47 results about "Stable transfection" patented technology

The invention provides a SynNotch system delivery method as well as a delivery vector and application thereof. The method comprises the following steps: constructing a stably transfected SIN-gamma-RV vector by using a PiggyBac transposon system, expressing a SynNotch system by using a PSR vector, and combining a GAL4 induction vector and a UAS effect vector of the SynNotch system on one PSR vector, so that the SynNotch system is delivered by one PSR vector. The PSR.SynNotch system disclosed by the invention not only solves the problem that the transduction efficiency of double-carrier downstream effector molecules of an original system is difficult to detect, but also solves the problem that the efficiency of preparing the double-positive cells by double-carrier transduction target cells is low, most importantly, a stable transfection packaging cell line can be constructed for expanded production, and the application of the SynNotch system is very facilitated.

The invention discloses a recombinant protein for detecting an anti-myelin sheath related glycoprotein MAG antibody and a detection reagent of the recombinant protein, and belongs to the technical field of neural immunodiagnostics, five amino acid sites in MAG protein are subjected to specific amino acid point mutation replacement to obtain an MAG protein mutation sequence, the MAG protein mutation sequence is fused with GlcATP enzyme and HNK-1ST enzyme full-length sequences to construct a lentiviral expression vector, and the recombinant protein and the detection reagent are used for detecting the anti-myelin sheath related glycoprotein MAG antibody. According to the present invention, three genes are constructed, and are stably transfected in a host cell so as to stably express the three genes in one cell at the same time, such that the constructed recombinant protein and the wild type MAG protein have similar anti-MAG IgM antibody binding domain, and on the premise of not using GlcATP enzyme and HNK-1ST enzyme small molecule agonists, the sensitivity and the specificity of the detection of the MAG autoimmune antibody by using the CBA method are improved; the method is suitable for preparing related detection reagents for in-vitro detection of the IgM type anti-MAG antibody.

The invention belongs to the technical field of medicines, and discloses application of hyperforin in preparation of a medicine for preventing or treating amyotrophic lateral sclerosis. The hyperforin can improve the survival rate of stable transfection cell lines of amyotrophic lateral sclerosis related cell models (SOD1G93A / TDP43M337V), and the hyperforin is prompted to play a protective role in the two cell models. After the hyperforin is given, the expression levels of HO-1 and Drp1 proteins in SOD1G93A cells are increased, which indicates that the hyperforin has certain antioxidant activity; and mitochondrial division in cells can be inhibited, so that cell apoptosis is inhibited and retarded, and a protection effect is achieved. Hypericin can help to improve the SOD1G93A cell morphology and promote synaptic generation, and it is prompted that hypericin has a positive effect on maintaining normal functions of cells. In conclusion, the hyperforin plays a protective role in an SOD1G93A cell model and a TDP43M337V cell model, and the mechanism of the hyperforin is possibly related to the antioxidant activity, the mitochondrial function improvement and the like of the hyperforin.

The invention provides a preparation method of a Syn-CAR-T cell, the Syn-CAR-T cell and application of the Syn-CAR-T cell, and the method comprises the following steps: constructing a stably transfected SIN-gamma-RV vector, namely a PSR vector, by using a PiggyBac transposon system, preparing a Syn CAR plasmid vector, expressing a SynNotch system by using the PSR vector, combining a GAL4 induction vector and a UAS effect vector of the SynNotch system on the PSR vector, and preparing the Syn CAR plasmid vector. Enabling the SynNotch system to be delivered by one PSR vector, and constructing a plasmid of the Syn CAR retrovirus vector; and the plasmid of the retroviral vector is used for transduction of a T cell, and the Syn-CAR-T cell is prepared. Results of the invention show that the induction efficiency of the target spots BCMA, CD38 and GPC3 is extremely low, and the target spots are not suitable for the SynNotch system of the invention; the induction efficiency of the target CD19 is highest, and the target CD19 is most suitable for being applied to the SynNotch system.

The invention discloses a chimeric gene engineering nano vesicle for removing pathogenic antibodies as well as a preparation method and application of the chimeric gene engineering nano vesicle, and belongs to the technical field of biology. According to the invention, a stable transfection cell line of a protein A / G gene is constructed through lentiviral particle transfection, so that the protein A and the protein G can be stably expressed. The fusion protein A / G can overcome the defect of weak binding capacity of pure protein A and IgG3, and the adsorption effect of the antibody IgG is improved. The PLGA nanoparticles with relatively uniform particle size are adopted as the core of the target vesicle and are mixed with the engineered cell membrane broken by extrusion to promote self-assembly of the cell membrane and form an extracellular vesicle simulant, so that the stability of the target nano vesicle is improved. The prepared and separated nano vesicles can be applied to in-vivo adsorption and antibody removal, the biological safety is good, and the dialysis indwelling catheter operation risk in the use process of an existing immunoadsorption column and the risks of bleeding, allergy and the like in the treatment process are avoided.

The invention discloses a stably transfected cell strain for evaluating fibroblast autophagy flux and a construction method of the stably transfected cell strain. The construction method comprises the following step: infecting fibroblasts by using HBAD (Hepatitis B Antigen)-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus. The construction method can be combined with a super-resolution microscope to dynamically observe the autophagy intensity change in the primary fibroblasts through an imaging method.

The invention discloses a monoclonal cell strain screening method based on fluorescence intensity, which comprises the following steps: determining a cell line, and stably transfecting fluorescent protein to cells of the cell line; a microwell plate is selected, a datum hole is set, the cells stably transfected with the fluorescent protein are separated and put into the microwell plate, a plurality of cells are put into the datum hole, and only one cell is put into the other holes; screening out single-cell pores with only one cell from the rest of pores except the reference pore; screening out clone group holes capable of growing clone groups; respectively digesting cells in the cloning group pores and then transferring the cells into new pores for culturing; screening out fluorescent screening holes; splitting proliferation holes are screened out; and determining the cells in the division proliferation hole as the screened monoclonal cell strain. According to the monoclonal cell strain screening method provided by the invention, the monoclonal cell strain which is high in fluorescence intensity and can proliferate at a normal speed can be screened, compared with fluorescence microscope observation and imaging, the method is convenient and rapid, and errors possibly generated by comparing the fluorescence intensity with human eyes are reduced.

The invention belongs to the technical field of biomedicine, and particularly relates to application of SOX4 in preparation of a preparation for improving sensitivity of cervical cancer to chemotherapeutic drugs, and the amino acid sequence of the SOX4 is as shown in SEQ ID NO.1. An SOX4 knocked-down stable transfection cell line is constructed in a Hela cell, then carboplatin and oxaliplatin are respectively added into the SOX4 knocked-down Hela cell, and the IC50 of the cell to the carboplatin and the oxaliplatin is obviously reduced, which indicates that the sensitivity of the Hela cell to the carboplatin and the oxaliplatin is enhanced by SOX4 knock-down. By reducing the expression of SOX4, the sensitivity of Hela cells to chemotherapeutic drugs such as carboplatin and oxaliplatin is enhanced, so that the treatment effect of the chemotherapeutic drugs such as carboplatin and oxaliplatin is effectively improved. Results of the invention prove that inhibition of SOX4 is beneficial to recovery of chemosensitivity of cervical cancer cells to drugs such as carboplatin and oxaliplatin, and a new treatment strategy is provided for treatment of drug-resistant cervical cancer.

The invention relates to the technical field of bioengineering, and aims to provide a CHO stably transfected cell strain capable of expressing a recombinant antibody capable of specifically recognizing CSFV2 type Erns protein. The cell strain is preserved in the China Center for Type Culture Collection, the preservation name is Chinese hamster ovary cell gs weak strain CHO-9B1-C, and the preservation number is CCTCC NO: C2024393. The invention also provides application of the cell strain in preparation of a diagnostic kit for specifically detecting the CSFV2 type hog cholera virus. The CHO stably transfected cell strain provided by the invention has the advantages of short monoclonal antibody production cycle, high consistency and high repeatability, is suitable for large-scale production, and is easy to transform.

The invention discloses a Betti alkali derivative as well as a synthesis method and application thereof. According to the present invention, the substituent group on the molecular formula of the Betti base is regulated so as to obtain the Betti base derivative with high EGFR inhibition activity; the betati base derivative has a relatively strong inhibitory activity effect on an H3255 cell strain highly expressing EGFRL858R, a PC-9 cell strain of EGFRdel19, an H1975 cell strain of EGFRL858R / T790M or a stably transfected Ba / F3-EGFRL858R / T790M / C797S cell strain, can effectively inhibit the growth of lung cancer cells, and can be applied to preparation of drugs for treating, preventing, delaying, assisting in treating or treating diseases related to EGFR activity passing and drugs for treating tumor diseases.

The invention provides an organoid for simulating a dormancy and recurrence process of breast cancer cells as well as a construction method and application of the organoid. The construction method comprises the following steps: culturing breast cancer cells, and carrying out flow sorting on surface markers CD44 and ITGB4 to obtain an ITGB4 low-expression (ITGB4low) cell population; packaging a lentiviral vector, infecting a cell population with low expression of ITGB4, and carrying out flow sorting to obtain a stably transfected cell population; and carrying out induction treatment on the stably transfected cell population and culturing to obtain the organoid. The organoid provided by the invention provides powerful support for prevention and / or treatment of metastatic breast cancer, and shows extraordinary application prospects in accurate and efficient screening of drugs for targeting dormancy-recurrence process.

A polynucleotide is provided for the stable transfection of an animal cell. A host cell transfected with the polynucleotide according to the invention exhibits very high expression of at least one target gene and very high production of the gene product of the at least one target gene (peptide or protein). Furthermore, a kit for transfecting a host cell and a host cell transfected with the polynucleotide according to the invention are provided according to the invention. The host cell is characterized by strong production of at least one target gene. In addition, the use of the polypeptide and the kit according to the invention for transfecting a host cell is proposed. Furthermore, the use of the host cell according to the invention for expressing at least one target gene is proposed.

The present invention discloses a recombinant protein for detecting anti-myelin associated glycoprotein (MAG) antibody and a detection reagent thereof, belonging to the technical field of neuro-immunodiagnosis. By performing specific amino acid point mutation substitutions at five amino acid positions in the MAG protein, a MAG protein mutant sequence is obtained, and then it is fused with the full-length sequences of GlcATPase and HNK-1ST enzyme to construct a lentiviral expression vector, which is stably transfected into host cells, enabling the simultaneous stable expression of the three genes in one cell. The constructed recombinant protein has a similar anti-MAG IgM antibody binding domain to the wild-type MAG protein, and without using small molecule agonists of GlcATPase and HNK-1ST enzyme, it improves the sensitivity and specificity of detecting MAG autoantibodies by the CBA method, and is suitable for preparing related detection reagents for in vitro detection of IgM-type anti-MAG antibodies.

The present invention relates to methods for the selection of a stable producer cell line expressing one or more protein(s) of interest (POIs), comprising the stable transfection of cells with a gene encoding a constitutively active variant of a growth factor receptor, respective methods for the expression of one or more POIs in such stable producer cell lines, and uses of a constitutively active variant of a growth factor receptor as selection marker in the cultivation of cells.

This invention relates to a detection kit and preparation method for enhancing the detection efficacy of autoimmune encephalitis (CBA) antibodies. The kit comprises genetically engineered 293T cells stably integrating and co-expressing the eIF2α S52A mutant and HSP70, and transiently transfected with CBA-associated antigens. The preparation method includes constructing a co-expression vector, establishing a stable transfection cell line, stress preconditioning treatment, antigen transfection, and kit assembly. A method for assessing antibody affinity maturation using this kit is also provided. By synergistically modifying detection cells at three levels—translational regulation, protein folding, and anti-apoptosis—the invention significantly increases antigen expression, reduces apoptosis, and improves detection sensitivity and specificity. Furthermore, it integrates antibody affinity assessment into CBA detection for the first time, providing auxiliary information for clinical staging.

The application relates to a cell line stably expressing an orthogonal aminoacyl tRNA synthetase / tRNA pair, a HEK293T cell line capable of stably expressing an EcTyr orthogonal aminoacyl tRNA synthetase / BsTyr tRNA pair is constructed, and a construction method of the cell line is disclosed. The construction method is to stably integrate the orthogonal EcTyr aminoacyl tRNA synthetase and BsTyr tRNA genes into the HEK293T cell genome by means of a PiggyBac transposon system and plasmid stable transfection. The constructed cell line genome is integrated with the EcTyr orthogonal aminoacyl tRNA synthetase and BsTyr tRNA genes, and provides a stable platform for efficiently introducing unnatural amino acids at any site of a protein. The application has the effects of further laying an experimental foundation for the exploration of novel protein structures and functions and the research and development of protein drugs containing certain specific functions.

The present invention provides a transfection reagent based on blank lipid nanoparticles, a preparation method thereof and applications, belonging to the technical field of biomedicine. The transfection reagent based on blank lipid nanoparticles of the present invention comprises: (1) blank lipid nanoparticles; (2) biologically active components; the composition of the blank lipid nanoparticles includes: ionizable lipids, phospholipids, cholesterol and polyethylene glycol-conjugated lipids. The preparation processes of the blank lipid nanoparticles and the transfection reagent are simple and can be completed without the aid of equipment. The transfection reagent based on blank lipid nanoparticles is prepared from blank lipid nanoparticles and nucleic acids, can flexibly adjust the dosage according to the needs of users, has a stable transfection effect, can transfect a variety of cells (such as 293T, Hela, HepG2, TWO3, MCF7), has low cytotoxicity and good transfection effect.

The invention belongs to the technical field of medicine, and particularly relates to a preparation method and application of a recombinant human blood coagulation factor VII, and the preparation method comprises the following steps: transforming a nucleotide sequence of the recombinant human blood coagulation factor VII into a vector, and constructing a recombinant expression plasmid; transfecting the recombinant expression plasmid into a eukaryotic cell to obtain a recombinant expression cell; screening the recombinant expression cells to obtain stable transfected cells; domesticating the stably transfected cells to obtain monoclonal suspension engineered cells; and performing amplification culture on the monoclonal suspension engineered cells, collecting supernate, and extracting target protein in the supernate to obtain the recombinant human blood coagulation factor VII. The recombinant human blood coagulation factor VII produced by using the method disclosed by the invention is high in yield and good in procoagulant activity, so that the production efficiency and the application effect of the recombinant human blood coagulation factor VII can be improved by using the preparation method disclosed by the invention.

The invention provides a method for constructing a multi-copy eukaryotic expression vector based on a programmable enzyme Argonaute and application of the multi-copy eukaryotic expression vector based on the programmable enzyme Argonaute, and belongs to the technical field of biological medicines.The method comprises the following operation steps that DNA assembly is conducted on recombinant plasmids In-pc-CDs based on Argonaute and resistance gene reconstruction, and expression units in the plasmids obtained through assembly are transferred to plasmids Lamp; in R-Arm, the multi-copy expression vector is obtained. A set of in-vitro seamless DNA assembly technology which is low in mutation rate, convenient to operate, easy to screen, short in assembly period, wide in base sequence universality and low in both economic cost and time cost is developed by starting from a novel programmable enzyme Argonaute and combining a resistance reconstruction strategy, and the in-vitro seamless DNA assembly technology is used as an eukaryotic expression platform DNA assembly tool to realize the in-vitro seamless DNA assembly of the eukaryotic expression platform. The invention provides a method for rapidly constructing a multi-copy expression vector and a signal peptide expression plasmid library, so that the construction of the CHO stable transfection cell strain is realized.

The embodiment of the invention provides a library design and analysis method for high-throughput analysis of non-classical open reading frame coding capability and function, and belongs to the technical field of data analysis. The invention discloses a library design method for high-throughput analysis of non-classical open reading frame coding capability and function. The library design method comprises the following steps: acquiring target non-classical open reading frame (ncORFs) data; designing an overexpression library vector according to the ncORFs data of the target; and obtaining the overexpression library according to the overexpression library vector. The analysis method for analyzing the coding capability and function of the non-classical open reading frame at high throughput comprises the following steps: obtaining an overexpression library; obtaining stable transfected cells; according to the stably transfected cells, obtaining the translation ability information of the ncORFs; acquiring the influence information of the ncORFs on the survival of the cells; and acquiring information of the regulation effect of the ncORFs on the cells. According to the invention, the coding capability and function of ncORFs can be efficiently screened and evaluated, and novel functional protein products can be found.

The present invention belongs to the field of genetic engineering technology, and specifically relates to a method for improving the expression level of recombinant proteins in CHO cells. The method comprises the following steps: (1) combining a target gene recombinant vector with a gene containing cell cycle regulation-related genes; p18 and / or genes that regulate cell growth aFGF The co-transgenic recombinant vector is co-transfected into CHO cells; the gene p18 The sequence of is shown in SEQ ID NO: 1; the gene aFGF The sequence is shown in SEQ ID NO:2; (2) co-transfecting the co-transgenic recombinant vector and the target gene recombinant vector into CHO cells, culturing and screening to obtain a CHO cell expression system; (3) culturing the transient and stable transfected CHO cell lines in suspension serum-free culture to obtain the target protein. The method of the present invention can significantly improve the expression level of the target gene in CHO cells and achieve transient and stable high-level expression of the target protein in CHO cells.

The invention discloses a preparation method of a DNA vaccine for preventing and treating echinococcosis multilocularis. The preparation method comprises the following steps: (1) preparing a nucleic acid vaccine plasmid (pVAX1T / B); (2) synthesizing chitosan selenium nano particles (CS-SeNPs); (3) obtaining a stable transfection cell strain for expressing a DEC 205 antibody on the surface of a cell membrane through a cell membrane display technology, crushing the cell through an ultrasonic crusher, and then separating through a density gradient centrifugation method to obtain cell membrane nano-vesicles; (4) mixing the pVAX1T / B obtained in the step (1) with the CS-SeNPs obtained in the step (2) under the condition of room temperature, so as to obtain a chitosan selenium nanoparticle loaded nucleic acid vaccine compound (pVAX1T / B coated CS-SeNPs); and (5) carrying out ultrasonic mixing on the cell nano-vesicles extracted in the step (3) and the pVAX1T / B-coated CS-SeNPs obtained in the step (4), and carrying out membrane extrusion on the cell nano-membrane vesicles so that the pVAX1T / B-coated CS-SeNPs are entrapped by the cell nano-membrane vesicles, thereby obtaining the chitosan selenium nanoparticles (NVs pVAX1T / B-coated CS-SeNPs) entrapped by the cell membrane vesicles. The DNA vaccine can be used for preventing and treating alveolar echinococcosis.

The invention discloses an application of a construction method of a novel stably transfected cell pool in degradation research of a virulent pathogen. Belongs to the technical field of stable transfection cell pool application, and the application can be used for carrying out virulent pathogen research in a low-grade laboratory or an unprotected environment, so that the research and development cost is reduced, and the application value and strategic value are further improved.

Provided herein systems and methods for processing cryopreserved cells without an expansion phase after thawing, including transfecting cells for gene editing purposes and cryopreserving transfected cells. Systems and methods for high-throughput processing of frozen cells are also provided. The systems and methods provided can be used to modify cells, including gene editing, in cell arrays.

The invention discloses a preparation method of a CA125 recombinant antibody, which comprises the following steps: electrically transferring a plasmid DNA fragment of a recombinant expression CA125 antibody into a CHO-GS cell by adopting a stable transfection technology, carrying out pressurized screening by using MSX to obtain a positive cell strain of a high expression CA125 antibody, establishing a positive cell pool, detecting the expression of the antibody, and carrying out subcloning on the positive cell strain to obtain a single cell line. Finally, a cell strain which can specifically and highly express the CA125 recombinant antibody and is qualified in stability is obtained, and a production process of the CA125 recombinant antibody is established. Compared with conventional CHO expression, the expression quantity of the recombinant antibody can be remarkably improved, and the recombinant antibody can be applied to high-yield expression of various recombinant antibodies, can be suitable for various sources and has wide application value.

The invention discloses a human renal tubular epithelial cell immortalized cell strain as well as a construction method and application thereof, and belongs to the technical field of cell biology. In the process of constructing the renal tubular epithelial cell immortalized cell strain, a high-quality renal cortex sample is firstly obtained, high-purity primary cells are separated out, the cell activity and proliferation are guaranteed in the culture process, the problem of cell heterogeneity caused by traditional manual operation is solved, and a foundation is laid for subsequent research. The infection efficiency is improved by optimizing virus infection conditions, and stable transfection is ensured in combination with optimal BSD concentration screening. According to the construction method disclosed by the invention, the virus infection efficiency and the gene integration stability are remarkably improved, the immortalized cell strain capable of infinitely proliferating is successfully obtained, the problem that primary cell amplification is limited is solved, and a cell source with stable functions is provided for the bioartificial renal tubule.

The invention discloses a cell line for stably expressing porcine delta coronavirus S protein, a porcine delta coronavirus IgA antibody detection antigen and a kit. The method comprises the following steps: based on an S protein sequence of a CH / XJYN / 2016 strain, selecting an extracellular domain part D20-N1077, adding a human source septoglobin family 1D meber 1 signal peptide sequence MRLSVCLLLTLALCCYRANA to an amino terminal of the S protein, adding a 6 * His tag sequence to a carboxyl terminal, carrying out codon optimization according to a CHO expression system, and constructing into a pCDNA3.1 (+) eukaryotic expression vector to obtain a recombinant eukaryotic expression plasmid pcDNA3.1 (+)-PDCoV-S; then transfecting CHO cells with the plasmid, and carrying out two rounds of heredity mycin G418 pressurized screening, limited dilution cloning and continuous subculture to obtain an expiCHO-PDCoV-S stably transfected cell line through screening; and finally, constructing the PDCoV IgA antibody detection kit based on the cell line. The kit has no cross reaction with other common porcine disease virus positive serum, has intra-batch and inter-batch variation coefficients of less than 10%, has good specificity, repeatability and sensitivity, and can be used for early prevention and control of PDCoV.

The invention discloses a human lung cancer fibroblast stably transfected strain for knocking down PTPN11 and a construction method thereof. The construction method comprises the following steps: obtaining an in-vitro human lung cancer tissue operation specimen, and dissociating primary lung cancer tissue; carrying out digestion separation and purification to obtain primary human lung cancer fibroblasts; inoculating into a 24-pore plate for subculture, and transfecting human lung cancer fibroblasts with lentivirus containing PTPN11 target gene fragments according to the use amount of MOI 50; puromycin with the concentration of 4.0 mu g / mL is used for screening the transfected human lung cancer fibroblast to obtain a human lung cancer fibroblast stable transfected strain with PTPN11 knocked down, and the preservation number of the human lung cancer fibroblast stable transfected strain is GDMCC No: 64707. Compared with the prior art, the human lung cancer fibroblast stable transfection strain knocking down PTPN11 can be rapidly and efficiently constructed, and the human lung cancer fibroblast stable transfection strain can be applied to models of PTPN11 genes of fibroblasts in a lung cancer tumor microenvironment and downstream signal channels of the PTPN11 genes and basic and clinical researches of malignant clone evolution, pathogenesis, drug resistance mechanisms and biological targeted drugs.

The invention relates to application of RNA (Ribonucleic Acid) binding protein SAMD4A or a coding gene thereof in differentiation from stem cells to myocardial cells. The importance of SAMD4A in the myocardial cell differentiation process is elaborated by inducing human embryonic stem cells to differentiate into myocardial cells, and in the myocardial directional differentiation process, SAMD4A expression is gradually up-regulated; an SAMD4A knock-down and overexpression plasmid is constructed, a human embryonic stem cell is infected by lentivirus to establish an SAMD4A knock-down and overexpression stable transfection cell line, and the key effect of the SAMD4A on myocardial differentiation is explored; the function of the FGF2mRNA specifically combined with the SAMD4A is explored; and a molecular mechanism that SAMD4A targets FGF2mRNA to influence myocardial cell differentiation is explored. The SAMD4A has important significance in specific high expression in myocardial cells and differentiation and function of the myocardial cells, and plays a key role in inducing stem cells to differentiate into the myocardial cells.

The application discloses a monoclonal cell strain screening method based on fluorescence intensity, and the method comprises the following steps: determining a cell line, and stably transfecting cells of the cell line with fluorescent proteins; selecting a microwell plate and setting a reference hole, and separating the cells stably transfected with the fluorescent proteins into the microwell plate, wherein the reference hole is filled with multiple cells, and the remaining holes are filled with only one cell; screening single-cell holes with only one cell in the remaining holes except the reference hole; screening clone group holes capable of growing clone groups; transferring the cells in the clone group holes to new holes for culture after being respectively digested; screening fluorescence screening holes; screening division and proliferation holes; and determining the cells in the division and proliferation holes as screened monoclonal cell strains. The monoclonal cell strain screening method provided by the application can screen monoclonal cell strains with strong fluorescence intensity and normal proliferation speed, is convenient and fast compared with fluorescence microscope observation and imaging, and also reduces errors possibly caused by human eye comparison of fluorescence intensity.