The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used analytical immunochemistry assay based on the specific binding between the antigen and the antibody. The application of this test has significantly changed the practice of medical laboratories, where it is used to detect and quantify molecules such as hormones, peptides, antibodies and proteins. Various technical variants of this test can detect antigen (native or foreign) or antibodies that determine the intensity of the immune response, whether pathological or not; the type of immune response induced as well as the innate immunity potential; and much more.
These capabilities, along with the test’s high sensitivity and robustness, and low price allow it to diagnose diseases quickly and reliably in most laboratories. Besides, ELISA is a test that is also used in veterinary medicine, toxicology, allergology, food industry, etc. Despite the fact that it has been around for almost 50 years, different ELISA tests are still being developed with different technical solutions that improve and expand the application possibilities of this exceptional test. The goal of this chapter is to enable the driver to optimize, standardize and validate an enzyme-linked immunosorbent assay.
introduction
Enzyme Linked Immunosorbent Assay (ELISA) has been around for 50 years and ELISAs with different technical solutions are still being developed, improving and expanding the scope.
The test was first described by Engvall and Perlmann in 1971 and was based on the work of Avrameas who used enzyme-linked antibodies in histochemistry. The method was quickly developed for the serodiagnosis of trichinosis and antibodies against Plasmodium vivax and P. falciparum to be used in epidemiological studies of malaria.
- Since its discovery, there have been numerous applications of ELISA, used to detect both antigens and antibodies. In addition to the detection of protein antigens, ELISAs have been optimized that enable the determination of antibodies against native and denatured DNA, polysaccharide antigens and phospholipids.
- In fact, the name ELISA is sometimes used for tests that do not have antibodies present, but instead use specific protein-protein interactions. From the point of view of optimization, validation and standardization, such tests can be treated the same. In the case of protein antigens, the sensitivity of the ELISA is usually in the pg/ml range.
- When developing a diagnostic test, precise and optimal performance conditions must be found for all steps within the test protocol. This ensures that the entire process is optimal. Before it can be used routinely, for example in diagnostics, the newly developed or newly modified method must prove to be accurate, precise and reproducible.
- In addition, in order to measure the values obtained with the test, it is necessary to standardize the test. Therefore, the Optimization, Validation and Standardization (OVS) of ELISA are extremely important and necessary, especially if they are to be used in clinical or veterinary medicine. In this chapter, the procedures that characterize ELISA are presented in a clear and concise manner.
- When reviewing the literature, we noticed that the described boundaries between optimization, standardization and validation are not clear enough.
- The reason for this is that in certain situations performing a single ELISA can lead to a completion of both validation and optimization features, which is fully valid. Before going into more detail, and to avoid confusion, it is appropriate to clearly define these three terms.
ACCORDING TO THE MERRIAM-WEBSTER DICTIONARY,
Optimization is: „ An action, process, or method to make something such as a design, system, or decision as completely perfect, functional, or effective as possible.“
Validation is: „ an action, process or instance of validation, in particular : determining the degree of validity of a measuring device.“
Standardization is: „to bring into line with a standard, particularly to ensure consistency and regularity… to compare to a standard: to determine the strength, value, or quality of (something) by comparison to a standard.“
ELISA is mostly used to measure the presence or amount of antibodies or antigens or, in general, biomolecules that can be recognized by antibodies. In biological matrices (such as serum, plasma, blood, urine and saliva) ELISA is an important diagnostic tool for the detection of various antigens and antibodies. Indirect or direct ELISAs are used in medical device development, especially for testing vaccines and new drugs. ELISA with specific antibodies can be developed to measure impurities in the medical devices resulting from the manufacturing process.
Antibody assays against these contaminants should also be developed and validated to test contaminant levels, which should be kept to a minimum to avoid adverse immune responses. The sandwich ELISA is used for immunogenic substances with expected low concentrations such as cytokines, hormones, toxins, etc.
Shikari (Q-AFA) Filgrastim Quantitative ELISA kit
FIL-QNS-FIL
Matriks Biotek
1x96-wells test plate
1217.4 EUR
Shikari (Q-ATI) Infliximab (Remicade) Quantitative ELISA kit
INF-QNS-REMI
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (Q-ATI) Infliximab (Remicade) Qualitative ELISA kit
INF-QLS-REMI
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATB) Bevacizumab (Avastin) Quantitative ELISA kit
BEV-QNS-AVA
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATC) Cetuximab (Erbitux) Quantitative ELISA kit
CET-QNS-ERB
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATA) Adalimumab (Humira) Quantitative ELISA kit
ADA-QNS-HUM
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-AIR) Infliximab (Remsima) Quantitative ELISA kit
INF-QNS-REMS
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (Q-ABA) Abatacept (Orencia)Free drug ELISA kit
ABA-FD-ORE
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-AF) Aflibercept (Eylea)Free drug ELISA kit
AFL-FD-EYL
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-BEVA) Bevacizumab (Avastin)Free drug ELISA kit
BEV-FD-AVA
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-ATE) Atezolizumab (Tecentriq)Free drug ELISA kit
ATE-FD-TEC
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-AVE) Avelumab (Bavencio)Free drug ELISA kit
AVE-FD-BAV
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-CERT) Certolizumab (Cimzia)Free drug ELISA kit
CER-FD-CIM
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (Q-CET) Cetuximab (Erbitux)Free drug ELISA kit
CET-FD-ERB
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-DAR) Daratumumab (Darzalex)Free drug ELISA kit
DAR-FD-DAR
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-DEN) Denosumab (Prolia)Free drug ELISA kit
DEN-FD-PRO
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-CAN) Canakinumab (Ilaris)Free drug ELISA kit
CAN-FD-ILA
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-ADA) Adalimumab (Humira)Free drug ELISA kit
ADA-FD-HUM
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-DUR) Durvalumab (Imfinzi)Free drug ELISA kit
DUR-FD-IMF
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-OMA) Omalizumab (Xolair)Free drug ELISA kit
OMA-FD-XOL
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-NIVO) Nivolumab (Opdivo)Free drug ELISA kit
NIV-FD-OPD
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-NAT) Natalizumab (Tysabri)Free drug ELISA kit
NAT-FD-TYS
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-GOL) Golimumab (Simponi)Free drug ELISA kit
GOL-FD-SIM
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-ETA) Etanercept (Enbrel)Free drug ELISA kit
ETA-FD-ENB
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (Q-INFLIXI) Infliximab (Remicade)Free drug ELISA kit
INF-FD-REMI
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-REMS) Infliximab (Remsima)Free drug ELISA kit
INF-FD-REMS
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-IPI) Ipilimumab (Yervoy)Free drug ELISA kit
IPI-FD-YER
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-PEM) Pembrolizumab (Keytruda)Free drug ELISA kit
PEM-FD-KEY
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-RAN) Ranibizumab (Lucentis)Free drug ELISA kit
RAN-FD-LUC
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-TOC) Tocilizumab (Actemra)Free drug ELISA kit
TOC-FD-ACT
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-UST) Ustekinumab (Stelara)Free drug ELISA kit
UST-FD-STE
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-VEDO) Vedolizumab (Entyvio)Free drug ELISA kit
VED-FD-ENT
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (Q-RAM) Ramucirumab (Cyramza)Free drug ELISA kit
RAM-FD-CYR
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (S-ATR) Trastuzumab (Herceptin, Herclon) Quantitative ELISA kit
TRA-QLS-HH
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATR) Rituximab (Rituxan, Mabthera) Quantitative ELISA kit
RIT-QNS-RM
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (Q-ATIDUO) Infliximab (Remicade) Free/Total semiquantitative ELISA kit
INF-QNFT-REMI
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (Q-TRAS) Trastuzumab (Herceptin, Herclon)Free drug ELISA kit
TRA-FD-HH
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (Q-RITUX) Rituximab (Rituxan, Mabthera)Free drug ELISA kit
RIT-FD-RM
Matriks Biotek
1x96-wells test plate
971.7 EUR
Filgrastim (rHuG-CSF)
rHuG-CSF
Amyotop
300µg/vial
270 EUR
Rabbit AFA ELISA Kit
ERTA0435
Abclonal
96Tests
625.2 EUR
Rat AFA ELISA Kit
ERA0435
Abclonal
96Tests
625.2 EUR
Mouse AFA ELISA Kit
EMA0435
Abclonal
96Tests
625.2 EUR
Monkey AFA ELISA Kit
EMKA0435
Abclonal
96Tests
625.2 EUR
Porcine AFA ELISA Kit
EPA0435
Abclonal
96Tests
625.2 EUR
Human AFA ELISA Kit
EHA0435
Abclonal
96Tests
625.2 EUR
Goat AFA ELISA Kit
EGTA0435
Abclonal
96Tests
625.2 EUR
Canine AFA ELISA Kit
ECA0435
Abclonal
96Tests
625.2 EUR
Chicken AFA ELISA Kit
ECKA0435
Abclonal
96Tests
625.2 EUR
Anserini AFA ELISA Kit
EAA0435
Abclonal
96Tests
625.2 EUR
Bovine AFA ELISA Kit
EBA0435
Abclonal
96Tests
625.2 EUR
Sheep AFA ELISA Kit
ESA0435
Abclonal
96Tests
625.2 EUR
BioSim? anti-Filgrastim (Neupogen®) (Human) ELISA Kit
E4399-100
Biovision
each
2082 EUR
Guinea Pig AFA ELISA Kit
EGA0435
Abclonal
96Tests
625.2 EUR
Custom Antibody titration by ELISA up to 2 rabbits and 1 bleed
ELISA-1
Alpha Diagnostics
1
242.4 EUR
Human Anti-Filaggrin Antibody (AFA) ELISA Kit
abx053209-96tests
Abbexa
96 tests
943.2 EUR
Monkey Anti-Filaggrin Antibody (AFA) ELISA Kit
abx359742-96tests
Abbexa
96 tests
990 EUR
Rabbit Anti-Filaggrin Antibody (AFA) ELISA Kit
abx362441-96tests
Abbexa
96 tests
990 EUR
Sheep Anti-Filaggrin Antibody (AFA) ELISA Kit
abx364233-96tests
Abbexa
96 tests
1111.2 EUR
Pig Anti-Filaggrin Antibody (AFA) ELISA Kit
abx361458-96tests
Abbexa
96 tests
990 EUR
Human Anti-Filaggrin Antibody (AFA) ELISA Kit
abx351230-96tests
Abbexa
96 tests
943.2 EUR
Chicken Anti-Filaggrin Antibody (AFA) ELISA Kit
abx356311-96tests
Abbexa
96 tests
990 EUR
Human anti-filaggrin antibody,AFA ELISA KIT
201-12-2097
SunredBio
96 tests
528 EUR
Human anti-filaggrin antibody, AFA ELISA Kit
CSB-E09079h-24T
Cusabio
1 plate of 24 wells
198 EUR
Human anti-filaggrin antibody, AFA ELISA Kit
1-CSB-E09079h
Cusabio
Human anti-filaggrin antibody,AFA ELISA Kit
CN-04352H1
ChemNorm
96T
571.2 EUR
Human anti-filaggrin antibody,AFA ELISA Kit
CN-04352H2
ChemNorm
48T
391.2 EUR
Rat anti-filaggrin antibody(AFA)ELISA Kit
GA-E0834RT-48T
GenAsia Biotech
48T
380.4 EUR
Rat anti-filaggrin antibody(AFA)ELISA Kit
GA-E0834RT-96T
GenAsia Biotech
96T
595.2 EUR
Human anti-filaggrin antibody(AFA)ELISA Kit
QY-E02547
Qayee Biotechnology
96T
433.2 EUR
Rat anti-filaggrin antibody(AFA)ELISA Kit
QY-E10818
Qayee Biotechnology
96T
433.2 EUR
Mouse anti-filaggrin antibody(AFA)ELISA Kit
QY-E20121
Qayee Biotechnology
96T
433.2 EUR
Human anti-filaggrin antibody,AFA ELISA KIT
YLA1906HU-48T
Shanghai YL Biotech
48T
435 EUR
Human anti-filaggrin antibody,AFA ELISA KIT
YLA1906HU-96T
Shanghai YL Biotech
96T
562.5 EUR
Shikari (S-ATAB) Abatacept (Orencia) Qualitative ELISA kit
ABA-QLS-ORE
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATAF) Aflibercept (Eylea) Qualitative ELISA kit
AFL-QLS-EYL
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATB) Bevacizumab (Avastin) Qualitative ELISA kit
BEV-QLS-AVA
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATAT) Atezolizumab (Tecentriq) Qualitative ELISA kit
ATE-QLS-TEC
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATAV) Avelumab (Bavencio) Qualitative ELISA kit
AVE-QLS-BAV
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATC) Cetuximab (Erbitux) Qualitative ELISA kit
CET-QLS-ERB
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATDAR) Daratumumab (Darzalex) Qualitative ELISA kit
DAR-QLS-DAR
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATD) Denosumab (Prolia) Qualitative ELISA kit
DEN-QLS-PRO
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATCAN) Canakinumab (Ilaris) Qualitative ELISA kit
CAN-QLS-ILA
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATA) Adalimumab (Humira) Qualitative ELISA kit
ADA-QLS-HUM
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATDUR) Durvalumab (Imfinzi) Qualitative ELISA kit
DUR-QLS-IMF
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATO) Omalizumab (Xolair) Qualitative ELISA kit
OMA-QLS-XOL
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATN) Nivolumab (Opdivo) Qualitative ELISA kit
NIV-QLS-OPD
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATNAT) Natalizumab (Tysabri) Qualitative ELISA kit
NAT-QLS-TYS
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATG) Golimumab (Simponi) Qualitative ELISA kit
GOL-QLS-SIM
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATE) Etanercept (Enbrel) Qualitative ELISA kit
ETA-QLS-ENB
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-AIR) Infliximab (Remsima) Qualitative ELISA kit
INF-QLS-REMS
Matriks Biotek
1x96-wells test plate
971.7 EUR
Shikari (S-ATI) Ipilimumab (Yervoy) Qualitative ELISA kit
IPI-QLS-YER
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATP) Pembrolizumab (Keytruda) Qualitative ELISA kit
PEM-QLS-KEY
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATRAM) Ramucirumab (Cyramza) Qualitative ELISA kit
RAM-QLS-CYR
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATOC) Tocilizumab (Actemra) Qualitative ELISA kit
TOC-QLS-ACT
Matriks Biotek
1x96-wells test plate
999 EUR
Shikari (S-ATU) Ustekinumab (Stelara) Qualitative ELISA kit
UST-QLS-STE
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Shikari (S-ATV) Vedolizumab (Entyvio) Qualitative ELISA kit
VED-QLS-ENT
Matriks Biotek
1x96-wells test plate
1053.6 EUR
Tiamulin ELISA Kit, Quantitative
DEIA036
Creative Diagnostics
96T
1414.8 EUR
hnRNP Q Cell ELISA Kit
abx595670-96tests
Abbexa
96 tests
764.4 EUR
Guinea pig Anti-Filaggrin Antibody (AFA) ELISA Kit
abx357337-96tests
Abbexa
96 tests
990 EUR
Guinea pig anti-filaggrin antibody, AFA ELISA Kit
CSB-EQ028009GU-24T
Cusabio
1 plate of 24 wells
198 EUR
Regardless of the ELISA design (indirect, direct, or sandwich), the OVS principles are the same. Of paramount importance for any bioanalytical method is that it is well characterized, fully validated and documented to a satisfactory standard in order to produce reliable results.
The first step in ELISA development is optimization, followed by standardization and finally validation.
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