| With the policies carried out to encourage the birth of a second child in China and the gradual aging of the population,there is an increasing clinical demand for nutrition screening for infants,pregnant women,and the elderly.The anemia diagnostic and vitamin D testing are the main test items of nutrients testing.Vitamin D testing for infants and young children has not been widely accepted because of venous blood sampling is not acceptable for parents.During the development of folic acid and vitamin B12 test kits,it is difficult to obtain raw proteins,such as folate binding proteins and intrinsic factor,and high technical difficulties in releasing procedure and conjugates preparation,the market in China monopolized by imported manufacturers.In order to break the situation of foreign medical device companies occupying the domestic market,in recent years,many national departments have released signals to accelerate the localization of medical devices.The Ministry of Industry and Information Technology puts forward the industrial development goals of 50%,70% and 95% of domestically produced high-end medical devices in county-level hospitals by 2020,2025 and 2030.In response to our country's policies,this paper was carried out the studies on the development and application of automatic immunoassay techonology for human nutritional status assessing,pichia pastoris expression system was used for the first time to express biologically active intrinsic factor,and an efficient extraction method was developed to purify the folate binding protin from cow's milk.These methods break the core technology mastered by international giants,developing three anemia detection methods,and debugging and verification the kits adjusts to the developed automatic chemiluminescence instrument.Meanwhile,this article also developed the whole blood pretreatment technology for the first time,which laid the foundation for the comprehensive promotion of 25-hydroxyvitamin D detection for infants and young children.This article is mainly divided into the following parts.(1)Preparation of the biological materials for nutrients testing.The gene sequences of intrinsic factor and folate binding protein were optimized by Pichia species and constructed into the vector p PICZ?A.After plasmid linearization,homologous recombination was introduced into Pichia pastoris to construct expression strains p PICZ?AIF/GS115 and p PICZ?A-FBP/GS115,the intrinsic factor was successfully expressed by methanol induction,the expression level was 0.14g/L,and the purified intrinsic factor activity was about 115% reported abroad.For folate binding proteins that fail to express,an extraction method from cow's milk was carried out.Folic acid molecules coupled to the surface of agarose gel was used to adsorb the folate binding protein in milk.After the gel column was eluted and purified,the folate binding protein with high activity was successfully obtained,the biological activity was 137% higher than the imported material,and 0.34 mg folate binding protein can be extracted from 1 liter fresh milk.(2)Development of highly sensitive nutrient immunoassay methods and single-serve automatic chemiluminescence instrument.A competitive method was used to establish a quantitative detection method for folic acid in human serum.The experiment optimized the release process of serum folic acid,the labeling process of Biotin-FBP and FA-HRP,the concentration of Biotin-FBP and FA-HRP,the incubation time and other factors.The blank limit of this method is 0.41ng/m L,the deviation of in house control 1 and 2 are less than 5%,the intra-day and inter-day precision are less than 15%,the specificity is good,the cross-reaction rates of methotrexate,aminopterin and leucovorin are less than 0.1%.The thermal stability of the reagent is good,and the signal value decrease for less than 15% after being placed at 37? for seven days.A competitive method was used to establish a quantitative detection method of vitamin B12 in human serum.The experiment optimized the release process of vitamin B12 in serum,the labeling process of Biotin-IF and VB12-HRP,the working concentration of Biotin-IF and VB12-HRP,and the incubation time and other factors.The blank limit of the established method is 42 pg/m L,the deviations of the in house control 1 and 2 are less than 8%,the intra-and intraday precision are less than 15%.The cross-reaction with hydroxycobalamin is 45.96%,and vitamin B1,Vitamin B2,and Vitamin C are all less than 0.1%.The reagents have good thermal stability,and the signal value decrease for less than 15% after being incubated at 37? for seven days.A double-antibody sandwich method was used to establish a quantitative detection method for ferritin in human serum.Biotinylated capture antibodies and enzyme-labeled capture antibodies were prepared and their concentration were optimized.The blank limit of the method is 1.53 ng/m L.The detection deviation of in house control 1 and 2 are less than 5%.The intraday and inter-day precision are less than 10%.The reagents show good thermal stability,and the signal value drop less than 10% after being placed at 37? for seven days.Compared with the test results of Beckman Coulter,the correlationship is good.A competitive method was used to establish a quantitative detection method for 25-hydroxyvitamin D.The antibody-coated plates and SA-HRP were prepared experimentally,and the working concentration and incubation time of Biotin-VD and SA-HRP were optimized.The blank limit of this method is 3.16 ng/m L.The detection deviation of the in-house control 1 and 2 is less than 6%.The precision within the day and day is less than 15%.The thermal stability of the reagent is good,and the light signal values has a slightly upward trend in seven days at 37?,but the change is less than 15%.The fully automatic single chemiluminescence immunoassay analyzer developed by this subject is accurate and has good repeatability.The optical noise of the reading device is low;the temperature control is accurate and stable,and the fluctuation is 0.7?.The washing method is good,and the residual volume is small.The optimized reading waiting time and exposure time of HRP system were 60 seconds and 0.5 seconds,respectively.The detection reagent format is a single detection reagent strip.The product is stable by avoiding repeated opening,which effectively solves the serious waste of traditional reagents caused by the small number of samples in primary hospitals.The instrument and supporting reagents have the advantages of small and rapid attributes of point of care testing devices and the high accuracy performance of a large chemiluminescence instrument,which effectively promotes the relevant policies for the sinking of domestic medical equipment and the localization of imported products.(4)Research on the matching and debugging of nutrient detection method and single-serving automatic chemiluminescence instrument.Adjusted the three detection procedures,the three items can be randomly sampled,simultaneously tested,and the results are synchronized.Designed reagent strips for the items.After the three items evaluated on the machine,the accuracy and precision met the clinical needs,and the test results were also highly correlated with the imported control products.The later clinical evaluation studies also showed that the performance of the product developed can fully met the requirements of domestic product registration.After matching with the automatic chemiluminescence instrument,25-OH VD test accuracy and precision were good,and the results were also highly correlated with the imported control products.The clinical studies showed that the develoed products have a good correlationship with the import products.In addition,the whole blood pretreatment method developed by capillary centrifugation solved the problems of sample collection and quantitative determination of infants and young children;the accuracy and precision of whole blood samples were highly consistent to plasma test results which can fully meet the clinical needs.(5)Clinical evaluation studies of nutrients detection reagents and supporting equipment.The clinical evaluation results of the four nutrients detection reagents and the supporting single-serving automatic chemiluminescence instrument show that the test results were consistent with the test results of the reference reagent and met the clinical evaluation standards for product registration,and the test results of the reagents and instruments has high reliability and safety. |
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