J BlOLUMlN CHEMILUMIN 1995; 10: 219-27

TM The Development of Luminomaster a Fully Automated Chemiluminescent Enzyme lmmunoassay System

Takashi Ikegami, Masatoshi Yamamoto, Koichi Sekiya, Yoshihiro Sat0 and Yukio Saito Sankyo Co., Ltd, 2-7-1 2 Ginza, Chuo Tokyo 104, Japan

Masako Maeda and Akio Tsuji" School of Pharmaceutical Sciences, Showa University, 1 -5-8 Hatanodai, Shinagawa, Tokyo 148, Japan

We have developed a fully automated discrete chemiluminescent heterogeneous enzyme immunoassay system called LuminomasterTM . The characteristics of this analy- ser are: 120 test/h throughput, 14 test menu, wide dynamic range, automated sample dilution, automatic retest, communication with a host central processing unit (CPU) and connection with sample transfer system.

Keywords: LuminomasterTM; chemiluminescent enzyme immunoassay; automated analyser

I NTRO D U CTl ON

We have developed a fully automated chemilumi- nescent enzyme immunoassay (CLEIA) system called LuminomasterTM. The analyser automates an EIA employing GOD as the enzyme label and a highly sensitive chemiluminescence detection of the label. Furthermore, following the trend towards full automation of clinical test systems in laboratories and hospitals, we have developed the analyser to be compatible with an automatic sam- ple transfer system.

MATERIALS AND METHODS

GOD was purchased from Toyobo Co. (Tokyo, Japan). Luminol was purchased from Tokyo Che- mical Industry Co. (Tokyo, Japan). Microperoxi- dase (m-POD) was obtained from Sigma Chemical Co. (St Louis, MO). Other chemicals were of analytical grade. The substrate comprised

* Author for correspondence.

0.1 mol/L glucose in 0.05 mol/L acetate buffer (pH 5.8). The chemiluminescent reagent solution contained 20 mmol/L luminol and 60 pmol/L m- POD in 0.1 mol/L 2-(cyclohexylamino) ethane sul- phonic acid (pH 9.5). The assay buffer is 0.01 mol/L phosphate buffer saline in 0.1 YO BSA (pH 7.0). All reagents are stable for one year when stored at a temperature below 10°C.

Flow chart of LuminomasterTM

The analyser automates a heterogeneous chemilu- minescent EIA based on discrete solid phase anti- body-coated tubes. The block diagram of the analytical steps (dispensing, immune reaction (antigen-antibody reactions), enzymatic reaction, chemiluminescent reaction and data processing) in the analyser is shown in Fig. 1.

Principle of CLEIA

Fig. 2 shows the principle of the chemiluminescent

CCC 0884-3996/95/0402 19-09 0 1995 by John Wiley & Sons, Ltd.

Received 23 November 1994 Revised I 1 January 1995

220 T. IKEGAMI E T A .

4 1 Data processing I

Figure 1. Block diagram of analytical steps in a Lumino- masterTM

Enzymatic Reaction Labeled Enzyme : GOD Substrate : Glucose

CHzOH

OH OH

Luminescent Reaction Luminescent Reagent : Lurninol+rnicro-Peroxidase

g:: +Light

NH2

Figure 2. Principle of chemiluminescent EIA

[Integrating Sphere

PMT : Photomultiplier tube AMP: Amplifier AID : AnaloglDigital Converter CPU : Central processing unit

Figure 3. Structure of the chemiluminescence detection unit

EIA. GOD is used as the marker enzyme, and H 2 0 2 produced by enzymatic reaction with a glu- cose substrate is assayed according to the chemilu- minescence method using luminol and m-POD (1,2). The structure of the chemiluminescence detection unit is shown in Fig. 3.

Improvement in the reproducibility of the CL detection was realized by the combination of a spa- tial integration method, that measures chemilu- minescence in an integrating sphere, and a time integration method, which uses a chemilu- minescence integration value of 10 seconds. Using spatial integration, the amount of chemilu- minescence is independent of both the position of the cell and the direction of emission caused by the variation of the glass thickness. Detection is made at a rate of 20 times per second. Before mix- ing the sample solution with the chemilu- minescence reagent, a blank test is performed for 3 seconds. After mixing the chemiluminescence is measured for 10 seconds, from which 260 pieces of data per assay are obtained. The peak-height, the half-width of chemiluminescence and time-inte- gration were compared as methods for converting such measurements to analytical data. The time integration method was chosen because of its high reproducibility. The sum of 200 pieces of data (time-integration value) after subtraction of the blank, is considered to be intensity of the lumi- nescence. By using the combination of time integra- tion and spatial integration, chemiluminescence measurements can be used for the detection of enzyme activity for immunoassay. A CLEIA has a wider dynamic range than EIAs with colori- metric or fluorescent end-points. As shown in the Fig. 3, the combined use of two detectors (photo- multipliers) for both high and low sensitivity allows for a wide dynamic range.

Procedure

This analyser allows for a number of analytical methods: (1) one-step sandwich (ISS), (2) two- step sandwich (2SS), (3) one-step competitive (ISC) and (4) a two-step competitive (2SC) or any combination of them simultaneously. Incuba- tion times are 15 minutes; for example, in the 2SS method, incubation times for the first immune reac- tion, the second immune reaction, and the detec- tion reaction are each 15 minutes, resulting in a total assay time of 45 minutes. In the sandwich assay, the reagent is designed to be used according

AUTOMATED CLEIA SYSTEM 221

to the 2SS method in order to prevent the prozone phenomenon. The analyser has been designed for a competitive method so that low molecular antigens can be analysed by 1SC. The e and c antibodies of hepatitis B normally determined by the inhibition method can be analysed by the 2SC method. In mixed analyses with different reaction times, reac- tions with similar reaction times are grouped together and are monitored in ascending order of reaction times.

Fig. 4 shows an exterior view of the analyser. It consists of the main unit on the left (1180mm (width) x 770mm (depth) x 1250mm (height), 320kg), and the operation unit on the right (415mm (width) x 770mm (depth) x 1250mm (height), 100 kg). Analysis of the dispensed sample is performed in the main unit. The apparatus must be maintained at an ambient temperature of 20- 25°C and at 40-80% relative humidity. A total of 110 serum samples, including 100 for routine tests and 10 for urgent tests, can be loaded onto the ana- lyser. Disposable tips (total of 384 tips; 4 trays of 96 tips) are used for sampling to prevent carry-over of samples. The volume of the test samples can be either 30,50 or 100, depending on the analyte. Dur- ing automatic dilutions, the volume is either 10,20 or 30pL, and a pressure indicating system is employed to detect the liquid surface level. Under this system, compressed air is emitted from the end of a tip, and the pressure increases whenever the tip touches the liquid surface. Immune and enzymatic reactions are performed in a dry-type 90-well incubator. The incubator maintains reac- tion tubes at 37 f 0.5"C with three thermistors and three heaters. A total of 500 tubes coated with immobilized antibody (20 trays of 25 tubes each) can be loaded onto the analyser, together with a further 256 tubes for dilution of the sam- ples. Enzyme-labelled antibody reagent sufficient for 2000 tests (20 bottles for 100 tests each), the substrate solution and chemiluminescent reagent for 2000 tests (two bottles for 1000 tests each) can be housed in the reagent cooler. All reagents are controlled by means of bar codes that record each analytical item, lot number and stock bal- ance. This information is also displayed on the sys- tem's monitor.

CLEIA system

The chemiluminescent peak emission wavelength of luminol is about 425nm. As shown in Fig. 3,

Figure 4. Exterior view of LuminomasterTM

+ a a a 0 a, 5

m a, n:

+

.- + -

8- 7- 6 - 5- 4 - 3 - 2- 1 - 0- -

Log CLI)

Figure 5. Dynamic range of the two detection systems com- bined in the analyser

luminescence is detected after screening through a bandpass filter to remove extraneous luminescence at other wavelengths. The photomultiplier for high-sensitivity measurements is cooled to reduce dark current which improves the S/N ratio enabling detection of low intensity luminescence. Fig. 5 indicates the method for combining two detection systems with the values for current out- put in relation to chemiluminescence. Both photo-

222 T. IKEGAMI ET AL.

multipliers with high and low sensitivity can measure chemiluminescence over six orders of magnitude. The sensitivity of the two photomultipliers differ by a factor of 1000. Chemiluminescence over nine orders of magnitude can be reliably measured by combining the two photomultipliers.

Automatic dilution and retesting

Serum samples (10 to 30pL) are automatically diluted in a one-step (10-10,000~) or a two-step (200- 10,000 x) procedure by adding an appropri- ate amount of assay buffer.

The analyser’s wide dynamic range reduces retesting. For samples with results outside the mea- surement range, a dilution ratio is determined and the solutions are rediluted and retested.

Calibration curves and throughput

Multiple point regression curves or two-point cali- brations are used depending on the analyte to be measured. The calibration curve corresponding to the lot of reagent being used is automatically selected and concentrations calculated.

This analyser samples every 30 seconds, enabling 120 tests to be conducted per hour. However, ana- lytes and samples diluted by 200 times or more take one minute each resulting in only 60 tests per hour to be performed.

Interface

This analyser is able to communicate with a host CPU. Output to the host CPU is made by reading the sample ID number on the bar code, and trans- ferring the information in real-time. The host CPU then transfers the test requests corresponding to the sample ID number and any previous values, to the analyser in real-time. Thus, the sample and dilution ratio are recorded automatically. The results of assays can be output to the host CPU in real-time, for further processing.

Connection with an automatic sample transfer system

Automatic sample transfer systems assist in the auto- mation of laboratory work. Such systems classify samples after centrifugation and transfer them to

their respective units automatically. Testing data is also sent to the analyser from the host compu- ter. In this study the automatic sample transfer sys- tem for biochemical testing (Hitachi type) used at Akita University, converted for immune reactions (Hitachi type) (Fig. 6 ) was linked to the analyser (3). The automatic sample transfer system consists of four parts, a main unit, an operation unit, an ele- vator unit, and the sample transfer unit. In order to connect this system to the analyser, the following improvements were made.

Height adjustment. An elevator unit was installed to adjust the height of the automatic sample transfer line. During transfer the elevator adjusts the height of every rack entering and leaving the main unit.

Throughput adjustment. The speeds of the sample transfer unit and other measurement units upstream from the analyser were adjusted by the analyser sample transfer unit (buffer action). The analyser sample transfer unit is able to store up to 20 racks entering or exiting the main unit, allowing for speed to be adjusted to fit this analyser throughput. A bar code reader reads the rack number and other information on racks coming into the analyser sample transfer unit. Urgent samples may be added to the sample transfer unit which are then transferred to the main unit.

Installation of sample transfer line in LuminomasterTM. Since this analyser did not have sample transfer lines installed, we had to adjust the locations of the sample and disposable tips to enable the racks to pass through the analyser. We intend to install similar sample transfer systems, including the Toshiba (IDS) and A&T’s version in other analysers in the future.

Constitution of reagents

The reagents used in this analyser consist of the tubes coated with immobilized antibody (one for each analyte to be measured) and enzyme labelling antibodies (enzyme labelling antigen and primary antibody in case of the competitive method), as well as the buffer solutions, substrate solutions, chemiluminescence reagent and washing fluids (used for every analyte). These reagents (14 different analytes) are ready to use and can be put straight

AUTOMATED CLEIA SYSTEM 223

1 . Sample Inlet 2 . Cap Opener 3 . Centrifuge Station 4 . On-line Dispenser Station 5 . Off-line Dispenser Station 6. Bar code Labeler 7 . Sample Inlet

8 . 2nd Sample Store 9 . Sample Store

10. HlTACHl Model 7070 11. KYOWA MEDEX Model EL-1200 12. SANKYO LUMINOMASTERTM 13. System Control Unit 14. Sample Store

Figure 6. The automatic sample transfer system at Akita University

~~

Table 1. Analytical performance of the analyser Analyte Sample Measurement range Detection Reference standard Other

volume (pL) limit

AFP 30 0.5-1 000 ng/mL 0.048 National Institute of

CEA 30 FER 30 0.5-1 000 ng/mL 0.089 WHO 1st IS 80/602

TBG 30 T4 30 2.0-400 ng/mL 0.820 TS H 100 0.05-1 00 pIU/mL 0.01 6 WHO 2st IRP 801558 LH 30 0.5-1000mlU/mL 0.299 WHO 2st IS 80/552 FS H 30 0.5-1 000 mlU/mL 0.308 WHO 2st IRP 781549 hCG 100 0.5-1 000 mlU/mL 0.079 WHO 3rd IS 751537 PRL 30 0.5-1 000 ng/mL 0.085 WHO 3rd IS 841500 M LCI 100 0.5-1 000ng/mL 0.1 90 In house IR I 100 0.63-320 pIU/mL 0.1 70 WHO 1st IRP 66/304

100 0.1 -10001U/mL 0.01 7 WHO 2nd IRP 75/502 I9E

MLCI: myosin light chain-I. IRI : immunoreactive insulin.

Health, Japan (Lot 3) 0.5-1 000 ng/mL 0.050 WHO 1st IRP 73/601

30 0.5-1 000 ng/mL 0.01 6 In house Sample x 1000 0.5-1 00 ng/mL 0.1 83 In house Sample x 1000

PMG

In house Sample x10

224 T. IKEGAMI H A L .

Table 2. Reproducibility of the 14 assays currently configured for the analyser

Analyte Intra-assay ( n = 10) Inter-assay ( n = 10)

Average SD CV% Average SD CV%

AFP (ng/mL) 30.3 1.3 4.3 31.2 1.3 3.8 153.6 6.5 4.2 150.3 6.8 4.5

68.7 1.8 2.7 65.2 2.3 3.6 FER (ng/rnL) 135.8 6.1 3.2 133.2 6.4 4.8

330.2 12.9 2.7 330.6 14.4 4.4 PZMG (vg/mL) 3.6 0.2 5.5 3.2 0.2 6.3

20.2 0.8 4.0 19.5 1 .o 5.1 TBG (pg/mL) 14.8 0.4 2.7 14.9 0.9 6.0

21.5 0.5 2.3 21.1 1.2 5.7 T4 (Pg/mL) 3.6 0.1 2.8 3.8 0.2 5.3

16.4 0.4 2.4 17.2 0.8 4.7 TSH (mlU/mL) 8.5 0.2 2.4 8.9 0.5 5.6

21.7 0.7 3.2 22.1 0.6 2.7 LH (rnlU/mL) 6.8 0.3 4.4 7.4 0.4 5.4

31.6 1.2 3.8 33.1 1.6 4.8 FSH (mlU/mL) 20.4 1 .o 4.9 21.5 1.2 5.6

98.3 4.3 4.4 104.1 4.4 4.2 hCG (mlU/mL) 25.9 1.1 4.2 27.3 1.2 4.4

393.8 7.1 1.8 409.7 12.3 3.0 PRL (ng/mL) 8.8 0.3 3.4 9.1 0.2 2.2

56.3 1.2 2.1 59.5 1.9 3.2 MLCl (ng/mL) 5.4 0.3 5.6 5.1 0.3 5.9

81.6 2.2 2.7 79.8 2.3 2.9 I R I (pIU/mL) 9.9 0.3 3.0 105.7 4.8 4.5

10.2 0.5 4.9 101.5 4.4 4.3 IgE (IU/mL) 323.1 10.6 3.3 82.5 2.5 3.0

79.7 2.3 2.9 31 6.4 11.0 3.5

CEA (ng/rnL) 24.3 0.8 3.2 24.4 1.2 4.9

into the unit after unpacking, allowing for fast and efficient analysis.

Performance o f the system

Sensitivity and measurement range. Table 1 shows the measurement range and limit of detection for the current menu of 14 analytes. Statistical detec- tion limit was defined as the concentration that produced chemiluminescence equal to that of ana- lyte free serum plus 2SD. The measurement covers a range of 3.5-4orders ofmagnitude foreachanalyte.

Reproducibility. Table 2 shows the intra-assay reproducibility (n = 10) for each analyte and the inter-assay reproducibility (10 days).

Correlation. Table 3 shows correlation data for

each analyte with reference assays (EIA, FEIA). The correlation coefficients were all > 0.95.

Frequency of retesting. Table 4 compares the frequencies of retesting on this analyser and an ES-600 (Boehringer Mannheim) for AFP, CEA and ferritin. The broad dynamic range of this system allows the frequency of retesting to be markedly reduced.

Effects of interferents. We used ‘Interference Check A Plus’ (Kokusai Shiyaku Co.) to check the effects of haemoglobin, bound bilirubin, free bilirubin and chylomicrons. No effect was detected for each analyte up to 450mg/dL of haemoglobin, 2450 degrees of chylomicron (formazin turbidity), 17.7mg/dL of free bilirubin, and 18.7mg/dL of bound bilirubin. The insulin assay was affected by haemoglobin and is thought to be due to the

AUTOMATED CLEIA SYSTEM 225

Table 3. Correlation data for the assays currently configured for the analyser

Analyte n Regression equations Correlation coefficient Reference method

AFP C EA FER

P2MG urine TBG T4 TS H LH FS H hCG PRL M LCI IR I IgE

serum

FEIA: Abott IMx EIA: Yamasa. RIA: Hoechst.

75 150 75 66 55 75 75 82 75 82 69 65 40 80 95

y = 1.006~ + 6.1 01 y = 0.988~ - 1.1 46 y = 0.985~ + 0.080 y = 1 .I 4 0 ~ + 0.423 y = 1.108~ - 10.28 y = 1.01 4~ - 2.495 y = 0.962~ - 0.955 y = 0.828~ + 0.820 y = 0.81 6x + 1.029 y = 0.844~ + 0.721 y = 1.027~ + 2.205 y = 1 .I 51 x + 0.785 y = 0.954~ + 0.976 y = 1.076~ + 3.267 y = 0.927~ - 11.89

0.995 0.979 0.999 0.987 0.994 0.958 0.973 0.995 0.989 0.991 0.983 0.972 0.987 0.996 0.987

FElA FElA FElA

FElA

RIA FElA FElA FElA FElA FElA FElA EIA FElA FElA

~~ ~~~ ~ ~~

Table 4. Frequency of retesting comparisons

1 Analyte

Measurement range (ng/mL) i

~~ I Rate (%)

degradative effect of insulinolytic enzymes in red blood cells.

Sampling normal individuals. Table 5 shows the levels of analytes (except LH, FSH, hCG and PRL) detected in normal individuals (normal ranges). The levels obtained using a kit supplied by a different manufacturer are also shown for purposes of comparison.

Carry-over. No contamination was anticipated

during dispensing of samples, as disposable tips are used. No contamination was detected in the chemiluminescence cell despite this one cell being used for every measurement. Using AFP as a test assay, samples from a patient (AFP = 1,300,000 ng/mL) and a normal individual (AFP = 1.5 ng/mL) were measured 10 times alternately, followed by 15 different samplings of the normal individual. The mean value of the sample analysed alternately from the normal individual was 1.475 ng/mL and 1.466 ng/

226 T. IKEGAMI ETAL.

Table 5. Normal ranges

Analyte n Average SD Comparison range

AFP (ng/mL) CEA (ng/mL)

female 40s female 50s

TBG (pg/mL) T4 (PLg/dL) TSH (pIU/mL) MLCl (ng/mL) IRI (pIU/mL) IgE (IU/mL)

284 290 199 76 38 55 20

294 309 309 103 113 31 4 301

3.20 2.90

147.8 37.3 38.9 38.3 60.4 1.68

24.2 7.8 3.2 0.76 7.01

11 4.81

1.18 1.46

93.28 27.66 28.60 34.1 8 42.58

0.48 9.30 1.70 1.60 0.62 6.73

154.28

1 .O-6.8 0.9-9.1 5.2-448.7 3.2-1 26.9 4.2-1 03.5 4.4-1 64.7 6.2-1 82.4 0.4-3.0 0.8-48.2 2.2-1 4.6

0.34-4.7 0.4-3.4

3.25-41.4 0.23-836.3

mL for the continually sampled normal individual, which resulted in a carry-over of 6 . 9 2 ~ lop7%. A difference of 0.1% was noted as the level of significance, but the carry-over detected was not enough to affect the normal value.

Labour-saving intercommunication system. Fig. 7 shows the flow of samples and information employed by Hamamatsu University School of Medicine which uses an interfacing system described previously (4). The hospital host CPU records the analytes to be sampled and issues a bar code for each analyte. The samples are then sorted by the sample dispenser and transferred manually to each sampling unit together with the bar-coded information on the sample. When linked to this analyser, the system enhances the labour-saving advantages of the CLEIA analyser. The new system is very fast, requiring only 90 minutes from the collection of blood samples to the production of the final read-out. The computer linkage function also helps to reduce labour cost sand the timeneeded to perform the tests.

Labour-saving transfer function. It is a great advantage if after collection of the sample, tests can be performed without further intervention by the operator. The sample transfer system described previously (Fig. 6) allows sampling to take place without human assistance. The development of such a system paves the way for completely automated systems which will allow

tests to be run with only the simple confirmation of reagent stocks before starting the test. The sample transfer line at Akita University is equipped with a shunting loop, allowing the automatic resampling function of this analyser to be used with the automatic transfer system, eliminating the need for human assistance in the resampling process. Accordingly, in addition to the reduction of labour costs and the increase in processing speed, full automation of the processing has been made possible.

Conclusion

This analyser is a highly sensitive and fully auto- mated enzyme immunoassay system. The combina- tion of two detection ranges permits detection over a wider dynamic range which enables a reduction in the frequency of retesting, and leads to shorter test time, and decreased labour and reagent costs. In addition, by connecting to a host computer the analyser can judge whether the expected value of samples would be within the assay range or not from the previous data and automatically dilute the samples before proceeding with the assay.

There is a general trend in laboratories towards full automation. We have modified the analyser so that it can be connected with automatic sample transfer lines in laboratories without compromis- ing the advantageous characteristics of this analy- ser. We believe that this development is a

AUTOMATED CLElA SYSTEM 227

PID T. R Hospital

+ Test Request

Information

Results

T. R

PID Patient Identification

RNP : Rack Number and Position IPD : Immediate Past Data

RNP T.R : Tests Requested

Figure 7. Flow of test information and test samples at Hamamatsu University School of Medicine

significant step towards the full automation of clin- ical immunoassay testing in laboratories in the future.

Acknowledgements

The authors express their appreciation to Professor Takashi Kanno, Hamamatsu University School of Med- icine, Professor Kunihide Gomi, Showa University, Pro- fessor Hiroaki Okabe, Kumamoto University, Dr Hazime Naka, Director, Mitsui Memorial Hospital, Dr Masayuki Totani, Director, National Research Institute of Health and Nutrition for Mothers and Children, Assistant Professor Katsuyoshi Tabata, Division of Clin- ical Chemistry of Medical Technology Kyoto University, and Professor Shiro Uesugi of Akita University, for their assistance in the development of this analyser.

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