Flow-ELISA: Binding assays for process control

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The editing and rearranging of graphics are carried out according to the habitual manner of data documentation. Headlines and additional text can be arranged in an arbitrary amount and at any position. Full documentation of every step in numerical data analysis is available on-line, both on screen as well as on printers and files. It is possible for all laboratory staff to perform routine work because every single step, from the capture of the data, its analysis

and graphical documentation, can be programmed in advance. References for every numerical algorithm are given in an extensive handbook. The illustrations (Figs. l-3) show examples of everyday laboratory work. Further information is obtainable from the author. Dr. Gerolf Kraus is at the Institute of Physical and Theoretical Chemistry, University of Tiibingen, Auf der Morgenstelle 8, W-7400 Tiibingen, Germany.

biotechnology focus

Flow-ELISA: Binding assays for process control Bo Mattiasson, Mats Nilsson, Per Berdbn and Hhkan HAkanson Lund, Sweden The combination of jlow injection analysis (FIA) and en.Zyme-linked immunosorbent assay (ELISA) gives a powerful immunoanalytical: technique that can be automated for bioprocess monitoring and control.

Introduction The desire to obtain high productivities in biotechnological processes has led to technical developments in at least three different directions: l process integration; l development of new reactor configurations to allow for operation at high catalyst densities; l process control through applications of specific biosensors as well as pattern recognition procedures. All these three approaches have demonstrated that much can be gained by proper design of the process. Furthermore, the different strategies can operate very well in synergy, e.g. process control may be applied with any of the other two approaches taken. A prerequisite for successful process control is to have access to suitable sensors. Biosensors have long been regarded as being potentially very useful; however they have only recently started to meet the expectations. This statement is valid for enzyme based sensors used for measuring low-molecular-weight 01659936/90/$03.00.

compounds; when it comes to determining macromolecules, fairly little has been achieved. A flow injection binding assay to quantify macromolecules in a continuous flow system was first introduced from our laboratorylP2. It was later modified to shorten the time for a cycle of analysis from 12 to 6 minutes3 and later to 70 seconds4. The present article deals with the development of a fully automated binding assay that is designed to meet the requirements for process control. When designing a binding assay to be used for process control a few questions arise: e.g. what is its analytical sensitivity? Does one have to sacrifice sensitivity for speed in analysis? If an intermittent assay is going to be used for monitoring a bioprocess, what is the frequency of analysis required? Is the stability of the immobilized affinity binding material sufficient? Is it sufficiently reproducible and reliable to be used as a signal for controlling a process? What are the molecules that can be measured with a sufficient accuracy? The present review is aimed at addressing some of these questions on the basis of what is known today about immunobased biosensors and other biosensors that exploit binding reactions. Many attempts have been presented in the last five years to design sensors based on biospecific recognition reactions. A broad spectrum of binding reactions have been tried in conjuction with construction of a biosensor. In this context, however, it should be mentioned that the dead end type of analysis, exemplified by traditional dip-stick technology, OElsevier

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is not classified among biosensors - which are designed for continuous or intermittent reading. What can be measured? Any molecule giving rise to an antibody can in principle be quantified in a binding assay. Even small molecules interacting specifically with receptors, enzymes etc. can also be quantified with the same technology. During the production phase of a biotechnological process it may be very interesting to monitor the target molecules directly in order to know production rate, degradation etc. During the first stages in downstream processing, when there are still many different molecules present in the preparation, it may be worthwhile to follow the concentrations of the target protein. However, as the purity of the product increases, it becomes more interesting to monitor the presence of impurities, such as DNA, endotoxins, proteases, etc.

1 1 0

1 2

1 4

1 6

Time (mini

Fig. 2. An example on an assay cycle in a j7ow injection enzyme linked immunosorbent assay procedure.

new assay cycle. (Fig. 2 shows the procedure in 2 schematic form.) To be fully useful for process con trol there is a need for automation and compute] control.

Measuring technologies Equipment Flow injection binding assay

The basis for this assay has been presented earlier1p3. In short, it is carried out as follows: a small column filled with immobilized antibodies is placed in a continuous flow of buffer. A sample containing the antigen to be quantified is injected via a sample injection loop into the flow system (Fig. 1). The sample is premixed with a fixed amount of enzyme labelled antigen prior to injection into the loop. Upon passage through the column, which takes from a few seconds up to a minute depending on the conditions, binding between the antigen and the antibody takes place. The amount of enzyme label that is bound in the antibody column is quantified by administering a pulse of substrate through the same flow system. The affinity-bound enzyme converts substrate to product that is transported with the flow system to a detector. The amount of product formed gives the amount of enzyme trapped in the column and from this value it is possible to deduce the amount of native antigen in the sample. After the assay cycle, a pulse of dissociating medium is passed through the system in order to rinse the affinity column and make it ready for a

Fig. 1. Schematic presentation of the FZA system

The flow system consists of a peristaltic pump precision valves, a sample loop of the type usually used in high-performance liquid chromatographic systems, a small column (C-250 pl), with thin tub. ing connecting the units and the detector. At this stage of development it is very difficult tc have a clear idea about the time constants needed foi future applications. A duration of an assay of l/10( to l/1000 of the time needed for the whole process can be used as an initial estimate. This would allow for 100 to 1000 measuring points during the process studied. The work so far has mainly been devoted tc finding out how well the assay works in the laboratory. As the system is now being implemented with real processes, information will soon be available concerning these issues. In an effort to speed up the assay, the sample mixed with labelled antigen was injected into a liquid stream that already contained substrate for the marker enzyme4. As the pulse passes the affinity column, binding takes place and the free antigen and enzyme-labelled antigen were washed out. The affinity bound enzyme label is still converting substrate and forming product. The graphs registered by a recorder are as shown in Fig. 3. The descending part of the curve is interesting since the behaviour here is a function of the amount of enzyme label trapped by the affinity sorbent. Readings are collected by a computer and evaluated with respect ta the behaviour of standard solutions. When the standard is set, a fairly short part of the descending curve is efficient for the determination of the amount of enzyme-labelled target molecule trapped. From this, the amount of native target molecule can be de-

trends in analyticalchernktry,vol. 9, no. lo,1990

319 3 Cont. (PM) 1

0

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40 Sample number

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TIME (s)

Fig. 3. A typicalpeak from an experiment with substrate included in the buffer flow. The enzymatic reaction normally produced peaks out of the range of the photometer which is represented by the dashed portion of the curve. For a manual evaluation of the curves, a measurement (Ah) was made at a fixed time (At) after the curve passed the 100% limit.

duced. As soon as this is achieved, dissociation is initiated and a new assay can be run. The time taken for ,one cycle is cu. 70 s. Another approach was to use the more conventional flow-ELISA procedure, but to automate it fully. Details of this process configuration will be published elsewhere’. The experimental set-up is schematically presented in Fig. 4. The equipment is the same as described above but supplemented with a computer and electrically sup-

r-l

F7 F8

F9

Computer

Fig. 4. Schematic presentation of the automated flow injection ELISA system. FI = excess flow of main buffer; IQ = main buffer flow; F3 = washing buffer; F4 = excess flow of washing buffer; F5 = substrate solution; F6 = sample solution; F7-9 = waste streams; PI and P2 = peristaltic pumps; VI and V2 = j-way valves; V3 and V4 = injector valves. The computer logs and evaluates the data as well as controls the flow through the whole system by valves and pumps.

Fig. 5. Recorded () and calculated (-) concentration of horse radish peroxidase in a reactor monitored by intermittent sampling and analysis of the enzyme activity bound to an affinity column with immobilized concanavalin A (for further details see ref. 5).

plied and monitored valves etc. (Epipactis AB, Lund, Sweden). Repeated runs were carried out for a 24-h period. However, it is possible to run the assays for a much longer period of time, a prerequisite being that microbial infection in the column is avoided. In the present assay, where one assay is run every 10 min and horse radish peroxidase serves as label, the column is treated with phenol and hydrogen peroxide 6 times per hour. Even if the concentrations used are insufficient for sterilization, the level of microbial growth will be kept down. The time constant is an important factor for a biosensor. So far the assays have been used only for monitoring, but we plan to use the signals for control. The monitoring reaction was performed by varying the concentration of the target molecules, e.g. by successive dilution with buffer or by drastically changing the concentrations step-wise. Yet another experiment that was carried out dealt with continuously increasing/decreasing the concentration in the solution of target molecules. This is a good way to study the response of the assay system to a continuously changing titer of the target molecule (Fig. 5). Flow injection ELISA or similar procedures have been used in a number of cases. Table I gives examples of systems studied and transducers used to read the signals. Sensitivity The flow-ELISA operates under conditions far from equilibrium and is therefore not as sensitive as an assay based on equilibria. Furthermore, since reversibility in binding is required, a lower binding strength can be applied than in a dip-stick test. These two factors, the non-equilibrium and the need for reversibility, contribute to lowering the sensitivity of

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TABLE I. Examples of analyses performed binding assays and the detectors used

in flow injection

Metabolite

Detector

Comment

Ref.

Humanserum albumin Gentamicin

Calorimeter

Macromolecular target Hapten

6

Insulin Proinsulin Methyl-a-mannopyranoside Transferrin Methyl-a-mannopyranoside Immunoglobulin G

Spectrophotometer Polarograhic oxygen electrode Calorimeter Polarographic oxygen electrode Spectrophotometer Spectrophotometer Electrochemical detector

The first immuno electrode Automated assay Lectin-carbohydrate interactions Flow-injection equipment used In-line substrate administration Sandwich assay

2 7 8 3 4 9

the assay. If higher sensitivities are needed, sandwich-modifications of the assay may be used’. However, this would lengthen the assay cycle. Stability Since the competitive binding assay is characterized by competition between two molecular species at a certain ratio for binding to an immobilized ligand, one can estimate the amount of each species bound by the ratio between the two. If the number of ligand molecules is reduced, e.g. by denaturation, the competitive binding will be directed towards the active ligand molecules. In other words, the ratio between the bound quantities of the two entities remains constant even if the ligand is denaturated. When operating with a fixed amount of enzyme-labelled antigen, it is useful to run one assay where no native antigen is present. The enzyme-labelled antigen will bind to the immobilized antibody and give a ‘loo-%’ value. A calibration curve is thus valid throughout the lifetime of an antibody column, provided the readings are adjusted for the capacity of the adsorbent, which has to be monitored intermittently.

TABLE II. Techniques studied for monitoring tion between antigen and antibody

direct interac-

Technique

Ref.

Surface plasmon Piezoelectric crystals Reflectometry Ellipsometry Immuno-field effect transistor Streaming potential

10,ll 12 13 14-16 17 18,19

Reproducibility The experimental error in traditional ELISAs is quite high, even if recent developments have reduced the chances of errors. Flow-ELISA contains very few manual steps and instead is performed under strict control. Therefore, variations between assays of l-1.5% are realistic. The discrete repeatable analyses have certain advantages over units based on continuous readings. The former can be based on established technology utilizing antibodies of medium binding strength and detectors, etc. that already are available on the market. Calibration is also fairly easy to perform. The continuous binding assays, on the other hand, are tapping the potential of new physicochemical methods to monitor immunological (and other) binding reactions directly. When a true continuous reading is expected one must secure both the on and the off reactions. Therefore, fairly low affinities must be used for the interacting entities, or a regeneration step must be employed outside the reading unit. Various techniques are presently being studied in this context. Some of these are listed in Table II. There is no doubt that immunobased biosensors hold great potential in bioprocesses. It will, however, take time both to develop measuring devices that are reliable under operational conditions and to find routines for their applications in process control. Most developmental work in biosensors has so far been focused on medical applications. The situation may be different for binding assays as process control exploits higher concentrations of the target molecule and thus puts less demands on sensitivity. The fact that clinical chemistry may not be the primary market may work against the commercialization of such biosensors. It should, however, be stressed that in the application of enzyme immunoassays for measuring different metabolites, one basic technology can be maintained for all. Thus, it should be possible to use a working immunosensor for a broad spectrum of analyses. In this regard, at its present level of development flow-ELISA offers a simple, operable concept based on an already well established technology. Acknowledgements This project was supported by The National Swedish Board for Technical Development and The Swedish Council for Forestry and Agricultural Research (SJFR). References 1 B. Mattiasson, C. Borrebaeck, B. Sanfridsson and K. Mosbath, Biochim. Biophys. Acta, 483 (1977) 221-227. 2 B. Mattiasson and H. Nilsson, FEBS Lett., 78 (1977) 251-254.

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3 B. Mattiasson and K. M. Larsson, in 0. M. Neijssel, R. R. van der Meer and K. Ch. A. M. Luyben (Editors), Proc. 4th Europ. Congr. Biotechnol. Amsterdam, The Netherlands, June 14-19, 1987, Vol. 4, Elsevier, Amsterdam, 1987, pp.

517-522. 4 B. Mattiasson, P. Berdtn and T. G. I. Ling, Anal. Biochem., 181(1989) 379-382. 5 M. Nilsson, H. Hakanson

and B. Mattiasson, in peparation. 6 B. Mattiasson, K. Svensson, C. Borrebaeck, S. Jonsson and G. Kronvall, Clin. Chem., 24 (1978) 1770-1773. 7 S. Bimbaum, L. Biilow, K. Hardy, B. Danielsson and K. Mosbach, Anal. Biochem., 158 (1986) 12-19. 8 C. Borrebaeck and B. Mattiasson, Anal. Biochem., 107 (1980) 446-450. 9 W. U. de Alwis and G. S. Wilson, Anal. Chem., 57 (1985) 2754-2756.

10 P. B. Daniels, J. K. Deacon, M. J. Eddowes and D. G. Pedley, Sens. Actuators, 15 (1988) 11-18. 11 M. T. Flanagan and R. H. Pantell, Eletric Lett., 20 (1984) 968-970.

12 J. E. Roederer and G. J. Bastiaans, Anal. Chem., 55 (1983) 2333-2336.

13 C. F. Mandenius,

K. Mosbach, S. Welin and I. Lundstrom,

Anal. Biochem., 157 (1986) 283-288. 14 C. F. Mandenius and K. Mosbach, Anal. Biochem., 70 (1988) 68-72. Biochim. Biopeys. Acta, 632 (1980) 15 M. Horisberger, 298-309. 16 P. A. Cuypers, W. T. Hermens and H. C. Hemker, Anal. Biochem., 84 (1978) 56-57. 17 S. Collins and J. Janata, Anal. Chim. Acta, 136 (1982) 93-99. 18 C. Glad, K. Sjodin and B. Mattiasson, Biosensors, 2 (1986) 89-100. 19 B. Mattiasson and A. Miyabayashi, Anal. Chim. Acta, 213 (1988) 79-89. Professor B. Mattiasson, Drs. M. Nilsson, P. Berdkn and H. H& kanson are at the Department of Biotechnology, Chemical Center, Lund University, P. 0. Box 124, S-221 00 Lund, Sweden.

trends

Laser desorption ionization mass spectrometty of large biomolecules M. Karas and U. Bahr Miinster, Germany The development of new ionization techniques bus revolutionized the field of mass spectrometry of large biomolecules within the lust two years. Matrix-assisted W-laser desorption ionization is a new technique which today enables molecular ion generation and molecular weight determination of proteins with high sensitivity up to several hundred thousand daltons.

Introduction The development of new mass spectrometry (MS) techniques capable of studying high-molecularweight biopolymers represents one of the most exciting achievements of research in structural biology in the last two years. As is typical for MS, this progress is due to the discovery of new ionization techniques, namely matrix-assisted UV-laser desorption ionizaUV-LDI)‘-5 tion and electrospray-ionization (ESI)634. The ‘established’ techniques, fast atom bombardment and plasma desorption MS, find their practical upper mass limit in the 10 or 20 kDa range respectively and require increasing quantities of 01659936/90/$03.00.

sample with increasing mass. However, in dramatic contrast to this situation matrix UV-LDI yields high sensitivity molecular ion signals up to at least 300 000 Da and ES1 up to cu. 100 000 Da, respectively. One of the first matrix LDI mass spectra was reported four years ago in this journa18. It was a spectrum of an oligopeptide, mellitin, with a chemical molecular weight of 2846.5 Da. The concept of matrix-assisted laser desorption was developed as early as 19859 and summarized by the authors in 1987l. A different type of matrix desorption was reported by Tanaka et uZ.~, who used protein analytes dissolved in a liquid glycerol matrix with the admixture of small metal particles enabling energy transfer of a nitrogen laser pulse into the non-UV-absorbing matrix-analyte liquid phase. In the following discussion the principle features and experimental conditions are described for the method and illustrated with some examples. Principles of matrix UV-laser desorption ionization The main previous limitations of UV-LDI of neat 0

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