Chemiluminescent Image Detection of Haptoglobin Phenotyping after

May 4, 2004 - Chemiluminescent Image Detection of Haptoglobin. Phenotyping after .... by mixing 10.0 mL of gel stock solution, 10.0 mL of Tris-HCl (1...
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Anal. Chem. 2004, 76, 2997-3004

Chemiluminescent Image Detection of Haptoglobin Phenotyping after Polyacrylamide Gel Electrophoresis Guangming Huang,† Jin Ouyang,*,† Joris R. Delanghe,‡ Willy R. G. Baeyens,§ and Zhongxin Dai†

Department of Chemistry, Beijing Normal University, Beijing 100875, P. R. China, Department of Clinical Chemistry, Microbiology and Immunology, University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium, and Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium

The development of an enhanced chemiluminescence detection method for the rapid detection of haptoglobin phenotyping after polyacrylamide gel electrophoresis is described in this paper. The enhanced chemiluminescence detection is based upon chemiluminescent reaction between luminol and hydrogen peroxide. Increased sensitivity and dynamic range are achieved by employing ammonium persulfate to enhance the chemiluminescence signal. Detection of haptoglobin phenotypes in human blood serum was easily achieved even without the addition of hemoglobin. Different polyacrylamide gel electrophoresis results were found between pure serum and hemoglobin-supplemented serum. Applying the suggested enhanced chemiluminescence detection, the original combining forms of haptoglobin and hemoglobin can be detected. The linear range of haptoglobin is 0.1-13.3 µg/mL, with a detection limit of 1.21 ng (sample loading volume 15 µL). Other proteins, such as catalase and ferritin, can also be detected using enhanced chemiluminescence detection. All detections after polyacrylamide gel electrophoresis were completed within 15 min. The proposed detection is very fast, compared to traditional methods using staining detection (minutes versus hours). Haptoglobin (Hp) is a hemoglobin-binding acute plasma protein and is characterized by a genetic polymorphism.1,2 Three phenotypes (Hp1-1, Hp2-1, Hp2-2) are known in human serum.3 Hp phenotypes show a molecular variation in their R subunits and are characterized by differences in molecular mass: Hp1-1 has a molecular mass of 98 kDa, whereas Hp2-1 and Hp2-2 show a range of molecular masses (Hp2-1, 86-300 kDa: Hp2-2, 170900 kDa) due to the existence of polymeric forms.2 Hp phenotypes also show important functional differences, which have pertinent clinical consequences,4,5 mainly in infectious diseases and cardio* To whom correspondence should be addressed: (e-mail) jinoyang@ bnu.edu.cn; (tel.) +86-010-62795561; (fax) +86-10-62799838. † Beijing Normal University. ‡ University Hospital. § Ghent University. (1) Smithies, O. Biochem. J. 1955, 61, 629-641. (2) Langlois, M.; Delanghe, J. Clin. Chem. 1996, 46, 1589-1600. (3) Ouyang, J.; Delanghe, J.; Baeyens, W. R. G..; Langlois, M. Anal. Chim. Acta 1998, 362, 113-120. (4) Kohler, W.; Prokop, O. Nature 1978, 271, 373. 10.1021/ac035109e CCC: $27.50 Published on Web 05/04/2004

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vascular risk stratifications.6,7 Recently, attention has been given to the role of Hp polymorphism as a determining factor in disease evolution and prognosis of liver transplantation.7 Clinical application and introduction into daily practice would request fast determination of Hp phenotype, which cannot be guaranteed by the currently used methods based on electrophoretic separation in starch gel.1 Currently, Hp phenotyping becomes possible due to the use of polyacrylamide gel electrophoresis (PAGE). However, the traditional Coomassie Brilliant Blue (CBB) staining technique after PAGE is time-consuming and confined by its detection limit. Hence, chemiluminescence (CL) detection was applied to PAGE, and chemiluminescent immunoanalysis has greatly improved the sensitivity due to the high sensitivity of the CL reaction.3 As polyacrylamide gel is impenetrable to antibodies and therefore cannot be immunoprobed directly, the electrophoresis pattern is to be transferred to a nitrocellulose or other protein-binding membrane to form a “Western blotting".3 However, the transfer efficiency of different proteins varies;8 therefore, the replicated pattern may be an inaccurate representation of the originating electropherogram, particularly with regard to large proteins.9 Further studies using radiolabeled antigens have shown that as much as 60% of the antigen may be lost from the membrane during immunoprocessing.10 This is important especially for the larger molecular mass of Hp2-2 (170-900 kDa),2 since current data about hemoglobinbinding capacity (HBC), the lowest found in Hp2-2 subjects, of various phenotypes are based on immunomethods. In these methods, the serum Hp concentration is generally underestimated because the diffusion of immune complexes is impaired in gels.11,12 Recently, CL signals of free radicals of proteins have been (5) Langlois, M.; Delanghe, J.; De Buyzere, M. L.; Bernard, D. R.; Ouyang, J. Am. J Clin. Nutr. 1997, 66, 606-610. (6) Delanghe, J.; Cambier, B.; Langlois, M.; De Buyzere, M. L.; Neels, H.; Bacquer, D. D.; Van Cauwelaert, P. Atherosclerosis 1997, 132, 215-219. (7) Langlois, M. R.; Martin, M. E.; Boelaert, J. R.; Beaumont, C.; Taes, Y. E.; De Buyzere, M. L.; Bernard, D. R.; Neels, H. M.; Delanghe, J. Clin. Chem. 2000, 46, 1619-1625. (8) Smejkal, G. B.; Hoff, H. F. Biotechniques 1994, 16, 68-70. (9) Millipore Corporation, Immunostaining Techniques and their Application to Protein Blotting Applications from a Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis System; Millipore Corp.: Danvers, MA 1990; Immobilon Tech Protocol 7, pp 1-7. (10) Hollander, D.; Befus, D. J. Immunol. Methods 1989, 122, 129-135. (11) Javid, J. Vox Sang 1965, 10, 320-325. (12) Braun, H. J.; Aly, F. W. Clin. Chim. Acta 1969, 26, 588-590.

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reported,13 which provides a potential route for exploring the enhancement of CL signals instead of horseradish peroxidase that is used in CL immunoanalysis. We investigated the enhancement by ammonium persulfate of the CL detection of Hp without Western blotting. Loss of immune complexes may hence be avoided and tedious immunization procedures including employment of expensive antibodies and markers may also be avoided, as no immunoprocess occurs. No data are available on the Hp concentration in pure serum due to the low sensitivity of traditional detection means, all previous studies being based on the analysis of hemoglobin (Hb)-supplemented serum, in which Hb is added to sera to form a Hp-Hb complex. Therefore, Hp in sera was detected at high concentrations of Hb, comparing to in vivo circumstances where Hp2-2 is found to be less efficient for removing free Hb and to prevent Hb-iron stimulated peroxidation of lipids.14,15 Taking advantage of the high sensitivity offered by the enhanced CL detection, we also tried to find out the differences of Hp-Hb combining form of different Hp phenotypes in sera where a lower Hb concentration exists. The results of these investigations are reported. EXPERIMENTAL SECTION Materials. All reagents were of A.R. grade. Tris(hydroxymethyl)aminomethane (Tris), aminoacetic acid (glycine), N,Nmethylenebisacrylamide (Bis), acrylamide, ammonium persulfate, and tetramethylethylenediamine (TEMED) were obtained from Sino-American Biotechnology Co. (Beijing, China). NaOH, H2O2, 3′,3′′,5′,5′′-tetrabromophenolsulfonephthalein (Bromophenol Blue), and glycerin were purchased from Beijing Fine Chemical Factory (Beijing, China). Molecular weight markers (ferritin (440 kDa) and catalase (232 kDa)) for proteins were purchased from Amersham Pharmacia Biotech Inc. 3-Aminophthalic hydrazide (Luminol) was obtained from Acros Organics. CBB-R 250 was from Fluka. Purified water was prepared by passing house-distilled water through a Millipore Simplicity 185 water purification system. Gel Preparation. Gel stock solution (30%, w/v) contained 29.2 g of acrylamide and 0.8 g of Bis, which were dissolved in 100 mL of H2O and then filtrated. The separating gel solution was prepared by mixing 10.0 mL of gel stock solution, 10.0 mL of Tris-HCl (1.5 M, pH 8.80), 200-800 µL of (NH4)2S2O8 (10%) (w/v), and 20-80 µL of TEMED and then diluting the mixture to 40.0 mL. For the stacking gel preparation, 1.33 mL of stock gel solution was mixed with 2.5 mL of Tris-HCl (0.5 M, pH 6.80), 50 µL of (NH4)2S2O8 (10%, w/v), and 10 µL TEMED, and then the mixture was diluted with water to 10.0 mL. The solution of (NH4)2S2O8 was used throughout at a concentration of 10% (w/v). This solution was daily prepared to ensure the stability. Sample Preparation. Fresh blood samples of 128 healthy people were obtained from the affiliated hospital of the Beijing Normal University. Fresh blood was allowed to stand for up to 5 h. The supernatant part was extracted as serum with a minisample collector and then centrifuged at 10 000 prm for 5 min. Sera were kept for 3 days at 4 °C in a refrigerator. Sera were stored at -20 °C for 60 days if the analysis was delayed. (13) Ionescu, G.; Merk, M.; Bradford, R. Forsch Komplementarmed 1999, 6, 294300. (14) Gutteridge, J. M. C. Clin. Chem. 1995, 41, 1818-1828. (15) Gutteridge, J. M. C. Biochim. Biophys. Acta 1987, 917, 219-223.

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Electrophoresis Procedure. The electrophoresis system consisted of a DYY-6B vertical electrophoresis tank and a DYCZ24D electrophoresis instrument of steady voltage (Liu yi Instrument Factory, Beijing, China). The electrophoresis buffer was made of 25 mM Tris and 192 mM glycine, adjusting the pH to 8.30. The vertical discontinuous polyacrylamide gel system was chosen, consisting of separating (7.5%, w/v) and stacking gel (4.0%, w/v). The loading sample volume was 15 µL. The voltage was of 130 V and was kept constant for 2 h and 10 min. Detection after PAGE. When enhanced CL detection was applied, the gel was taken out of the mold and washed with water. All the following procedures were accomplished in a dark room. The gel was spread on an even glass. Hydrogen peroxide (1.2% v/v) solution was blown onto the gel at ∼16 µL/cm2 (for 15 s), and luminol (1.0 × 10-3 M) was blown onto the gel at ∼80 µL/ cm2 (for 50 s). Next the gel was rinsed briefly with redistilled water and then covered with a sheet of transparency film, above which an X-ray film was placed. The X-ray film was exposed for ∼2 min, then developed for 2 min, and submerged into the fixing agent for 4 min. The exposure time was recorded from the beginning of the X-ray film being covered onto the gel. These developed X-ray films were then scanned, and bands were quantified with a dual-wavelength flying spot scanning densitometer (CS-9301PC, Shimadzu, Japan) and a VIS-7220 spectrophotometer (Ruili Co., Beijing, China). The optimal exposure time was obtained by recording the CL intensities utilizing a CL detector. After the luminescence reagent was sprayed onto the gel, the integral CL signal from emitting bands was recorded. The maximum intensity was obtained at ∼1.7 min. When the traditional staining detection was used, after taking out of the mold and washing, the gel was stained in CBB R250 (0.1%)/41.7% methanol/16.7% acetic acid solution for 2 h. It was washed and submerged into 10% methanol/7% acetic acid solution to destain for 10 h. The gel was scanned, and the results were transferred to the computer. RESULTS AND DISCUSSION Enhancement by Ammonium Persulfate of the CL Signal. (1) Effect of Extra Ammonium Persulfate Added to the gel before Electrophoresis. Commonly, in PAGE experiments, ammonium persulfate is used as an initiating agent of free radical reaction to fabricate polyacrylamide gels. Under different temperatures, various concentrations of ammonium persulfate are required during the course of gel fabrication. Hence, different amounts of ammonium persulfate were added to the gel at 20 °C to find the best concentration at this temperature. The gel would be polymerized in 50 min when the ammonium persulfate concentration was set at 50 µL/10 mL. Furthermore, increase of ammonium persulfate was found to increase the CL signal of Hp, especially when the concentration of ammonium persulfate was above 50 µL/10 mL. The effect of ammonium persulfate inside the gel upon the CL signal is shown in Figure 1. As illustrated in Figure 1A, featuring the normal concentration of ammonium persulfate (50 µL/10 mL), proteins produced only ultraweak CL signals that could hardly be detected by direct CL image detection. In Figure 1B, in which a higher concentration of ammonium persulfate (80 µL/10 mL) was added, the CL signal was greatly enhanced and several bands could be easily detected. The experiments above showed that ammonium persulfate could

Figure 1. Effect of ammonium persulfate added to the gel. (A) Normal gel with ammonium persulfate 50 µL/10 mL and TEMED 5 µL/10 mL. (B) Gel with ammonium persulfate 80 µL/10 mL and TEMED 8 µL/10 mL. Sample information: (1, 2, Hp2-2; 3, Hp1-1; 4, Hp2-1). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

enhance the CL signal of Hp, the mechanism of the enhancement to be discussed further. To find the relationship between the ammonium persulfate concentration in the gel and the CL signal, a series of ammonium persulfate concentrations was tested. During the course of gel fabrication, the concentration of ammonium persulfate was varied from 50 to 150 µL/10 mL, and accordingly, the TEMED concentration was varied between 5 and 15 µL/10 mL. The effect of these concentrations upon the CL signal is shown in Figure 2. From Figure 2A to Figure 2D, it can be seen that higher concentrations of ammonium persulfate and TEMED lead to higher sensitivity of Hp detection. It may be concluded that extra ammonium persulfate added to the gel clearly enhances the CL signal of Hp. The enhancement by ammonium persulfate may possibly be caused by the free radicals degraded from this reagent. Free radical degraded from ammonium persulfate may oxidize Hp in sera to form other free radicals, which contain fragments of Hp. Although we did not find the reference to directly support the oxidation of Hp by free radical degraded from ammonium persulfate, there are literature data that report on the oxidation of sulfhydryl-containing proteins such as Hp by ammonium persulfate.16 The formation of free radicals from persulfate has also been demonstrated by some reports in the literature.17,18 Therefore, we can conclude that enhanced CL signals are attributed to the free radicals oxidized from Hp due to the sensitization of free radicals upon the CL reaction of luminol and hydrogen peroxide. Further investigations as mentioned further in this paper will explain these results. The experiments clearly indicated that the CL signal of proteins increase with increasing ammonium persulfate concentrations. Once the concentration of ammonium persulfate is above 120 µL/ 10 mL, the background also increases rapidly. To increase the signal-to-noise ratio, the concentration of ammonium persulfate should be chosen below 120 µL/10 mL. In addition, the increase (16) Dirksen, M. L.; Chrambach, A. Sep. Sci. 1972, 7, 747-772. (17) Okaya, T.; Kikuchi, K.; Morii, Y. Polym. J. 1997, 29, 545-549. (18) Willett JL, Finkenstadt, V. L, Polym. Eng. Sci. 2003, 43, 1666-1674.

of ammonium persulfate concentrations and TEMED will not only lead to higher CL intensities but also to a relative shorter polymerizing time of the gel. The quality of the polyacrylamide gel proved to be fine when the gel polymerized within 30-50 min. At 20 °C, the polymerizing time of the gel varied from 25 to 50 min when ammonium persulfate varies from 150 to 50 µL/10 mL. To avoid poor gel quality caused by too long or too short polymerizing times of the gel, the proper concentration of ammonium persulfate should be set from 80 to 120 µL/10 mL. Taking both the signal-to-noise ratio and the quality of the gel into account, at 20 °C ammonium persulfate concentration was chosen as 120 µL/10 mL. (2) Effect of Immersion into Ammonium Persulfate Solution after Electrophoresis. We also tested the immersion of the gel into the solution of ammonium persulfate after electrophoresis. It was found that the CL signal could be enhanced not only by including ammonium persulfate in the gel, when fabricating the latter before electrophoresis, but also by submerging the gel into the solution of ammonium persulfate after electrophoresis. To study the effect of ammonium persulfate after electrophoresis, gels that contain normal concentrations (50-60 µL/10 mL) of ammonium persulfate were submerged into the solution of ammonium persulfate after electrophoresis. The results are shown in Figure 3, which shows that ammonium persulfate also possesses the ability to enhance the CL signal after electrophoresis. As shown in Figure 3A, no obvious CL signal could be detected in the gel that had not been submerged into the solution of ammonium persulfate. In Figure 3B, it can be seen that the CL signal was greatly enhanced when the gel was submerged into the solution of ammonium persulfate. This indicates that the immersion of gel into the solution of persulfate reagent after electrophoresis could also enhance the CL emission. Different concentrations of ammonium persulfate were tested to find a proper concentration for enhancing the CL signal of Hp. The gels were submerged into solutions of ammonium persulfate (0.01, 0.02, and 0.03%), and the results are illustrated in Figure 4. The CL signal increased with increasing ammonium persulfate concentrations; therefore, higher concentrations of ammonium persulfate were preferred. On the other hand, higher concentration of ammonium persulfate produced some irregular spots as could be seen on the X-ray film, leading to a lower signal-to-noise ratio. From the results above, a proper concentration of 0.02% was chosen for obtaining relatively higher CL light to be collected and higher signal-to-noise ratios to be achieved under these conditions. (3) Enhancement by Ammonium Persulfate on Cellulose Acetate Membrane. All polyacrylamide gels used in the present experiments contained different amounts of ammonium persulfate added during gel fabrication. Although we only partially proved the enhancement by ammonium persulfate of the CL signal from proteins, we still wanted to prove this feature by employing a cellulose acetate membrane, chosen as it contains no ammonium persulfate and it is easy to use without preparing the gel. Different amounts (1, 2, and 5 µL) of serum were spotted on two cellulose acetate membranes using slide glass. One of them was submerged into the solution of ammonium persulfate (0.02%); next, luminol and hydrogen peroxide were spread on it. On the other membrane, hydrogen peroxide and luminol were spread without submergence into ammonium persulfate solution. The Analytical Chemistry, Vol. 76, No. 11, June 1, 2004

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Figure 2. Effect of ammonium persulfate concentration added to the gel. (A) Normal gel with ammonium persulfate 50 µL/10 mL and TEMED 5 µL/10 mL. (B) Gel with ammonium persulfate 80 µL/10 mL and TEMED 8 µL/10 mL. (C) Gel with ammonium persulfate 120 µL/10 mL and TEMED 12 µL/10 mL. (D) Gel with ammonium persulfate 150 µL/10 mL and TEMED 15 µL/10 mL. Sample information: (1, 2, Hp2-2; 3, Hp1-1; 4, Hp2-1). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

Figure 3. Effect of ammonium persulfate after electrophoresis. (A) Normal gel with no immersion into the solution of ammonium persulfate. (B) Normal gel with 10-min immersion into the solution of ammonium persulfate of 0.01%. Sample information: (1, 2, Hp1-1; 3, Hp2-1; 4,5, Hp2-2). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

comparison of the two membranes is shown in Figure 5. From Figure 5A it can be seen that no obvious CL signal was detected when hydrogen peroxide and luminol were directly spread on the membrane. When immersing into the solution of ammonium persulfate, the CL signal of serum is greatly enhanced, which is illustrated on the other membrane as shown in Figure 5B. This indicates that ammonium persulfate (0.02%) can sensitize the CL reaction of proteins with hydrogen peroxide and luminol. (4) Application of Enhanced CL Detection on Hp after PAGE. From the experiments above, it was found that ammonium persulfate may enhance the CL signal of Hp, which makes it possible to directly detect Hp using enhanced CL detection after PAGE. Enhancement by ammonium persulfate on the CL signal of Hp by adding it to the gel before electrophoresis and submerg3000

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ing the gel into the solution after electrophoresis have been discussed in the former part of this paper. Submerging the gel into a solution of ammonium persulfate (0.02%) after electrophoresis was chosen by considering the interaction of ammonium persulfate in the electrophoresis. Bands of Hp could be easily detected using enhanced CL detection, while they could hardly be detected using the traditional CBB R250 staining method. The comparison of enhanced CL detection with traditional staining detection is shown in Figure 6. The gel was submerged into the solution of ammonium persulfate (0.02%) after electrophoresis. From the comparison above, it can be seen that the sensitivity of enhanced CL detection is much higher than traditional staining detection. Furthermore, it took less than 10 min to collect the results by enhanced CL detection while to obtain good results with the stain, 6-8 h is required (2 h to stain at room temperature and 4-6 h to destain while heated at 50 °C). Enhanced CL detection may hence be considered a fast and sensitive means to detect Hp in human serum. In the literature, three major Hp phenotypes can only be detected when Hb is added to form the Hp-Hb complex.1,2,4 As can be seen from Figure 6A, with traditional CBB R250 staining detection, no obvious differences were found between the three major phenotypes of Hp in serum without addition of Hb. In contrast, with enhanced CL detection, three major phenotypes of Hp in serum without addition of Hb could be detected as illustrated in Figure 6B. The different results of staining detection and enhanced CL detection may be derived from the sensitivity differences. There is some Hb in human serum, while in our research, Hb of 1.21-3.2 µg/mL is detected in sera using benzidine spectrophotometry.19 Hp in human serum is to combine with Hb due to the function of Hp. The concentration of the HpHb complex is lower than the detection limit by the CBB R250 staining detection. Hence, with traditional CBB R250 staining detection, Hp phenotypes can only be detected by adding Hb to (19) Yu, Z. G., Yang, M. Y., Feng, W. L., Yang, H., Feng, T. B., Zhang, S. P., Pan, E. T., Eds. Experimental Guidance to Haematology and Haematology Analysis; The People’s Medical Publishing House: Beijing, China, 1999; pp 61-62.

Figure 4. Effect of concentration of ammonium persulfate after electrophoresis. (A) 10-min immersion into the solution of ammonium persulfate of 0.01%. (B) 10-min immersion into the solution of ammonium persulfate of 0.02%. (C) 10-min immersion into the solution of ammonium persulfate of 0.03%. Sample information: (1, 2, Hp1-1; 3, Hp2-1; 4,5, Hp2-2). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

Figure 5. Enhancement by ammonium persulfate on cellulose acetate membrane. (A) Membrane without immersion into the solution of ammonium persulfate. (B) Membrane with 20-min immersion into the solution of ammonium persulfate (0.02%).

the serum to increase the concentration of the Hp-Hb complex. Since enhanced CL detection offers high sensitivity, it is possible to directly detect Hp phenotypes in human serum without the addition of Hb. Detection results are shown in Figure 7A corresponding to standard phenotypes of Hp.20 An interlaboratory stock standard serum with the Hp concentration of 1.33 mg/mL was used to prepare calibration curves. The standard serum was diluted successively by using NaCl solution (0.85%, w/v). The linearity of the Hp response based on a dilution ratio of 0.1-13.3 µg/mL can be acquired, with a detection limit of 1.21 ng (sample loading volume 15 µL) by using the flying spot scanning densitometer. Relative standard deviation is 3.27% at 1.00 µg/mL (n ) 9). Enhanced CL detection clearly improved the sensitivity of Hp detection. In view of the lower reference range for Hp2-2 (0.381.50 g/L)2, the improved sensitivity of enhanced CL detection is particularly important since in a number of cases “anhaptoglobinaemia” was reported in earlier studies, based on starch electrophoresis methods.1,21 Furthermore, literature data about the (20) Wu, J. M.; Liu, M. J.; Wang, B. J. Science of Forensic Evidence; The People’s Medical Publishing House: Beijing, China, 1996; p 80-83.

Figure 6. Comparison of enhanced CL detection and CBB R250 staining detection. (A) Result of CBB R250 staining detection. (B) Result of PAGE-enhanced CL detection. Sample information: (1, 2, Hp2-2; 3, Hp1-1; 4, Hp2-1). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

relative HBC of the various Hp phenotypes are based on earlier immunodiffusion methods, in which serum Hp immune complexes are impaired in gels (typically in the analysis of Hp 2-2).11,12 Up to now, all previous research about Hp in human serum is based on the analysis of Hb-supplemented serum, which cannot identify the original Hp-Hb combining form in human serum. Analysis of Hp without addition of Hb has not been reported due to the low sensitivity of detection. In our research, we detected the original combining form of Hp-Hb in human serum due to the increased sensitivity offered by enhanced CL detection. Figure (21) Sutton, H. E., Steinberg, A., Bean, A., Eds. Progress in Medical Genetics, The Haptoglobins; W. Heinemann Medical Books Ltd.: London, 1970; Vol. VII, pp 163-216.

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Figure 7. Results of the detection of Hp by PAGE-enhanced CL detection. (A) Results of enhanced CL detection. (B) Standard phenotypes of Hp. Sample information: 1-3 and 11, Hp 2-2; 4-6 and 12, Hp 2-1; 7-10, Hp 1-1; 3, 6, and 9, Human serum diluted 1:300 in the solution of 6.67% glycerin and 0.05% bromophenol blue; sample loading volume, 15 µL. 2, 5, and 8, human serum diluted 1:150 in the solution of 6.67% glycerin and 0.05% bromophenol blue; sample loading volume, 15 µL; 1, 4, and 7, human serum diluted 1:60 in the solution of 6.67% glycerin and 0.05% bromophenol blue; sample loading volume was 15 µL.

8 illustrates the comparison of the results from pure serum and from Hb-supplemented serum. Sera of 128 healthy people (Hp2-1, 71; Hp2-2, 44; Hp1-1, 13) were detected in this study. We observed three additional bands (Figure 7A, b-d; Figure 8B, b-d) for Hp2-1, and one additional band (Figure 7A, a; Figure 8B, a) for Hp 1-1 in the pattern of pure sera. These new bands demonstrate different combining forms of Hp-Hb for Hp2-1 and Hp1-1, which has not been reported so far. Hp1-1 and Hp2-1 are more inclined to combine with Hb as they can form complexes as illustrated in Figure 8B, a-d, especially under in vivo circumstances as in serum where the concentration of free Hb is relatively lower (below 4.0 mg/100 mL). HBC of Hp1-1 and Hp2-1 are higher than that of Hp2-2 besides the lower concentration of Hp2-2, which has been demonstrated by high-pressure gel permeation chromatography.22 Hp is capable of rapidly removing free Hb from the circulation and thereby prevents the hemoglobiniron stimulated peroxidation of lipids,14,15 while Hp2-2 is a less efficient antioxidant.2 So Hp phenotypes will affect the metabolism of vitamin C5 and coronary lesions6 in consequence of lower HBC of Hp2-2. We also found from Figure 8C that, along with the addition of higher concentrations of Hb to the samples, the additional bands for Hp 1-1 and Hp 2-1 gradually disappeared, but the band of Hb (Figure 8A and C, e) appeared. This can be explained by the fact that when Hb is insuffient, Hp can be detected as a complex of Hp-Hb. A similar result was also obtained by Liau et al.,23 when the authors purified the Hp by using Hp-Hb complex. However, (22) Delanghe, J.; Allcock, K.; Langlois, M.; Claeys, L.; De Buyzere, M. L. Clin. Chim. Acta 2000, 291, 43-51. (23) Liau, C. Y.; Chanf, T. M.; Pan, J. P.; Chen, W. L.; Mao, S. J. T. J. Chromatogr., B 2003, 790, 209-216.

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Figure 8. Comparison of enhanced CL detection and direct CL detection. (A and C) result of direct CL detection; (B) result of enhanced CL detection. (a) Additional band of Hp 1-1 detected by enhanced CL imaging; (b-d) additional bands of Hp 2-1 detected by enhanced CL imaging; (e) band of Hb detected by direct CL imaging. Sample information: 1 (Hp1-1), 2 (Hp2-2), and 3 (Hp2-1), human serum (80 µL) and Hb (16 mL); 4 (Hp1-1), 5 (Hp2-2), and 6 (Hp2-1), human serum (5 µL); 7 (Hp2-1), human serum (80 µL) and Hb (30 mL); 8 (Hp2-1), human serum (80 µL) and Hb (16 mL); 9 (Hp2-1), human serum (80 µL) and Hb (10 mL); 10 (Hp2-1), human serum (80 µL) and Hb (5 mL); 11 (Hp2-1), human serum (80 µL) and Hb (2 mL); 12 (Hp2-1), human serum (80 µL); 13 (Hp2-1), human serum (16 µL). Every sample contains 0.05% bromophenol blue/6.67% glycerin and water was added until the total sample volume reached 300 µL; sample loading volume was 15 µL.

further increase in Hb concentrations may lead to the formation of multiple coordinated complexes of Hb and Hp, decreasing the mobility sequentially. This assumption needs to be further demonstrated since the molecular weight may not be a judgment in native PAGE. Thus, an explanation of the two bands that disappeared at higher concentrations of Hb remains a question for study. In summary, the present study suggests that people carrying an Hp2-2 phenotype are more likely to accumulate coronary atherosclerotic lesions6 despite serum lipid concentration and that they more likely have lower vitamin C concentration in serum.5 In the present study, we demonstrated the higher HBC of Hp2-1 and Hp1-1, due to their unique combining form with Hb, especially in serum where the concentration of Hb is quite low. Further studies are required to explain the difference of the combining forms of Hp2-1 and Hp1-2 between higher and lower Hb concentration levels, as well as to understand how the differences affect the HBC. (5) Detection of Standard Proteins. During these investigations, we observed that some standard proteins could also be detected using enhanced CL detection after PAGE. For example, catalase (232 kDa) and ferritin (140 kDa) can be detected by PAGE-enhanced CL detection. The results of enhanced CL detection after PAGE for catalase and ferritin can be seen in Figure 9. Bands of catalase and ferritin are very clear using enhanced CL detection. Catalase can greatly sensitize the CL reaction of luminol and hydrogen peroxide; hence, its band produced a

Figure 9. PAGE result of standard proteins using enhanced CL detection. 1, solution of standard proteins (15 µL); 2, solution of standard proteins (5 µL).

Figure 10. Effect of pre-electrophoresis. (A) Normal gel (ammonium persulfate 60 µL/10 mL and TEMED 6 µL/10 mL) without preelectrophoresis. (B) Normal gel (ammonium persulfate 60 µL/10 mL and TEMED 6 µL/10 mL) with 30-min pre-electrophoresis. (C) Improved gel (ammonium persulfate 120 µL/10 mL and TEMED 12 µL/10 mL) without pre-electrophoresis. (D) Improved gel (ammonium persulfate 120 µL/10 mL and TEMED 12 µL/10 mL) with 30-min preelectrophoresis. Sample information: (1, Hp2-2; 2, Hp2-1; 3, Hp11). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

massive CL signal. Ferritin contains the ferric ion, which can sensitize the CL reaction; hence, it can also be detected by enhanced CL detection. Possible Mechanism of the Enhancement by Ammonium Persulfate on the CL Signal. (1) Mechanism of Enhancement by Extra Ammonium Persulfate Added to the Gel. According to our experiments, the addition of extra ammonium persulfate to the gel before electrophoresis can enhance the CL signal. It is presumed that the free radicals decomposed by the extra ammonium persulfate enhance the CL signal. These free radicals could oxidize the complex of Hp-Hb to form other free radicals. These free radicals oxidized from the proteins could then catalyze the CL reaction of luminol and hydrogen peroxide. To prove this mechanism, we tried to decrease the concentration of extra ammonium persulfate before electrophoresis. According to the literature,24 pre-electrophoresis can decrease the ammonium persulfate concentration in the gel, which means electrophoresis without sample at 150 V for 30 min. We chose pre-electrophoresis at 140 V for 30 min, and the results are shown in Figure 10. Panels C and D of Figure 10 demonstrate that when pre-electrophoresis occurred with higher concentration of ammonium persulfate, the CL signal of Hp-Hb decreased. By decreasing ammonium persulfate concentrations during preelectrophoresis, less free radicals would be formed by the decomposition of this reagent and less free radicals would be oxidized from Hp-Hb. The decrease of free radicals oxidized from Hp-Hb leads to lower CL signals. As illustrated in Figure 10A and B, with pre-electrophoresis, the CL signal in the gel with lower concentration (60 µL/10 mL) of ammonium persulfate also decreased. This experiment has partially proved the suggested assumption. Although pre-electrophoresis could decrease the CL signal, the apparent CL signal shown in Figure 10D was still stronger than that in Figure 10B. This shows the concentration of ammonium persulfate in Figure 10D is still higher than that in Figure 10B. This may be explained as following.

First, pre-electrophoresis for 30 min could not completely remove the ammonium persulfate from the gel. A relatively longer pre-electrophoresis time may completely remove the ammonium persulfate from the gel, but this will lead to lower gel quality. Second, ammonium persulfate can enhance the CL signal at very low concentrations. Pre-electrophoresis could not decrease the concentration of ammonium persulfate under such conditions. (2) Mechanism of Enhancement by Ammonium Persulfate after Electrophoresis. It has been reported that ammonium persulfate can sensitize the CL reaction of luminol and hydrogen peroxide.25 Both the free radicals decomposed from ammonium persulfate and free radicals oxidized from Hp-Hb can sensitize the CL reaction of luminol and hydrogen peroxide. Hence, it is more important to find out whether the enhanced CL signal results from Hp-Hb or simply from ammonium persulfate action. The influence of exposure time upon CL signal was observed, and the results are shown in Figure 11. It was found that the background increased with longer exposure times when the normal gel was submerged into the solution of ammonium persulfate after electrophoresis. As shown in Figure 11, with the same gel, during the first 2.5 min proteins yielded CL signals with a relatively lower background. After 2.5 min, the CL signal of Hp-Hb dropped increasingly; the background however produced strong CL emission. The best exposure time was considered 2-2.5 min for normal gel submerged into the solution of ammonium persulfate after electrophoresis. The results above also proved our assumption. Both free radicals decomposed from ammonium persulfate, and free radicals oxidized from Hp-Hb can sensitize the CL reaction. For free radicals oxidized from Hp-Hb, it is relatively fast to sensitize the CL reaction. Hence, during the first 2.5 min, Hp-Hb emitted

(24) Nevaldine, B.; Hahn, P. J.; Rizwana, R. Radiat. Res. 1999, 152, 154-159.

(25) Li, J. Z.; Dasgupta, P. K. Anal. Chim. Acta 1999, 398, 33-39.

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Figure 12. Curve of exposure time vs integral CL intensities. Gel was incubated in the 0.02% ammonium persulfate solution, and luminescence reagents were then sprayed onto the gel. Next, the integrative CL signal from emitting bands was recorded.

effects. Hence, a best signal-to-noise ratio should be set at the exposure time of 2.3 min in order to avoid the high background caused by the free radicals decomposed from ammonium persulfate.

Figure 11. Effect of exposure time. Exposure time (A) 1.0, (B) 1.5, (C) 1.7, (D) 2.5, and (E) 3.5 min. Sample information: (1, 2, Hp1-1; 3, Hp2-1; 4, 5, Hp2-2). Human serum diluted 1:60 in 6.67% glycerin and 0.05% bromophenol blue; loading volume was 15 µL.

obvious CL light. It takes more than 2.5 min for free radicals decomposed from ammonium persulfate to sensitize the CL reaction. Thus, the background increased quickly after 2.5 min. This can also be explained by Figure 12, a curve recording the optimal exposure time. This curve was plotted according to the procedure mentioned in the Experimental Section. We can observe from Figure 12 that the free radicals oxidized from HpHb produce effects within 2.5 min while at 1.7 min the maximum signal of proteins could be observed. On the other hand, background increases rapidly after 2.5 min when free radicals decomposed from ammonium persulfate were starting to cause

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CONCLUSIONS Enhanced CL detection after PAGE is a simple and rapid means for most sensitively detecting Hp phenotype in human sera. High sensitivity is achieved without any expensive antibodies or tedious procedures of Western blotting. With the proposed method, even the original combining forms of Hp-Hb in serum can be detected, where a low concentration of Hb exists. It was found that there are differences of Hp-Hb combining forms between higher and lower Hb concentration levels, especially in the cases of Hp1-1 and Hp2-1. The unique combining forms of Hp1-1 and Hp2-1 with Hb are believed to be the answer to why Hp2-2 is less efficient in removing free Hb in serum. Further studies are still needed to demonstrate the structural difference of Hp-Hb between higher and lower Hb concentration levels and the mechanism on how this difference affects the HBC. Furthermore, since there exist various kinds of CL systems, enhanced CL detection after PAGE has the most potential to detect more proteins apart from Hp. ACKNOWLEDGMENT We gratefully acknowledge the support from the National Nature Science Foundation of China (20375005) and the Bilateral Scientific and Technological Cooperation Flanders Belgiums China (011S0503). Received for review September 22, 2003. Accepted March 1, 2004. AC035109E