What exactly is a specific protein analyzer?

Nowadays, many hospitals' laboratories and outpatient departments are equipped with one or more dedicated specific protein analyzers, and there are special testing staff to continuously test blood/urine samples on the machine. Perhaps most ordinary people often hear the most about hospital examination items such as blood routine, urine routine, and biochemical examinations, and may not know much about specific protein testing. What is a specific protein analyzer? Why are more and more hospitals equipped with this instrument? What is the difference between a specific protein analyzer and other instruments?

1. What is a specific protein?
Specific proteins, also called special proteins, refer to a series of proteins present in human serum or other body fluids. They are derived from tissue cells and perform different functions in body fluids. They usually increase or decrease to varying degrees during diseases, thus providing important diagnostic information for clinicians. Specific proteins can be classified into different categories according to their functions in the body, such as immune system specific proteins, rheumatism and rheumatoid specific proteins, urine specific proteins, acute phase response specific proteins, nutritional status monitoring specific proteins, nervous system specific proteins, and lipid specific proteins;
Special proteins come from tissue cells, with high content, complex composition, and wide functions. Analyzing the changes in the quantity and quality of special proteins is of great value for understanding the disease, clarifying its pathogenesis, and early diagnosis and early treatment of clinical diseases.

2. What are the detection methods of specific proteins?
There are various specific proteins in different body fluids such as serum, cerebrospinal fluid, and urine in the human body. Quantitative detection of them has become an important means for clinical diagnosis of diseases, monitoring of the course of disease, and evaluation of efficacy. Specific proteins have good antigenicity and are usually detected using the principle of antigen-antibody reaction. The detection of specific proteins in body fluids has always been a direction of great concern to people:

The development stage of the classic immunoprecipitation test:
The measurement range is narrow, the sensitivity is low, and the operation is cumbersome
●1897: Kraus found that a visible precipitation reaction occurred after the bacterial culture fluid was mixed with the corresponding antiserum;
●1902: Ascoli established the ring precipitation test;
●1905: Bechhold mixed the antibody in gelatin and then added the corresponding specific antigen to it. The specific binding of the antigen and antibody can cause precipitation in the gelatin;
●1964-1965: Oudin reported the test tube unidirectional immunodiffusion test, and Mancini proposed the plate unidirectional immunodiffusion test (i.e. the single diffusion method in the medical service fee standard);
●1953: Grabar and W Williams reported immunoelectrophoresis (countercurrent immunoelectrophoresis, rocket immunoelectrophoresis and immunofixation electrophoresis);

The qualitative leap of immunochemical analysis:
Accurate, fast, wide linear range
●1959: Schultze et al. reported the turbidimetric method, which quantitatively analyzed the amount of antigen-antibody complex by measuring the reduction of transmitted light (a common method used in biochemical analyzers);
●1967: Ritchie et al. proposed the use of laser scattering to measure the antigen-antibody complex formed by complement C3 and haptoglobin, and called it turbidimetric method. This method shortened the classic gel immunoprecipitation method that took tens of hours to a few hours to obtain the measurement results (the predecessor of the specific protein analyzer methodology);
●1977: Sternberg et al. proposed a faster turbidimetric method, called rate turbidimetric method;
●After 1977: turbidimetric method derived from timed turbidimetric method.

People are paying more and more attention to the development of specific protein-related detection methods and instruments. From the beginning, there was no special specific protein analyzer instrument. Some items could be detected on the biochemical analyzer, but only large molecular proteins could be detected. Due to the limitation of the method, the measurement accuracy of the biochemical analyzer could not be guaranteed. Later, many clinical diagnostic manufacturers began to develop semi-automatic specific protein analyzers. Although the methodological principles were diverse (scattering turbidimetry, gold label method, immunofluorescence method, etc.) and the speed was fast, the number of channels was small and manual operation was required. Today, the technology of fully automatic specific protein analyzers is mature, which can integrate many features such as full automation, high throughput, full detection items, dedicated machine and even online joint inspection.

The detection method of fully automatic specific protein analyzer is mainly immunoturbidimetry, which is mainly divided into the following three types:
Immunotransmission turbidimetry: After the antigen and antibody combine, an immune complex is formed, and the complex aggregates and turbidity appears within a certain period of time. When light passes through the solution, it can be absorbed by the immune complex. The more immune complexes there are, the more light is absorbed. The amount of light absorbed is positively correlated with the amount of immune complexes within a certain range. The content of the object to be tested is converted by the change in the intensity of the transmitted light;

Immunoscattering turbidimetry: When a certain wavelength of light is irradiated along the horizontal axis and passes through the solution, it encounters the antigen-antibody complex particles. The light is refracted by the particles and deflected. The angle of light deflection is closely related to the wavelength of the emitted light and the size and number of the antigen-antibody complex particles. The intensity of the scattered light is positively correlated with the content of the complex, that is, the more antigens to be tested, the more complexes are formed, and the stronger the scattered light. The content of the object to be tested is converted by the change in the intensity of the scattered light;

Latex-enhanced immunoturbidimetry: This method uses tiny latex particles to link antibodies, and the corresponding antigens in the liquid phase are combined to produce changes in scattered light/transmitted light to determine the antigen content. The detection accuracy of plasma protein can be greatly enhanced by combining latex particles.

It should be noted that the intensity distribution of scattered light in nephelometry depends on the relationship between the particle size and wavelength of the antigen-antibody complex, and is divided into Rayleigh or Mie scattering [2]:
●Rayleigh scattering: If the particle diameter is smaller than the wavelength of the incident light, Rayleigh scattering will occur, that is, the scattered light will be almost symmetrically scattered in all directions, resulting in a decrease in the light efficiency of the measurement;
●Mie scattering: If the particle diameter is larger than the wavelength, Mie scattering will occur. As the particle size increases, the scattered light will be mainly concentrated in the forward direction;
When latex particles are used, the diameter of the antigen-antibody complex is generally d>1000nm, and the particles are usually larger than the wavelength. Therefore, Mie scattering is mainly generated, and the corresponding signal value is also higher, and the measurement is more accurate.

3. What are the components of a specific protein analyzer?
A specific protein analyzer is an analytical system used to quantitatively detect proteins in body fluids such as human serum, plasma, and urine. Like most in vitro diagnostic instruments, it consists of a sample system, a reagent system, a reaction system, a temperature control system, a liquid system, an operating system, and a data processing system.
It is worth mentioning that the sample injection position usually includes sample turntable injection and sample track injection, among which the turntable type can keep the sample tube/cup and other containers in the center, but it is more common on biochemical analyzers; the track type is currently widely used in fully automatic specific protein analyzers. Through standardized tracks, batch processing of sample racks can be realized. At the same time, in order to better detect whole blood samples, fully automated technologies such as automatic barcode scanning, closed-cap puncture sampling, and automatic shaking of original tubes are gradually integrated, which greatly saves the workload of inspection workers. In addition, standardized tracks also provide hardware foundation for assembly line or online detection.

4. What is the reaction principle of antigen-antibody and protein detection?

The detection of special proteins utilizes the reaction of samples and reagents to form antigen-antibody complexes, and then uses scattering turbidimetry to detect the corresponding concentration. The Heidelberger-Kendall p curve shows the relationship between antigen level and measurement signal under a constant antibody level condition, which can be divided into three stages:

▲ Antibody excess: There is an excess of antibody in the reaction cup, and there are many idle binding sites. Each added antigen will immediately bind and induce further cross-linking. The relationship between the antigen level and the measurement signal at this stage is proportional;
▲Equivalent interval: When the concentrations of antigen and antibody in the reaction cup are basically equal, the cross-linking reaction here is the strongest and the solubility of the antigen-antibody complex is the lowest;
▲Antigen excess: When the amount of antigen in the reaction cup exceeds the antibody, because the number of antibodies is not enough to bind all the antigens, the solubility of the immune complex increases again, which means that the measurement signal is smaller instead, and there is a situation where the test result does not show too high but indicates a low level (also known as the HOOK after-band effect).
In order to solve the problem of excessive antigen causing result deviation when testing high-concentration samples, some specific protein analyzers have begun to launch the functions of antigen excess detection and automatic dilution of high-value samples, which include three steps: pre-reaction, turbidity check, and sample dilution: In the pre-reaction stage, a part of the sample is first reacted with the full amount of reagent. If the threshold is not exceeded in the pre-reaction stage, a normal amount of sample will be added for measurement. The calculated value is evaluated using a reference curve. If the original value signal growth significantly exceeds the threshold, the measurement will be automatically re-measured with the second-level dilution. During the turbidity check, if the original value of a certain measurement signal exceeds the defined threshold, the measurement result will be marked with a prompt. The analyzer will then dilute the sample according to a preset or different ratio before testing to avoid the HOOK effect.