The current best method of measuring Human Chorionic Gonadotropin is ElLIZA immunoassay method. Most ELIZA tests employ a monoclonal antibody, which is specific to the β-subunit of hCG (β-hCG). This is high specific method in which ensure that tests do not make false positives by confusing hCG with LH and FSH.

Good luck.

There are several types of immunoassay for this hormone, and they work in serum and urine , so there is no expected problem with media supernatant. Immunoassay are usually sensitive.

But if the glycosylation status of the hCG is a concern you should be careful with methods selection.

Structure of Human Chorionic Gonadotrophin (hCG):

The structure of human chorionic gonadotrophin (hCG) is so similar to that of luteinizing hormone (LH) that a variety of assay techniques have been devised to differentiate between these two hormones.

What should be measured in an bCG assay?

· Intact hCG

· Free β subunit of hCG

· Alpha subunit of hCG

· Beta core of hCG

· Other hCG"fragments

· Combination of intact hCG, free β subunit and β core fragment

Assays for hCG:

The first assays for hCG were based upon bioactivity of the molecule when injected into animals. More recent bioassays have been devised that rely on in vitro stimulation of steroidogenesis from gonadal cells. As neither of these groups of assays are used in routine laboratories they will not be considered further. The first reliable in vitro qualitative tests of pregnancy were based on haemagglutination inhibition assays of urine hCG and subsequent modifications have led to specific and sensitive qualitative hCG assays.

Quantitative assays have been widely used for the measurement of hCG in serum and are usually based on RIA or IRMA methods. The large number of antibodies and different techniques often leads to confusion as to the most appropriate assay for a laboratory.

Amended on 24 October 2019

I have to sadly declare that Follicle stimulating hormone/FSH, Gonadotropin releasing hormone/GnRH/Luteinizing hormone releasing hormone/LH-RH, Luteinizing hormone/LH, Chorionic gonadotrophin/hCG, Thyroid-stimulating hormone/TSH, Activin, Inhibin, and Erythropoietin/EPO are absent in Humans (please see files; HepG2 Fucoidan and The Fascio Effect). These hormones are resistant to be hydrolised by the Human Serum Biotinidase (please see file; Enk serBIN). Therefore, Human Serum Biotinidase, which is richly present in human blood, is surely working in the Metabolism of Human peptide hormones in the Human blood.

This determination method for presence or absence of proteins and peptides in Humans is depended on the substrate specificity of Human Serum Biotinidase/BIN; i.e., Human Serum Biotinidase can not hydrolyze or metabolize these N-terminal structures, (1) XP- , (2) pyrE- and pyrD- , (3) D- , (4) X-D-Amino acids- such as X-D-Ala- , (5) Acetyl-X-/AcX- (X is not Ala, Met, and Ser; in non-cancer tissues), and Formyl-X-/fX-, and (6) molecules, which have internal or intra Cys-Cys bonds (within 6 position from N-terminal) such as Insulin and Avidin (please see again the file; Enk serBIN). Further, Humans has Lipoamidase/LIP, and it cannot hydrolyze the chemical structure of Arg-Glu/RE and Arg-Asp/RD (please see file; Multiple hydrolase LIP). Lipoamidase/LIP can hydrolyze anchoring structure of Myristoyl-X/MyrX- at N-terminal (please see file; Release of Anchored). These characteristics of LIP have also been utilized to determine the absence of proteins in Humans.

Bee venom Melittin of Apis mellifera (Honeybee) has N-terminal Formyl-GIGA- or f

GIGA-, and Human serum biotinidase cannot hydrolyze it.

Frog's neuropeptide of Dermorphin has N-terminal Y-D-Ala-F-G-Y-P-Samide, and Human serum biotinidase cannot hydrolyze it.

Snake venom Metalloproteinase BaP1/Hemorrhagic metalloproteinase BaP1 of Bothrops asper (Terciopelo) has N-terminal pyrEQRFS-, and has internal Cys-Cys bonds. Therefore, Human serum biotinidase cannot hydrolyze it.

Phospholipase A2 of Micrurus mipartitus (Red-tailed coral snake) has N-terminal amino acid D- and/or internal Cys-Cys bonds (with molecular mass less than 14,000), and it cannot be hydrolyzed by Human serum biotinidase.

On the other hand, Cytosolic phospholipase A2 of Humans has no Cys-Cys bonds, and has N-terminal sequence of MpiSFID-. Therefore, Human serum biotinidase can hydrolyze human Cytosolic phospholipase A2.

Platelet-activating factor (PAF) acetylhydrolase 2, cytoplasmic/Serine-dependent phospholipase A2/SD-PLA2/hSD-PLA2 of Humans has N-terminal sequence of Myrstoyl-GVNQS- or Myr-GVNQS- , which cannot be hydrolyzed by Human serum biotinidase. However, such an anchored membrane protein can be hydrolyzed by Human lipoamidase, and hSD-PLA2 is surely present in Humans (please see the file; Release of Anchored).

Toxin peptides Haplotoxin-2/Beta/kappa-theraphotoxin-Hlv1a of Cyriopagopus lividus (Cobalt blue tarantula) (Haplopelma lividum) and Phrixotoxin-3/Kappa-theraphotoxin-Ps1a/Kappa-TRTX-Ps1a of Paraphysa scrofa (Chilean copper tarantula) (Phrixotrichus auratus) have internal Cys-Cys bonds (with molecular mass less than 14,000), and Human serum biotinidase can not hydrolyze them.

Thus, Humans has no Bee venom proteins, Snake venom proteins, and Spider venom proteins.

Humans has Acylamino-acid-releasing enzyme/Acyl-peptide hydrolase/APH gene, which cleaves AcAla-, AcMet, and AcSer. APH is expressed HCC tissue (with PBC) and Pseudo-liver cancer. APH seems to be induced or expressed by Human cytomegalovirus/HHV-5. Protein-expression phenomena seem to be under the control of virus. Thus, human Hepatocellular carcinoma/HCC is surely induced by the HIV-1 and HCV (please see file again; The Fascio effect).

The human proteins, which can be hydrolyzed or metabolized by Human serum Biotinidase, are considered to be present in Humans. Further, the results of PDMD (Protein-Direct-Microsequencing-Deciphering) method are also utilized.

A glycoprotein hormone of Erythropoietin/EPO is also not present in Humans. It's N-terminal sequence is APPRL-, which can not hydrolyze by Human serum biotinidase. It does also not been found in our protein database derived by PDMD method.

Follicle stimulating hormone/FSH has N-terminal sequence of DLVYK-, which Human serum biotinidase can not handle with.

Gonadotropin releasing hormone/GnRH/Luteinizing hormone releasing hormone/LH-RH has N-terminal sequence of pyrEHWSY-, which Human serum biotinidase can not handle with.

Chorionic gonadotrophin subunit alpha/Luteinizing hormone alpha chain/Thyroid-stimulating hormone alpha chain has N-terminal sequence of APDVQ-, which Human serum biotinidase can not handle with. By the way, Humans seems to utilize Plancental-specific 1-like protein/Placenta-specific protein 1 instead of hCG.

Human growth hormone and Human prolactin are surely present in Humans. It is noteworthy that both Human growth hormone and Human prolactin have been purified from Human autopsies (pituitary glands) by Chinese-American Biochemist Dr. Chou Hao Li (University of California, Berkeley, CA, USA). Human growth hormone has N-terminal sequence of MATGSRTSLL- , which can be hydrolyzed by Human serum biotinidase. Human prolactin have N-terminal sequence of MNIKGSPWKA GSLLLLLVSN LLLCQSVA / PLPI- (presented by Dr. Hiraoka Y., Dept. of Microbiology, Keio University School of Medicine, 35 Shinanomachi, Shinijuku-ku, Tokyo, Japan), and both precursor and possible mature chain can be hydrolyzed by Human serum biotinidase.

The above listed hormones, except Insulin, may be present in Anterior Pituitary and/or Hypothalamus of Sheep (Ovis aries) and/or Pig (Sus scrofa domesticus). Beta cells of Islets of Langerhans/Langerhans' islet/Pancreatic islets may not be present in Human Pancreas.

Further, I have found that Macrophage migration inhibitory factor/MIF and CD74 are absent in Humans, but may be present in Guinea pigs (my unpublished observation).

Furthermore, I have found that Activin and Inhibin are absent in Humans as estimated from Human serum biotinidase. Inhibin, beta A (Activin A, activin AB alpha polypeptide) has N-terminal sequence of SPTPG- , Inhibin beta A chain preproprotein has N-terminal sequence of SPTPG- , Inhibin beta B chain preproprotein has N-terminal sequence of SPTPP-, and Inhibin beta C chain proprotein has N-terminal sequence of TPRAG-, respectively. All these N-terminal sequences can not be hydrolyzed by Human serum biotinidase.

On the other hand, Activin receptor type-2A and Activin receptor type-2B are present in Humans. Activin receptor type-2A has N-terminal sequence of AILGR- and Activin receptor type-2B has N-terminal sequence of SGRGE-, respectively. These N-terminal sequences can be hydrolysed by Human serum biotinidase, and these receptors are surely present in Humans. Therefore, these receptors seem to be physiologically working as BMP receptor and Growth differentiation factor/GDF receptor. Then, the name of Activin receptor should be changed to BMP-GDF receptor.

This is the representative and famous Species Differences.

Furthermore, I must sadly say that notorious ELISA, RIA, MS, HPLC-MS, NMR, Direct-utilization of Photometer and/or Fluorometer without purification and Electrogenerated Chemiluminescence/ECL, Western blotting, Northern blotting, Southern blotting, and PCR are not quantitative methods at all. Immunological ECL (Electrochemiluminescence/ Electrogenerated chemiluminescence) method is also not quantitative.

Very interesting and important finding has previously been kindly informed from Dr. Chihiro Yabe-Nishimura, M.D., Ph.D. (National Children’s Medical Research, Center, Setagaya-ku, Tokyo; now Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto) that "Monoclonal antibody is less specific than Polyclonal antibody".

Therefore, protein/peptide quantitative analysis should be performed by our high-recovery RP-HPLC-photometric method (please see file; Lysozyme by RP-HPLC) and PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see files; HepG2 Fucoidan and JMBT Alopecia).

Kou Hayakawa dear sir i have not mentioned that Insulin, Luteinizing hormone (LH) is present at human chorionic gonadotrophin (hCG). I just mentioned the structure of human chorionic gonadotrophin (hCG) is so similar to that of luteinizing hormone (LH) that mean not present in hCG.

Thank you.

Further amended on 24 October 2019

I have further studied on Hormones and Proteins of Humans.

Thus, I have found that such Hormones and Proteins in addition to above mentioned Peptide Hormones are also absent in Humans; i.e., Pituitary adenylate cyclase-activating peptide/PACAP, Interleukin-6, Histamine, Histamine receptor, Leucokinin, Dermorphin, Fetuin, Transforming growth factor beta/TGF-beta, Insulin, Insulin receptor, Amylin, Mesothelin/Pre-pro-megakaryocyte-potentiating factor, Galectin-1, Ghrelin/Appetite-regulating hormone/Motilin-related peptide, Motilin, Orexin, Leptin/Obese protein/Obesity factor, Secretin, Gastrin, Gastric inhibitory peptide/Gastric Inhibitory Polypeptide/GIP/Glucose-dependent insulinotropic polypeptide, Glucose-dependent insulinotropic polypeptide receptor, Gastrin-releasing peptide/GRP, Neuromedin B-32, Neuromedin C (bombesin-like), Neuromedin K, Neuromedin L (tachykinin-like), Neuromedin N (neurotensin-like), Neuromedin U, Neuromedin S (NmU-like), Glucagon, Cholecystokinin/CCK/PZ, Kininogen-1, Kallikrein, Endothelin-1, Renin, Angiotensin, Thyroglobulin, Myoglobin, Immunoglobulin E/IgE, Avidin, β- Lactoglobulin, α- S2 Casein, Glyceraldehyde-3-phosphate dehydrogenase/GAPDH, Pyruvate carboxylase/PC, Acetyl-CoA carboxylase/ACC, Actin, Aldosterone, Testosterone, and Estrogen. Absence of these steroid hormones is estimated from the absence of receptors for these three steroid hormones. These upper results have been obtained by the specific and reliable proteomics of the PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see files; HepG2 Fucoidan and JMBT Alopecia).

Interestingly, no effect of Secretin to Humans has been recently reported (please see file; Human Secretin Autism).

Further, Human breast milk biotinidase (please see file; compa mBIN) is present in Human breast milk, but biotinidase is absent in Cow's milk (please see file; The Fascio effect). Biotinidase is present in many Animals including Chicken egg yolk and Insect of American cockroach (Periplaneta americana), but is absent in Human umbilical cord (without Umbilical cord blood）and Nematode (Roundworm) of Anisakis (our unpublished observation and please see file; J Chrom B Rat BIN LIP Km).

These clear results also are due to the famous Species Differences, though Racial Differences/Ethnic Differences among Humans has not yet been studied. It has been found by Dr. Claudio De Felice that the Excretion of Biotinidase into Human Urines has shown the presence of Racial Differences/Ethnic Differences (please see file; Race BIN Urine).

Furthermore, Human serum biotinidase (Thiol-type; purified from Japanese bloods; please see file, Thiol-type BIN) can hydrolyze Proctolin (neuropeptide present in insects and crustaceans, and possibly in Humans; RYLPT), Morphine-modulating neuropeptide (FMRFamide-related peptide; AGEGLSSPFWSLAAPQRF-amide), and Delta Sleep-inducing peptide/DSIP (this gene has not been found yet; WAGGDASGE), and those peptides may surely be present in the Humans. Therefore, these peptides including Kyotorphin (Tyr-Arg or YR) seem to be realy working in the Humans (please see file again; Enk serBIN). I consider that Pain and Sleep are common between Humans and Animals, and Opioid neuropeptides are similarly utilized among Humans and Animals. It is noteworthy that Humans have higher Cerebrum biotinidase activity than Pig, Rat, and Guinea Pig (please see file; Brain-BIN Pig). The Species Differences of the active center of Biotinidase between Bacteria (can not be inhibited by p-hydroxymercuribenzoate/PHMB) and Pig (Thiol-type; can be inhibited by p-hydroxymercuribenzoate/PHMB) has firstly indicated by the German Biochemists Drs. J. Knappe, W. Brümmer, & K. Biederbick (Knappe J, Brümmer W, Biederbick K. Reinigung und Eigenschaften der Biotinidase aus Schweinenieren und Lactobacillus Casei. Biochem Z 1963; 338: 599-613.).

It is noteworthy that Human serum biotinidase can not hydrolyze chemotactic factors derived from Bacteria (Formyl-MLF, Formyl-MF, Formyl-MFF-OMe; please see file; Enk serBIN). However, Humans has fMet-Leu-Phe receptor/fMLF receptor and fMLF-related receptor II/fMLF-R-II (please see file; The Fascio effect). Therefore, I am now considering that fMet-Leu-Phe receptor/fMLF receptor and fMLF-related receptor II/fMLF-R-II may have the hydrolyzing ability for ligands of Formyl-MLF, Formyl-MF, and Formyl-MFF-OMe, or Fetal-Inflammatory-biotinidase (please see file again; HepG2 Fucoidan) can hydrolyze Formyl-MLF, Formyl-MF, and Formyl-MFF-OMe.

Interestingly, Agrobacterium tumefaciens co-infected with Mycobacterium leprae seems to be able to hydrolyze Formyl-peptides; i.e., LC tissue with leprosy does not have the fMet-Leu-Phe receptor/fMLF receptor.

HCC and LC tissue of named No.6 have the DNA-directed RNA polymerase alpha chain of Salmonella enterica and proteins of various vira, and this liver have not fMet-Leu-Phe receptor/fMLF receptor. DNA-directed RNA polymerase alpha chains of Enterobacteria such as Escherichia coli O-157, Escherichia coli K-12, and Salmonella enterica have N-terminal sequence of fMQGS-, and Enterobacteria's amino-exopeptidase seems to be able to hydrolyze this N-terminal portion of this protein. Then, the presence of Salmonella enterica in the liver of No.6 can not up-regulate fMet-Leu-Phe receptor/fMLF receptor. Other bacterial DNA-directed RNA polymerase alpha chains have such N-terminal sequences of fML-, fMR-, fMI-, and fMS- (not fMQ-).

HCC tissue with PBC and liver of Pseudo-liver cancer have fMet-Leu-Phe receptor/fMLF receptor, and do not possess Enterobacteria.

Cultured liver cells such as Hc and HepG2 have no fMet-Leu-Phe receptor/fMLF receptor. Therefore, another cultured-cell lines also do not seem to express fMet-Leu-Phe receptor/fMLF receptor, and the culture medium should be added some antibiotics against Bacterial invation.

In summary, C-terminal portion and N-terminal portion of peptide hormones are strongly linked to physiological or biochemical activity and metabolic possibility or turn-over of Peptide Hormones, respectively.

Effects of Glyco-chains of Peptide Hormones and Hormone receptors seem to be also important. Changed Glyco-chain structures in Human serum biotinidase, which induce the changed substrate specificity and the changed thermo-stability, is the cause of Biotin Deficiency (please see file; Wide Range of Biotin).

Thus, possible sequence of Human Glycoprotein hormones alpha chain/Anterior pituitary glycoprotein hormones common subunit alpha/Choriogonadotropin alpha chain/Chorionic gonadotrophin subunit alpha/CG-alpha/Follicle-stimulating hormone alpha chain/FSH-alpha/Follitropin alpha chain/Luteinizing hormone alpha chain/LSH-alpha/Lutropin alpha chain/Thyroid-stimulating hormone alpha chain/TSH-alpha/Thyrotropin alpha chain (N-terminal APDVQ-), Callithrix jacchus (white-tufted-ear marmoset) Chorionic gonadotrophin subunit alpha (N-terminal LPDGE-), Pig sequence (N-terminal FPDGE-), and Sheep sequence (N-terminal FPDGE-) can not be hydrolyzed or metabolized by the thiol-type Human Biotinidase. Animals' biotinidases may have different substrate specificities from Human biotinidase; i.e., Bovine biotinidase seems to be able to hydrolyse β-Casomorphin (YPFPGPI), which human serum biotinidase can not hydrolyse.

I am grateful to Dr. Leilani Santos (Department of Medicine, Monash Medical Centre, Centre for Inflammatory Diseases, Monash University, Clayton, Victoria, Australia) for leading me to these important conclusions.

I have further considered the reason why some proteins are not present in Humans.

Avidin, Myoglobin, and GAPDH are not present in Humans.

Avidin gene of Humans' is not found in PubMed database, and Avidin protein is also absent in our human protein-database derived from PDMD method.

Avidin of Gallus gallus (Chicken) is a molecule having intra Cys-Cys bond with the the position at 4 amino acids from N-termnus, and it can not be hydrolysed or metabolized by Human serum biotinidase. Thus, the Egg-White disease expresses Skin Allergy. Healing effect by Biotin may be occurring via normalizing the membrane structure (please see again file; HepG2 Fucoidan). We have already found that chicken Avidin is a Lipoic acid/Biotin/Amino acid-binding protein.

Myoglobin of Rhincodon typus (whale shark) has N-terminal sequence DWENVNKV-, which can not be hydrolysed or metabolized by Human serum biotinidase.

However, mature chain of Haemoglobin subunit alpha has N-terminal VLpiS- and mature chain of Haemoglobin subunit beta has N-terminal VHL- sequences, respectively, which can be metabolised by Human serum biotinidase. Therefore, Humans surely has Hemoglobin A/HbA (heterotetramer; α2β2) instead of Myoglobin.

GAPDHs of Pigs and Rats have N-terminal sequence of VdimethylKVGVNGFG-, and which seems to be non-metabolisable by Human serum biotinidase. Therefore, GAPDH gene seems to be absent in Humans, and we cannot find GAPDH protein among our human-protein-database derived from PDMD method.

αS2-Casein gene and β-Lactoglobulin genes are not present in Humans. αS2-Casein is present in the PubMed only in Bovine, Horse, and Bubalus bubalis (water buffalo). β-Lactoglobulin gene of Humans is surely absent in the UniProt.

Interestingly, bovine β-Casomorphin (YPFPGPI) sequence, which can not be hydrolysed by Human serum biotinidase, is surely present in bovine β- Casein, but this sequence is absent in human β- Casein.

Interleukin-6 has N-terminal sequence of VPPGE-, and Human serum biotinidase cannot metabolise it.

Fetuin-A/Alpha-2-HS-glycoprotein chain A has N-terminal sequence of APHGP-, and Human serum biotinidase cannot metabolise it. However, N-terminal sequence Fetuin-B/Gugu has no such difficulty, and it may be present in Humans. However, Humans has Cystatin-SA/Cystatin-2/Cystatin-S5 or Cystatin-SN/Cystain-SA-I/Cystatin-1/Salivary cystatin-SA-1 or Cystatin-S/Cystatin-4/Cystatin-SA-III/Salivary acidic protein 1 instead of Fetuin-B/Gugu. Therefore, Fetuin has not been found in our Human-database as determined by the PDMD method.

Transforming growth factor beta/TGF-beta has N-terminal sequence of RPAAG-, and Human serum biotinidase cannot metabolise it. Interestingly, Transforming growth factor alpha/TGF-alpha has N-terminal sequence of VVSHF-, and Human serum biotinidase can metabolise it. Therefore, Transforming growth factor alpha/TGF-alpha or Protransforming growth factor (TGF) alpha is present in Humans, and it may bind either to EGF receptor 5/EGFR-5, Epidermal growth factor receptor/EGFR/Proto-oncogene c-ErbB-1/Receptor tyrosine-protein kinase erbB-1, or Receptor tyrosine-protein kinase erbB-2/Metastatic lymph node gene 19 protein/Proto-oncogene c-ErbB-2/Proto-oncogene Neu/CD340.

Actin, alpha skeletal muscle has N-terminal sequence D-, and Human serum biotinidase cannot metabolise it. Actin of Strongylocentrotus purpuratus (purple sea urchin) has N-terminal structure of VAALV-.

Immunoglobulin E/IgE has N-terminal sequence D-, and Human serum biotinidase cannot metabolise it.

Endothelin genes are obtained from notorious cDNA cloning method. Propeptide of Endothelin-1 is APETA-, which can not be metabolized by the Human serum biotinidase. Therefore, Endothelin-1 is absent in Humans. However, Endothelin-2 and Endothelin-3 are present in Humans, and Preproendothelin-2 and Preproendothelin-3 can be safely processed by Human serum biotinidase.

Kininogen-1 heavy chain has sequence of pyrEESQ-, Kallikrein-1 Activation peptide has sequence of PPIQ-, and Kallikrein-2 Activation peptide has sequence of VPLI-, and these proteins are not able to be handled with by Human serum biotinidase.

Renin has N-terminal D- and/or Activation peptide (24-) has LP-, and Human serum biotinidase cannot metabolise putative human Renin.

Thyroglobulin has N-terminal sequence of mature chain DIFEY-, and Human serum biotinidase cannot metabolise it.

Insulin/INS structure 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. Further, Insulin/INS is a unique hormone molecule having the internal Cys-Cys bond at 6th position from N-terminus, which Human serum biotinidase cannot metabolise. Or, Humans may not have putative human Insulin/INS precursor gene.

Amylin is absent in Humans due to similar reason to Insulin.

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 not have putative human Glucagon preproprotein gene.

Humans has no Gastrin. N-Terminal sequences of Gastrin-52, Big Gastrin and Gastrin of Humans are DLELP-, pyrELGPQ- and pyrEGPWL-, respectively, and Human serum biotinidase cannot process or metabolise them. Or, Humans may not have putative human Gastrin gene, although Pigs have Gastrin gene

Humans has no Gastric inhibitory peptide/Gastric Inhibitory Polypeptide/GIP/Glucose-dependent insulinotropic polypeptide. N-Terminal sequence of GIP seems to be pyrE-, and Human serum biotinidase cannot process or metabolise it.

Humans also does not have Glucose-dependent insulinotropic polypeptide receptor. N-Terminal sequence of precursor of this receptor is MPLR-, and Human serum biotinidase cannot process or metabolise this precursor.

Humans does not have Gastrin-releasing peptide/GRP. N-Terminal sequence of this peptide is VPLPA-, and Human serum biotinidase cannot process or metabolise this peptide.

Humans also does not have Neuromedin B, Neuromedin B-32, Neuromedin C (bombesin-like), Neuromedin K, Neuromedin L (tachykinin-like), Neuromedin N (neurotensin-like), Neuromedin U, and Neuromedin S (NmU-like). Nuromedins seem to be part of the GRP-related genes, and GRP-related peptides also do not metabolivcally produce these Neuromedins via Human serum biotinidase.

Humans has N-terminal sequences of Neuromedins as follows; i.e., Neuromedin B-32 has APLSWD-, Neuromedin C is on GRP peptide, Neuromedin K has DMHDF-, Neuromedin L has DADSSI-, Neuromedin N has IPYIL, Neuromedin U (NmU; Bombina maxima (large-webbed bell toad) has DSSGI-, Neuromedin U receptor has MPICA-, Neuromedin S has FPQPL-. Therefore, Human serum biotinidase cannot metabolise them. Or, Humans may have no Neuromedin-containing genes.

It is interesting that Toad, Frog, and Insect can do the metamorphosis, but Humans cannot.

Humans does not have Histamine receptor. N-terminal sequence of precursor is MP-, and Human serum biotinidase cannot process or metabolise this Histamine receptor.

Humans does not have Leucokinin and Dermorphin. N-terminal sequences of Leucokinin and Dermorphin are DP- and Y-D-Ala-F-, respectively, and Human serum biotinidase cannot process or metabolise these neuropeptides.

Pyruvate carboxylase/PC of Humans' mitochondria seems to have N-terminal

sequence of pyrEYKPIKKV- (according to Uniprot and PubMed), and Human serum biotinidase cannot metabolise it. Or, Humans may have no Pyruvate carboxylase/PC gene.

Acetyl-CoA carboxylase/ACC of Fungi of Crucibulum leaves has N-terminal sequence of precursor of MPEYDHSRVQ- (according to PubMed), and Human serum biotinidase cannot metabolise this sequence. Then, Humans may not have ACC, and the ACC has not been detected in my Human protein-database derived from our PDMD method.

Humans' mitochondria has N-terminal sequence of Propionyl-CoA carboxylase/PCC of MVSRNLGSVG-, which can be handled with by Human serum biotinidase.

Humans' mitochondria has N-terminal sequences of Methylcrotonyl-CoA carboxylase/MCC subunit alpha and Methylcrotonyl-CoA carboxylase/MCC subunit beta of TTATG- and YHGDS-, respectively, which can be handled with by Human serum biotinidase. However, bacteria of Pseudomonas sp. GM41(2012) has N-terminal sequence of Methylcrotonyl-CoA carboxylase/MCC of MPGLRKILIA-, which can not be handled with by Human serum biotinidase.

Therefore, symbiosis with bacteria (origin of mitochondria) has been occurred only with bacteria, which produce metabolizable protein by the Human serum biotinidase. Thus, Humans has only PCC and MCC.

Further added on 14 October 2019

γ-Glu-Gln and Glutathione (γ-Glu-Cys-Gly) seem to be present in Humans, since Pig Brain Lipoamidase (and putative Human Brain Lipoamidase) can hydrolyze these peptides, although human serum biotinidase can not (please see files; Purify Brain Lipoamidase and Multiple Hydrolase LIP).

Human cell has Acylamino-acid-releasing enzyme/Acyl-peptide hydrolase/APH in Cytoplasm, and can liberate N-acetyl and/or N-formyl amino acid from the N-terminus of a polypeptide. Therefore, Humans can metabolize Mitochondria-derived N-formyl polypeptide. Thus, Human cells possess the ability to hydrolyze N-terminal acetylated and/or formylated proteins/peptides. Then, Humans may have Leupeptine/Acetyl-Leu-Leu-Arginal (a peptidase inhibitor). Amino acid sequence at 297-299 of Human Arylsulfatase A/ASA/Cerebroside-sulfatase/Arylsulfatase A component A/Arylsulfatase A component C has sequence of Leu-Leu-Arg.

Only Metallothionein-1F is present in Humans. There is no report to purify Metallothionein-1F. Other Metallothioneins seem to be not present in Humans, although present in Horse; i.e., Signal peptide AcMDPNCS- may possibly be present. Then N-terminal sequence of Metallothionein-1F becomes to be CAAGV-, and this can be metabolised by Human serum biotinidase and Human lipoamidase. Interestingly, Metallothionein-1F gene is present in Humans, but absent in Horse. Another Horse Metallothionein-1A protein have N-terminal sequence CPTGG-, and this protein can not be metabolised by Human serum biotinidase and Human lipoamidase (please see files again; HepG2 Fucoidan and JMBT Alopecia). Further, the clam Ruditapes decussatus Cd-Metallothionein-1 has been purified and N-terminal sequence is GDPCNVAETGQCVCAQCCK- by Edman-degradation analysis, which is very different sequence obtained from cDNA cloning method.

Glutathione-related enzymes are present; i.e.,

Human breast milk has Glutathione S-transferase theta-2/GSTT2 at 4.4 μg/mg milk protein.

Fetal hepatocyte Hc has Glutathione S-transferase A4/GST class-alpha member 4 at 13.6 μg/mg cell protein.

Cancer hepatoma HepG2 (cultured without Fucoidan) has Glutathione peroxidase 1/Cellular glutathione peroxidase/GSH Px-1 at 0.14, Glutathione peroxidase 6 at 0.13, and Glutathione synthetase at 0.055 μg/mg cell protein, respectively. On the other hand, healed hepatocyte HepG2 (cultured with Fucoidan) does not have Glutathione-related enzymes.

Since liver-tissue specimens (n=5) and non-cancer serum (n=13) have no Glutathione-related enzymes, Glutathione may be working in actively developing human cells.

By the way, we have detected 4-kind of Carboxylases' activities in Guinea pig and Rat by using reliable HPLC-photometric enzyme-assay method; i.e., Pyruvate carboxylase/PC, Acetyl-CoA carboxylase/ACC, Propionyl-CoA carboxylase/PCC, and Methylcrotonyl-CoA carboxylase/MCC are present in Guinea pig and Rat (our unpublished observation for Rat and please see file for Guinea-pig kidney; 4-kind of carboxylases). We have previously tested Effect of Avidin (Egg-white injury) in foodstuff using Rat, and found that all 4-kind of Carboxylases in liver and kidney have become to be Storage labile (our unpublished observation).

On the other hand, we have now firstly found that Humans has only 2-kind of carboxylases of Propionyl-CoA carboxylase/PCC and Methylcrotonyl-CoA carboxylase/MCC, however the Egg-white injury is surely present in Humans (please see file; Feed by Measure).

I am also grateful to Dr. Foysal Ahammad (Department of Biotechnology Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia) for leading me to these interesting results.

Further amended on 16 October 2019

I must sadly add the truth that Humans does not have the Tubulin. Tubulin alpha-1A chain of Humans has N-terminal sequence of RECISIHVG- and Tubulin beta chain of Humans has N-terminal sequence of REIVHIQAG-, respectively, and both of these proteins cannot be hydrolyzed or metabolyzed by Lipoamidase (please see file; Multiple hydrolase LIP). Thus, Tubulin is not detected by our Human-protein database derived from the PDMD method.

Tubulin and Actin genes have been estimated using notorious cloning, hybridization, and computer-aided "In Silico" methods. Thus, presence and/or absence of proteins and peptides should be determined by utilizing reliable and quantitative PDMD (Protein-Direct-Microsequencing-Deciphering) method and/or Microsequencing/Edman degradation with covalently Bonded Protein onto the glass-filter after fractionation of the protein peak by our high-recovery and quantitative RP-HPLC-photometric method.

I must further say that notorious cloning, hybridization, and computer-aided "In Silico" methods are not indicating the truth of protein expression and/or protein biosynthesis.

We have surely isolated or purified Human serum biotinidase 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 purified and Edman-degradation method.

Sequence from cDNA method was

SAGS NPQSCRWNMGVCIPISCPGNMRQIGTCFGPRVPCCRRW

Sequence from Protein sequencing method was

SGIS GPLSCGRNGGVCIPIRCPVPMRQIGTCFGRPVKCCRSW

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

By the way, beta-Defensin 1/Defensin, beta 1 is absent in Humans; i.e., N-terminal sequence of it is DHYNC-, and Human serum biotinidase cannot metabolize it.

On the other hand, alpha-Defensins of Neutrophil defensin 1/Defensin, alpha 1 and Neutrophil defensin 2 are present in Humans; i.e., N-terminal sequences of them are ACYCR- and CYCRI-, respectively, and Human serum biotinidase can metabolize them.

Further amended on 24 September 2019

I must add the answer of my previous declaration that "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."

I have measured (1) Translation-related proteins utilizing ribosome and ATP in accordance with notorious Central dogma, (2) Protein degradation/metabolism system by utilizing Ubiquitin-system, and (3) Protein synthetases/Reversed-reaction system of Proteases including Biotinidase in my Human protein database (please see file; HepG2 Fucoidan) and bacterium protein database (data not shown; my unpublished result).

A bacterium (Lactobacillus casei (Shirota)) has 9.31% of (1), 0.0% of (2), and 0.095% of (3) among cell proteins (100%).

Fetal hepatocyte Hc has 0.27% of (1), 1.20% of (2), and 2.363% of (3).

Hepatoma HepG2 (cultured without Fucoidan) has

1.708% of (1), 1.916% of (2), and 6.845% of (3).

Cured hepatocyte HepG2 (cultured with Fucoidan at 0.102 mg/mL for 3 days) has

0.147% of (1), 1.002% of (2), and 3.37% of (3).

Therefore, bacteria (procaryote) performs Translation-related protein-synthesis, and has no Ubiquitin-related proteins.

On the other hand, Humans (a representative eucaryote) seems to synthesize proteins mainly via (3) Protein synthetases/Reversed-reaction of Proteases system as Dr. Hans Neurath has suggested.

Normal human cells do not utilize Translation-related protein-synthesis system, but Cancer cells of humans seem to also utilize Translation-related protein-synthesis system in addition to Protein synthetases/Reversed-reaction of Proteases system.

Protein-degradating-Ubiquitin-metabolic system seems to be two-fold activated in Cancer cells as compared to normal cells and healed HepG2 (Normalization has been induced by Fucoidan).

Therefore, I must sadly declare that the Central dogma of molecular biology firstly proposed by Dr. Francis Harry Compton Crick is not true in Humans at all. Further, I must sadly declare that the coclusion of “A Black is inferior in intelligence to a White (as estimated from DNA technology)” presented by Dr. James Dewey Watson is completely wrong, since Human proteins are not produced via the procaryotic Central-dogma system.

Further added on 14 September 2019

I have further measured (1) Translation-related proteins utilizing ribosome and ATP in accordance with notorious Central dogma, (2) Protein degradation/metabolism system by utilizing Ubiquitin-system, and (3) Protein synthetases/Reversed-reaction system of Proteases including Biotinidase in my Human liver-tissue-protein database (our unpublished observation).

Normal liver with Pseudo-liver cancer has 2.05% of (1), 3.80% of (2), and 5.14% of (3).

LC tissue with Leprosy has 2.37% of (1), 2.81% of (2), and 6.44% of (3).

LC tissue (named as No.6) has 1.14% of (1), 2.25% of (2), and 4.96% of (3).

HCC tissue (No.6) has 0.961% of (1), 0.768% of (2), and 5.86% of (3).

HCC tissue with Primary biliary cholangitis/Primary biliary cirrhosis/PBC has 6.36% of (1), 0.940% of (2), and 4.66% of (3).

The Translation-related proteins utilizing ribosome and ATP in accordance with notorious Central dogma in PBC patient seems to be induced by the infection of Hog cholera virus/Classical swine fever virus/CSFV and HAV. HAV seems to specifically infect to Human Female (our unpublished observation). Therefore, PBC may also be cured by the edible Japanese Fucoidan (please see file; Feed by Measure). Serum of PBC patient has elevated Anti-Mitochondrial Antibodies; i.e., such mitochondrial proteins as ADP/ATP translocase 3/Adenine nucleotide translocator 3/ANT 3/SLC25A6, NADPH:Adrenodoxin oxidoreductase, mitochondrial, Cytochrome P450 11B2, mitochondrial/Cytochrome P450 Aldo, and Uracil-DNA glycosylase 2 may be up-regulated or excreted from the mitochondria of PBC patients into blood.

It seems interesting that (2) Protein degradation/metabolism system by utilizing Ubiquitin-system is ca. 3.3-fold lower in HCC tissue than normal and LC tissue. This is reversed observation as compared to Cultured cells. Cultured cells may have changed the Ubiquitin-Proteasome protein-degradation system as compared to the cells in the tissue specimens. Cultured cells seems to become to similar in Ubiquitin system to Procaryote Bacterial cells. Normal tissue cells seem to increase the (2) Protein degradation/metabolism system by utilizing Ubiquitin-system as compared to Cancer cells.

By the way, HCV, Dengue virus/DENV, and Human herpesvirus/HHV up-regulate Apolipoprotein B-100/Apo B-100, and Hog cholera virus/Classical swine fever virus/CSFV, HGV, and HBV down-regulate Apo B-100 (our unpublished observation). Repressed state of Apo B-100 by CSFV and HGV in HCC (Hepatocellular carcinoma; caused by HIV-1 and HCV; please see file, The Fascio effect) patient is very important. Suppressed Cholesterol metabolism induces HCC patient to die immediately (our unpublished observation). Therefore, edible Japanese Fucoidan

can safely cure HCC without any side effect (please see file; Rat DEN Np-Fuco).

HAV regulates protein expression of Homeobox protein OTX1, Homeobox/POU domain protein RDC-1/Brain-specific homeobox/POU domain protein 3A, and Homeobox protein MSX-1/Hox-7, and possibly causes cleft lip and cleft palate when infected in pregnant period (our unpublished estimation). Then, prevention of cleft lip and cleft palate seems to be also possible by the edible Japanese Fucoidan.

Further added and amended on 18 September 2019

I have compared relative molecular-mass distribution between Procaryote (Lactobacillus casei (Shirota)) and Eucaryote (Humans).

Procaryote has Median 59,000, Average 74,000 , (range; 24,000 ~ 264,000),

largest protein; Wall-associated protein Mr 264,000.

Eucaryote (Human liver tissue with Pseudo-liver cancer) has Median 68,000, Average 132,000, (range; 16,700 ~ 3,880,000),

largest protein; Connectin/Titin Mr 3,880,000 (ca. 15-fold longer than procaryotic Wall-associated protein).

Eucaryotic proteins usually have Glyco-chains, and differences in relative molecular mass seem to be more increased. Interestingly, only eucaryotes have glycoproteins.

Therefore, protein synthetic system seems to be different between Procaryote and Eucaryote.

Analyzer-read chromatographic immunoassay (CIA) tests that determine human chorionic gonadotropin (hCG) in specimens. Also see the link: Article Comparison of newly developed immuno-MS method with existing...