The Lateral Flow Assay – An almost perfect POCT
The need for cost-effective, highly sensitive and reliable analysis options that can be used close to the patient (point-of-care, POC) is increasing both in the western world and in developing and emerging countries. In the case of diseases with high mortality rates in particular, a quick and quantitative determination of the sample material is often required to make a therapy decision. But the need for high-quality POCT is also very high in other areas of application such as home tests for self-use, the examination of food or environmental samples or pharmaceutical production.
Lateral flow tests (LFA) offer an ideal platform for quick and easy application in the point-of-care area. Above all, the very short analysis times, simple handling, scalable production with very low manufacturing costs and the possibility of storing the tests without a cold chain for long periods are advantageous. The combination of manufacturing costs and the ability to easily carry them out anywhere make them the best-selling POCT in the world.
ELISA as a pioneer for the LFA
The success of the LFA is based on the success of the immunoassays. These measurement methods, which use the antibody-antigen interaction to detect an analyte from a complex sample, began their triumphal march in the 1970s. The special feature of immunoassays is the very good specificity in combination with the high sensitivity in extremely complex sample matrices. To date, there are no other analytical methods that can even begin to compete with the immunoassay in this regard.
Two independent research groups developed the idea of the ELISA for the first time in 1971 and showed the results of the feasibility of this method. This discovery led to a whole series of test and device developments, as we still know them today in the laboratories. The classic 96-well format was already established for the first commercial ELISA, which Organon Teknika launched in 1976. Among other things, this resulted in the development of the 8-channel pipette, ELISA washers and readers. The first fully automated device for performing ELISA by Boehringer Mannheim appeared on the market as early as 1980. Today, the ELISA is an integral part of everyday clinical and laboratory work. The positive effects of the ELISA on patient well-being and the health system are practically unsurpassed in the area of diagnosis.
First therapeutic use of antibodies
The antibodies with the ability to specifically bind an antigen form the basis of all immunoassays. For immune defense against pathogens or pathogens, they are formed by the immune system (vertebrates) and released into the blood. Nowadays, these properties are used, for example, when vaccinating or for treating various diseases. Antibodies were first used therapeutically in 1890 by Emil Adolf von Behring. He used so-called antisera to treat the diphtheria discovered in 1988. He obtained the antisera from the blood of patients with diphtheria and recovered patients who, as we know today, have specific antibodies against the diphtheria pathogen. The immunologist received the Nobel Prize in Medicine for the first time in 1901.
Structure and mode of operation
In the period before the Second World War, knowledge about antiserum was steadily expanded. The antibodies were first isolated from sera in 1926 and identified as protein in 1930. In the same year, John Marrak first described the theory of antigen-antibody interaction. The correct structure of the antibody, however, was only correctly postulated by Rodney Porter in 1961. Until his research results, it was assumed that the antibodies consist of a long amino acid chain. Porter found that the enzyme papain breaks down the antibody into three fragments. With the knowledge of researcher Gerald M. Edelmann, who found that four different antibody fragments are obtained when the disulfide bridges are cleaved by reduction, the antibody structure was correctly described for the first time.
Nowadays, the antibodies that are used for diagnostic purposes are mostly produced recombinantly. We owe this achievement to César Milstein and Georges Köhler who in 1975 were able to produce monoclonal antibodies for the first time using hybridoma technology. The three researchers were awarded the Nobel Prize in Medicine in 1984 for this milestone in human history (perhaps better “medicine”).
Foundation stones for the establishment of ELISAs in today’s laboratory
Jacques M. Singer and Charles M. Plotz first used specific antibodies to establish reliable diagnostic methods in 1960. These described the latex agglutination test known today, which alongside the ELISA is one of the widespread diagnostic tests. To do this, they synthesized latex nanoparticles and mixed them with γ-globulins. Rheumatism could be diagnosed by adding patient sera to the latex particle mixture. In rheumatism patients, the latex particles aggregated and sedimented, which cleared up the otherwise cloudy solution. When sera from healthy patients were added, the solution remained milky cloudy. The cause is specific antibodies, which occur in the serum of rheumatism patients and interact with the γ-globulins, which resulted in the cross-linking between antibodies, γ-globulins and latex particles and this mixture failed.
The scientific milestone and the first description of the immunoassays known today was achieved in 1961 by Rosalyn Yalow. For the first time, it managed to make the antibody-antigen interaction directly quantifiable by means of a label. By labeling insulin with the radioactive iodine isotope I 125 and using antisera (from pigs), she succeeded in quantitatively detecting insulin. For this purpose, the sample, labeled insulin and the antiserum were incubated in a homogeneous reaction and then separated by size by chromatography. The radioactivity of the fractions, on the one hand free insulin and on the other hand antibody-bound insulin, was measured, from which the insulin concentration in the sample could then be calculated. In 1977 she received the Nobel Prize in Medicine for the discovery of the radioimmunoassay.
This was carried out in clinical laboratories for a long time and revolutionized diagnostics. However, the radioimmunoassay was gradually replaced by the ELISA and has been almost non-existent in everyday clinical practice since the 2000s. The reasons are, on the one hand, the health-endangering properties of the radioactive labels and the fact that radioactive labels sometimes disintegrate quickly (half-lives of approx. 30 days) and are therefore not suitable for commercial use, where storage stability of several months is expected.
From homogeneous to heterogeneous immunoassay – the ELISA
In order to get from the first described immunoassays to the ELISA, further outstanding researchers and scientifically valuable discoveries were required. For example, Leif Wide and Jerker Porath succeeded in specifically and consciously binding antibodies to the surface of Sephadex particles for the first time. The first heterogeneous immunoassay for the detection of an antigen was born. The possibility of removing the homogeneous sample mixture of antiserum, sample and labeled antigen by simply separating the solid phase made the implementation of the immunoassays drastically easier. A short time later (1967) it was described that antibodies also bind to the materials used today such as polystyrene or polymethyl methacrylate. The foundation stone for the well-known 96 well format was laid.
What was missing was a simpler and, above all, easier to detect label on the antibody. Many researchers tried to bind enzymes as a label to antibodies, whereby the problem of cross-linking and inactivation of the enzymes was first solved by this cross-linking reaction in 1969 by Stratis Avrameas. He used the reagent glutardialdehyde to cross-link the two components, which is still used today for the conjugation of various proteins. With these antibody-enzyme conjugates, he detected various antigens intracellularly in cell suspensions. All components were now available for the development of the ELISA.
To carry out the ELISA, a wide variety of reagents and devices as well as professionally trained personnel were still required today. A large number of companies and developers have therefore established countless automated assay formats on the market that make the diagnostic process possible almost everywhere with ever smaller devices and shorter analysis times. What remains is a large number of devices with different handling, corresponding purchase costs of the devices, the dependence on a reliable power supply and mostly a need to store the individual test in a cool place. The ELISA does not meet the requirement to provide a diagnostic test for everyone or for use at the point of care.
The main application that drove the early development of rapid test technology was clearly the human pregnancy test, which was a continuing historical interest in urine tests for medical diagnostic purposes.
One of the earliest records of an urine diagnostic pregnancy test can be found in ancient Egypt. In this test – mentioned in the “Berliner Papyrus” – a potentially pregnant woman was able to urinate on wheat and barley seeds for several days. The result: if neither grows, she is not pregnant. If barley grows it is a boy, if wheat grows it means a girl. Further rather dubious tests were also described in the Middle Ages – here, for example, the color of the urine should diagnose pregnancy.
In 1927, the pregnancy hormone hCG in urine was used for the first time for the diagnosis of pregnancy. The urine of potentially pregnant women was spiked in female mice, the existing hCG causing ovulation in the mouse. This could be demonstrated by an autopsy 48 hours later. A little later the mouse was replaced by a frog. The hCG triggered the spawning of the frog, which could be found even without the animal’s autopsy. It is surprising that this frog test was carried out regularly until the 1960s, when the immunoassay was invented.
All these records show that the interest in simple and quick diagnostic tests (POCT), especially for the diagnosis of pregnancy, has existed for a long time. The first lateral flow test, which was launched in 1988 by the Unilever company under the Clearblue brand, was a pregnancy test for every woman.
The nitrocellulose membrane as the core of LFA technology
Nitrocellulose is a universal polymer that has been widely used since the 19th century. Also known as cellulose nitrate (or gun cotton), it gained importance during this period. Porous membranes were first described in 1916 by Richard Zsigmondy, who used them for filtration.
Heute werden Nitrocellulosemembranen durch Phasenumkehr hergestellt, bei der Nitrocellulose in einem organischen Lösungsmittel gelöst wird, das in Gegenwart eines Nichtlösungsmittels verdampft, wodurch die Nitrocellulose als Membran mit hoher Porosität zurückbleibt. Die Porosität und Porengröße der Membran kann durch die verwendeten Lösungsmittel, die Verdampfungsgeschwindigkeit, die Temperatur und die Feuchtigkeit gesteuert werden. Das Ergebnis ist ein Material mit der einzigartigen Kombination von einstellbarer Porengröße, hohen Verhältnissen von Oberfläche zu Volumen und sehr niedrigen Herstellungskosten.
The knowledge that nitrocellulose membranes have other outstanding properties in addition to filter technology was then gained in the 1960s. Agnar P. Nygaard and Ben Hall showed in 1963 that RNA-DNA complexes adsorb on the nitrocellulose, whereas free nucleic acids passed through it. This property was then used by other researchers to investigate the interaction of immobilized DNA with target molecules.
In 1975 Edwin M. Southern demonstrated the ability to transfer DNA from polyacrylamide gels to nitrocellulose membranes and then stain them on the membrane. This groundbreaking technique, known as the “Southern Blot”, made it possible to detect certain nucleic acid fragments. The Southern blot inspired the “Northern blot” – for RNA transfer as well as the “Western blot” – for protein transfer – on nitrocellulose membranes. These blotting techniques are widely used in biological research and take advantage of nitrocellulose’s unique ability to interact with the three main classes of biomolecules (proteins, DNA, RNA).
The idea of allowing liquid sample material to migrate together with color particle-labeled antibodies through porous nitrocellulose membranes to which specific capture antibodies were applied in a linear manner was then described in various patents in 1986. The labeled antibodies bind the antigen in the sample and form an antibody-antigen complex. As a result of the migration of the liquid, this complex passes the capture antibody, which in turn also binds the antigen (a so-called sandwich assay, in which the antigen is caught between two antibodies). If the antigen accumulates on the line with the catcher antibodies, the marking of the detection antibody also accumulates and a colored line is formed. The lateral flow immunoassay was born.