Forensti Science Internationul, 35 (1987) 253-265 Elsevier Seientific Publishers Ireland Ltd.

253

A HIGHLY SPECIFIC AND SENSITIVE SANDWICH ENZYME IMMUNOASSAY FOR HUMAN HEMOGLOBIN A

NOBUHIRO YUKAWAa, TAKEYUKI KOHNOb. EIJI ISHIKAWAb and KEIICHI TAKAHAMAa’*

aDepartment of Legal Medìcine and b Department of Biochemistry, Medical College of Miyazaki Kiyotake, Miyaxaki 889-16 (Japad

(Received March 16th, 1987) (Accepted July 13th, 1987)

Summary

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A (Hb A) is described. A rabbit anti-human Hb A IgGeoated polystyrene bal1 was incubated with human Hb A and subsequently with affinity-purified anti-human Hh A Fab’-horseradish peroxidase conjugates, which had been prepared before and after absorption with Japanese monkey Hb-Sepharose 4B and dog Hh-Sepharose 4B. Bound peroxidase activity was measured by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. The detection limits of human Hb A using the eonjugate before and after the absorption were 0.65 pgltube (3 x lOlo- fold dilution of whole bloed) and 2.0 pgltube (1 x 10’O-fold dilution of whole bloed), respectively. Human Hb A could be discriminated from Hb of animals sueh as Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow by measuring bound peroxidase activity in the presence and absente of the conjugates prepared before and after the absorption. Human Hb A in bloodstains on cotton gauze could be discriminated from Hb of animals described above even after seven washings. Human Hb A in 220 OOO-fold diluted bloodstains on cotton gauze could also be discriminated from Hb of animals described above.

Key wowis: Hemoglobin: Enzyme immunoassay; Fab’; Peroxidase; Bloodstain

Introduction

For the discrimination of human blood from blood of animals using min- ute bloodstains, which is important in forensic investigations, hemoglobin (Hb) is a suitable marker, since Hb is the most abundant protein in blood. Many sensitive immunological methods for the discrimination of human Hb from Hb of animals have been described, including counter immunoelec- trophoresis [1,2], micro-thin layer immunoassay [3], absorption test using latex particles [4,5], radioimmunoassay [6] and enzyme immunoassay [7]. The detection limits of Hb using these methods were 1 x 104-8 x 105- fold dilution of whole blood.

This paper describes a highly specific and sensitive sandwich enzyme immunoassay, which can discriminate human Hb A from Hb of animals. The

+To whom correspondence should be addressed.

037S-O738187/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

254

detection limit of human Hb A is as little as 0.65 pg or 3 x lOlo-fold dilution of whole bloed.

Materials and Methods

Buffers The regularly used buffers were 10 mmol/l sodium phosphate buffer (pH

‘7.01, containing 1 g/l bovine serum albumin (fraction V, Armour Pharmaceutical Co., Kankakee, Illinois1 (buffer Al and 10 mmol/l sodium phosphate buffer (pH 7.01, containing 0.1 mol/l NaCl (buffer Bl.

Enzymes Horseradish peroxidase (EC 1.11.1.71 (grade 1, lyophilized RZ = 3.01 and

pepsin from porcine gastric mucosa (EC 3.4.23.11 were obtained from Boehringer Mannheim GmbH, Mannheim, F.R.G.

Assay of peroxidase activity Peroxidase activity was assayed at 30°C by fluorimetry using 3-(4-

hydroxyphenyllpropionic acid (Aldrich Chemical Co., Inc., Milwaukee, Wisconsinl as a substrate [8]. The fluorescente intensity was measured relative to 0.2 mg/I quinine in 50 mmol/l H,SO, using 320 nm for excitation and 405 nm for emission in a Shimadzu spectrofluorophotometer (RF-510, Shimadzu, Seisakusho Ltd., Kyoto, Japan).

Hemoglobin Red cells from various sources (human, Japanese monkey, horse,

cow, pig, sheep, dog, cat and chickenl were washed 10 times with 20 volumes of 10 mmol/l sodium phosphate buffer (pH 7.31, containing 0.14 mol/1 NaCl, and the packed red cells were lysed with 2 vol. of deionized water at 0°C. The hemolysate was centrifuged at 33 000 x g for 2 h, and used as Hb preparation without further purification except for human Hb A, since 90% of proteins in the hemolysate is Hb [9]. The concentration of Hb in the hemolysate was calculated from the absorbance at 540 nm by taking the molecular weight and the extinction coefficient to be 64 500 and 44 000 mol-’ * 1 - cm-’ (0.682 g-’ - 1 - cm-‘, respectively [lol.

Purification of human Hb A Human Hb A was purified from the hemolysate by the method of

Abraham et al. [ll] with modifications. The hemolysate (0.4 ml) obtained as described above was dialyzed against 0.2 mol/1 glycine -NaOH buffer (pH 7.81, containing 0.1 g/l KCN and applied to a column (1.5 x 25 cm) of diethylaminoethyl-cellulose (Whatman Chemical Separation Ltd., Kent, U.K.) using the same buffer. After washing of the column with the same buffer containing 5 mmol/l NaCl, human Hb A was eluted using a linear NaCl gradient (500 ml each of the same buffer containing 10 mmol/l NaCI and 0.2 mol/l NaCIl at a flow rate of 18 ml/h. The fraction volume was 5 ml. The

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human Hb A preparation was shown to be homogeneous by isoelectric focusing in 4% polyacrylamide gel containing 2O/b Ampholine (pH 5-8, LKB, Stockholm, Swedenl [12].

Preparation of protein-Sepharose 4B Human Hb A, Japanese monkey Hb, dog Hb and normal rabbit serum

protein (10 mg eachl were dialyzed against 0.1 mol/l sodium borate buffer (pH 8.01, containing 0.5 mol/l NaCl and coupled to CNBr-activated Sepharose 4B (1 g, Pharmacia Fine Chemicals AB, Uppsala, Swedenl according to the instructions of Pharmacia. Human Hb A was coupled to CNBr-activated Sepharose 4B in the presence of sodium dodecyl sulfate (1 g/ll.

AntGhuman Hb A Rabbit anti-human Hb A IgG was obtained from Dako-immunoglobulins al

s, Copenhagen, Denmark. F(ab’1, was prepared by digestion of IgG with pepsin [13]. Fab’ was prepared by reduction of F(ab’1, with 2- mercaptoethylamine [13]. The amounts of IgG and its fragments were calculated from the absorbance at 280 nm [13].

A bsorption of antkhuman Hb A Flab’), with Hb of Japanese monkey and dog Rabbit anti-human Hb A F(ab’1, (14 mgl in 0.1 mol/l sodium phosphate

buffer (pH 7.01, containing 1 g/l NaN, (2.4 ml1 was passed through a column (6 x 30 mm) of Japanese monkey Hb-Sepharose 4B and a column (3 x 2 mm)

of dog Hb-Sepharose 4B using the same buffer at a flow rate of 0.6 ml/h. The amount of the F(ab’1, obtained was 13 mg.

Preparation of ant&human Hb A Fab’-peroxidase conjugate Anti-human Hb A F(ab’), before and after the absorption with Japanese

monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B was reduced with 2- mercaptoethylamine and conjugated to horseradish peroxidase using N- succinimidyl-6-maleimidohexanoate (Dojindo Laboratories, Kumamoto, Japan) [14]. The amount of the conjugate was calculated from peroxidase activity 1131.

Affinity purafication of anti-human Hb A Fab’-peroxidase conjugate Anti-human Hb A Fab’-peroxidase conjugates (4.7- 13 mgl before and

after the absorption in (0.57 -0.81 ml1 of buffer A containing 0.1 mol/l NaCl were applied to a column (3 x 11 mm1 of human Hb A-Sepharose 4B using the same buffer at a flow rate of 0.4 ml/h. After washing of the column with the same buffer, the specific Fab’-peroxidase conjugates were eluted with 0.5 ml of 3.2 mmol/l HCl, pH 2.5 at a flow rate of 6.0 ml/h, and the eluates (0.5 ml) were immediately neutralized by addition of 0.055 ml of 1.0 mol/l sodium phosphate buffer (pH 7.01, containing 10 g/l bovine serum albumin. The amounts of affinity-purified conjugates before and after the absorption were 58 pg and 22 pg, respectively.

256

The affinity-purified conjugates before and after the absorption were applied to a column (1.5 x 100 cm) of Ultrogel AcA 44 (LKB, Stockholm, Sweden) using 10 mmol/l sodium phosphate buffer (pH 7.0), containing 0.1 g/l bovine serum albumin and 0.1 mol/l NaCl [15]. Fractions containing the conjugates were pooled and concentrated by centrifugation in a microconcentrator (Centricon 30, Amicon Corporation, Danvers, Massachusetts). Finally, the conjugates before and after the absorption (4.5 pg each) were passed through a eolumn (0.4 x 6.5 cm) of normal rabbit serum protein-Sepharose 4B using buffer A containing 0.1 mol/l NaCl at a flow rate of 0.6 ml/h [16].

Preparation of anti-human Hb A IgG-coated polystyrene balls Polystyrene balls (3.2 mm in diameter, Precision Plastic Bal1 Co., Chicago,

Illinois) were coated with rabbit anti-human Hb A IgG (0.1 gil of 0.1 mol/1 sodium phosphate buffer (pH 7.51, containing 1 g/l NaNJ by physical adsorption [17].

Sandwich enzyme immunoassay for Hb A rabbit anti-human Hb A IgG-coated polystyrene bal1 was incubated with

Hb in a total volume of 0.15 ml at 37OC for 4 h with continuous shaking and at 4OC for 16 h without shaking. Hb was diluted with buffer A containing 0.1 mol/I NaCl and 1 g/l NaN, to a final volume of 0.1 ml and mixed with 0.05 ml of buffer A containing 1 mol/l NaCl and 1 gil NaN,. The polystyrene bal1 was washed twice by addition and aspiration of 2 ml of buffer B after removal of the reaction mixture and incubated with 5 ng of affinity-purified anti-human Hb A Fab’-peroxidase conjugate in 0.15 ml of buffer A containing 0.1 mol/1 NaCl at 20°C for 4 h with continuous shaking. The polystyrene bal1 was washed twice as described above and transferred to another clean test tube. Peroxidase activity bound was assayed for 30 min as described above.

Calculation of the specific binding of anti-human Hb A Fab’- peroxidase conjugate

The specific binding of anti-human Hb A Fab’-peroxidase conjugate was calculated by subtraction of fluorescente intensity for peroxidase activity non-specifically bound in the absente of Hb (the non-specific binding) from that bound in the presence of Hb.

Expression of the detection limit of Hb The detection limit of Hb by sandwich enzyme immunoassay was taken as

the minima1 amount of Hb or the maxima1 dilution of whole blood which gave a bound peroxidase activity significantly in excess of that non-specifically bound in the absente of Hb (background). The existente of a significant differente from the background was confirmed by the t-test (P < 0.001, n = 5).

Assay of peroxìdase activity of hemoglobins bound to polystyrene balls Hb was diluted and incubated with an anti-human Hb A IgG-coated

polystyrene bal1 as described above. The polystyrene bal1 was washed twice as described above and incubated in 0.15 ml of buffer A containing 0.1 mol/l NaCl in the absente of the conjugate as described above. The polystyrene bal1 was washed twice as described above and transferred to another clean test tube. Peroxidase activity of Hb bound to the polystyrene bal1 was assayed for 30 min as described above.

Preparation of human bloodstains on cotton gauze Human bloodstains were prepared in two different ways: (11 An aliquot (2.7

ml) of heparinized human blood (166 g Hb/ll from normal subjects was placed on a piece of cotton gauze (8 x 10 cm, 290 mg dry wt.1 and dried at room temperature for 24 h. The cotton gauze was washed with a commercially available synthetic detergent (TopR, LION Corporation, Tokyo, Japan) for 6 min, rinsed for 6 min and spun for 3 min in a washing machine (ES-M365, SHARP Corporation, Osaka, Japan), and dried at room temperature for 1 h. The washing process was repeated up to seven times. (21 An aliquot of heparinized human blood (166 g Hb/l) from normal subjects was diluted with deionized water to various extents. The diluted blood (2.7 ml) was placed on a piece of cotton gauze (8 x 10 cm, 290 mg dry wt.1, dried at room temperature and stored at room temperature for 3-7 days prior to test.

Enzyme immunoassay for bloodstains A 1-cm fiber of cotton gauze with bloodstain was incubated in 0.15 ml of

buffer A containing 0.4 mol/l NaCl and 1 g/l NaN, at room temperature for 2 h. The incubation mixture with the fiber was incubated with a rabbit anti- human Hb A IgG-coated polystyrene bal1 at 37OC for 4 h with continuous shaking and at 4OC for 16 h without shaking. The fiber was washed twice with 2 ml of buffer B, and placed on a filter paper for a few seconds to remove remaining buffer around fiber. The polystyrene bal1 was also washed twice as described above. The fiber and the polystyrene bal1 were incubated separately with 5 ng of affinity-purified anti-human Hb A Fab’-peroxidase conjugates in 0.15 ml of buffer A containing 0.1 mol/l NaCl at 20°C for 4 h with continuous shaking. After washing twice as described above, peroxidase activity bound to the polystyrene bal1 and the fiber was assayed for 30 min as described above.

Assay of peroxidase activity of hemoglobins bound to cotton gauze fibers A 1-cm fiber of cotton gauze with bloodstain was incubated in 0.15 ml of

buffer A containing 0.4 mol/l NaCl and 1 g/l NaN, at room tempersture for 2 h and at 37OC for 4 h with continuous shaking and at 4OC 16 h without shaking. The fiber was washed twice as described above and incubated in 0.15 ml of buffer A containing 0.1 mol/l NaCl in the absente of the conjugate at 20°C for 4 h with continuous shaking. After washing twice as described above, peroxidase activity of Hb bound to the fiber was assayed for 30 min as described above.

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Results and Discussion

Detection limit of human Hb A Affinity-purified anti-human Hh A Fab’-peroxidase conjugates were

prepared before and after absorption of anti-human Hb A F(ab’1, with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. The detection limits of human Hb A by sandwich enzyme immunoassay with the conjugates before and after the absorption were 0.65 pgltube (3 x lOlo-fold dilution of whole bloodl and 2.0 pgltube (1 x lOlo-fold dilution of whole bloodl, respectively (Fig. 11. The detection limit with the conjugate before the absorption was lOO-fold less in terms of pgltube than that by the previous radioimmunoassay [lg] and 10 OOO-fold less in terms of pgltube than that by the previous enzyme immunoassay [7]. A major reason for this may be that the previous assays were based on the competitive binding of human Hb A and 3H-labeled human Hb A or human Hb A-coated polystyrene balls to anti- human Hb A antibodies. In the previous radioimmunoassay, human Hb A was incubated with sheep anti-human Hb A antiserum in the presence of 3H- labeled Hb A followed by precipitation with burro (anti-sheep IgGl

Hb (gltube)

Fig. 1. Dose-response curves by sandwich enzyme immunoassay for human Hb A (O), Japanese monkey Hb (0). dog Hb (A), cat Hb (B) and pig Hb (AI. Curves obtained using affinity-purified anti-human Hb A Fab’-peroxidase conjugates prepared before (- 1 and after (- - -) the absorption, respectively, with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B.

259

antiserum. In the previous enzyme immunoassay, human Hb A was successively incubated with rabbit anti-human Hb A, human Hb A-coated polystyrene balls and alkaline phosphatase-labeled goat (anti-rabbit IgG) IgG.

Specificity of sandwich enzyme immunoassay for human Hb A The cross-reaction with Hb of various animals (Japanese monkey, dog, cat,

pig, horse, sheep, chicken and cow) was examined using affinity-purified anti- human Hb A Fab’-peroxidase conjugates prepared before and after the absorption of anti-human Hb A F(ab’1, with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. By using the conjugate after the absorption, the cross-reaction with Hb of Japanese monkey, dog, cat and pig was remarkably reduced, and the specificity of sandwich enzyme immunoassay for human Hb A was greatly improved (Table 1 and Fig. 1). When the conjugate was prepared before the absorption, the detection limits of Hb of various animals ranged from 2.0 pgltube to 6.5 pgltube. Using the conjugate after the absorption, the detection limits of Hb of various animals were increased to a great extent (0.65-20 pgltube).

Dticrimination of human Hb A from other Hb Human Hb A could be discriminated from Hb of various animals by three

different assays measuring bound peroxidase activities in the absente and presence of affinity-purified anti-human Hb A Fab’-peroxidase conjugates prepared before and after the absorption of anti-human Hb A F(ab’1, with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B.

TABLE 1

DETECTION LIMIT OF HUMAN Hb A AND Hb OF VARIOUS ANIMALS

Expressed as pgltube.

Source of Hb Anti-human Hb A Fab’-peroxàduse conjugate Ratio lafter/beforel

Before After absorption” absorpttin’

Human (Hb A) 0.65 Japanese monkey 2.0 Dog 65 Cat 200 Pig 65 000 Horse 650 000 Sheep 650 000 Chicken 2 000 000 cow 6 500 000

650 000 6 500 000 6 500 000

20 000 000 20 000 000 2 000 000

20 000 000 20 000 000

3 300 000 100 000 30 000

300 30

3 10 3

*The conjugates were prepared before and after the absorption with Japanese monkey Hb- Sepharose 4B and dog HbSepharose 4B.

260

Specifically bound peroxidase activities obtained by sandwich enzyme immunoassay using affinity-purified anti-human Hb A Fab’-peroxidase conjugates before and after the absorption differed only 3-fold for human Hb A but 300-300 OOO-fold for Hb of Japanese monkey, dog, cat and pig (Table 1 and Fig. 1). Therefore, human Hb A could be discriminated from Hb of these animals by using the two conjugates before and after the absorption. Specifically bound peroxidase activities for Hb of horse, sheep, chicken and cow only slightly (3-30-fold) differed with the conjugates before and after the absorption (Table 1 and Fig. 2).

When sandwich enzyme immunoassay using the conjugate after the absorption was performed for Hb of pig, horse, sheep, chicken and cow, bound peroxidase activities were almost equal to those measured in the

? % l-

G L 1 ' 1 / 'I'JJ" ' 1""1' 10+ 10-5 10-4

Hb (gitube)

Fig. 2. Dose-response curves by sandwich enzyme immunoassay for horse Hb (El), sheep Hb (A), chicken Hb (0) and cow Hb (AL Curves obtained using affinity-purified anti-human Hb A Fab’-peroxidase conjugates prepared before ( ----) and after (- - -) the absorption, re- spectively. with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B.

261

Hb (gltube 1

Fig. 3. Peroxidase activity bound in the presence and absente of the conjugate. - - - -, fluorescente intensity of speci$cally bound peroxidase activity obtained by sandwich enzyme immunoassay using affinity-pwified anti-human Hb A Fab’-peroxidase conjugate prepared af’ter the absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. - fluorescente intensity for bound peroxidase activity in the absente of conjugate. Curves wik human Hb A (0). Japanese monkey Hb (O), dog Hb CA), cat Hb (M) and pig Hb (A).

absente of the conjugate, which were apparently due to the non-specific binding of Hb to the solid phase (Figs. 3 and 4). With human Hb A, bound peroxidase activity obtained with the conjugate after the absorption was much higher than that in the absente of the conjugate. Therefore, human Hb A could be discriminated from Hb of pig, horse, sheep, chicken and cow by measuring bound peroxidase activity in the presence and absente of the conjugate after the absorption.

Detection of human Hb A on cotton gauwe The detection limit of human Hb A on cotton gauze was examined in two

different ways: (1) Human blood was dried on cotton gauze at room temperature for 24 h.

After washings, a 1-cm fiber of cotton gauze with bloodstain was incubated in a buffer, and human Hb A both on the fiber and in the extract was measured using affinity-purified anti-human Hb A Fab’-peroxidase

262

??,

10-6 10-5

Hb (gitubel 10-4

Fig. 4. Peroxidase activity bound in the presence and absente of the conjugate. - - - -, fluorescente intensity for specifically bound peroxidase activity obtained by sandwich enzyme immunoassay using affinity-purified anti-human Hb A Fab’-peroxidase conjugate prepared after the absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. -, fluorescente intensity for bound peroxidase activity in the absente of the conjugate. Curves with horse Hb (O), sheep Hb (AI, chicken Hb (MI and cow Hb (AL

conjugates prepared before and after the absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B (Fig. 5). Even after seven washings, the amount of human Hb A on the fiber was sufficient to confirm that differente in peroxidase activities bound to the fiber in the presence of the conjugates prepared before and after the absorption was approx. 2-3- fold. Peroxidase activity bound to the fiber in the absente of the conjugate was negligible. These results indicated that human Hb A in bloodstains on cotton gauze even after seven washings could be discriminated from Hb of animals including Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow.

(2) Human blood was serially diluted with deionized water and dried on cotton gauze. A 1-cm fiber of cotton gauze with diluted bloodstain was

263

\ ‘\ 10 \ \ ’ \ \

i ’ ‘, ‘\

’ ‘1 \ \ \ < 3 ‘\ 0

1 0 t: y ~ 1 2 3

3

4 5 6 7

Wash ing (times 1

Fig. 5. Detection of human Hb A in human bloodstains on cotton gauxe. Human blood was dried on cotton gauze at room temperature for 24 h. After washings, a 1-cm fiber of cotton gauze with bloodstain was incubated in a buffer, and human Hb A both on the fiber ( -) and in the extract (- - - -1 was measured using affinity-purified anti-human Hb A Fab’-peroxidase conjugates prepared before (0) and after (ml the absorption with Japanese monkey Hb- Sepharose 4B and dog Hb-Sepharose 4B. - 0 -, peroxidase activity bound to the fiber in the absente of the conjugate.

incubated in a buffer, and human Hb A both on the fiber and in the extract was measured using affinity-purified anti-human Hb A Fab’-peroxidase conjugates prepared before and after the absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B (Fig. 6). Human Hb A could be detected more easily on the fibers than in the extracts. When blood was diluted 220 OOO-fold with water and placed on cotton gauze, the amount of human Hb A on the fiber was sufficient to confirm that differente in peroxidase activities bound to the fiber in the presence of the conjugates prepared before and after the absorption was approx. 2-fold. Peroxidase activity bound to the fiber in the absente of the conjugate was negligible.

264

Dilution of Whole Blood (fold)

Fig. 6. Detection of human Hb A on cotton gauze with diluted human bloodstains. Human blood was serially diluted with deionized water and dried on cotton gauze. A 1-cm fiber of cotton gauze with diluted bloodstain was incubated in a buffer, and human Hb A both on the fiber

(--- ) and in the extract (- - - - ) was measured using affinity-purified anti-human Hb A Fab’-peroxidase conjugates prepared before (0) and after (W) the absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. - o-, peroxidase activity bound to the fiber in the absente of the conjugate.

These results indicated that human Hh A in 220 OOO-fold diluted bloodstains on cotton gauze could be discriminated from Hb of animals including Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow.

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