Screening raw materials account for 70%, do you believe it?
According to the "Guiding Principles for Analytical Performance Evaluation of In Vitro Diagnostic Reagents", the main analytical performance evaluation items are: detection limit, linear range, reportable range, accuracy (recovery experiment), accuracy (methodological comparison), precision, interference Experimental, Stability, Reference Values. At present, it is usually based on the relevant standards of the American Clinical Laboratory Standardization Institute (CLSI) in the world.
Product performance evaluation is an important technical support research process for product development, product standard formulation and other processes, and may have a certain impact on product quality. The entire R&D process involves raw materials, processes and ideas. Because the industry is in the golden stage of development, little attention is paid to and optimize the entire R&D process. If the relationship between the three is not straightened out, the entire R&D process will fall into blindness and unsustainability.
When you are new to research and development, you usually use the existing system, including a stable formula system (coating buffer, blocking buffer, enzyme labeling buffer, calibrator dilution, luminescent substrate solution) and process flow, everything is ready According to the principle, use the existing formula, follow the existing process, and operate according to the process. Most of the work is used for screening and matching, and analyzing data.
This entry stage generally lasts for at least 2-3 years, because the formula system and process system (hereinafter referred to as process, including formula and system) are established by predecessors through a large number of research results, and are generally relatively stable and rarely modified (even if they need to be improved) , the authority is also in the hands of the R&D director), and the entire R&D thinking has been streamlined, so there is no need to think too much. Many new technicians, after this pairing screening, will select a more suitable pairing from a certain number of pairs, and carry out the next step of research and development. It is easy to conclude that the quality of a kit basically depends on the quality of the raw materials. If good raw materials are not selected, or difficult projects are encountered, there is no way to start.
We disassemble and analyze raw materials, processes and ideas in detail, with the purpose of clarifying which items are determined by raw materials, which items are determined by process, and which items are jointly affected by the two in the performance evaluation items, so as to promote research and development ideas The formation and maturity of the development, hope to be able to throw bricks to attract jade.
NO.1 raw material
We extract the key raw materials in immunological reagents and divide them into two parts: antigen antibody and special components.
1. Antigen and antibody
Immunological reagents can generally be divided into two processes: immune reaction and chromogenic reaction. The immune response is the core, and the specific separation of the analyte from the complex sample, the quality of this link directly determines the core quality of the entire reagent.
In industrial production, antibodies for immunoassays are generally expressed by engineering bacteria to express antigenic fragments, immunized animals, and polyclonal or monoclonal antibodies are prepared, and then the obtained antibodies are paired and screened to select a suitable pair for use in immunoassays . In this process, because the antigens to be tested are generally natural antigens (a small number of scientific researches detect recombinant antigens), and the preparation and screening of antibodies generally use recombinant antigens, which will bring many application problems. For antibody manufacturers, worthy of attention.
2. Special ingredients
As a fragile non-covalent bonding process, the immune response is affected by time, interferers, bridges, structural analogs, etc. In order to eliminate these adverse effects, some special components are generally added to the buffer system. Special ingredients we define as minor raw materials, detailed in the process.
NO. 2 Craft
Process includes formula system and process flow, both of which complement each other. All formulas and processes are designed to ensure and improve product performance. When raw materials cannot meet product performance requirements, how to improve through formula technology, or in other words, which properties can be improved through formula technology, how to improve, which It can only be determined by raw materials, which must be continuously accumulated and improved during our R&D career.
1. Formula system
The formulation system can be disassembled into a buffer system and an improvement system.
(1) Buffer system The general buffer system can be seen in textbooks, product brochures, and papers. It mainly includes ion pair type, ion concentration, pH value, etc. The non-confidential formula is relatively easy to understand and master.
(2) Improved system Because of the fragility of non-covalent bonding of the immune response, it is difficult to obtain good reaction results by relying on a simple buffer system. To solve this problem, scientists have designed various improved systems. Under these systems, there are various methods to produce.
A. Reduce the background system
a. Use appropriate coating and labeling concentration, generally determined by checkerboard titration.
b. Add substances that increase the dissolution rate and reduce non-specific adsorption, such as Triton and Tween.
B. Reduce interference system
a. Add normal mouse serum or mouse IgG to reduce the impact of human anti-mouse antibody
b. Add blocking agent to reduce the interference of rheumatoid factor
C. Reduce the prozone effect system
a. Appropriately increase the concentration of coating and labeling, taking into account the background
b. Add detection antibody and naked antibody, taking into account the sensitivity
D. Increase the sensitivity system
a. Add Triton, Tween, etc.
b. Add special substances, such as S19
E. Improve the detection rate system
a. Combined use of multiple antibodies, such as the CnTI project
F. Improve the stability system
a. Add protective proteins, sugars, and small molecular compounds to the buffer
G. Other systems, etc. With the deepening of research, this directory will continue to increase, and the corresponding methods will also continue to increase.
2. Process flow
The process flow also includes two aspects: the production process flow and the kit detection operation process flow.
(1) Production process The production process has a great impact on the overall product performance, such as the preparation process of pre-coated boards, the temperature and time of coating, sealing, and drying have a great impact on the entire performance, which requires research and development groping.
(2) Detection operation process Compared with the production process, the detection operation process of the kit has a greater impact on the performance, one-step method, two-step method, sample volume, sample addition sequence, incubation temperature, and incubation time.
NO.3 Train of thought
We analyze raw materials and processes in order to find a suitable R&D idea, guide the R&D process, and improve R&D efficiency. The entire R&D idea is divided into two issues: product R&D and improvement R&D.
1. Product development
Product research and development is a process from scratch, most of which are based on the existing reagents on the market to develop products that the company does not have. This process accounts for the bulk of research and development, and we subdivide it into three processes: preliminary research and development, pre-clinical trials, and stability testing. For all research and development, we must know how to evaluate a performance item, what factors will affect this performance, and how to improve it. Let us take the double antibody sandwich as an example.
(1) Preliminary research and development During preliminary research and development, three active ingredients are generally emphasized: coating antibody, detection antibody, and calibrator. The coating antibody and detection antibody will have an empirical concentration. The calibrator generally uses a suitable matrix to gradiently dilute the antigen or high-value samples, and the obtained gradient concentration is assigned by a third party, which can be traced back to international, national or enterprise standards.
Most of the initial research and development is doing pair screening, and only need to use the three performance evaluation items of detection limit, linear range, and reportable range to screen out candidate pairs. For example, if you get 10 strains of antibodies, you can make 100 pairs, screen them, and select a number of candidate pairs that are initially available.
The precision, accuracy (recovery test), and interference test evaluations are performed on the candidate pairs of the primary screening, and the candidate pairs are screened again through these experiments.
(2) Pre-clinical experiment After the preliminary research and development that does not involve clinical serum samples is completed, a certain candidate pairing is obtained, and a pre-clinical experiment is carried out, mainly to evaluate the accuracy (methodological comparison) and the reference value range.
(3) Stability test Through the above two steps, the kit is preliminarily formed, and the stability of the active components (including pre-coated plates, calibrators, and enzyme-labeled antibodies) needs to be evaluated and improved one by one until the requirements are met.
2. Improve R&D
The development of the entire kit is ultimately to detect clinical serum samples, and all analytical performance evaluation items are also designed for this purpose. The amount of serum samples is very limited, at most a small trial production can be completed. When the real product is launched and clinically applied, with the expansion of the sample size, many problems that have not been exposed before will be exposed intensively, and sufficient data must be collected for improved research and development.
Generally, at this stage, for samples that do not meet clinical requirements, especially false positive and false negative samples, it is necessary to compare the samples with reagents from different manufacturers, analyze components, etc. This process will be done in both pre-clinical and formal clinical trials. In clinical and formal clinical trials, samples with serious discrepancies in test values, but which did not lead to false positives or false negatives, should be analyzed.
