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Luminous reaction in chemical analysis

New techniques enable cheaper and more efficient laboratory examinations

Two researchers at the University of São Paulo (USP) have developed technology unprecedented in Brazil for clinical diagnosis based on the use of light emitting reactions, enabling the measurement of the level of various enzymes of interest in the laboratory. The presence and the amount of these enzymes determine whether there is a disease related to them. Under the new techniques the enzymes grow and cause chemical reactions in organic substances, called substrata, the central point of the studies carried out by the teams of professors Luiz Henrique Catalani, of the Chemistry Institute, and Ana Campa, of the Pharmaceutical Sciences School.

Through the research, it was possible to synthesize the substrata using simpler and cheaper raw materials than the imported products available in the market. Ana also emphasizes that the object is to create less toxic substrata for professionals working in the field of clinical analysis and for discharging them into effluent, also more practical, sensitive and stable. According to Catalani, these substrata are molecules whose main structure is fixed but with a variable part depending on the type of enzyme acting on them. “The enzyme’s action on the substratum triggers off to a series of reactions culminating in light production”.

The emission of light by the system is not visible to the naked eye. It is measured by a piece of equipment called an illuminometer. The result can be seen on a chart where the intensity of the light given off is compared to a standard curve, enabling information to be obtained about the amount of enzyme present, for example, in the portion of blood analyzed. The enzyme acts as a marker deliberately connected to an antibody, in a process called enzyme-immunoassays (EIA), which can indicate the presence of a disease.

Pancreatic enzyme
Enzyme action can be seen in an examination of body fluids – blood serum or any other – to identify the activity of an enzyme indicating the occurrence of a disease. One example is pancreatitis, which leads to an increase in the level of the lipase enzyme in the blood. “This increase can be detected if a substratum that can be recognized by the lipase is used”, says Catalani.

Where the doctor has any doubts about any pathology of the pancreas, he usually asks for an examination of the pancreatic lipase. “Perhaps only one laboratory in São Paulo can carry out this examination based on the new procedures. Most use what are called secondary markers – other enzymes marking the pathology.” To do this, they carry out the test based on a mixture of olive oil and water. When the lipase is added to the mixture there is a difference in turbidity that can determine the activity of the lipase in the serum. “Our process is much more sensitive and accurate.”

Beyond radioisotopes
Another use of the enzyme technology in clinical analysis is the replacement of the radioisotopes as markers to check for the presence of an antigen in the organism – a virus or bacteria, for example. In radio-immunoassays (RIA), in use since the 50s, antibodies are produced in advance and marked with a radioactive element. During the test, they link to the antigen forming a complex that is separated from the analysis agent. According to Catalani, this technique has gradually lost out over the last 20 years to enzyme-immunoassays. “There has been great progress for a long time in radio-immunoassays, including making the work easier. However, this technique is very expensive in various ways, such as the production and discharge of the radioisotope, the use of special security apparatuses for the laboratory, and labor rules to be complied with, among other factors”.

The researcher exemplifies this change with the team’s work on the enzyme ALP (alkaline phosphatase), which hydrolyzes (reacts with the water) phosphates: “Part of the variable substratum is a phosphate, whose structure has been altered so as to enable the enzyme to recognize the substratum. Now we can develop a method which, instead of a radioisotope, uses a simple, cheap and efficient substratum to quantify the enzyme’s action in an immunoassay with chemical light measurement. This type of technology has wiped out the radioimmunoassay from the market”, says Catalani.

Marking antibodies
Ana explains that the third use of the techniques developed by the group is the marking of antibodies by enzymes in various procedures. In the techniques of identifying proteins and fragments of DNA and RNA, prepared in an electrophoresis gel (organic polymer), the revelation is a crucial stage. In spite of the inconveniences, this revelation has been done with radioisotopes: “An alternative technique is the use of enzymes as markers and the consequent emission of light to assess the results”.

Catalani and Ana have undertaken other studies with Luiz Marcos da Fonseca and Iguatemy Lourenço Brunetti, of the Pharmaceutical Sciences School of the São Paulo State University (Unesp) in Araraquara, and Roberto Passetto Falcão, of USP’s Medicine School at Ribeirão Preto, which has a collection of leukemia cells for research. In one of the works, it was observed that a substratum developed by the group can be used to classify some types of leukemia.In identifying these diseases, in the first instance, the laboratory analyzes the shape of the blood’s white cells and, then, tests of the characteristics and chemical actions of these cells are carried out. For some types, the esterase enzyme becomes an important marker. According to Ana, one of the substrata created by the team enables a differentiation of the action of the esterase on monocyte (a type of white cell) in relation to other types of cell. “Based on this, we are proposing a method of classifying certain types of leukemia.”

No patents
The work that disclosed the ability to identify types of leukemia has already been published, as well others on disease marking enzymes, which makes it impossible to patent the processes. Catalani regrets: “For some time, we tried to patent it, but at the time FAPESP had no Patents and Licensing Technology Nucleus (Nuplitec). In addition, we had to present the results of the project”. Nonetheless, the group has other lines of research underway, able to be patented. They are a continuation of the development of new substrata that will serve new types of clinical examination.

The Project
Clinical Biotechnology: Development of Chemical illumination Systems Triggered by Enzymes and their Application in Clinical Diagnosis (nº 97/04957-3); Type Normal line of relationship support; Coordinator Luiz Henrique Catalani; Investment R$ 79,596.66 and US$ 81,340