Molecular and Cellular Probes (1991) 5, 81-95

REVIEW

Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the sensitivity Eiji Ishikawa,* Seiichi Hashida and Takeyuki Kohno Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan (Received 13 September 1990, Accepted 24 October 1990)

Development of ultrasensitive enzyme immunoassays for antigens, haptens and antibodies is reviewed with emphasis on factors which limit the sensitivity. One of the most important conditions for ultrasensitive immunoassays is the use of non-competitive solid phase assay systems rather than competitive ones . Although non-competitive immunoassays are available for antigens and antibodies, there are only competitive immunoassays for hapten molecules which can not be bound simultaneously by two different antibodies . In order to overcome this difficulty, methods have been developed to derivatize haptens with amino groups so that the derivatized haptens may be measured by two-site assays. The other condition for ultrasensitive immunoassays is to minimize the nonspecific binding of labelled reactants . This has been achieved by developing methods to transfer the complex of analytes and labelled reactants from solid phase to solid phase without dissociation . Thus, the sensitivity for antigens, haptens and antibodies has been markedly improved . However, the sensitivity for the detection of label enzymes remains to be improved . KEYWORDS: Enzyme immunoassay, antigen, antibody, hapten, (3-D-galactosidase, fluorometry

INTRODUCTION The use of enzymes as a label in immunoassay was suggested in 1968 .' Three years later, its first trials were reported ." For many years following, the potential of enzyme immunoassay especially concerning the sensitivity, was disputed with no convincing conclusion . In 1976, a positive view that enzyme immunoassays would overtake radioimmunoassays in various fields was published .' One month later, arguments against this view were also published .',"

from rat liver with a molecular weight of 170,000, was reported .' During the ensuing decade, enzyme immunoassay was successfully applied to the measurement of various antigens at attomole levels, which are below those possible using radioimmunoassay . 8-1Y Very recently, the sensitivity of enzyme immunoassay not only for antigens 13 but also for antibodies" and haptens 15 has been remarkably improved . This paper reviews the development of ultrasensitive enzyme immunoassay with emphasis on the factors which limit the sensitivity .

One argument stated that the suggestion that enzyme labelling would replace radioisotopic techniques (particularly in assays demanding highest sensitivity) was questionable .' The other stated that for analysis of femtomole (1 x 10 -15 mol) amounts of steroids, hormones, and so on to aid patient diagnosis and treatment there is only radioimmunoassay." Less than 4 months after these arguments, detection by enzyme immunoassay of 1 attomole (1 x 10 -78 mol; 600,000 molecules as calculated from Avogadro's number) of an antigen, ornithine 8-aminotransferase

ANTIGENS AND HAPTENS Competitive and non-competitive immunoassays for antigens and haptens In 1977, 1 amol of an antigen, orinithine 8-aminotransferase with a molecular weight of 170,000, was

a ` Author to whom correspondence should be addressed 0890-8508/91/020081 +15 $03 .00/0

81

© 1991 Academic Press Limited

E . Ishikawa et al.

82

50

I00

40

30

10

C O

E L

0.5 0 Human growth hormone (amol/tube)

Dose-response curves of human growth hormone (hGH) by competitive radioimmunoassay using 15 1-labelled hGH and non-competitive (two-site) enzyme immunoassay using anti-hGH Fab'-horseradish peroxidase conjugate and anti-hGH IgG-coated polystyrene balls . The diameter of polystyrene balls used was 3 . 2 mm, and the reaction mixture volume used in the enzyme immunoassay was 0 . 15 ml . Fig. 1 .

detected by enzyme immunoassay' and during the ensuing decade many antigens were measured at attomole levels (Fig . 1) . 12 One of the reasons that this was possible was the use of a non-competitive assay system instead of a competitive one which has been widely used in radioimmunoassay . In a competitive assay system, a certain amount of labelled antigen is reacted with the corresponding amount of antibody in the absence and presence of antigen to be measured . The amount of antigen to be measured is correlated to the amount of labelled antigen bound to the antibody, which is measured only with a certain range of error (approximately 5%) . The lower the concentration of labelled antigen and antibody used the higher the sensitivity is . However, the concentration of labelled antigen and antibody should be sufficiently high so that more than 50% of labelled antigen and antibody used are in bound form . In other words, the minimal concentration of labelled antigen that can be used is limited by the affinity of the antibody . Thus, the detection limit of antigens by competitive immunoassay is at femtomole or higher levels in most cases . By contrast, in non-competitive two-site immunoassay, the antigen to be measured is trapped onto antibody-coated solid phase and reacted with labelled antibody . The amount of antigen to be measured is correlated to the amount of labelled antibody bound to the solid phase . Since excess of labelled antibody is effectively eliminated by washing, the amount of labelled anti-

body non-specifically bound to the solid phase in the absence of antigen (background) can be reduced, providing a high degree of sensitivity . The detection limit of antigens by non-competitive two-site enzyme immunoassay with appropriate techniques is at attomole levels ."'

Molecular size of antigens and haptens for two-site immunoassay As evident from the principle of two-site enzyme immunoassay, antigen and hapten molecules to be measured have to have two or more epitopes, which are sufficiently separated from each other to allow simultaneous binding of two antibody molecules . The smallest peptide that has been measured with attomole sensitivity by two-site enzyme immunoassay is human a-atrial natriuretic hormone, a single chain polypeptide consisting of 28 amino-acids with a ring structure formed by an intramolecular disulphide bond ." Ten amol of this peptide was measured by two-site enzyme immunoassay using peroxidaselabelled Fab' directed to the C-terminus of the peptide and immobilized IgG against N-terminal half of the ring structure (Fig . 2). This was 100-fold more sensitive than competitive radioimmunoassay . 17 The distance between the two epitopes recognized by the two antibodies appeared to correspond to 12-15 amino-acid single chain peptides . From this, peptides



Ultrasensitive enzyme immunoassay

83

6000

100

N

t e v w

X

2

C, '0

1000

C 0

a

60 O U U

100 40

0 T y C

s N u C

10

20

5

u N N O LL

-ii ~

10 2

10

10 3

10 4

10 5

0

Human a-atrial natriuretic hormone (amot tube')

Fig. 2 . Dose-response curves of human a-atrial natriuretic hormone (a-hANH) by competitive radioimmunoassay using 151-labelled a-hANH and non-competitive (two-site) enzyme immunoassay using anti-a-hANH (C-terminus) Fab'-horseradish peroxidase conjugate and anti-a-hANH (ring) IgG-coated polystyrene balls . The diameter of polystyrene balls used was 3 . 2 mm, and the reaction mixture volume used in the enzyme immunoassay was 0 . 15 ml .

Hetero-two-site immunoassay for haptens

measured may be performed chemically using excess of labels activated with appropriate functional groups such as N-hydroxysuccinimide esters, anhydride groups and aldehyde groups which are reactive with haptens to be measured . Alternatively haptens may be labelled by direct conjugation to enzymes . Subsequently, excess of the labels partly unreacted and partly bound to substances other than haptens to be measured should be eliminated prior to two-site assay . This may be performed using solid phase coated with antibodies directed to the structure of haptens to be measured . Finally, two-site assay can be performed using anti-hapten antibodies and bind-

Very recently, a novel non-competitive two-site enzyme immunoassay (hetero-two-site enzyme immunoassay) has been developed for smaller peptides or haptens with amino groups ." , " The principle of this method is as follows . First, haptens to be measured are labelled with an appropriate substance, so that one antibody molecule directed to the haptens to be measured and another binding molecule for the label may be simultaneously bound to the labelled hapten molecules, thus allowing for a two-site assay . An appropriate label and its binding substance may be chosen from a variety of combinations such as biotin-avidin, hapten-anti-hapten antibody (distinct from those to be measured), antigen-antibody, hormone-receptor and nucleotide hybrids . Labelling of haptens to be

ing substances for the labels . Anti-hapten antibodies are used for trapping labelled haptens onto solid phase, and binding substances for the labels are conjugated with enzymes . Alternatively, anti-hapten antibodies are conjugated with enzymes, and binding substances for the labels are used for trapping labelled haptens onto solid phase . Enzymes may be replaced by fluorescent or luminescent substances . Alternatively, haptens to be measured may be labelled directly with fluorescent and luminescent substances such as-europium and acridinium which can be measured with high sensitivity . Feasibility of this principle has been demonstrated using biotin as the label and angiotensin I, a 10 amino-acid single chain peptide with no lysine residue, as the model hapten ." The procedure used is schematically shown in Fig . 3 . Anti-angiotensin I IgG

consisting of more than 12-15 amino-acids are strongly suggested to be measurable at attomole levels using antibodies with sufficiently high affinity . However, smaller peptides may not be measured with attomole sensitivity by two-site enzyme immunoassay . Namely, there is only competitive assay for measuring smaller peptides and haptens with similar or smaller sizes that can not be bound simultaneously by two antibody molecules . The sensitivity of competitive immunoassay is at femtomole levels.

E . Ishikawa et al.

84

Hapten Incubate hapten with N-hydroxysuccinimidobiotin

Incubate the reaction mixture with anti-hapten IgG-coated solid phase (Anti-Hapten-SP) Hapten Anti-Hapten-SP Bio tin After washing, incubate the solid phase at pH 1 . 0, remove the solid phase and neutralize the medium Hapten Biotin Incubate the medium with dinitrophenylated rabbit anti-hapten IgG (Rb Anti-Hapten-DNP) and (anti-dinitrophenyl group) IgG-coated solid phase (Anti-DNP-SP) T Hapten Rb Anti-Hapten-DNP - - - Anti-DNP-SP Biotin After washing, incubate the solid phase with avidin(streptavidin)-enzyme conjugate (Avidin-Enz) Hapten Rb Anti-Hapten-DNP - - - Anti-DNP-SP Biotin Avidin-Enz After washing, incubate the solid phase with dinitrophenyl-L-lysine, remove the solid phase and incubate the medium with (anti-rabbit IgG) IgG-coated solid phase (Anti-Rb IgG)-SP

1

Hapten Rb Anti-Hapten-DNP --- (Anti-Rb IgG)-SP Biotin Avidin-Enz

3 . Hetero-two-site enzyme immunoassay for haptens with amino groups using dinitrophenylated anti-hapten IgG and (strept)avidin-enzyme conjugate . Fig.

used was specific for the C-terminus of the peptide, which was confirmed by the finding that there was no significant cross-reaction with angiotensin II which is produced from angiotensin I by deletion of two Cterminal amino-acids . Angiotensin I was biotinylated through N-terminal amino group with N-hydroxysuccinimidobiotin . Biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls . The polystyrene balls were washed to eliminate unreacted biotin and other biotinylated substances and were treated at pH 1 . 0 to elute biotinylated angiotensin I . Biotinylated angiotensin I eluted was reacted with affinity-purified dinitrophenyl rabbit anti-angiotensin I IgG and was trapped onto affinity-purified (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls . The polystyrene balls were washed to completely eliminate unreacted biotin and other biotinylated substances and were reacted with avidin-f3-D-galactosidase conjugate . The complex of affinity-purified dinitrophenyl rabbit anti-angiotensin I IgG, biotinylated angiotensin I and avid in-(3-D-galactosidase conjugate was eluted from the polystyrene balls with dinitrophenyl-L-lysine

and was trapped onto affinity-purified goat (antirabbit IgG) IgG-coated polystyrene balls . In this step, the complex was transferred from affinity-purified (anti-dinitrophenyl bovine serum albumin) IgGcoated polystyrene balls, to which avidin-/3-D-galactosidase conjugate had been adsorbed non-immunologically, to clean polystyrene balls coated with affinity-purified (anti-rabbit IgG) IgG (immune complex transfer) . After washing the polystyrene balls, bound R-D-galactosidase activity was assayed by fluorometry . The detection limit of angiotensin I was 10 amol per tube . This level of detection can be further lowered by additional transfer to the clean solid phase of the complex consisting of anti-angiotensin I IgG, biotinylated angiotensin I and avidin-(3-D-galactosidase conjugate . However, the procedure was time-consuming . An alternative, simpler method has also been developed (Fig. 4). 1518 Biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls. The polystyrene balls were washed to eliminate unreacted biotin and other biotinylated substances and were subsequently treated at pH 1 . 0



Ultrasensitive enzyme Immunoassay

85

Hapten Incubate hapten with N-hydroxysuccinimidobiotin i Hapten Biotin Incubate the reaction mixture with anti-hapten IgG-coated solid phase (Anti-Hapten-SP)

1 Hapten Anti-Hapten-SP Biotin

After washing, incubate the solid phase at pH 1 .0, remove the solid phase and neutralize the medium Hapten Biotin Incubate the medium with anti-hapten Fab'-enzyme conjugate (Anti-Hapten Fab'-Enz) Hapten Anti-Hapten Fab'-Enz 1

Biotin Incubate the medium with avidin(streptavidin)-coated solid phase (Avidin-SP) Hapten Anti-Hapten Fab'-Enz Biotin Avidin-SP Fig. 4 . Hetero-two-site enzyme immunoassay for haptens with amino groups using anti-hapten Fab'-enzyme conjugate and (strept)avidin-coated solid phase.

100

T

8 I O

X

1000

80

0 CL

T

0u

100

u

a

40

0

C N d

N C

C a U

10

C

u d. LL

5 L-41 -

10

10 10

2

3

' 0 10 10

4 -

5

Angiotensin I (amot tube ')

Fig. 5 . Dose-response curves of angiotensin I by competitive radioimmunoassay using 1251-labelled angiotensin I and non-competitive (hetero-two-site) enzyme immunoassay using anti-angiotensin I Fab'-horseradish peroxidase conjugate and streptavidin-coated polystyrene balls . The diameter of polystyrene balls used was 3 .2 mm, and the reaction mixture volume used in the enzyme immunoassay was 0 . 15 ml .



86

E . Ishikawa et al .

to elute biotinylated angiotensin I . Biotinylated angiotensin I eluate was reacted with anti-angiotensin I Fab'-horseradish peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls . Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a hydrogen donor . The detection limit of angiotensin I was 13 fg (10 amol) per tube, which was 100-fold lower than that by competitive radioimmunoassay using the same antiserum and 125 1angiotensin I (Fig. 5) and 80 to 480-fold lower than those previously reported by competitive radioimmunoassay" and competitive enzyme immunoassay .", " The detection limit of plasma angiotensin I using 5 pl of plasma was 2 .6 ng per litre ." When angiotensin I was extracted from 50 µl of plasma using anti-angiotensin I IgG-coated polystyrene balls, the detection limit of plasma angiotensin I was 0-8 ng per litre ." A similar technique has been applied to the measurement of arginine vasopressin ([Arg']-vasopressin), a nine amino-acid single chain peptide with an intramolecular disulphide bridge .22 Maleimide groups were introduced into the peptide molecules using N-succinimidyl-6-maleimidohexanoate and subsequently reacted with thiol groups of glutathione molecules . Amino groups of glutathione residues bound to arginine vasopressin molecules were reacted with N-hydroxy-succinimidobiotin . Biotinylated arginine vasopressin was measured using antiarginine vasopressin Fab'-peroxidase conjugate and streptavidin-coated polystyrene balls as described above for angiotensin I . As a result, the detection limit of arginine vasopressin was 10 amol, which is 50fold lower than that by competitive enzyme immunoassay using the same antibody and 23 to 400-fold lower than those by previously reported competitive immunoassays .23, 24 This suggested that other haptens with amino groups could also be measured with high sensitivity in a similar manner . However, techniques for labelling haptens without amino groups remain to be developed .

Solid phase and liquid phase immunoreactions in two-site enzyme immunoassay for antigens and haptens In two-site enzyme immunoassay, antigen to be measured is reacted sequentially or simultaneously with antibody-coated solid phase and enzymelabelled antibody . The immunoreactions on the solid phase take place with high efficiency, providing high sensitivity . For some antigens, however, the immunoreactions on the solid phase may be less effective than those in solution . This possibility was tested for

some antigens as follows . Antigens to be measured were reacted simultaneously with dinitrophenylated antibody IgG and enzyme-labelled antibody Fab' . The complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls . [Dinitrophenylated antibody IgG and (anti-dinitrophenyl group) IgG-coated polystyrene balls can be replaced by biotinylated antibody IgG and (strept)avidin-coated polystyrene balls .] Enzyme activity specifically bound to (anti-dinitrophenyl group) IgG-coated polystyrene balls was significantly higher than that specifically bound to antibody IgG-coated polystyrene balls in the conventional two-site enzyme immunoassay . However, non-specifically bound enzyme activity was also higher to similar extents, and the sensitivity was improved only slightly . 13,25

Various attempts to reduce non-specific binding of enzyme-labelled antibody in two-site enzyme immunoassay for antigens and haptens In two-site enzyme immunoassay for antigens, enzyme-labelled antibody non-specifically (non-immunologically and physically) adsorbs to antibodycoated solid phase to various extents . This is one of the major obstacles to improvement in sensitivity, and various attempts have been made to reduce the non-specific bincling. 9-13 Fc portion of IgG molecule is hydrophobic, causing high non-specific binding of enzyme-labelled IgG . This was avoided by using Fab obtained after papain digestion of IgG or Fab' obtained after pepsin digestion and reduction of IgG . 9-1 ' ,26 Furthermore, Fab' was conjugated to enzymes by selective use of thiol groups in the hinge of Fab' which is remote from the antigen-binding site of Fab' (the hinge method) .' 1126 The conjugate prepared by the hinge method provides not only lower non-specific binding but also higher specific binding than that prepared by other methods, in which Fab' or Fab is conjugated to enzymes through amino groups' -1,16 IgG in antiserum consists of specific IgG and nonspecific IgG, and the proportion of non-specific IgG is generally greater than 90% . Non-specific Fab'-enzyme conjugate gives no specific binding and causes non-specific binding. Consequently, the presence of non-specific Fab'-enzyme conjugate limits the sensitivity of two-site enzyme immunoassay for antigens . This was overcome by using affinity-purified Fab'enzyme conjugate, and affinity-purification of Fab' improves the sensitivity 10 to 100-fold in most cases . 9-12 The non-specific binding of Fab'-enzyme conju-

Ultrasensitive enzyme immunoassay gate was lowered to some extent by the addition of non-specific Fab' or F(ab')2 27 and by changing pH, ion, temperature and so on ."

body IgG and enzyme-labelled antibody Fab' . The complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, eluted with dinitrophenyl-L-lysine . Elution of the complex was

It is theoretically possible to eliminate the nonspecific binding of enzyme-labelled Fab' and reduce the background, if the complex of antibody IgG, antigen and enzyme-labelled antibody Fab' on solid phase is eluted without desorption of non-specifically bound enzyme-labelled antibody Fab' . This possibility was tested as follows . Antigen to be measured was reacted simultaneously with dinitrophenylated anti-

Table 1 .

Table 2. serum

87

efficient, but enzyme-labelled Fab' non-specifically bound was also desorbed significantly . Therefore, the sensitivity was improved only 2- to 3-fold . Even if the non-specific binding of enzyme-labelled antibody is reduced by combination of all the means described above, the sensitivity using polystyrene

Detection limit of antigens and peptides by various enzyme immunoassays

Analyte

Enzyme immunoassay (EIA)

Approximate level of detection limit

Antigens and peptides

Competitive EIA

Femtomole

Antigens

Two-site EIA

Attomole

Peptides consisting of more than 12-15 amino-acids

Two-site EIA

10 attomoles

Peptides consisting of less than 12-15 amino-acids

Hetero-two-site EIA

10 attomoles

Antigens

Immune complex transfer two-site EIA

Milliattomole

Sensitivity of various enzyme immunoassays for anti-insulin antibodies and anti-thyroglobulin antibodies in

Improvement of sensitivity (-fold) Enzyme immunoassay (EIA)

Solid phase (SP)

Conventional EIA

SP-Ag

Anti-Ig-Enz

Two-site EIA

SP-Ag

Soluble two-site EIA

SP-anti-DNP

Soluble two-site EIA with assay of eluted enzyme activity

SP-anti-DNP

Immune complex transfer EIA

Conjugate

Anti-insulin antibodies

Anti-thyroglobulin antibodies

1

1

Ag-Enz

20

20

DNP-Ag

Ag-Enz

200

65

DNP-Ag

Ag-Enz

600

200

SP-anti-DNP SP-avidin

DNP > Ag Biotin

Anti-Ig-Enz

150

SP-anti-DNP SP-anti-Ag

DNP-Ag

Anti-Ig-Enz

-

40

150

20 2,000-

SP-anti-DNP Sp-anti-Ig

DNP-Ag-Enz

SP-anti-DNP SP-anti-Ig

DNP-Ag

Ag-Enz

4,000'

SP-anti-DNP SP-streptavidin

DNP >Ag Biotin

Ag-Enz

4,000

Ag: antigen; 1g: immunoglobulin; Enz: enzyme; DNP: dinitrophenyl group . ' The detection limits of anti-insulin IgC and anti-thyroglobulin IgG in serum were 50 ng/l and 100 ng/l, respectively .



88

E. Ishikawa et al .

balls of 3 . 2 mm in diameter in a reaction mixture of 150µl cannot be improved beyond attomole levels for most of antigens . The use of a smaller solid phase surface may further reduce the non-specific binding . By using glass balls of 1 mm in diameter in a reaction mixture of 5 pl, one milliattomole (1 x 10 -21 mol ; 600 molecules) of human ferritin was detected ." In this small scale two-site enzyme immunoassay, however, the volume of samples such as serum, plasma and urine that can be used has to be reduced proportionally . Consequently, the sensitivity cannot be improved in terms of g or mol per litre of serum, plasma or urine . Immune complex transfer two-site enzyme immunoassay for antigens Recently, a new two-site enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) has been developed to reduce further the nonspecific binding without reducing the reaction mixture volume ." Human thyroid-stimulating hormone (hTSH) was reacted simultaneously with dinitrophenylated mouse monoclonal (anti-hTSH) IgG and rabbit anti-hTSH Fab' labelled with P-D-galactosidase from Escherichia coli . The complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls . After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to (antimouse IgG) IgG-coated polystyrene balls . By transfer of the complex, P-D-galactosidase activity non-specifically bound to (anti-dinitrophenyl group) IgG-coated polystyrene balls was effectively eliminated, and the

Ab-solid phase

detection limit of hTSH was improved to 20 milliattomoles . More recently, one milliattomole (600 molecules) of human ferritin has been detected by immune complex transfer two-site enzyme immunoassay (Figs 6 and 7) . Ferritin was reacted simultaneously with affinity-purified dinitrophenylated biotinylated antiferritin IgG and affinity-purified anti-ferritin Fab' labelled with (3-D-galactosidase from Escherichia coli . The complex formed of the three components trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls, was eluted with dinitrophenyl-Llysine and was transferred to streptavidin-coated polystyrene balls . (3-D-galactosidase activity non-specifically bound to streptavidin-coated polystyrene balls in the absence of ferritin (background) was 0 . 50 . 7% (n = 6) of that non-specifically bound to (antidinitrophenyl group) IgG-coated polystyrene balls in the absence of ferritin before incubation with dinitrophenyl-L-lysine . (3-D-galactosidase activity specifically bound to streptavidin-coated polystyrene balls in the presence of ferritin was 43-66% (n = 6) of that specifically bound to (anti-dinitrophenyl group) IgGcoated polystyrene balls in the presence of ferritin with dinitrophenyl-L-lysine . before incubation Namely, the non-specific binding (background) was markedly lowered with much less decrease in the specific binding . As a result, the detection limit of ferritin was 1 milliattomole (1 x 10 - 21 mol, 600 molecules as calculated from Avogadro's number) (Fig . 7) . This was 30-fold smaller than that by the conventional two-site enzyme immunoassay, in which an anti-ferritin IgG-coated polystyrene ball was incubated with ferritin and, after washing, with affinity-

Anti-DNPsolid phase

Ag

Non-specifically bound Ab-Enz

Ab-Enz

I 13

Non-specifically bound Ab-Enz

DNP Biotin-Ab

00o DNP-lysine (Strept) avidin - solid phase

VMOIEC

Conventional two-site enzyme immunoassay and immune complex transfer two-site enzyme immunoassay for antigens using dinitrophenylated biotinylated antibody IgG and antibody-enzyme conjugate. Ab: antibody; Ag: antigen ; Enz : enzyme ; DNP : dinitrophenyl . Fig . 6.



Ultrasensitive enzyme immunoassay T

phenyl biotinyl bovine serum albumin-anti-ornithine 8-aminotransferase Fab' conjugate and anti-ornithine 8-aminotransferase Fab'- P-D-galactosidase conjugate without affinity-purification . On the other hand the detection limit of this enzyme by the conventional two-site enzyme immunoassay was 0 . 3 amol (Fig . 7) . Affinity-purification may further lower the detection limit. In the same way, the detection limit of hepatitis B surface antigen (HBs Ag) is 2 milliattomoles using dinitrophenyl biotinyl mouse monoclonal anti-HBs Ag IgG 3 and mouse monoclonal anti-HBs Ag Fab'-j3-D-galactosidase conjugate, while its detection limit by the conventional two-site enzyme immunoassay using the same antibody is 0 . 1 amol (Fig . 7) .

U °

3000

O 72 0

1000

U °

0

0

1

0 CC

100

O T

0

U

10 N

0

N W O 7 LL

_

89

Serum interference in two-site enzyme immunoassay for antigens 0 .1-il 0 .001 0.01 0 .1 1 10 Ferritin (0-0), ornithine 8-aminotransferase (o-o) or hepatitis B surface antigen (a--a) tamot tube') i I

~ I ~ I

Fig. 7 . Dose-response curves of human ferritin, rat ornithine 5-aminotransferase and hepatitis B surface antigen by immune complex transfer two-site enzyme immunoassay using dinitrophenylated biotinylated antibody IgG and antibody Fab'- (3-D-galactosidase (E. coli) conjugate. Antibody IgG and Fab' were affinity-purified for ferritin but not for ornithine 8-aminotransferase . For hepatitis B surface antigen, mouse monoclonal IgG, was used. purified anti-ferritin Fab'- 3-D-galactosidase conjugate (Fig. 6: left) . From (3-D-galactosidase activity specifically bound to streptavidin-coated polystyrene balls in the presence of 0. 001-10 amol of ferritin, the average number of anti-ferritin Fab'- (3-D-galactosidase conjugate molecules bound per ferritin molecule added was calculated to be 0 . 95-1 .4. In addition, loss of specifically bound /3-D-galactosidase activity during elution and transfer of the complex was only 34-57% as described above . Therefore, another transfer of the complex (double transfers) was strongly suggested to further improve the sensitivity, although the sensitivity of assay of label enzyme remains to be improved considerably, and an efficient method for another transfer of the complex also remains to be developed . As suggested by the above results, the immune complex transfer two-site enzyme immunoassay technique may be capable of detecting milliattomole amounts of other antigens, which are measurable at attomole levels by the conventional two-site enzyme immunoassay . In a preliminary experiment, the detection limit of ornithine 8-amino-transferase from rat kidney was 10 milliattomoles by immune complex transfer two-site enzyme immunoassay using dinitro-

The detection limit of antigens in terms of g or mol 1 -1 of samples such as serum, plasma and urine depends on their detection limit in terms of g or mol per assay as well as the volume of samples that can be used without interference. The volume of serum used, for example, has to be reduced, depending on the severity of serum interference . This enhances the detection limit of antigens in terms of g or mol I -1 of serum . Therefore, various attempts have been made to alleviate serum interference . Less interference was obtained by incubation of reagents with serum in the presence of 0. 3-0 •4 mol I -1 NaCl than 0 . 1 mol I -1 NaCl.", " Incubation with serum at lower temperatures (4-20° C) also resulted in less interference ." Coating of solid with acid-treated antibody IgG alleviated some degree of serum interference . 31 Dilution of serum with an appropriate buffer is the easiest way to eliminate serum interference . Three to 15-fold dilution is sufficient for most antigens .

ANTIBODIES

Competitive and non-competitive immunoassays for antibodies As described for antigens and haptens, non-competitive immunoassays also potentially provide higher sensitivity for antibodies than competitive ones .

Interference by non-specific immunoglobulins In the most widely used conventional enzyme immunoassay for antibodies in serum, antigen-coated solid phase is incubated with test serum to trap



90

E. Ishikawa et al .

specific immunoglobulins to be measured and, after washing, with enzyme-labelled anti-immunoglobulin antibodies to measure the specific immunoglobulins which are trapped . This assay is simple and has been widely used . However, the sensitivity is seriously limited by non-specific (non-immunological and physical) binding to the solid phase of non-specific immunoglobulins in test serum, which tremendously enhances the background ." In another widely used conventional enzyme immunoassay for antibodies in serum, anti-immunoglobulin IgG-coated solid phase is incubated with test serum to trap both specific and non-specific immunoglobulins and, after washing, with enzyme-labelled antigen to measure specific immunoglobulin trapped . The sensitivity of this assay is limited by the capacity of anti-immunoglobulin IgG-coated solid phase to trap immunoglobulins . The capacity becomes larger with increasing surfaces of solid phase . However, larger surface of solid phase suffers from higher nonspecific binding of enzyme-labelled antigen, enhancing the background and limiting the sensitivity ."

Various attempts to overcome interference by non-specific immunoglobulins The non-specific binding of non-specific immunoglobulins, which seriously limits the sensitivity of the most widely used conventional enzyme immunoassay described above, can be reduced to some extent by addition of non-specific immunoglobulins from other animals 32 or detergents . However, the sensitivity has been improved only less than 10-fold by these modifications . A large amount of non-specific immunoglobulin in test serum was successfully removed using enzymelabelled antigen to improve the sensitivity ." Firstly, enzyme-labelled antigen was incubated with specific antibodies in the test serum and the immune complex formed was precipitated with anti-enzyme F(ab') Z. Secondly, the precipitates were washed to eliminate non-specific immunoglobulins and solubilized by reduction of anti-enzyme F(ab') z . The solubilized immune complex was precipitated with antiimmunoglobulin F(ab') z . (Alternatively, the solubilized immune complex may be trapped onto anti-immunoglobulin antibody-coated solid phase .) Finally, the precipitates were washed to eliminate uncomplexed

100 4-4

0 T

N

C d

C U) U UU N d O 0 LL

0-2---4, 1 0 1

0 10

100

1000

10000

Guinea pig anti-insulin IgG 1n serum (Fig (-)

Fig. 8 . Dose-response curves of guinea pig anti-insulin IgG in serum by two-site enzyme immunoassay using insulin-bovine serum albumin-coated polystyrene balls and insulin-horseradish peroxidase conjugate (0), two-site enzyme immunoassay using dinitrophenylated non-specific rabbit IgG-insulin conjugate and insulin-horseradish peroxidase conjugate (W and the conventional enzyme immunoassay using insulin-bovine serum albumin-coated polystyrene balls and (antiguinea pig IgG) Fab'-horseradish peroxidase conjugate (0). In all the methods, the volume of serum used was 20µl .

0

1

10

100

1000

10 000

Human anti-thyroglobulin IgG in serum (,ug I -1 )

Fig. 9 . Dose-response curves of human anti-thyroglobulin IgG in serum by two-site enzyme immunoassay using dinitrophenylated thyroglobulin and thyroglobulin-(3-D-galactosidase (E . coli) conjugate (0), two-site enzyme immunoassay using dinitrophenylated thyroglobulin and thyroglobulin-(3-D-galactosidase (E . coli) conjugate coupled with assay of eluted (3-D-galactosidase activity (0) and the conventional enzyme immunoassay using thyroglobulin-coated polystyrene balls and (anti-human IgG y-chain) Fab'-horseradish peroxidase conjugate ( •) . In all the methods, the volume of serum used was 20 µl .



Ultrasensltive enzyme immunoassay enzyme-labelled antigen and were solubilized by reduction of anti-immunoglobulin F(ab') Z . The solubilized complex was again precipitated to more completely eliminate uncomplexed enzyme-labelled antigen . Using 1 ml of test serum, the sensitivity was improved 100-fold as compared with that by the conventional enzyme immunoassay . However, the procedure was tedious and not practical . An alternative enzyme immunoassay to overcome the interference by non-specific immunoglobulins described above has been available, although not frequently used . Antigen-coated solid phase is incubated with test serum to trap specific immunoglobulins to be measured and subsequently reacted with enzyme-labelled antigen .", ", " The sensitivities by this method for anti-insulin antibodies and anti-thyroglobulin antibodies are approximately 20-fold higher than those by the conventional enzyme immunoassay using antigen-coated solid phase and enzymelabelled anti-immunoglobulin antibodies (Fig . 8) . 35 As in enzyme immunoassay systems for the quanti-

immune complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to avidin-coated polystyrene balls . Finally, the immune complex transferred was reacted with enzymelabelled anti-immunoglobulin Fab' . For anti-thyroglobulin IgG, the immune complex of dinitrophenylated thyroglobulin and anti-thyroglobulin IgG was transferred to rabbit anti-thyroglobulin IgG-coated polystyrene balls ." The sensitivities for anti-insulin IgG and anti-thyroglobulin IgG in serum were improved 40- to 150-fold as compared with that by the conventional enzyme immunoassay, in which antigencoated polystyrene balls were incubated with test serum and, after washing, with enzyme-labelled antiimmunoglobulin Fab' (Fig . 10) . This new methodimmune complex transfer enzyme immunoassayhas been modified in different ways . A modification has been made using enzymelabelled dinitrophenylated antigen ." , " Antibodies in

tations of antigens the sensitivity can be improved, when the immunoreactions are allowed to take place in solution . For example, antibodies can be reacted simultaneously with dinitrophenylated antigen and enzyme-labelled antigen . The immune complex formed of the three components is trapped onto (anti-dinitrophenyl group) IgG-coated solid phase . In this way, the sensitivities for anti-insulin antibodies and anti-thyroglobulin antibodies can be improved 3to 10-fold (Fig. 8) . 34 In addition, elution of bound enzyme activity with dinitrophenyl-L-lysine further improved the sensitivity approximately three-fold (Fig . 9) . 74 As a result, the sensitivity for anti-thyroglobulin antibodies in serum was improved 200-fold as compared with that by the conventional enzyme immunoassay using antigen-coated solid phase and enzyme-labelled anti-immunoglobulin antibodies . 14 However, the class of immunoglobulins to be measured cannot be identified .

3 0

enzyme immunoassay Recently, a practical method has been developed to eliminate non-specific immunoglobulins in test serum ." Antibodies in test serum were reacted simultaneously with dinitrophenylated biotinylated antigen . The immune complex formed was trapped on to (anti-dinitrophenyl group) IgG-coated polystyrene balls .The polystyrene balls were washed to eliminate non-specific immunoglobulins in the test serum. The

v t 0. 1 1 10 100 1000 10000 Guinea pig anti-insulin IgG in serum (µg 1 -1)

Dose-response curves of guinea pig anti-insulin IgG in serum by immune complex transfer two-site enzyme immunoassay using dinitrophenylated biotinylated non-specific rabbit IgG-insulin conjugate and (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate (0), immune complex transfer two-site enzyme immunoassay using dinitrophenylated non-specific rabbit IgG-insulin conjugate and insulin-3-D-galactosidase (E. coli) conjugate (0), immune complex transfer two-site enzyme immunoassay using dinitrophenylated biotinylated non-specific rabbit IgG-insulin conjugate and insulin-p-D-galactosidase (E. coli) conjugate (A) and the conventional enzyme immunoassay using insulin-bovine serum albumin-coated polystyrene balls and (anti-guinea pig IgG) Fab'horseradish peroxidase conjugate ( •). In all the methods, the volume of serum used was 20 µl . Fig. 10.

Practical and sensitive method to overcome interference by non-specific immunoglobulins-immune complex transfer

91



92

E. Ishikawa et al .

test serum was reacted with enzyme-labelled dinitrophenylated antigen. The immune complex formed was trapped onto (anti-dinitrophenyl group) IgGcoated polystyrene balls . The polystyrene balls were washed to eliminate non-specific immunoglobulins in the test serum . The immune complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to (anti-immunoglobulin) IgG-coated polystyrene balls . The sensitivity was improved 20- to 150-fold for anti-insulin IgG 18 and anti-thyroglobulin lgG39 as compared with that by the conventional enzyme immunoassay, in which antigen-coated polystyrene balls are incubated with test serum and, after washing, with enzyme-labelled anti-immunoglobulin Fab' . Another modification has been made using dinitrophenylated antigen and enzyme-labelled antigen (Fig . 11) . 4° Antibodies in test serum were reacted simultaneously with dinitrophenylated antigen and enzyme-labelled antigen . The immune complex formed by the three components was trapped onto (antidinitrophenyl group) IgG-coated polystyrene balls . The polystyrene balls were washed to eliminate nonspecific immunoglobulins in the test serum and the excess of enzyme-labelled antigen . The immune complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to (anti-immunoglobulin) IgG-coated polystyrene balls . By elimination of non-specific immunoglobulins, efficient transfer of the immune complex to (anti-immunoglobulin) IgG-coated polystyrene balls was possible . By

WI I +

+

0I „ 0 0 .1 1

I

Ab

1000

12 . Dose-response curves of human anti-thyroglobulin IgG in serum by two-site enzyme immunoassay using dinitrophenylated thyroglobulin and thyroglobulin-enzyme-conjugate (0), immune complex transfer two-site enzyme immunoassay using dinitrophenylated thyroglobulin and thyroglobulin-enzyme conjugate (A) and the conventional enzyme immunoassay using thyroglobulin-coated polystyrene balls and (anti-human IgG y-chain) Fab'-horseradish peroxidase conjugate ( •) . Solid and broken lines with open symbols indicate assays with j3-D-galactosidase from Escherichia coil and peroxidase from horseradish, respectively . In all the methods, the volume of serum used was 20µl .

+

.@ Ag-Enz

100

Fig.

a Anti - DNPsolid phase

10

Human anti-thyroglobulin IgG in serum (Fig t ')

Anti-DNPsolid phase

DNP, Biotin-Ag

TA Ab

+

9 Ag-Enz

l

DNP-lysine c--, =- DNP-lysine Anti-Ig-solid phase

(Strept)dvidin-solid phase

I .40 'I

A Immune complex transfer two-site enzyme immunoassay for antibody immunoglobulins using dinitrophenylated antigen and antigen-enzyme conjugate . DNP: dinitrophenyl ; Ag : antigen ; Ab : antibody; Enz : enzyme; Ig : immunoglobulin . Fig. 11 .

Immune complex transfer two-site enzyme immunoassay for antibodies using dinitrophenylated biotinylated antigen and antigen-enzyme conjugate . DNP : dinitrophenyl ; Ag : antigen ; Ab : antibody ; Enz : enzyme . Fig. 13 .



Ultrasensitive enzyme immunoassay transfer of the immune complex, non-specific binding of enzyme-labelled antigen (background) was markedly reduced . This method was simpler and more sensitive than the initially developed immune complex transfer immunoassay described above . The detection limits of anti-insulin IgG and anti-thyroglobulin IgG in serum were 50 ng I -1 and 100 ng I -1, respectively, which were 4,000-fold and 2,000-fold, respectively, lower than those by the conventional enzyme immunoassay (Figs 10 and 12) . This method has been further modified as follows (Fig . 13) . 41 Antibodies in test serum was reacted simultaneously with dinitrophenylated biotinylated antigen and enzyme-labelled antigen . The immune complex 'formed by the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, transferred to streptavidin-coated polystyrene balls . This format was as sensitive as the above method using dinitrophenylated antigen and enzyme-labelled antigen, although the class of immunoglobulins to be measured is not specifically identified (Fig . 10) . By transfer of the immune complex, the nonspecific binding of enzyme-labelled antigen was markedly reduced with less decrease in the specific binding (Fig. 12).4Q42 The non-specific binding of thyroglobulin-3-D-galactosidase conjugate to (anti-human IgG y-chain) IgG-coated polystyrene balls was 1 . 4% of that to (anti-dinitrophenyl group) IgG-coated polystyrene balls, while the specific binding to (antihuman IgG y-chain) IgG-coated polystyrene balls was 50% of that to (anti-dinitrophenyl group) IgG-coated polystyrene balls .42 Therefore, multiple transfers of the immune complex from solid phase to solid phase may further improve the sensitivity of immune complex transfer immunoassay, although efficient methods for multiple transfers remain to be developed .

DETECTION LIMIT OF LABEL ENZYMES The detection limit of horseradish peroxidase is 0.5 amol by fluorometric assay for 100 min using 3-(4hydroxyphenyl) propionic acid as hydrogen donor ." More than 100 min of assay does not improve its detection limit, since the enzyme is inactivated with time by its substrate, hydrogen peroxide . The detection limit of antigens by two-site enzyme immunoassay using horseradish peroxidase as label is higher than 0 . 1 amol, since the number of horseradish peroxidase-labelled Fab' molecules bound per antigen molecule to be measured is mostly less than 5-10 . 10 The detection limit of 3-D-galactosidase from Escherichia coli is 0 . 1-0•2 amol by fluorometric assay for 10 min using 4-methylumbelliferyl-(3-D-galacto-

93

side as the substrate . 10 This makes it possible to measure attomole amounts of many antigens by twosite enzyme immunoassay . 10 Fifteen to 40 h assay improves the detection limit to 1-2 milliattomoles . 10 As a result, 1 milliattomole of antigens (e .g . human ferritin) is detectable, provided that antibodies with sufficiently high affinity are used and provided that the non-specific binding of enzyme-labelled Fab' is sufficiently lowered, for example, by immune complex transfer methods described above . Less than one attomole of alkaline phosphatase from calf intestine is detectable either by colorimetry coupled with enzymatic cycling using NADP as substrate43 or by luminescent assay using luciferin phosphate"," or 1,2-dioxetane phosphate . , " Thus, peroxidase, (3-D-galactosidase and alkaline phosphatase are useful as labels for the detection of attomole or even milliattomole amounts of antigens . For the detection of less than one milliattomole of antigens, however, the sensitivity for the detection of label enzymes remains to be further improved . An attempt has been made to detect one molecule of (3-D-galactosidase from Escherichia coli by bioluminescent assay coupled with enzymatic cycling .' By the catalytic action of (3-D-galactosidase, D-galactose was released from 1,2-dinitrophenyl-o-D-galactoside as substrate . Subsequently, NADH was produced by galactose dehydrogenase and measured by bioluminescent assay using NADH :FMN oxidoreductase and luciferase." The amount of NADH produced per p-D-galactosidase molecule was 023 amol (1 . 4 x 105 molecules) min -1 , and the detection limit of NADH was 10 fmol . As a result, the detection limit of P-D-galactosidase by 1,000 min assay was 0 . 2 milliattomoles (2 x 10 -22 mol, 120 molecules), although the reaction mixture volume had to be reduced to only 2 pl to lower the background . Therefore, the detection of one molecule of f3-D-galactosidase was considered to be possible by more than 120-fold amplification of NADH using enzymatic cycling reaction . However, it was not possible due to the fact that the amount of 1,2-dinitrophenyl-o-D-galactoside spontaneously hydrolysed in 2 µI of the reaction mixture containing 0. 6 pmol of the substrate was 476-fold larger than that hydrolysed by one molecule of R-Dgalactosidase. Alternative methods to detect one molecule of label enzymes remains to be developed . One of the greatest obstacles limiting the sensitivity of non-competitive solid phase enzyme immunoassays is the non-specific binding of enzyme-labelled reactants to solid phase. In order to overcome this difficulty, a novel method (immune complex transfer enzyme immunoassay) has been developed initially for the measurement of antibodies in serum, improv-



94

E . Ishikawa et al .

ing the sensitivity more than 1,000-fold as compared with conventional methods . Its usefulness is being demonstrated in the detection of auto-antibodies for diagnosis of autoimmune diseases and anti-retrovirus antibodies for both diagnosis and prevention of retrovirus infection . The principle of the immune complex transfer immunoassay method has been successfully applied to measure milliattomole amounts of antigens . This method may be useful as an alternative to the polymerase chain reaction for' the detection of some micro-organisms in terms of more rapidity, lower cost, less chance for contamination and lack of need for a thermocycler . A non-competitive enzyme immunoassay (hetero-two-site enzyme immunoassay) very recently developed, has made it possible to measure directly peptide hormones such as vasopressin and atrial natriuretic hormone in plasma, which have been measured by radioimmunoassay only after extraction and concentration . Practical use of these ultrasensitive enzyme immunoassays remains to be facilitated by partial or full automation especially for the immune complex transfer process, and further improvement of the sensitivity should be preceded by development of a method to detect label enzyme with higher sensitivity within a shorter time .

REFERENCES 1 . Miles, L . E . M . & Hales, C . N . (1968) . Labelled antibodies and immunological assay systems . Nature 219, 186-9 . 2 . Engvall, E . & Perlmann, P . (1971) . Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G . Immunochemistry 8, 871-4 . 3 . van Weenmen, B . K. & Schuurs, A . H . W . M. (1971) . Immunoassay using antigen-enzyme conjugates . FEBS Letters 15, 232-6 . 4 . Editorial . (1976) . ELISA: a replacement for radioimmunoassay? Lancet ii, 406-7 . 5 . Ekins, R . (1976) . ELISA : a replacement for radioimmunoassays? Lancet ii, 569-70 . 6 . Watson . D . (1976) . ELISA : a replacement for radioimmunoassays? Lancet ii, 570 . 7 . Kato, K., Hamaguchi, Y ., Okawa, S ., Ishikawa, E ., Kobayashi, K . & Katunuma, N . (1977). Enzyme immunoassay in rapid progress . Lancet i, 40 . 8 . Ishikawa, E . & Kato, K . (1978) . Ultrasensitive enzyme immunoassay . Scandinavian Journal of Immunology 8 (Suppl . 7), 43-55 . 9 . Ishikawa, E ., Imagawa, M . & Hashida, S . (1983) . Ultrasensitive enzyme immunoassay using fluorogenic, luminogenic, radioactive and related substrates and factors to limit the sensitivity . Developments of Immunology 18, 219-32 . 10 . Ishikawa, E . (1987) . Development and clinical application of sensitive enzyme immunoassay for macromolecular antigens-a review . Clinical Biochemistry 20, 375-85 .

11 Ishikawa, E ., Hashida, S ., Kato, Y . & Imura, H . (1987) . Sensitive enzyme immunoassay of human growth hormone for clinical application : a review . Journal of Clinical Laboratory Analysis 1, 238-42 . 12 . Ishikawa, E ., Hashida, S., Tanaka, K . & Kohno, T . (1989) . Ultrasensitive enzyme immunoassay for antigens : technology and applications-a review . Clinical Chemistry and Enzymology Communications 1, 199-215 . 13 . Hashida, S ., Tanaka, K ., Kohno, T . & Ishikawa, E. (1988) . Novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens . Analytical Letters 21, 1141-54 . 14 . Ishikawa, E . & Kohno, T . (1989) . Development and applications of sensitive enzyme immunoassay for antibodies : a review . Journal of Clinical Laboratory Analysis 3, 252-65 . 15 . Tanaka, K ., Hashida, S ., Kohno, T ., Yamaguchi, K . & Ishikawa, E . (1989) . Novel and sensitive noncompetitive enzyme immunoassay for peptides . Biochemical and Biophysical Research Communications 160, 40-5 . 16. Hashida, S ., Ishikawa, E ., Nakao, K ., Mukoyama, M . & Imura, H . (1988) . Enzyme immunoassay for or-human atrial natriuretic polypeptide-direct measurement of plasma level . Clinica Chimica Acta 175, 11-8 . 17 . Mukoyama, M ., Nakao, K ., Yamada, T ., Itoh, H ., Sugawara, A ., Saito, Y., Arai, H ., Hosoda, K ., Shirakami, G ., Morii, N ., Shiono, S . & Imura, H . (1988) . A monoclonal antibody against N-terminus of ct-atrial natriuretic polypeptide (o(-ANP) : a useful tool for preferential detection of naturally circulating ANP . Biochemical and Biophysical Research Communications 151, 127784 . 18 . Ishikawa, E ., Tanaka, K . & Hashida, S . (1991) . Novel and sensitive noncompetitive (two-site) immunoassay for haptens with emphasis on peptides . Clinical Biochemistry (in press) . 19 . Fyhrquist, F ., Soveri, P ., Puutula, L . & Stenman, U-H . (1976) . Radioimmunoassay of plasma renin activity . Clinical Chemistry 22, 250-6 . 20 . Scharpe, S ., Verkerk, R ., Sasmito, E . & Theeuws, M . (1987) . Enzyme immunoassay of angiotensin I and renin . Clinical Chemistry 33, 1774-7 . 21 . Aikawa, T ., Suzuki, S ., Murayama, M., Hashiba, K ., Kitagawa, T . & Ishikawa, E . (1979) . Enzyme immunoassay of angiotensin I . Endocrinology 105, 1-6. 22 . Tanaka, K ., Hashida, S ., Uno, T ., Yamaguchi, K . & Ishikawa, E . (1991). Novel and sensitive noncompetitive enzyme immunoassay for arginine vasopressin . Clinical Chemistry and Enzymology Communications (in press) . 23 . Uno, T ., Uehara, K ., Motomatsu, K ., Ishikawa, E . & Kato, K . (1982) . Enzyme immunoassay for arginine vasopressin . Experientia 38, 786-7 . 24. Morton, J . J ., Padfield, P . L . & Forsling, M . L . (1975) . A radioimmunoassay for plasma arginine-vasopressin in man and dog: application to physiological and pathological states . Journal of Endocrinology 65, 411-24 . 25 . Hashida, S ., Ishikawa, E., Mukoyama, M ., Nakao, K. & Imura, H . (1991) . Highly sensitive two-site enzyme immunoassays for human atrial natriuretic polypeptides . Peptides (in press) . 26 . Ishikawa, E ., Imagawa, M ., Hashida, S ., Yoshitake, S ., Hamaguchi, Y . & Ueno, T . (1983) . Enzyme-labelling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining . Journal of immunoassay 4, 209-327 .

Ultrasensitive enzyme immunoassay

27 . Hashida, S . & Ishikawa, E . (1985) . Use of normal IgG and its fragments to lower the non-specific binding of Fab'enzyme conjugates in sandwich enzyme immunoassay . Analytical Letters 18, 1143-55 . 28 . Imagawa, M., Yoshitake, S ., Hashida, S . & Ishikawa, E . (1982). Effect of temperature on the sensitivity of sandwich enzyme immunoassay with Fab'-horseradish peroxidase conjugate . Analytical Letters 15, 1467-77. 29 . Ruan, K-H ., Hashida, S ., Tanaka, K ., Ishikawa, E., Niitsu, Y ., Urushizaki, I . & Ogawa, H . (1987) . A small scale sandwich enzyme immunoassay for macromolecular antigens using O-D-galactosidase from Escherichia coli and horseradish peroxidase as labels . Analytical Letters 20,587-601 . 30 . Hashida, S ., Nakagawa, K ., Imagawa, M., Inoue, S ., Yoshitake, S ., Ishikawa, E., Endo, Y., Ohtaki, S ., Ichioka, Y . & Nakajima, K . (1983) . Use of inorganic salts to minimize serum interference in a sandwich enzyme immunoassay for human growth hormone using Fab'horseradish peroxidase conjugate . Clinica Chimica Acta 135, 263-73 . 31 . Ruan, K-H ., Hashida, S ., Yoshitake, S ., Ishikawa, E., Wakisaka, 0 ., Yamamoto, Y ., Ichioka, T. & Nakajima, K . (1986) . A more sensitive and less time-consuming sandwich enzyme immunoassay for insulin in human serum with less serum interference. Annals of Clinical Biochemistry 23, 54-8 . 32. Kohno, T ., Hashida, S . & Ishikawa, E . (1985) . A more sensitive enzyme immunoassay of anti-insulin IgG in guinea pig serum with less non-specific binding of normal guinea pig IgG . Journal of Biochemistry 98, 37984. 33 . Ishikawa, E., Yoshitake, S., Endo, Y . & Ohtaki, S . (1980) . Highly sensitive enzyme immunoassay of rabbit (antihuman IgG) IgG using human IgG-0-D-galactosidase conjugate. FEBS Letters 111, 353-5 . 34. Kohno, T ., Mitsukawa, T ., Matsukura, S . & Ishikawa, E . (1988) . Novel and sensitive enzyme immunoassay for anti-thyroglobulin antibodies in serum using dinitrophenyl thyroglobulin and thyroglobulin-peroxidase conjugate. Analytical Letters 21, 2033-48 . 35 . Kohno, T . & Ishikawa, E. (1988) . Novel and sensitive enzyme immunoassays for antibodies . Journal of Miyazaki Medical Association 12, 177-82 . 36 . Kohno, T. & Ishikawa, E . (1987) . A novel enzyme immunoassay of anti-insulin IgG in guinea pig serum . Biochemical and Biophysical Research Communications 147, 644-9 . 37 . Kohno, T., Mitsukawa, T., Matsukura, 5 ., Tsunetoshi, Y . & Ishikawa, E . (1989). Measurement of antithyroglobulin IgG in serum by novel and sensitive immune complex transfer enzyme immunoassay . Clinical Biochemistry 22, 277-84 .

95

38 . Kohno, T. & Ishikawa, E. (1988) . Novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-insulin IgG in guinea pig serum . Analytical Letters 21, 1019-31 . 39 . Kohno, T., Mitsukawa, T ., Matsukura, S . & Ishikawa, E . (1990). Sensitive time-resolved fluorimetric immunecomplex-transfer immunoassay for antithyroglobulin IgG in serum . Journal of Clinical Laboratory Analysis 4, 224-30. 40 . Kohno, T., Mitsukawa, T ., Matsukura, S ., Tsunetoshi, Y . & Ishikawa, E . (1989) . More sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum . Journal of Clinical Laboratory Analysis 3, 163-8 . 41 . Kohno, T . & Ishikawa, E . (1991) . Ultrasensitive measurement of antibodies in blood by immune complex transfer enzyme immunoassay . Journal of Miyazaki Medical Association (in press). 42 . Kohno, T. & Ishikawa, E . (1990) . Development of novel and ultrasensitive enzyme immunoassay for antibodies and their application to the assay of human antithyroglobulin autoantibodies. Journal of Miyazaki Medical Association 14, 15-21 . 43 . Johannsson, A ., Ellis, D . E., Bates, D . L., Plumb, A. M. & Stanley, C . J. (1986) . Enzyme amplification for immunoassays . Detection limit of one hundredth of an attomole. Journal of Immunological Methods 87, 7-11 . 44 . Geiger, R ., Hauber, R. & Miska, W. (1989). New, bioluminescence-enhanced detection systems for use in enzyme activity tests, enzyme immunoassays, protein blotting and nucleic acid hybridization . Molecular and Cellular Probes 3, 309-28 . 45 . Miska, W. & Geiger, R . (1989) . Luciferin derivatives in bioluminescence-enhanced enzyme immunoassays . Journal of Bioluminescence and Chemiluminescence 4, 119-28 . 46. Bronstein, I ., Edwards, B . & Voyta, J . C. (1989). 1,2Dioxetanes : novel chemiluminescent enzyme substrates. Applications to immunoassays . Journal of Bioluminescence and Chemiluminescence 4, 99-111 . 47 . Bronstein, I ., Voyta, J . C ., Thorpe, G . H . G ., Kricka, L. J . & Armstrong, G . (1989) . Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin . Clinical Chemistry 35, 1441-6 . 48 . Tanaka, K . & Ishikawa, E . (1990) . Factors to hamper the detection of one molecule of (3-D-galactosidase from Escherichia coli by bioluminescent assay coupled with enzymatic cycling. Analytical Letters 23, 241-53. 49 . Tanaka, K . & Ishikawa, E . (1986) . A highly sensitive bioluminescent assay of (3-D-galactosidase from Escherichia coli using 2-nitrophenyl-o-D-galactopyranoside as a substrate . Analytical Letters 19, 433-44 .