and b). The optimum concentrations for the six commercial anti-A and B mAbs were determined by checkerboard titrations (1:400–1:25,600 dilutions) using an indirect ELISA and coating antigen concentration of 1.0 lg/mL. Fig. 2c and d represent the standard curves for all six commercial anti-A and B mAbs, respectively. These results demonstrated that Seraclone anti-A mAb was highly reactive against blood type antigen A (tri)-PAA, and that both Seraclone and Immucor anti-B mAb were highly reactive against blood type antigen B (tri)-PAA. The optimum (saturation)

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a

an-A or -B monoclonal anbody

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angen BSA

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Fig. 1. Development of an indirect competitive ELISA for the detection of ABO blood group antigens. (a) Samples were diluted with PBS (pH 7.5), centrifuged at 25,000g for 10 min, the supernatant recovered and divided in half. An equal volume (30 lL) of sample was combined with either anti-A or anti-B mAb and incubated at 4 °C for 12 h. (b) The wells of polystyrene ELISA plates were coated with blood group antigen probes in 50 lL PBS at 4 °C for 12 h. The wells were washed three times with 250 lL PBST. (c) The wells were blocked with 100 lL BSA–PBS and incubated at 37 °C for 1 h. At the completion of the incubation the wells were washed with 250 lL PBST three times. (d) A 50-lL aliquot containing sample extract combined with either anti-A or anti-B mAb was added to the wells. Plates were incubated at 37 °C for 1 h prior to washing with 250 lL PBST three times. (e) Anti-mouse IgM–HRP conjugate (50 lL) was added to each well. The plates were incubated at 37 °C for 1 h, prior to washing with 250 lL PBST three times. (f) o-Phenylenediamine solution (50 lL) was added to each well, the plates incubated at room temperature for 10 min, and the colorimetric reaction stopped by the addition of 100 lL 1 M sulfuric acid was to each well. The OD at 490 nm was measured using a spectrophotometer.

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Fig. 2. Standard curves obtained from checkerboard titrations of six mAbs and blood group antigens using indirect ELISAs. The optimum concentration of A and B antigens were determined by indirect ELISA against six mAbs; Gamma clone (open circle), Novaclone (open square), Wako (open triangle), Seraclone (closed circle), Immucor (closed square) and Ortho (closed triangle). Tests were performed in duplicate. Error bars represent the S.D. Panel (a) Detection of blood group A antigens using diluted anti-A mAbs (1:400 dilution). The highest O.D was observed for Seraclone (closed circle) when the concentration of blood group Type A (tri-PAA) was P1 lg/mL. Panel (b) Detection of blood group B antigens using diluted anti-B mAbs (1:400 dilution). The highest O.D was observed for Immucor (closed square) when the concentration of blood group Type B (tri-PAA) was P1 lg/mL. Panel (c) Effect of anti-A mAb concentration on assay sensitivity. Seraclone (closed circle) was the most efficient in binding to immobilized blood group A probes at an mAB dilution of 1:1600. Panel (d) Effect of anti-B mAb concentration on assay sensitivity. Immucor (closed square) was the most efficient in binding to immobilized blood group B probes at an mAb dilution of 1:1600.

concentration for anti-A mAb (Seraclone) and anti-B mAb (Immucor) was established as a 1:1600 dilution (Fig. 2c and d). The optimum concentration of blood type A and B (tri)-PAA were 1.0 lg/mL, and the optimum dilution for both anti-A mAb (Seraclone) and anti-B mAb (Immucor) were 1:1600. These optimized conditions were then used for the indirect competitive ELISA.

3.2. Detection of ABO blood group antigens in human samples by indirect competitive ELISA The optimized indirect competitive ELISA was used to detect ABO blood group antigens in human samples (blood, Se and se saliva, and blood stain).

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Fig. 3 shows the inhibition curves for blood. B/B0(%) values for Type A blood were reduced below 100% for anti-A mAb, but not anti-B mAb (Fig. 3a). Similarly, B/B0(%) values for Type B blood were reduced below 100% for anti-B mAb, but not anti-A mAb (Fig. 3b). B/B0(%) values for Type AB blood were below 100% for both anti-A and B mAbs (Fig. 3c). No reduction in B/B0(%) values were observed for either anti-A and B mAbs in Type O blood (Fig. 3d). Similar results were observed for the saliva and blood stain samples. Type A Se saliva (Fig. 4a), se saliva (Fig. 5a) and blood stain (Fig. 6a) reduced B/B0(%) values for anti-A mAb, but not anti-B mAb. Similarly, Type B samples (Figs. 4b, 5b and 6b) reduced B/B0(%) values for anti-B mAb, but not anti-A mAb. Type AB samples (Fig. 4c, 5c and 6c) reduced B/B0 values for both anti-A and B mAbs. Type O samples (Fig. 4d, 5d and 6d) failed to reduce B/B0 values for either anti-A or B mAbs. The detection limit for A antigen in type A blood was 1:800 (B/B0(%) = 81.1 ± 7.0%, Fig. 3a); B antigen in type B blood was 1:200 (B/B0(%) = 85.1 ± 3.6%, Fig. 3b); A antigen in type AB blood was 1:800 (B/B0(%) = 80.9 ± 6.6%, Fig. 3c) and B antigen was 1:200 (B/B0(%) = 68.6 ± 17.1%, Fig. 3c). The detection limit for A antigen in type A saliva (Se) was 1:1024 (B/B0(%) = 52.1 ± 27.1%, Fig. 4a); B antigen in type B saliva (Se) was 1:4096 (B/B0(%) = 46.9 ± 41.0%, Fig. 4b); A antigen in type AB saliva (Se) was 1:1024 (B/B0(%) = 59.6 ± 17.8%, Fig. 4c) and B antigen was 1:4096 (B/B0(%) = 81.0 ± 8.4%, Fig. 4c). The detection limit for A antigen in type A saliva (se) was 1:64 (B/B0(%) = 70.4 ± 16.1%, Fig. 5a); B antigen in type B saliva (se) was 1:32 (B/B0(%) = 72.2 ± 13.5%, Fig. 5b); A and B antigens in type AB saliva (se) were both 1:32 (A: B/B0(%) = 74.4 ± 10.7%, B: B/B0(%) = 55.3 ± 31.8%, Fig. 5c). The detection limit for A antigen in type A blood stain was 1:16 (B/B0(%) = 57.2 ± 16.1%, Fig. 6a); B antigen in type B blood stain was 1:16 (B/B0(%) = 55.6 ± 25.1%, Fig. 6b); A and B antigen in type AB blood stain were both 1:16 (A:B/B0(%) = 77.4 ± 1.7%, B: B/B0(%) = 81.1 ± 2.2%, Fig. 6c).

The specificity of the mAbs for their respective ABO blood type antigen was evaluated by indirect competitive ELISA. Type A samples failed to bind to anti-B mAbs (Figs. 3a, 4a, 5a and 6a). Similarly, type B samples failed to bind to anti-A mAbs (Figs. 3b, 4b, 5b and 6b). Type O samples failed to bind both anti-A and -B mAbs (Figs. 3d, 4d, 5d and 6d). 3.3. Effect of PK and heat treatment Fig. 7 illustrates the effect of PK and heat treatment on ABO antigen recovery from blood stains. Samples extracted from blood stains using PK and heat treatment reacted with mAbs (Fig. 7a), whilst samples extracted from untreated blood stains failed to react with mAbs (Fig. 7b). These results demonstrated that blood group antigens in blood stains can be solubilized using a mixture of PBS and PK.

4. Discussion 4.1. Immobilized antigens Indirect competitive ELISAs require the immobilization of capture antigen on the well surface of microtiter plates. However, ABO antigens in blood stains are unable to immobilize efficiently to the surface of the microplate [12]. Therefore ABO antigens in blood stains are to immobilize to the surface of the microplate with any other blood protein, competitively. The capture antigen is generally a low-molecular weight antigen (hapten) conjugated to a carrier protein such as BSA which is easy to immobilize [21–25]. In the current study we found that commercially available blood group antigens conjugated with polyacrylamide are easily immobilized onto the surface of microplate wells. At antigen concentrations of P1.0 lg/mL saturation was observed (Fig. 2a and b).

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Fig. 3. Analysis of ABO blood group antigens in human blood using an indirect competitive ELISA. Tests were performed in duplicate. Error bars represent the S.D. Panel (a) Human Blood (group A) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (b) Dilutions of human blood (group B) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (c) Dilutions of human blood (group AB) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (d) Dilutions of human blood (group O) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

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Fig. 4. Analysis of ABO blood group antigens in human secretor (Se) saliva using an indirect competitive ELISA. Tests were performed in duplicate. Error bars represent the S.D. Panel (a) Human saliva (group A, Se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (b) Dilutions of human saliva (group B, Se) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (c) Dilutions of human saliva (group AB, Se) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (d) Dilutions of human saliva (group O, Se) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

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Fig. 5. Analysis of ABO blood group antigens in human non-secretor (se) saliva using an indirect competitive ELISA. Tests were performed in duplicate. Error bars represent the S.D. Panel (a) Human saliva (group A, se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (b) Dilutions of human saliva (group B, se) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (c) Dilutions of human saliva (group AB, se) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (d) Dilutions of human saliva (group O, se) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

4.2. Blood group antigen extraction ELISAs generally require antigens to be soluble. Soluble ABO antigens, primarily glycoproteins, are found in body fluids such as saliva. In contrast, blood has very little soluble ABO antigens; exist-

ing as membrane-associated glycolipids. The extraction of antigen from blood stains usually requires the use of detergent [17,18] or an organic solvent [19], but the use of detergent and organic solvent have been proposed to interfere with the BSA in the blocking step or mAb absorption step. Saito and Tokiwa [12] found that ABO anti-

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Fig. 6. Analysis of ABO blood group antigens in human blood stains using an indirect competitive ELISA. Tests were performed in duplicate. Error bars represent the S.D. Panel (a) Human blood stain extracts (group A) were diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (b) Dilutions of human blood stain extract (group B) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (c) Dilutions of human blood stain extract (group AB) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). Panel (d) Dilutions of human blood stain extract (group O) were combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

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Fig. 7. Effect of PK treatment on the extraction of blood group antigens from blood stains. Tests were performed in duplicate. Error bars represent the S.D. Panels (a) and (b) Improved inhibition activity of capture anti-A and anti-B mAbs was observed for blood stains extracted with PK; comparisons made with non PK-treated blood stains. Panel c, PK treatment did not influence the inhibition activity of capture anti-A and anti-B mAbs for blood group O samples.

gens could be extracted from dehemoglobinized blood stain, however it was difficult to handle many samples at a time. We investigated whether PK, commonly used for proteolysis in DNA extractions from human samples including blood stains, could

assist in the recovery of ABO antigens from blood stains. We found that PK treatment resulted in dehemoglobinization and increased the level of ABO antigen recovered (Fig. 7). Heat treatment at 95 °C for 15 min inactivated PK.

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4.3. Indirect competitive ELISA for the detection of ABO blood types

References

The detection limit for type A blood was 1:800, whilst types B and AB reported a detection limit of 1:200. The detection limit for unknown blood was determined as 1:200. The detection limits for type A (Se), type B (Se) and AB (Se) in saliva were 1:1024, 1:4096 and 1:1024, respectively. The detection limit for type A (se) was 1:64, and 1:32 for both type B (se) and type AB (se). The detection limit for unknown saliva samples was therefore set at 1:32. The detection limit for type A, type B and type AB in blood stain was 1:16. The detection limit for unknown blood stains was determined to be 1:16. The specificity of the anti-A and -B type mAbs for their respective antigens were evaluated using the optimized indirect competitive ELISA. Type A samples failed to bind to anti-B mAbs (Figs. 3a–6a), and type B samples did not bind to anti-A mAbs (Figs. 3b–6b). Type O samples failed to react with either anti-A or -B mAbs (Figs. 3c–6c). Lower dilution of blood (61:400) gives high B/B0 (P100%) value (Fig. 3a, b, d) since B/B0 value is low thus, giving a higher blood dilution rate, suggesting that heat treatment is not enough conducive for high hemoglobin concentration. In the current study, we developed an indirect competitive ELISA for the detection of blood group antigens in human samples including blood stains. The ELISA was specific and sensitive for the detection of A and B type antigens, but failed to detect O type antigens. This was not unexpected as we did not include type O probe in this study. If this ELISA format is to be used for the identification of all ABO blood types, a method for the detection of O antigen must be devised. Moreover, it was difficult to test a large number of samples at a time using the conventional method such as absorption–elution and absorption–inhibition assays. In contrast, PK and heat treatments are simple to use with a thermomixer and were also capable to test for large number of samples. Furthermore, the use of PK and heat treatment in the extraction of antigens and subsequent blood grouping from bloodstain was made possible. This method require large amount of samples compared to the conventional method such as absorption–elution and absorption– inhibition assays. However, in this method, DNA typing is enabled to perform at a time because of PK and heat treatment are same as DNA extraction step. In DNA extraction, PK treatment is usually used containing detergent such as sodium dodecyl sulfate. It is thought that the sensitivity is still rise to select detergent. There is room for further improvement. Because the indirect competitive ELISA developed in this study can be handled with plate washer and multi-channel pipette, many steps can be automated. This method will be a useful tool for ABO blood grouping of large numbers of human samples derived from large-scale disasters such as the Great East Japan Earthquake. Thus, we conclude that the indirect competitive ELISA developed in this study can be performed with relative ease, for high throughput screening, and is a useful tool in ABO blood grouping of biological samples, particularly for blood stains.

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Acknowledgments We express our sincere appreciation to the Japanese Red Cross Society for providing human blood. We would also like to express our thanks to Dr. Hisayo Fukushima (Forensic Science Laboratory, Hokkaido Prefectural Police Headquarters) for her technical advice.

Development of an indirect competitive ELISA for the detection of ABO blood group antigens.

We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in part...
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