ANALYTICAL BIOCHEMISTRY 93, 267--271 (1979)

A LuminoI-Assisted, Competitive-Binding Immunoassay of Human Immunoglobulin G LEROY S. HERSH, W I L L I A M

P. V A N N , AND SALLY A . W I L H E L M

Coming Glass Works, Sullivan Park Research Building, Coming, New York 14830 Received July 13, 1978 Luminol (3-aminophthalhydrazide) has been covalently coupled to human immunoglobulin G (IgG). Chemiluminescence of the luminol-IgG conjugate was measured in the presence of heroin and hydrogen peroxide. The effective specific chemiluminescent activity of the conjugate is approximately one luminol molecule bound for every five molecules of IgG. The luminol-IgG conjugate was used as a label in a heterogeneous competitivebinding immunoassay of human IgG. Antibody-bound luminol-IgG was separated from free label by precipitin reaction with rabbit antibody. The precipitate was dissolved in alkali and monitored for chemiluminescent activity. A level of 5/~g of human IgG yielded a 13% decrease from zero dose binding after a 1-h incubation. A semilog displacement curve was obtained for the range 5 to 50/xg HlgG/tube. The mean slope was 13% for every doubling of dose. Results obtained with luminol-HIgG agreed with radial immunodiffusion values of low, medium, and high IgG in reference serum.

The direction for high-volume immunoassays is toward automated nonradiometric methods. A technology used presently is enzyme immunoassays using automatable instrumentation (1). Additional nonradiometric approaches are fluorometric or chemiluminescent tracers. The weak point of fluorometry is lack of sensitivity compared to radioactivity. Clinical fluorometric sensitivity limits are determined by method sensitivity rather than instrument sensitivity. In other words, the limiting factor is the fluorescence intensity of the serum blank. Chemiluminescence offers the potential of increased signal-to-blank ratios because of low concentrations of intrinsic chemiluminescent serum components. Recently, Schroeder et al. (2) have reported an immunoassay for total thyroxine using a thyroxine-luminol conjugate. We report on the use of luminol to synthesize a label used in an immunoassay for human immunoglobulin (IgG) in serum samples. The luminol-IgG label was used to assay IgG using a corn267

petitive precipitin reaction with rabbit antibody raised against human IgG.

METHODS Luminol (3-aminophthalhydrazide) was obtained from Aldrich (code No. 12,307-2, 97% pure) and used without further purification. Human IgG used for coupling to luminol was obtained from Miles Laboratory (lyophilized, code No. 64-145). Human IgG used as standards were obtained from Cappel (normal human controls, human fraction rich in IgG, no IgA or IgM). The samples for reference values of IgG were obtained from Hyland (multicomponent immunological reference serum I, 15.3 mg/ml; II, 7.4 mg/ml; and III, 2.64 mg/ml by radial immunodiffusion). Rabbit antihuman IgG (against the whole IgG molecule) was purchased from Miles-Yeda as the IgG fraction (code No. 65-155, lot No. $364, 2.6 mg antibody/ml). A glass flow cell was constructed based on the design of Seitz and Hercules (3); the 0003-2697/79/040267-05502.00/0 Copyright © 1979by AcademicPress, Inc. All fightsof reproductionin any form reserved.

268

HERSH, VANN, AND WILHELM

cell's volume was 1.5 ml. Three inlet tubes were used for: luminol, standard, or unknown (3.2 ml/min); 10-4M hemin in 0.4 M H3BO3/KOH bufferat pH 13.2plus 5 × 10-3 M H~O2 (1.6 ml/min); and oxygen gas at 4 cm3/ rain. Oxygen gas was used to generate convection currents as described by Seitz and Hercules (3). The buffer p H in the heroin line was adjusted to 13.2 in order to compensate for the low pH in the H20~ solution. H202 was kept at p H 5.5 to avoid self-oxidation. The hemin and HzO2 were kept in separate reservoirs and mixed in line just before entering the reaction cell. The outlet pH was 11 __+0.2. The flow cell was placed next to a RCA 1P21 photomultiplier (PMT). 1 An Aminco photomultiplier microphotometer (J10-280) supplied 1 KV to the PMT. A Sargent recorder (Model MR) was used to determine peak heights of light intensities. The luminol sample volumes ranged from 0.1 to 1.0 ml. The l u m i n o l - I g G label was synthesized using a periodate oxidation of IgG and subsequent Schiff base formation with luminol. The procedure was, first, to dissolve 1 mg luminol and l0 mg IgG in 5 ml water. Then, 3 ml of a solution containing 1.5 mg/ml of KI04 was added, and the p H adjusted to 4.7 with 4 N HC1. The solution was stirred in the dark for 45 min. An additional 1 ml of the KIO4 solution then was added, and the p H adjusted to 7.2 with 4 N N A O H . After 2 additional h of reaction in the dark, the solution was passed through a Sephadex G-25 Fine column to remove unreacted luminol. The fractions containing protein were pooled and then assayed for chemiluminescent activity. Measurements indicated a luminol activity equivalent to a concentration of 8 × 10-7 M luminol. Estimated protein content was about 4 × 10-6 M, and therefore, the complex had a luminol 1 Abbreviations used: PMT, photomultiplier; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PEG, polyethylene glycol; CE chemiluminescent efficiency; RID, radial immunodiffusion.

specific activity of about one luminol molecule per five IgG molecules. A rough indication of actual number of luminol molecules bound was obtained by absorption measurements (IE347 ~---7.1 x 10a M-1 cm -1 was used for luminol). This value indicated approximately one luminol per IgG molecule, and thus the effective chemiluminescence efficiency of bound luminol is approximately 20% of free luminol. The immunological activity of the lumin o l - I g G label was determined in a precipitin reaction with rabbit anti-IgG. A final concentration of 8 x 10-7 M (refers to IgG concentration) luminol-IgG was incubated with different amounts of rabbit anti-IgG overnight at 25°C in 0.03 M phosphate-buffered saline (PBS), 0.1% BSA, pH 7.5, at a final volume of 0.4 ml. The precipitates formed were centrifuged at 1000g for 45 min at 4°C and then washed in 0.15 M NaC1. Luminol activity was assayed in the precipitates after dissolution with 0.1 ml of 0.5 M K O H for 30 min and subsequent readjustment to pH 7.5 with 0.03 M PBS and 0.1 ml of 0.5 M HC1 to final volume of 1 ml. The 0.1 ml of the latter solution was assayed for luminol activity. A dose-response curve was generated by the competition of 0.05 ml of l u m i n o l - I g G (5 × 10-rM IgG) and varying concentrations of IgG (0.5, 10, 15, 20, 25, and 50/zg/ tube) for 0.05 ml of rabbit anti-human IgG. H u m a n IgG reference serums were assayed using this dose-response curve. Reference serums I and II were diluted 1:100 and reference serum III was diluted 1:20 in 0.03 M PBS, 0.1% BSA, p H 7.5. Then, 0.1 ml of these diluted serums were incubated with 0.05 ml of the l u m i n o l - H I g G label, 0.05 ml of rabbit anti-human IgG, and 0.2 ml of PBS, BSA, pH 7.5 buffer. An incubation time of 48 h at 4°C was used. The precipitate formed was centrifuged at 3000 rpm for 30 min, the supernatant aspirated off, and the precipitate washed once in 1 ml of 0.15 M NaC1. Luminol activity was determined as described above.

269

L U M I N O L - A S S I S T E D I M M U N O A S S A Y O F IgG 240

--

160 I'-" T W"I-

W (2_ 80

"•

I 4

2

i

I 6

t

I 8

,

I 10

LUMINOL, MOLES, x 1012

Fie. 1. Luminol calibration curve. Peak height of light response (arbitrary units) vs moles luminol introduced into flow cell.

RESULTS AND DISCUSSION

The results of precipitin reactions between the luminol-IgG label and rabbit anti-IgG is shown in Fig. 2. Nearly 100% of the luminol activity was observed in the immunoprecipitate phase. This fact indicates that the covalent attachment of luminol did not alter the antigenic character of IgG. The pre-

A luminol standard curve is shown in Fig. 1. Sample volumes of 0.1 ml of various dilutions ofluminol stock solution (10 -6 M) were introduced, and peak heights were measured. The limit of luminol sensitivity was approximately 10-1° mol/liter. IOC

/

9C

8C

•f

*

z 7c 0 m

6C

5(3

4O

J 5O RAH IgG F I

I

I

100

200

I 500

FIG. 2. Luminol chemiluminescent activity in precipitate phase plotted vs rabbit anti-human IgG' (RAH IgG) added in a precipitin reaction.

270

HERSH, VANN, AND WILHELM A

I00

B

80

0

_o 60 x o g3

~. 4 0

2C

io

5

I0

20

40 50 5 M.gH I g G / T U B E

I

I0

I

20

I II

4o 50

FIG. 3(A). Dose-response curves for competitive precipitin reaction using luminol-IgG label, IgG, and antiIgG. The percentage luminol activity in precipitate phase at various human IgG (HIgG) levels divided by percentage luminol activity at zero dose is plotted vs micrograms human IgG in each tube. Open circles, 48 h incubation at 4°C; closed circles, 1 h incubation at 25°C. (B) Same as A except 4% polyethylene glycol was added; 1 h incubation at 25°C was used.

cipitin results were used to determine the amount of anti-IgG required for the IgG assay. The maximum sensitivity in a competitive binding assay would be obtained by using a minimum amount of antibody. However, in order to obtain a pellet large enough to readily handle, 50/zl of rabbit anti-IgG was used. This yielded about 75% bound label. The dose response curves obtained with purified IgG are shown in Figs. 3A and B. The upper and lower curves in Fig. 3A were obtained using a 48 h at 4°C and 1 h at 25°C incubation, respectively. The 48 h incubation yielded a slight extension of dose range.

Figure 3B shows results obtained from a 1 h incubation at 25°C with the addition of 4% polyethylene glycol (PEG), a reported precipitin accelerator (4). The effect of adding PEG was to extend the range and slightly increase the drop in label binding at 5 /zg IgG per tube. The 1 h incubation results in Fig. 3A indicate a 13% decrease from zero dose binding for 5 /zg of IgG. The mean slope was 13% for every doubling of dose. Reference serums were tested and the results are presented in Table 1. Results obtained with the luminol assay agreed very well with radial immunodiffusion values of low, medium, and high IgG in the reference serum.

TABLE 1 COMPARISON OF CHEMILUMINESCENT IMMUNOASSAY RESULTS WITH RADIAL IMMUNODIFFUSION ANALYZED REFERENCE SAMPLES

Concentration ranges (reference serum)

Via present method (mg/ml)

Reference concentrations (mg/ml)

High (I) Average (II) Low (III)

16.5 7.5 2.2

15.3 7.4 2.6

SUMMARY The luminol-based chemiluminescent label can be employed as a substitute for radiolabels in immunoassays for serum components at concentrations greater than 10-9 mol/liter. The main factor limiting the sensitivity of the method is the relatively low overall chemiluminescent efficiency (CE) of the luminol tag. The CE of underivatized luminol is reported to be 1.5% (5). Our lu-

LUMINOL-ASSISTED IMMUNOASSAY OF IgG

minol-IgG label had a final efficiency of about 0.3%. It is possible that a more efficient means of coupling luminol, if found, would increase sensitivity by a maximum of 500%. The most efficient chemiluminescent system reported to date (not involving enzymes) is the hydrogen peroxide-oxalate ester reaction (6). This reaction has an overall chemiluminescence efficiency of 23%. The use of the oxalate ester as a chemiluminescent label would provide the more substantial gain of 1500% compared to the luminol system.

271

REFERENCES 1. Galen, R. S., and Forman, D. (1977) Clin. Chem. 23, 119. 2. Schroeder, H. R., Yaeger, F. M., Boguslaski, R. C., Snoke, E. O., and Buckler, R. T. (1977) Clin. Chem. 23, 1132. 3. Seitz, W. R., and Hercules, D. M. (1972) Anal. Chem. 44, 2143. 4. Hellsing, K. (1964)Acta Chem. Scand. 18, 1303. 5. Brundrett, R. B., and White, E. H. (1974)J. Amer. Chem. Soc. 96, 7497. 6. Rauhut, M. M., Bollyky, L. J., Roberts, B. G., Loy, M., Whitman, R. H., Iannotta, A. V., Semsel, A. M., and Clarke, R. A. (1967)J. Amer. Chem. Soc. 89, 6515.