Hi there! Was this particular kit validated for use with rat plasma? Did you have a known positive control on the plate with your samples? If so, did it come out ok? Is the color development proportional or inversely proportional to insulin concentration for the kit you used? If inversely proportional, perhaps your samples were much too concentrated and needed to be diluted? Lots of tricky things with ELISAs! Let me know if you have any further questions - I'd be happy to help!

Are you familiar with insulin oral rout problems?

Take it (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362257/)

And absolutely search more.

Regards.

I think immunoassays in general could be problematic in quantification. If blood glucose is affected as expected, then probably you have no problem with your preparation, so Danielle Dillon 's question are very important here.

Amended on 4 November 2019

I must sadly declare that Immunological methods such as notorious RIA and ELISA are all non-quantitative at all. Thus, peptide/protein determination should be performed via high-recovery RP-HPLC-photometric and HPLC-Surfactant-SEC-photometric methods using UV 210, 254, and/or 280 nm detection (please see files; Lysozyme by RP-HPLC and HPLC-Surf-SEC protein determination method).

By the way, I have recently found that Insulin and Insulin receptor and Glucagon and Glucagon receptor are absent in the Humans, although they may be present in Pig, Cattle, Sheep, Dog, Chicken, and Platypus (my unpublished observation). Humans seems to have no genes of these proteins. This is a representative Species Differences.

Chemical structure of Insulin/INS has been estimated by utilizing non-reliable or low recovery Sanger method (using 2,4-Dinitrofluorobenzene/DNFB), and non-reliable chemical structures have been published. N-Terminal sequence of Insulin B chain of Ornithorhynchus anatinus (Platypus) is FPNQH-, and Human serum biotinidase cannot process or metabolise it (please see file; Enk serBIN). Further, Putative Human Insulin is a molecule having internal Cys-Cys bond within 6 amino acid from the N-terminus, which cannot be handled with by Human serum biotinidase. Or, Humans may have no putative human Insulin/INS precursor gene.

Oxytocin/OXT, Vasopressin and Calcitonin are absent in Humans. These peptides have intra-chain Cys-Cys bond within the 6 amino acid from the N-terminus.

However, Vasopressin V1a receptor/Antiuretic hormone receptor 1a is surely present in Humans, and this name should be changed to Adrenalin/NO receptor.

Oxytocin receptor/OT-R is present in Humans, and this receptor's name should be changed to Cocaine/Olfactory receptor.

Calcitonin receptor/CT-R is also present in Humans, and this protein's name should be changed to Amyloid-beta/SAP receptor. Non-cancer humans has Amyloid beta A4 protein isoform a (with Ubiquitin) and Serum amyloid P-component/SAP (please see file; The Fascio effect).

The Insulin receptor precursor of Plutella xylostella (diamondback moth) has N-terminal sequence of MPDTYYLYAR- , which cannot be handled by Human serum biotinidase/hBIN. Therefore, Humans does not have putative human Insulin receptor gene.

Chemical structure of Glucagon has been estimated by utilizing non-reliable or low recovery DNS method (using Dansyl chloride/5-(Dimethylamino)naphthalene-1-sulfonyl Chloride), and non-reliable chemical structures have been published. Cloning method using cDNA is also non-reliable. N-Terminal sequence of Glicentin-related polypeptide/GRPP of Glucagon preproprotein isoform 2 of Gallus gallus (chicken) is NPLQD-, and Human serum biotinidase cannot process or metabolise glucagon preproprotein isoform 2 (please see file; Enk serBIN). Or, Humans may have no putative human Glucagon preproprotein gene.

Further, glycoprotein hormone of Erythropoietin/EPO is also not present in Humans.

It's N-terminal sequence is APPRL-, which can not be hydrolyzed by Human serum biotinidase. Erythropoietin/EPO has not been found in our Human-protein database derived via PDMD method.

As a reference, the Species Differences in Biotinidase has already been reported by us (please see file; J Chrom B Rat BIN LIP Km).

Furthermore, protein amino-acid-sequence problemn has been studied, since ELISA and RIA should obey the protein structures.

We have surely isolated or purified Human serum biotinidase from Human blood, and protein sequencing has been performed. The result is indicating the presence of many amino acids at the many positions of sequence (please see files; JMBT Alopecia and Structure BIN (Hayakawa K, Yoshikawa K, and Watanabe T, Vitamin (Kyoto) (1994) 68; 318-320, written in Japanese)). This result indicates that proteins are synthesized by reversed reaction of many proteases (firstly suggested by German-American Biochemist Dr. Hans Neurath), and are not synthesized from one unique DNA sequence. My Human protein database suggests that proteins and/or enzymes for protein synthesis, such as ribosomal proteins, amino acyl-tRNA synthetases, and ATP synthetases, are not abundant. By the way, I have compared the validity between DNA methods (such as cloning, hybridization, PCR, and DNA sequencer) and Edman degradation method (protein is covalently bound onto Glass fiber discs). Human serum biotinidase (Fetal-Inflammatory biotinidase) has been sequenced as in file "Fetal-BIN Cole". If the result of Edman-degradation is true, the result obtaind from DNA technology gives ca. 30% of errors (my unpublished new observation).

Further, I have compared the sequences of beta-Defensin of Cattle between the sequences obtained from cDNA cloning method and Protein-Purification-Edman-degradation method by utilizing the database of PubMed.

Sequence from cDNA method was SAGS NPQSCRWNMGVCIPISCPGNMRQIGTCFGPRVPCCRRW

Sequence from Protein sequencing method was SGIS GPLSCGRNGGVCIPIRCPVPMRQIGTCFGRPVKCCRSW

If sequence of Protein-Purification-Edman-degradation method is true, a 12 /42 = 29% of errors have been occurred in cDNA cloning method.

Human beta-Defensin sequence reported by Gene estimation method by PubMed is

DRCT KRYGRCKRDC LESEKQIDIC SLPRKICCTE KLYEEDDMF, and this sequence cannot be handled with by Human serum biotinidase.

Human beta-Defensin 1 sequence reported by Gene estimation method by Uniprot is DHYNCVSS GGQCLYSACP IFTKIQGTCY RGKAKCCK, which has N-terminal amino acid Asp/D and intra-chain Cys-Cys bond at 5th amino acid from N-terminus. This sequence also cannot handled with by Human serum biotinidase/hBIN, and this sequence seems to be absent in Humans.

Thus, I must sadly declare that "beta-Defensin is absent in Humans", although "beta-Defensin is present in the Cattle". Interestingly, alpha-Defensin seems to be present in Humans. Further, I must sadly declare that "beta-Lactoglobulin, α- S2 Casein, beta-Casomorphin, and Rennin/Chymosin are absent in Humans", although they are present in the Cattle.

Therefore, I am grateful to Miss Büşra Arpaç, BPharm (Department of Pharmaceutical Biotechnology, Ankara University, Ankara, Turkey) for leading me to these interesting and important findings.

By the way, regulation of glucose metabolism via Human Kidney Biotinidase in Humans has been researched (please see file; Dr. Terentyeva Urine BIN).

Humans has Insulin-like growth factor 1/IGF-I/Mechano growth factor/MGF/Somatomedin-C, Insulin-like growth factor binding-protein 3/IGFBP-3/IBP-3, Insulin-like growth factor-binding protein 5/IGFBP-5/IBP-5, Insulin receptor substrate 2/IRS-2, Growth hormone receptor/GH receptor/GHR, Somatoliberin/Growth hormone releasing hormone/GHRH/Growth hormone-releasing factor/GRF, Growth hormone-releasing hormone receptor /GRFR/GHRH receptor, Epidermal growth factor receptor/Proto-oncogene c-ErbB-1, Epidermal growth factor-like 2/Flamingo homolog 3/Multiple EGF-like domains protein 3, Pro-epidermal growth factor /Urogastrone/EGF, Fibroblast growth factor 13/Fibroblast growth factor homologous factor 2, Fibroblast growth factor receptor 1/Basic fibroblast growth factor receptor 1/BFGFR/Proto-oncogene c-Fgr/CD331, Fibroblast growth factor receptor 2/FGFR-2/Keratinocyte growth factor receptor/CD332, Somatostatin/Growth hormone release-inhibiting factor, Alpha-2C-1 Adrenergic receptor, subtype C4, Beta-3 Adrenergic receptor, and Prolactin receptor/PRL-R.

IDDM and/or DM seems to be occurred by Mumps virus/MuV (kind personal information from Dr. Ayako Tanae, M.D.; retired Department of Endoclinology and Metabolism, National Children's Hospital, Taishido, Setagaya-ku, Tokyo, Japan).

Liver tissue of Pseudo-liver cancer has Nucleoprotein/Nucleocapsid protein/Protein N (Mumps virus/MuV) at 1.8 μg/mg of tissue protein. This MuV seems to up-regulate 14-3-3 Protein Eta/Protein AS1, which binds to IRS-2, at 1.73 μg/mg of tissue protein.

Hepatoma HepG2 has Nucleoprotein/Nucleocapsid protein/Protein N (Mumps virus/MuV) at 0.06 μg/mg of cell protein. This MuV seems to up-regulate Glutathione synthetase at 0.055 μg/mg of cell protein.

Healed hepatocyte HepG2 (cultured with fucoidan for 3 days) has Protein L/RNA-directed RNA polymerase L (Mumps virus/MuV) at 2.2 μg/mg of cell protein. This MuV seems to up-regulate Glucocorticoid receptor/GR at 2.2 μg/mg of cell protein.

Dr. Ömer Kalayci (Pediatric Allergy and Asthma Unit, Hacettepe University School of Medicine, Ankara, Turkey) has published a nice review about "high ratio for GSH/GSSG is important for the protection of the cell from oxidative damage". Thus, I have been repeatedly saying that "Addition of Sulphurous acid/ Sulphite in Wine is very dangerous, and we should drink Additive-free Wine". Therefore, I have searched for the Human proteins linked to Oxidative stress.

Humans does not have Immunoglobulin E/IgE. Human IgE has N-terminal sequence D-, and Human serum biotinidase cannot metabolise it. Human serum biotinidase can not hydrolize GSH and GSSG, but Lipoamidase can hydrolize them (please see file; Multiple hydrolase LIP).

Humans has following proteins; i.e., Hypoxia-inducible factor 1 alpha, Mitogen-activated protein kinase kinase kinase 5/MEK kinase 5/MAPK/ERK kinase kinase 5, Mitogen-activated protein kinase kinase kinase 9 /Mixed lineage kinase 1/MAP3K9, Mitogen-activated protein kinase kinase kinase 15/MEK kinase 15, MAP (Mitogen-activated protein) kinase-activating death domain protein/MADD/Insulinoma glucagonoma clone 20, Tyrosine-protein kinase receptor ETK1/Ephrin type-A receptor 3, Mixed lineage kinase domain-like protein, Serine/threonine-protein kinase Chk2, Microtubule-associated serine/threonine-protein kinase 3/MAST3, Microtubule-associated serine/threonine-protein kinase 3, Tyrosine-protein kinase BTK/Bruton tyrosine kinase, Tyrosine protein kinase Lyn/Lck/Yes-related novel protein tyrosine kinase, Tyrosine-protein kinase ITK/TSK/T-cell-specific kinase/Tyrosine protein kinase LYK, G protein-coupled receptor kinase GRK5, Serine/threonine-protein kinase A-Raf/Proto oncogene Pks2, Tyrosine-protein kinase ABL1/Proto-oncogene c-Abl, Receptor tyrosine-protein kinase erbB-2/Proto-oncogene Neu, Casein kinase II, beta chain/Phosvitin, Serine/threonine-protein kinase B-raf/B-Raf proto-oncogene serine/threonine-protein kinase/P94,Receptor of activated protein C kinase 1/Guanine nucleotide-binding protein beta subunit-like protein, Connectin/Titin (marker of adult), Transcription factor HES-4, Transcription factor Dp-1, Transcription factor HIVEP3, Transcription factor AP-4/Class C basic helix-loop-helix protein 41, Transcription factor jun-D, Transcription factor-7/T-cell-specific transcription factor 1/TCF-1, Interleukin-32/Natural killer cells protein 4/IL-32/Tumor necrosis factor alpha-inducing factor, Interleukin-4/IL-4/B-cell stimulatory factor 1/Binetrakin/Lymphocyte stimulatory factor 1/Pitrakinra, Interleukin-1 alpha/IL-1 alpha/Hematopoietin-1, Interleukin-7 receptor alpha chain/IL-7RA/CD127, Interleukin-21/IL-21, Tumor necrosis factor receptor superfamily member 1A/p55/p60/TNF-R1/CD120a/Tumor necrosis factor receptor 1/Tumor necrosis factor receptor type I, TNF-alpha-stimulated ABC protein/ATP-binding cassette sub-family F member 1, Tumor necrosis factor (TNF) receptor superfamily member 8/CD30L receptor/Ki-1 antigen, TNF-related activation protein/CD40 ligand/T cell antigen GP39, and TNF receptor-associated factor 2/Tumor necrosis factor type 1 receptor-associated protein 2/26S proteasome non-ATPase regulatory subunit 2/Protein 55.11.

I am now considering that GSH is not related to the detoxification mechanism against heavy metals such as Hg, toxins, poisons, and venoms. Resistance to mercuric ions is occurring via binding reaction of hydrophobic membrane proteins (please see file; Resistance to Hg).

One famous mechanism against Strychnine and Brucine (Glycine receptor binding) is the Mithridatism (Mozart wrote a famous Opera "Mitridate, Re di Ponto"), which seems to be similar to Immunological idea such as Vaccine.

Furthermore, I would like to report about Insulin-degrading enzyme/Insulinase/IDE, Angiotensin-converting enzyme/ACE, and Angiotensin-converting enzyme 2/ACEH/Metalloprotease MPROT15, which are present in Humans, though their substrates of Insulin and Angiotensin are absent in Humans.

Human Insulin-degrading enzyme/Insulinase/IDE may be working on Transforming growth factor-alpha/TGF-alpha and EGF, and the name of IDE should be changed to EGF-TGF-alpha degrading enzyme.

Human Angiotensin-converting enzyme/ACE, and Angiotensin-converting enzyme 2/ACEH/Metalloprotease MPROT15 may be working on Amyloid-beta and Dynorphin, and the name of ACE should be changed to Dynorphin-Amyloid-beta converting enzyme.

I have further found that Humans does not have alpha-L-Fucosidase, which can hydrolyse Fucoidan of Brown Algae.

alpha-L-Fucosidase [Formosa algae; Flavobacteriaceae] has N-terminal sequence of DYM KVADDFNPSK-. Human serum biotinidase/hBIN can not hydrolyse this sequence, and Humans does not have alpha-L-Fucosidase. Therefore, Fucoidan has been taken up intactly via Humans' Gut, and excreted into the human urine as intact form (please see J. Chrom. B, 997 (2015) 187-193 and the file; JCB Fucoidan transport).

I agree with Iman Hassan Ibrahim and Kou Hayakawa . The ELISA is extremely sensitive for qualitative determination of whether a protein is present, but extremely poor at quantitating it. Too many factors can make the signal response disproportional to the amount of target protein.

I suppose you mean a kit that had been functioning well until recently. That will mostly be a storage problem. When the storage temperature keeps fluctuating and the kit goes through a series of freeze-thaw cycles. It definitely will fail.

Further amended on 29 October 2019

Humans has Lipoyl synthase, mitochondrial gene. This enzyme-protein is detected in normal Fetal hepatocyte Hc and Healed hepatocyte HepG2 (cultured with fucoidan for 3 days). Interestingly, hepatoma HepG2 (cultured without fucoidan) does not express this enzyme. Other adult liver tissues (n=5) and human serum (n=13) do not express this enzyme. Therefore, Lipoic acid is the vitamin of adult Humans.

Rare expression of Lipoyl synthase, mitochondrial gene seems to be induced by HCV, and to be repressed by GB virus C/GBV-HGV/GBV-C, Hog cholera virus/Classical swine fever virus/CSFV, Feline infectious peritonitis virus/FCoV, Bean yellow mosaic virus, Turnip yellow mosaic virus, Human coronavirus/HCoV, Murine coronavirus/Mouse hepatitis virus/MHV, Japanese encephalitis virus/JEV, and/or Porcine transmissible gastroenteric coronavirus/TGEV/PCoV.

By the way, I have further confirmed that the Collagen alpha-1(XV) chain/Restin is induced by the Epstein-Barr virus/EB virus/EBV/HHV-4.

Cultured adult human-cancer cell lines of HepG2 (Human liver cancer; 15y, male Caucasian), HuH-7 (Human liver cancer; 57y, male, Japanese), T24 (Human urinary bladder carcinoma; 82 y, female; Swedish; Caucasian), and 5637 (Human urinary bladder carcinoma; 68 y, male; Caucasian) showed a growth promoting effect by adding the growth factor of Lipoic acid (at 0.05 mg/mL). On the other hand, Rat cells of TRL-1215 (Liver cells; Fetal or spontaneously infected by some Murine virus) and NBT-T2 (Adult rat; Urinary bladder carcinoma) showed a toxic effect by Lipoic acid. Rats seem to biosynthesize Lipoic acid, and Lipoic acid is not the vitamin of Rat. This is also a representative Species Differences. Addition of Oxidized Lipoic acid to cultured Rat cells, which biosynthesize reduced-form of Lipoic acid, seems to induce the low ratio for GSH/GSSG and increase the oxidative damage in Rat cultured cells. Adult Humans cannot biosynthesize Lipoic acid and Fetal Humans can synthesize Lipoic acid, but toxic effect of Oxidized-form Lipoic acid does not occur in Human cell lines.

Humans may have Biotin synthase, mitochondrial gene, although it has not yet been discovered. The biosynthetic productions of Biotin/vitamin H in Human cancer HeLa cells and Human fetal hepatocyte Hc cells have been reported (please see files; Keranen Biotin and The Fascio Effect). Therefore, thiol-type Human serum biotinidase handles both with Biotin-amide and with Lipoic acid-amide (please see files; serum LIP and BIN-LIP). The addition of Biotin/vitamin H to culture medium at 5.0 μg/mL showed a remarkable growth-acceleration effect onto Hc, HuH-7, HuH-6, HepG2, Caco-2, JMSU1 (Human urinary carcinome; 75 y, male, Japanese or Mongoloid), HEK (Human Embryonic Kidney; female?)-293T (transformed by SV40), and rat TRL1215 (10 days young rat's liver; Fetal or Transformed/Infected) (our unpublished observation; as to Caco-2 cells, please see file; JCB Fucoidan transport). The addition of Biotin to culture medium seems not to induce the Oxidative stress and/or changing of GSH/GSSG ratio.

Thus, Human IDDM and/or DM is linked to Biotin and/or Lipoic acid which are present in Humans as Vitamins, but does not link to Insulin and Glucagon which are absent in Humans (please see file again; Dr. Terentyeva Urine BIN).

By the way, Humans, Haplorhini (Apes), Sparrow, Guinea pig/Cavia porcellus, and Bat have no L-Gulonolactone oxidase/GLO/Vitamin C synthase, and these animals and Humans cannot synthesize Vitamin C.

L-Gulonolactone oxidase/GLO/Vitamin C synthase of Gossypium arboreum (Cotton plant) has N-terminal sequence of MPAIP-, which cannot be handled with by Human serum biotinidase. Or, Humans may have no putative human Vitamin C synthase gene.