Simultaneous Bioluminescent Immunoassay of Serum Total and IgG

Article pubs.acs.org/ac

Simultaneous Bioluminescent Immunoassay of Serum Total and IgGBound Prolactins Alexander N. Kudryavtsev,†,‡ Vasilisa V. Krasitskaya,†,‡ Alexei I. Petunin,§ Andrey Y. Burakov,∥ and Ludmila A. Frank*,†,‡ †

Photobiology Lab, Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk 660036, Russia Siberian Federal University, Krasnoyarsk 660041, Russia § DIAS Ltd., Krasnoyarsk 660036, Russia ∥ Krasnoyarsk Regional Hospital No. 1, Krasnoyarsk 660022, Russia ‡

ABSTRACT: Novel dual-analyte single-well bioluminescence immunoassay (BLIA) for total and IgG-bound prolactins was developed on the base of Ca2+-regulated photoprotein obelin mutants with altered color and kinetics of bioluminescence signal as reporters. The mutants W92F-H22E and Y138F were chemically conjugated with monoclonal mouse anti-hPRL and anti-hIgG immunoglobulins and thus displayed signals from total prolactin and IgG-bounded prolactin (macroprolactin) correspondingly. Bioluminescence of the reporters was simultaneously triggered by a single injection of Ca2+ solution and discriminated via bioluminescent signal spectral and time resolution. The developed microplate-based immunoassay allows detection of two prolactin forms in crude serum without additional manipulations (e.g., gel chromatography or PEG-precipitation). Total prolactin bioluminescence immunoassay in standard, control, and clinical sera offers high sensitivity and reproducibility. The BLIA results show good correlation with those obtained by RIA and immunoassay after gel chromatography.

H

some analyzers, PEG has been reported to interfere immunoassay; freezing and thawing of sera samples (during storage) reduce macroprolactin concentration.3,6 The aim of our research was the development of the simultaneous detection of two prolactin forms using a bioluminescent immunoassay based on color mutants of Ca2+-regulated photoprotein obelin as reporters.8 Obelin is a stable complex of a single-chain polypeptide (22.2 kDa) and 2hydroperoxycoelenterazine (oxygen “preactivated” substrate), which is strongly but noncovalently immobilized in the protein hydrophobic cavity.9 The photoprotein has three Ca2+-binding sites. Upon Ca2+ addition, protein undergoes rapid conformational changes that cause substrate decarboxylation yielding CO2, coelenteramide, and blue light (λmax = 482 nm). Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution.8 The mutant W92F-H22E emits violet light (λmax = 385 nm), and the mutant Y138F emits green light (λmax = 498 nm), with small spectral overlap. Both proteins display high activity and stability and thus may be used as reporters. Besides the mutants’ bioluminescence kinetics differ significantly: the decay rates of violet and green signals are 0.6 s−1 and 6.1 s−1,

yperprolactinaemia (supraphysiological prolactin concentrations) is a biochemical marker of hypothalamicpituitary disfunctions, occurring in clinical practice of endocrinologists, gynecologists, urologists, and sexual pathologists.1,2 The measurements revealing hyperprolactinaemia are essential for the diagnosis, but a major problem the laboratories face is the correct differentiation of true hyperprolactinaemia and macroprolactinaemia. The matter is that the human serum prolactin (PRL) circulates in several forms: the predominant (85−95% in normal serum) monomeric form with a molecular mass of 23 kDa, big PRL (5−10%) with a molecular mass of 45−60 kDa, and big-big PRL or macroprolactin (macro-PRL) with a molecular mass of 150−170 kDa. In macro-PRL, the hormone is bound to IgG-type autoantibody3 and unlike monomeric prolactin is biologically inactive. Macroprolactinaemia comprises up to one-fourth of all cases of hyperprolactinaemia. Unfortunately, the routinely available immunoassay methods usually measure the circulating prolactin forms all together that commonly lead to diagnostic confusion, misdiagnosis, and mistreatment.4,5 The gold standard to determine prolactin forms in sera is gel-filtration chromatography (GFC), slow, labor-intensive, costly, and hence notsuited to routine use. An alternative approach is a polyethylene glycol (PEG) precipitation method which diagnostic applicability was repeatedly confirmed by researchers.1−7 This method, however, has some drawbacks: up to 35% of monomeric form may coprecipitate with macroprolactin; for © 2012 American Chemical Society

Received: October 29, 2011 Accepted: March 5, 2012 Published: March 20, 2012 3119

dx.doi.org/10.1021/ac300444w | Anal. Chem. 2012, 84, 3119−3124

Analytical Chemistry

Article

Figure 1. Bioluminescent signals of obelin violet and green mutants, transmitted through filter I (A) and filter II (B); signals of obelins’ mixture, transmitted through filter I and then through filter II (C). Dashed line, time for filter replacement.

green obelins were calculated at a signal-to-background ratio of 2, four replicates. Microtiter-Based Bioluminescent Immunoassay of Total and IgG-Bound Prolactin. The surface of the wells was activated with 100 μL of mouse anti-hPRL IgG (clone 5602), 10 μg/mL, in PBS (0.15 M NaCl, 50 mM K-Na phosphate buffer pH 7.0), overnight at 4 °C, washed (three times, PBS, 0.1% Tween 20, 5 mM EDTA), and blocked (150 μL per well of 1% BSA in PBS, for 1 h, 37 °C). After washing, 100 μL of standard (three replicates), control (three replicates), or clinical (two replicates) sera were placed into wells incubated with shaking for 1 h at 37 °C and washed thereafter. Then the mixture of conjugates, W92F-H22E-anti-hPRL IgG (clone 5601) and Y138F-anti-hIgG (clone XA-6) (100 μL, each 1 μg/ mL, in PBS, 0.1% BSA, 5 mM EDTA), was placed into the wells, incubated with shaking for 1 h at 23 °C, and washed thereafter. The bioluminescence intensity was measured with a plate reader Mithras immediately after rapid injection of CaCl2 solution (100 μL, 0.1 M in 0.1 M Tris-HCl, pH 8.8) into the plate well. The measurements were carried out as follows: during the first second, the photometer integrated a violet signal transmitted through filter I; the replacement for filter II took the next 0.3 s; the green light was integrated for 5 s. Bioluminescent Immunoassay of hIgG. The surface of the wells was activated with 100 μL of mouse anti-hIgG (to Fab fragment) solution (10 μg/mL, in PBS) as described above. Then 100 μL of hIgG standard solutions of 1000, 500, 250, 125, 62.5, and 0.0 ng/mL (three replicates) were placed into wells, incubated with shaking for 1 h at 37 °C, and washed thereafter. After that, the same mixture of conjugates W92FH22E-anti-hPRL IgG (clone 5601) and Y138F-anti-hIgG (clone XA-6) was placed into the wells, incubated with shaking for 1 h at 23 °C, and washed. The bioluminescence intensity was measured with a luminometer Mithras LB 940 as previously described. Gel-Filtration Chromatography (GFC). GFC was performed on a 1 cm × 48 cm column of BioGel P200 (BioRad), pre-equilibrated with 10 mM Tris-HCl, pH 7.0, 0.15 M NaCl, 1.25 mM CaCl2, 0.5 mM MgCl2, 0.01% NaN37 at room temperature. The column was calibrated with molecular weight markers including thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa) (Gel Filtration Standard, BioRad), and cytochrome C (12.4 kDa) from Serva (Germany). The volume of the sample (marker solution or serum) applied to the column was 300 μL; flow rate, 50 μL/min; the volume of fractions collected, 500 μL. The prolactin concentration in the fractions

correspondingly, this pointing to the possibility for their time resolution. On the basis of the obelin mutants as reporters, we developed a dual-analyte single-well bioluminescence immunoassay for total and IgG-bound prolactins in sera. The reporters conjugated with corresponding immunoglobulins (to prolactin or to human IgG) provide signals from total prolactin or IgGbounded prolactin. Bioluminescence of the reporters was simultaneously triggered by the single injection of Ca2+ solution and divided using optical filters and time resolution.

■

EXPERIMENTAL SECTION Chemicals and Reagents. The highly purified photoproteins (violet mutant W92F-H22E and green mutant Y138F of obelin) were obtained according to ref 8. Monoclonal mouse IgGs against hPRL (clone 5601) and (clone 5602) were from Medix Biochemica (Finland), monoclonal mouse IgG against hIgG γ-chain (clone XA-6) from Xema-Medica (Russia), and monoclonal mouse IgG against hIgG Fab fragment (clone L-5) from BIOSAN (Russia). The standard (calibrated according to hPRL standard WHO 84/500) and control prolactin sera were from DIAS (Russia). The standard sera of hIgG were prepared from control human serum, containing 11.31 mg/mL hIgG (SIC “Microgen”, Russia). Conjugates W92F-H22E-anti-hPRL IgG (clone 5601) and Y138F-anti-hIgG IgG (clone XA-6) were synthesized according to the method described in ref 10. The conjugates were stored at −18 °C in 20 mM Tris-HCl, pH 7.0, 0.25 M NaCl, 5 mM EDTA, 0.1% BSA over 1 year without loss of bioluminescent activity. The solid-phase immunoassay was carried out using 96-well opaque microtiter plates (Costar). At the dual-color immunoassay, the photometer registered signals through band-pass optical filters VB6 (I) and YB16 (II) (both from Len-ZOS, Russia). Sera samples were provided by the endocrinology diagnostic laboratory of Krasnoyarsk Regional Hospital No. 1. The total PRL and macroprolactin in the sera were previously quantified with RIA before and after PEG precipitation. Photoprotein Detection Limit. Serial proteins dilutions in 20 mM Tris-HCl, pH 7.0, 5 mM EDTA (50 μL) were placed into microtiter wells, and bioluminescence was initiated by CaCl2 solution (50 μL, 0.1 M in 0.1 M Tris-HCl, pH 8.8) rapid injection. The signals were integrated through optical filter I for 1 s and after filter replacement (0.3 s) through filter II for 5 s with a plate LB 940 Multimode Reader Mithras (Berthold, Germany). Detection limits for a highly purified violet and 3120



Article

Figure 2. (A) Schematic illustration of the simultaneous immunoassay of the total and IgG-bound hPRLs. Both forms, monomeric and IgG-bound, are captured on the IgG-activated surface. The conjugates mixture was placed into the wells, and the violet label was bound with all PRL, green one, with IgG-bound PRL. The bioluminescence of both labels was triggered with Ca2+ and measured: (1) through filter I for 1 s (violet signal) and then (2) through filter II for 5 s (green signal). (B) Data output of PRL bioluminescent immunoassay. The standard sera assay (in triplicates) is framed by a red color. The PRL concentrations are presented in row A1−A6. Other cells present clinical sera assays (in two replicates each). Clinical samples containing essential or a small amount of IgG-bound PRL are framed by the blue and green colors, respectively.

emission spectra maxima are separated by 113 nm, and the spectral overlap is small; (2) sharp distinction is observed in bioluminescence kinetics, it is 10 times slower in the case of green obelin as compared to that of violet obelin.8 These spectral and kinetic differences make possible the effective signals’ separation using the plate luminometer Mithras LB 940 (Figure 1). The device construction allows luminescence measurement through optical emission filters placed on the wheel which provides fast filter replacement. Filters transmit the corresponding light effectively, with adsorption being 8− 9%. At the same time, each filter strongly (for more than 90%) cuts off the adjacent bioluminescence (Figure 1A,B). Figure 1C shows the bioluminescent reaction of the protein mixture. To divide signals effectively, a fast-kinetic violet signal was registered through optical filter I during 1 s and a long-kinetic green signal through optical filter II during 5 s. Filters were replaced automatically during 0.3 s. This way of measurement provides detection limits of 14 and 18 amol for violet and green obelins, respectively, whereas in ref 8 the limits were detected for entire signals and correspondingly equaled 12 and 5 amol. The next step of our research was the development of a simultaneous bioluminescence immunoassay of total and IgGbound PRL in serum based on this method of obelins’ signals separation. The routinely available immunoassay methods measure the circulating prolactin forms all together. So to reveal macroprolactinaemia, sera with elevated total PRL concentrations (≥700 mU/L, as a rule) are analyzed for the second time after treatment with PEG. Macro-PRL is precipitated and discarded after centrifugation. PRL recovery is derived as a percentage ratio between PRL measured in the supernatant and that measured in untreated serum. However the PEG-precipitation method has some shortcomings, up to 35% of the monomer may coprecipitate with macroprolactin; for some analyzers, PEG has been reported to interfere with the immunoassay; freezing and thawing of sera samples (under storage) reduce the macroprolactin concentration.3,6 Determination of prolactin forms in sera using gel-filtration chromatography is the most reliable method, but the slow, laborious method is not applicable for routine use.

was detected using a bioluminescent immunoassay as described above.

■

RESULTS AND DISCUSSION Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were recently applied as the reporters for dual-color immunoassay of two targets in a sample.8 The mutants W92F-H22E (violet) and Y138F (green) are characterized by rather different bioluminescence: (1) the

Figure 3. (A) Bioluminescent microtiter-based immunoassay of total PRL in standard sera. Each point is an average ± 1 standard deviation (n = 3). (B) Correlation of total PRL concentrations in 83 clinical samples obtained with bioluminescent (BLIA) and isotopic 135I (RIA) labels (R2 = 0.92, slope = 0.94). Upper inset shows low-level PRL samples (R2 = 0.87, slope = 1.04). 3121



Article

Figure 4. (Left panel) The results of clinical sera gel filtration chromatography. The PRL curve is shown in violet. The green label bioluminescence is shown in green. The upper plot additionally displays the calibration curve (dashed line) for the BioGel P200 column; molecular weight markers: (a) thyroglobulin (670 kDa), (b) bovine γ-globulin (158 kDa), (c) ovalbumine (44 kDa), (d) myoglobin (17 kDa), (f) cytochrome C (12.4 kDa), and (g) vitamin B12 (1.35 kDa). The green bioluminescence axis is not shown. (Right panel) Prolactin forms in the same initial sera determined by the simultaneous bioluminescent immunoassay.

according to the data of 5 independent experiments, the corresponding values of slope and free term were 0.214 ± 0.027 and 27.7 ± 2.59. Using this dependence as a calibration curve, the total PRL in 83 clinical sera was determined. The data demonstrate good correlation between bioluminescent assay and RIA in the whole range of PRL concentrations (Figure 3B). The total PRL bioluminescence assay sensitivity was 3.54 ± 0.5 mU/L (calculated from three replicates of standard zero sera assay, as mean + 3 SD). It is close to that of the radioisotope immunoassay (3−5 mU/L, diagnostic kit from DIAS Ltd., Russia) and exceeds that of the colorimetric immunoassay (10 mU/L, diagnostic kit from XEMA, Russia). The control serum PRL concentration was 460 ± 20 mU/L, this fitting well in the range declared by manufacturer, 400−600 mU/L (DIAS Ltd., Russia). Unlike the standard, most of the clinical sera (Figure 2B, wells in area D1−H6) contain IgG-bound PRL to which a slow green signal testifies. Here, the green-framed samples demonstrate a high violet signal pointing to a high concentration of total PRL, and at that a low green signal indicative of a low macro-PRL concentration. It means that in these samples practically all prolactin is monomeric and its high concentration proves true hyperprolactinaemia. The blueframed samples of sera manifest two high signals, violet and green, which means that the high total PRL concentration is

To detect two targets, PRL and IgG-bound PRL, we synthesized two conjugates as labels, mouse anti-PRL immunoglobulin (clone 5602) with violet obelin and mouse anti-hIgG immunoglobulin (clone XA-6) with green obelin. The assay scheme is presented in Figure 2A. The well surface was activated with mouse anti-hPRL IgG (clone 5601), then the samples of standard, control, or clinical sera were placed into the wells and both PRL forms, monomeric and IgG-bound, were captured on a surface. Then (after the washing procedure) the conjugate mixture was placed into the wells and violet label got bound with all PRL on the surface, whereas the green one was only with IgG-bound PRL. Bioluminescence of both labels was triggered with Ca2+ injection and measured sequentially: during the first second, the violet signal was integrated through filter I. This signal correlates with total PRL concentration. After filter replacement, the green signal was measured for 5 s. This signal correlates with IgG-bound PRL concentration. Figure 2B presents the typically obtained results. The assay (in triplicate) of standard sera was performed in wells A1−C6 (shown in the red frame). These samples are artificial sera, containing only monomeric PRL of a given concentration. Therefore one can observe only violet signal from these wells. The dependence of violet bioluminescent signal upon PRL concentration (Figure 3A) is linear for the whole concentration range, with R2 = 0.994. The assay shows good reproducibility: 3122



Article

Figure 5. (A) Schematic illustration of model bioluminescent immunoassay. hIgG is captured on the surface activated with mouse immunoglobulins to hIgG Fab fragment. The conjugates mixture was placed into the wells. Only green label was immobilized on the surface due to affinity to the hIgG γ chain. Bioluminescence of the label was triggered with Ca2+ and measured (1) through filter I for 1 s (violet signal is absent) and then (2) through filter II for 5 s (green signal). (B) Immunoassay of hIgG in the standard sera data output (in triplicates). The hIgG concentrations are presented in row A1−A6. (C) Bioluminescent (green obelin-based) immunoassay of hIgG in standard sera. Each point is average ± 1 standard deviation (n = 3). In parentheses are the corresponding IgG-bound PRL concentrations (mU/L) calculated implying an equimolar IgG-PRL complex.

with macroprolactin-containing fractions. The percentage of macro-PRL, as determined by analysis of the area under the prolactin curve (violet line) is shown in the Table. Figure 4 (right panel) gives the results of PRL detection in the same initial sera using a simultaneous bioluminescent immunoassay. Judging from high green signals, the macroprolactin concentration in samples 1−3 is considerable, whereas sample 4 contains PRL in the monomeric form only. So, we observed good correlation between GFC and the bioluminescent assays results. Our further attempt was to find the way for quantitative determination of macroprolactin. The matter is that the calibration curve for total PRL based on the violet reporter is not applicable for macro-PRL determination based on the green reporter. As it was shown,8 the bioluminescence activity of Y139F is 7-fold as much as that of W92F-H22E. That is why the macro-PRL-associated green signal may be higher than the total PRL-associated violet signal (see samples 2 and 3, Figure 4, right panel). There is no way to obtain the macroprolactin calibration curve due to the absence of IgG-bound PRL standard sera. Here, for obtaining the curve, we propose a model macroprolactin immunoassay involving standard hIgG sample instead of hIgG carrying PRL as an antigen (Figure 5A). The well surface was activated with mouse antibodies to the Fab-fragment of the hIgG. Then samples of standard hIgG sera of known concentrations were placed into the wells, and after conventional incubation and washing procedures the labels mixture was placed into the wells. The violet label was not captured at the surface since there was no PRL in the samples, whereas the green one was bound with immobilized hIgG. Bioluminescence was triggered with Ca2+ injection and measured as it was indicated above. As a result, we observed only green signals (Figure 5B) correlating with hIgG concentrations (R2 = 0.987). With this dependence as a calibration curve (Figure 5C), the macro-PRL in the four untreated clinical sera under consideration was determined and then its percentage was calculated. Table 1 summarizes the

Table 1. Prolactin Forms in Clinical Sera bioluminescent immunoassaya

RIA

gel filtration

sample

total PRL, (mU/L)

macroPRL,b %

total PRL, (mU/L)

macroPRL, %

macroPRL, %

1 2 3 4 5 6 7 8 9 10

4375 1047 3650 4500 4157 210 199 582 259 2722

67 82.7 86.3 27 42 89 83.4 81.3 84.3 25.5

4450 1084 3323 4300 3756 316 169 494 276 2348

72 96 78.8 0 32 53 95 66.3 98 3

86 92.6 88.8 0

a

Both standard sera series (for total and macro-PRL determination) as well as clinical sera were placed in the same plate. bDetermined using the PEG-precipitation technique.

conditioned by the fair amount of macro-PRL. The fact points to the high possibility of macroprolactinaemia in these samples. Thus, our approach allows detection of total PRL concentration and visual identification of hyperprolactinaemia and possible cases of macroprolactinaemia. In order to detect and confirm the presence of prolactin forms, four clinical sera with supraphysiological PRL concentrations were subjected to gel filtration chromatography on a column of BioGel P200. With the simultaneous bioluminescent immunoassay applied, the obtained fractions were analyzed for prolactin (Figure 4, left panel) as it has been described above. Total PRL was detected with a violet label and displayed in the elution profile. In samples 1−3, the prevailing macroprolactin (high-molecular fractions) and some monomeric prolactin are observed, whereas sample 4 contains only monomeric prolactin. Simultaneously, macroprolactin was measured with a green label and the signals were displayed in the same plot. One may see that the green label bioluminescence is strongly associated 3123



Article

(5) Cavaco, B.; Prazeres, S.; Santos, M. A.; Sobrinho, L. G.; Leite, V. J. Endocrinol. Invest. 1999, 22, 203−208. (6) Beltran, L.; Fahie-Wilson, M. N.; McKenna, T. J.; Kavanagh, L.; Smith, T. P. Clin. Chem. 2008, 54, 1673−1681. (7) Olukoga, A. O.; Kane, J. W. Clin. Endocrinol. 1999, 51, 119−126. (8) Frank, L. A.; Borisova, V. V.; Markova, S. V.; Malikova, N. P.; Stepanyuk, G. A.; Vysotski, E. S. Anal. Bioanal. Chem. 2008, 391, 2891−2896. (9) Liu, Z. J.; Vysotski, E. S.; Chen, C. J.; Rose, J. P.; Lee, J.; Wang, B. C. Protein Sci. 2000, 9, 2085−2093. (10) Frank, L. A.; Petunin, A. I.; Vysotski, E. S. Anal. Biochem. 2004, 305, 240−246.

results on determination of prolactin forms applying RIA, developed bioluminescent methods, and GFC. As one may see the amounts of total PRL and macro-PRL calculated by the three techniques are close. At that GFC takes over 6 h to provide information on the macroprolactin proportion only, whereas the bioluminescent assay we designed lasts just 6 s and detects both diagnostically important values, total PRL and macroprolactin simultaneously. Conventional method RIAPEG precipitation involves an additional stage of sample treatment and has some drawbacks, monomeric form coprecipitation, PEG interference to immunoassay results, etc.3,6

■

CONCLUSIONS This study demonstrates how two obelin mutants can be used for simultaneous detection of two analytes in a single well based on the bioluminescent signal spectral resolution in combination with time resolution. As an example, we tested the immunoassay developed for detection of two forms, total and IgGbound prolactins, in crude serum. It is performed in a highthroughput microplate format and allows detection of two prolactin forms in crude serum without additional manipulation (chromatography or PEG-precipitation). The content of prolactins was easily calculated using two corresponding calibration curves. Determination of macroprolactin with the help of our model calibration curve as a diagnostic tool requires further investigations (e.g., statistical verification, choice of immunoglobulins, etc.). Nevertheless, the advantages of the assay are obvious, the high sensitivity of total PRL detection is close to the RIA one and the capability to visualize both prolactin forms like in the case of gel-filtration chromatography but in essentially shorter times. The approach allows intensification of the assay procedure especially when proper diagnostics needs the detection of two analytes in one sample, e.g., LH and FSH, pepsinogen I, and pepsinogen II; free and autoantibody-bound forms of an antigen; or simultaneous detection of target and internal standard, etc. Simultaneous determination is useful for large-scale investigation and offers advantages in terms of cost and labor savings.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+7-391) 2494430. Fax: (+7-391) 2433400. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported in part by Grant No. 76 of the Russian Academy of Sciences, Siberian Branch and by the Program of the Government of Russian Federation “Measures to attract leading scientists to Russian educational institutions” (Grant No 11. G34.31.058).

■

REFERENCES

(1) Toldy, E.; Löcsei, Z.; Szabolcs, I.; Góth, M. I.; Kneffel, P.; Szöke, D.; Kovács, G. L. Endocrine 2003, 22, 267−273. (2) Sadideen, H.; Swaminathan, R. Int. J. Clin. Pract. 2006, 60, 457− 461. (3) Hattori, N.; Ishihara, T.; Saiki, Y. Clin. Endocrinol. 2009, 71, 702− 708. (4) Suliman, A. M.; Smith, T. P.; Gibney, J.; McKenna, T. J. Clin. Chem. 2003, 49, 1504−1509. 3124


Simultaneous Bioluminescent Immunoassay of Serum Total and IgG

Recommend Documents