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Colloids and Surfaces B: Biointerfaces 54 (2007) 114–117

Short communication

Preparation of latex reagents combined with IgM and its F(ab′)2 fragmentfrom commercial ABO blood grouping reagent

Hiroaki Nakanishi a,b,∗, Takeshi Ohmori a,b,1, Tomoko Akutsu b,1, Koichi Sakurada b,1

a Forensic Science Laboratory of Yamanashi Prefectural Police H.Q., 312-4 Kubonakajima, Isawa-cho, Fuefuki-city, Yamanashi 406-0036, Japanb National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa-city, Chiba 277-0882, Japan

Received 3 February 2006; received in revised form 22 April 2006; accepted 21 May 2006Available online 26 May 2006

bstract

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouseonoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab′)2

ragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab′) fragment was able to detect the 1000-fold diluted saliva and

2

rovided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible fordsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette endsithin 30 min.2006 Elsevier B.V. All rights reserved.

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eywords: ABO blood grouping reagent; Mouse monoclonal IgM; F(ab′)2 frag

. Introduction

A monoclonal antibody-coated latex agglutination assayan be used to detect antigen sensitively and to visualizehe antigen–antibody reaction. Additionally, the assay is sim-le and rapid; commercial latex reagents have been widelymployed in clinical diagnosis since Singer’s report [1]. Thentibody adsorbed on commercial latex reagents is generallymmunoglobulin G (IgG) but the application of immunoglobulin

(IgM) to commercial latex reagents has not yet been reported.he ABO blood grouping remains very useful in narrowingown the group of unidentified people before DNA typing inorensic examination. To date, various methods, including forxample the absorption–elution test [2] and the mixed aggluti-ation test [3], have been developed for ABO blood grouping.owever, these methods are complicated and time-consuming.

herefore, we have developed a new simple and rapid for ABOlood grouping in forensic science examination using the latexeagent, although the anti-ABO blood group antibody is com-

∗ Corresponding author. Tel.: +81 55 262 0082; fax: +81 55 262 0082.E-mail address: nakanishi@384.jp (H. Nakanishi).

1 Tel.: +81 4 7135 8001; fax: +81 4 7133 9153.

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927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2006.05.010

; Latex agglutination

osed primarily of IgM. In the present study, we isolated IgMnd its F(ab′)2 fragment, which is dimeric disulfide linked pro-eolytic fragment consisting of two L-chains and two pieces of-chain [4], from commercial ABO blood grouping reagent and

ucceeded in the preparation of two reagents. In addition, wevaluated the applicability of these latex reagents in forensiccience practices.

. Materials and methods

.1. Isolation of IgM from commercial blood groupingeagent and preparation of its F(ab′)2 fragment

Mouse monoclonal anti-B antibody (Immucor Inc.) was pre-ipitated by the addition of a half volume of saturated solu-ion of ammonium sulfate. The precipitate dissolved in phos-hate buffered saline (PBS) was applied to gel filtration onSuperdexTM G-200 column equilibrated in PBS using the

MART systemTM (Amersham Bioscience) to isolate IgM. The

oid volume was collected and IgM in this fraction was verifiedy the red blood cells (RBCs) agglutination reaction and directnzyme-linked immunosorbent assay (ELISA) using anti-mousegM (H + L) antibody (American Qualex Antibodies).

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Fig. 1. (a) Gel filtration chromatogram of pepsin-digested IgM on a SuperdexTM

G-200 column equilibrated in PBS using the SMART systemTM. The columneluent was monitored for absorbance at 280 nm (solid line). Respective fractions(100 �l) were assayed by direct ELISA with the anti-mouse IgM (H + L) anti-body (closed circles with dotted line). The arrow indicates a peak containing the

H. Nakanishi et al. / Colloids and Sur

Pepsin digestion was performed using a modified version ofhe procedure described by Pascual and Clem [5] to obtain the(ab′)2 fragment. The IgM at 1–2 mg/ml was dialyzed against.02 M sodium acetate buffered saline (pH 4.0). Crystallineepsin (3200 U/mg; Sigma Chemical Co.) dissolved in the sameuffer was added to the IgM solution at ratios of 1:10, 1:25 and:50 (pepsin:IgM, w/w). Digestion proceeded with gentle stir-ing at 4 ◦C for 20 h, and was terminated by the addition of 1/10olume of 3 M Tris. The optimum amount of pepsin for diges-ion of IgM was determined based on the results of Westernmmunoblotting using anti-mouse IgM (H + L) antibody after2% sodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGE) under the reducing condition. The F(ab′)2 frag-ent was isolated by gel filtration on a SuperdexTM G-200

olumn equilibrated in PBS using the SMART systemTM. Theractions that collected every 100 �l were assayed by directLISA using anti-mouse IgM (H + L) antibody and the frac-

ion containing the fragment derived from IgM was exam-ned by 12% SDS-PAGE under reducing condition with silvertaining.

Concentrations of proteins were determined by the coppereduction/bicinchoninic acid (BCA) reaction using a BCA pro-ein assay kit (Pierce Biotechnology Inc.).

.2. Preparation of latex particles adsorbing IgMIgM-latex) and latex particles adsorbing F(ab′)2 fragmentF(ab′)2-latex)

Polystyrene latex particles (EN400, diameter of 0.4 �m; Sek-sui Chemical Co.) were suspended in 0.05 M phosphate bufferpH 7.2) at 0.5%. The latex suspension (0.2 ml) was mixed withntibody (0.8 ml of 0.125 mg/ml) dialyzed against the aboveuffer, and was stirred at 25 ◦C for 2 h. The latex–protein mixtureas then centrifuged for 20 min at 15,000 rpm. The latex par-

icles precipitated were suspended in 1 ml of 1% bovine serumlbumin (BSA) in 0.1 M phosphate buffer (pH 7.2), and the sus-ension was then stirred at 25 ◦C for 2 h. The latex particles wereashed three times with 1% BSA in 0.1 M phosphate buffer (pH.2), and were suspended in 0.4 ml of the same buffer; the con-entration of latex particles was 0.25%.

The total amount of antibody adsorbed on the latex particlesas calculated from decrease of antibody in the supernatant after

dsorption. The adsorbed amount of the antibody per 1 mg ofatex was calculated on the basis of the molecular weight of IgM900 kDa) and F(ab′)2 fragment (148 kDa that was estimated inhis study).

.3. Evaluation of sensitivity in latex reagents

The sensitivity of latex reagents was evaluated by nephelom-try [6]. The samples used were human whole saliva collectedrom six healthy donors (five donors with blood type B and oneith type O). The saliva samples were diluted 10-, 100-, 500- and

000-fold with PBS. Latex reagent (10 �l of 0.25%) was mixedith 90 �l of sample, and changes in absorbance at 570 nm for5 min were recorded. The degree of reaction was defined ashe difference in absorbance at 570 nm between 0 and 15 min.

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B: Biointerfaces 54 (2007) 114–117 115

ased on the results obtained by negative control examinationithout saliva, a positive result was defined as a value of more

han 0.01.

.4. Slide agglutination test

The conventional test was performed by slide agglutination.he sample used was RBCs from a type-B donor, and theBCs were suspended in PBS at 0.25%. The RBCs treatedith papain to remove electric charge were also prepared.riefly, 0.5 ml of the RBCs was mixed 1.0 ml of 0.1% papain

Wako Pure Chemicals) in PBS, and was incubated at 37 ◦Cor 30 min. The RBCs were washed three times with PBS toemove papain by centrifugation, and were prepared in PBSt 0.25%. Latex reagent (50 �l of 0.25%) was mixed with an

(ab′)2 fragments derived from IgM. (b) SDS-PAGE of the F(ab′)2 fragmentserived from pepsin-digests of IgM. SDS-PAGE proceeded under reducing con-itions. Lane 1: molecular size marker proteins; Lane 2: the pepsin-digests ofgM before separation; Lane 3: the F(ab′)2 fragments isolated from pepsin-igests of IgM. The bands were visualized by silver staining.

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.5. Applications for forensic science

Cigarette ends used as forensic material were collected fromhree smokers with blood type B. Saliva samples were firstxtracted by 120 �l of PBS from 5 mm × 10 mm of cigaretteaper cut off the cigarette end, and then examined by neph-lometry.

The institutional review board of the National Research Insti-ution of Police Science approved this study, and informed con-ent was obtained from all participants giving human samples.

. Results

.1. Preparation of F(ab′)2 fragment

Mouse IgM was digested with pepsin at ratios of 1:10, 1:25

nd 1:50 (pepsin:IgM, w/w). The band of approximately 82 kDa,hich corresponded to heavy chain of IgM, disappeared in aepsin concentration-dependent manner, and was completelyigested with pepsin at the ratio of 1:10 (data not shown). The

ig. 2. Comparison between F(ab′)2-latex and IgM-latex by nephelometry ofensitivity to human saliva of blood type B diluted by PBS. The degree of reactionas defined as the difference in absorbance at 570 nm between 0 and 15 min.he dotted line indicates the cut-off level.

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ig. 3. The microscopic images of two latex reagents mixed with blood type-B RB(ab′)2-latex. (c) The papain treated RBCs mixed with the F(ab′)2-latex.

B: Biointerfaces 54 (2007) 114–117

igest was separated by gel filtration as shown in Fig. 1. Theeptides derived from IgM were detected by direct ELISA inhe second peak (fraction number 15, Fig. 1(a)) and were foundo be approximately 45 and 29 kDa proteins corresponding to theragments derived from the H-chain and intact L-chain, respec-ively [5,7] (Fig. 1(b)).

.2. Sensitivity of the IgM-latex and the F(ab′)2-latex

The sensitivity of the IgM-latex and that of the F(ab′)2-latexere examined by nephelometry. Fig. 2 shows the increase in

he absorbance of the reaction mixture of diluted type-B salivand two latex reagents. In the case of the F(ab′)2-latex, when thealiva was diluted to 500-fold, all five samples showed positive.dditionally, three samples out of five showed positive even

fter 1000-fold dilution. On the other hand, the IgM-latex gavenegative result in three out of five type-B saliva samples even

t 10-fold dilution. Neither the F(ab′)2-latex nor the IgM-latexeagent showed a cross-reaction with saliva of blood type O inhe range of 10–1000-fold dilution.

The amounts of IgM and F(ab′)2 fragment adsorbed to 1 mgf the latex particles were approximately 0.51 × 10−10 mol45.6 �g) and 1.91 × 10−10 mol (28.3 �g), respectively.

.3. Reaction with RBCs in the IgM-latex and the(ab′)2-latex

Fig. 3 shows microscopic images of two latex reagentsixed with blood type-B RBCs. Some agglutinates were

bserved when the IgM-latex reagent was mixed with the RBCsFig. 3(a)). When the F(ab′)2-latex was mixed with RBCs, lit-le or no agglutination was observed (Fig. 3(b)). To examinehether or not an electric charge on the surface of RBCs dis-

urbs agglutination, the RBCs were treated with papain in ordero remove any charge from the surface. The RBCs thus treatedere agglutinated by the F(ab′)2-latex (Fig. 3(c)).

.4. Applications of F(ab′)2-latex in forensic science

The ABO blood type in samples extracted from cigarettends was examined using the F(ab′)2-latex. All three sampleshowed positive and their ABO blood types were determined.

Cs. (a) The RBCs mixed with the IgM-latex. (b) The RBCs mixed with the

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he increases in absorbance at 570 nm for 15 min in the threeamples were 0.0542, 0.1352 and 0.4216. This examination wasompleted within 30 min from sample preparation.

. Discussion

In the present study, we succeeded in preparing F(ab′)2 frag-ent by the pepsin digestion of IgM isolated from commercial

lood grouping reagent using gel filtration.The sensitivity of the F(ab′)2-latex to saliva was much bet-

er than that of the IgM-latex. The amount of adsorbed F(ab′)2ragment was approximately four-fold more than that of the IgMentamer, however, an IgM pentamer has five F(ab′)2 regions.hus, this result suggests that the total number of antigen bindingites in F(ab′)2-latex is smaller than that in IgM-latex. Therefore,he difference in sensitivity is not likely to be attributed to a dif-erence in the number of binding sites on the latex particles.

We speculated that the difference in sensitivity between thewo types of latex reagents was responsible for the orientationf the antigen binding site on the latex particle. A proteinithout specific physical adsorption regions commonly adsorbsorizontally on the surface of latex particles (“side-on” orienta-ion) [8], while IgG and its F(ab′)2 fragment with hydrophobicegions adsorb perpendicularly (“end-on” orientation) [9,10].

e suggest that the IgM pentamer, which has a planar structureike a starfish, might be adsorbed in a “side-on” orientation, andhat the F(ab′)2 fragment, which has a structure similar to thatf F(ab′)2 fragment of IgG, might be adsorbed in an “end-on”rientation. The F(ab′)2 fragment on the latex particles muste advantageous to the antigen–antibody reaction on the latexurface.

In contrast to the reaction with saliva samples, the F(ab′)2-atex was not able to agglutinate intact RBCs. However, whenhe RBCs were treated with papain, which is known to remove

n electric charge on the surface, they were successfully agglu-inated by F(ab′)2-latex, indicating that F(ab′)2 fragment onhe surface of latex particles is too short to form a cross link-ge between latex particles and intact RBCs having an electric

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B: Biointerfaces 54 (2007) 114–117 117

harge, and also that the F(ab′)2 fragment latex might be suit-ble not for RBCs, but for large molecular compounds suchs glycoproteins and glycolipids. It is well known that IgM cangglutinate intact RBCs due to its large molecular size. From thisesult, it is suggested that there has been some IgM adsorbed inend-on” orientation to latex particles and such an IgM mightontribute agglutination.

The F(ab′)2-latex was agglutinated with the extracts fromigarette ends. Generally, ABO blood grouping by traditionalethods requires over 4 h for detection, but the method using

he F(ab′)2-latex reagent requires only 30 min. Additionally,his new assay is very simple and may be automated. More-ver, the sensitivity and specificity of the F(ab′)2-latex has beennchanged for at least two months since latex reagent was pre-ared (data not shown). The present study provides a valuableasis for the preparation of latex reagent-adsorbing antibodyomposed primarily of IgM for use not only in clinical exami-ation but also in forensic science examination.

cknowledgments

The authors would like to express their gratitude to Mr.atoshi Obana and Mr. Toshiaki Kamiya (Sekisui Chemical Co.,td.) for their guidance and support on latex particles issues.

eferences

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ogy Inc., Rockford, 2004.[5] D.W. Pascual, L.W. Clem, J. Immunol. Meth. 146 (1992) 249.[6] M. Takahara, H. Kuroda, N. Nishida, Japanese Patent, P2000-346844A

(2000) (in Japanese).

[7] K. Inouye, K. Morimoto, J. Biochem. Biophys. Meth. 26 (1993) 27.[8] H. Shirahama, T. Suzawa, Colloid Polym. Sci. 263 (1985) 141.[9] P. Bagchi, S.M. Birnbaum, J. Colloid Interf. Sci. 83 (1981) 460.10] J. Buijs, J.W.Th. Lichtenbelt, W. Norde, J. Lyklema, Colloids Surf. B:

Biointerf. 5 (1995) 11.