Jan 16, 2026

Public workspaceQCSOP25-2: Identification of Anti-Rhesus IgA by Direct and Indirect ELISA

DOI
https://dx.doi.org/10.17504/protocols.io.3byl41m2zlo5/v1
QCSOP25-2: Identification of Anti-Rhesus IgA by Direct and Indirect ELISA
  • Yasmine IACONE1,
  • Vimala Gollamudi1,
  • Diogo Magnani1
  • 1NHPRR, UMass Chan Medical School, Worcester, MA (USA)
  • NHPRR
Yasmine IACONE
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl41m2zlo5/v1
External link: http://nhpreagents.org
Protocol Citation: Yasmine IACONE, Vimala Gollamudi, Diogo Magnani 2026. QCSOP25-2: Identification of Anti-Rhesus IgA by Direct and Indirect ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl41m2zlo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2025
Last Modified: January 16, 2026
Protocol Integer ID: 220641
Keywords: ELISA, Anti-IgA antibody, Rhesus Macaque, Nonhuman Primate (NHP), Immunoglobulin A (IgA), Mucosal Immunity, Monoclonal antibodies, Polyclonal Antibodies, Monomeric IgA, Dimeric IgA, NHPRR, Nonhuman Primate Reagent Resource, performing antibody identity testing, antibody identity testing for research, indirect elisa this elisa, standardized elisa method, antibody reagent, antibody specificity, antibody, indirect elisa, direct elisa, antigen specificity, immunoassay, nhprr reagent, identification of anti, reliable use of nhprr reagent, polyclonal goat, elisa, nhprr laboratory staff, nonhuman primate reagent resource,
Funders Acknowledgements:
NIH
Grant ID: AI126683
Abstract
This ELISA-based protocol confirms the identity and reactivity of Nonhuman Primate Reagent Resource (NHPRR) polyclonal goat anti-rhesus IgA and monoclonal mouse anti-rhesus IgA antibody reagents. Both Indirect and Direct ELISA formats are used to evaluate antibody binding specificity and lot-to-lot consistency. The procedure involves plate coating, blocking, sample incubation, detection, and data analysis, all performed according to defined acceptance criteria.
Objective:
To provide a standardized ELISA method for verifying the antigen specificity and identity of NHPRR anti-IgA antibodies.
Scope:
Applies to NHPRR laboratory staff performing antibody identity testing for research and quality control purposes.
Background:
Accurate identification of anti-IgA antibodies ensures reliable use of NHPRR reagents in immunoassays. The Indirect ELISA detects antibody binding through a secondary conjugate, while the Direct ELISA uses labeled anti-IgA for direct detection. Together, they confirm antibody specificity and support NHPRR’s quality testing program.
Image Attribution
Created in BioRender. de Vasconcellos Castro, J. (2026) https://BioRender.com/m2svlm2
Materials
Standard ELISA materials and reagents:
ItemPurposeVendorCatalog Number
TMB SubstrateSubstrate for HRP1-Step Ultra TMB ELISA Substrate Solution, Thermo Fisher Scientific34029
Corning® 96-well Clear Flat Bottom Polystyrene High Bind Microplate, NonsterileHigh-binding plates for ELISA assayCorning9018
Gibco™ [1X] DPBS, calcium, magnesium, glucose, pyruvateDiluent for reagents used for coating the working platesGibco™14-287-080
Gibco™ PBS (10X), pH 7.2Used in the preparation of the Washing BufferGibco™70-013-032
VWR® High-Performance [15 mL] Centrifuge Tubes with Flat or Plug Caps, PPUsed for the preparation/dilution of the test sample and other reagentsVWR or equivalent89039-668
ELISA Stop SolutionStops the enzymatic reactionInvitrogen™SS04
Non-fat Dried MilkUsed for the Blocking Buffer solutionLabScientific Biotin-free non-fat dry milkM0841
Low-binding platesDilution plateOf your choiceOf your choice
1-Step™ TMB ELISA Substrate SolutionsActs as the substrate for the HRP enzymeThermo Fisher Scientific34029
TWEEN® 20Used in the preparation of the Washing BufferSigma AldrichP6585-100ML
Fisherbrand™ Hexagonal Polystyrene Weighing DishesUsed for weighing dry milk during Blocking Buffer preparationFisher Scientific02-202-103
TimerTo keep track of incubation and developing timeof your choiceN/A
Rainin Classic™ Manual Single-Channel Pipettes and Allocated Pipette TipsUsed for the preparation, dilution, and transfer of the test sample and other reagentsRainin or equivalentN/A
Pipette Tips SR LTS 200µL F 960A/5Used for transferring the test sample and other reagents from the dilution plate to the working plateRainin17005859
PPE (Personal Protective Equipment)For analyst protection: lab coat, nitrile gloves, and safety gogglesof your choiceN/A
Plate SealersTo cover ELISA plates for incubationBioLegend423601
Elisa Plate Reader SoftwareAnalyze the OD valuesMolecular DevicesModel: SoftMax® Pro
Nalgene™ Rapid-Flow™ Sterile Filter Storage BottlesUsed for the preparation of the Blocking BufferThermo Fisher Scientific455-0250
Pipet-Lite™ XLS+ Manual 12-Channel Pipette, 20-200 μLUsed for transferring the test sample and other reagents from the dilution plate to the working plateRainin17013810
Sterile GRIPTIPS® INTEGRA 1250 μL Pipette TipsUsed for the preparation, dilution, and transfer of the test sample and other reagentsINTEGRA6544 (non-filtered) 6545 (filtered)
ELISA Plate WasherWash ELISA platesBioTekModel #: 405LSRS
ELISA Microplate ReaderMeasure the OD valuesMolecular DevicesModel: SpectraMax ABS
INTEGRA VIAFLO 12-Channel 50-1250 μL Electronic Pipette Used for the preparation, dilution, and transfer of the test sample and other reagentsINTEGRAModel: 12-channel VIAFLO
[Serological] Pipette, 10 mL and 5 mL, Graduated 1/10 mL, SterileUsed for the preparation/dilution of the test sample and other reagentsGreiner Bio-One606180 (5 ml) 607107 (10 mL)
Falcon® 96-well Polystyrene [Dilution] MicroplatesLow-binding plates for preparation of the test sample and other reagentsCorning353936
CalculatorCalculate the dilution volumesof your choiceN/A
Biohazard Waste BagsCollect used pipette tips and other waste from the experimentof your choiceN/A
Analytical BalanceFor weighing dry milk (part of the Blocking Buffer preparation)of your choiceN/A
VWR® [50 mL] Disposable Pipetting ReservoirsFor holding the test sample and other reagent solutions before transferVWR or equivalent89094-684

Assay-Specific ELISA reagents:
ReagentReference rhesus monomeric IgA [CC6.29]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00377
ReagentReference rhesus dimeric IgA [CC6.29]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00378
ReagentGoat Anti-Rhesus IgA [GARI]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00366

ReagentAnti-IgA [NHPMab001]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00423
ReagentAnti-IgA [NHPMab001]-HRPNonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00392
ReagentGoat Anti-Rhesus IgA [GARI]-HRPNonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00365

ReagentMouse Anti-Goat IgG Fc-HRP (SB115d)Southern BiotechCatalog #6158-05
ReagentPeroxidase AffiniPure® F(ab)₂ Fragment Goat Anti-Mouse IgG, Fcγ fragment specificJackson ImmunoResearch Laboratories, Inc.Catalog #115-036-071
AntibodyIsotypeSpeciesVendorCatalog NumberPurposeRRID
Reference rhesus monomeric IgA [CC6.29]IgArhesusNHPRRCAT-00377CoatingAB_3714921
Reference rhesus dimeric IgA [CC6.29]IgArhesusNHPRRCAT-00378CoatingAB_3714922
Goat Anti-Rhesus IgA [GARI]IgGgoatNHPRRCAT-00366Primary AntibodyAB_3086850
Anti-IgA [NHPMab001]IgGmouseNHPRRCAT-00423Primary AntibodyAB_3717991
Mouse Anti-Goat Fc-HRP (SB115d)IgGmouseSouthern Biotech6158-05Secondary AntibodyAB_2796222
Peroxidase AffiniPure® F(ab')₂ Fragment Goat Anti-Mouse IgG, Fcγ fragment specificIgGgoatJackson ImmunoResearch115-036-071Secondary AntibodyAB_2338524
Goat Anti-Rhesus IgA [GARI]-HRPIgGgoatNHPRRCAT-00365Primary Antibody already conjugatedAB_3086849
Anti-IgA [NHPMab001]-HRPIgGmouseNHPRRCAT-00392Primary Antibody already conjugatedAB_3105907







Protocol materials
ReagentMouse Anti-Goat IgG Fc-HRP (SB115d)Southern BiotechCatalog #6158-05
ReagentPeroxidase AffiniPure® F(ab)₂ Fragment Goat Anti-Mouse IgG, Fcγ fragment specificJackson ImmunoResearch Laboratories, Inc.Catalog #115-036-071
ReagentReference rhesus dimeric IgA [CC6.29]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00378
ReagentAnti-IgA [NHPMab001]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00423
ReagentGoat Anti-Rhesus IgA [GARI]-HRPNonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00365
ReagentReference rhesus monomeric IgA [CC6.29]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00377
ReagentGoat Anti-Rhesus IgA [GARI]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00366
ReagentAnti-IgA [NHPMab001]-HRPNonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00392
Troubleshooting
Problem
Blank Wells Show High Signal
Solution
The issue may be caused by incomplete washing, insufficient blocking, incorrect buffer volume, contamination of the washing buffer, or an inadequate number of wash cycles. To address this, repeat the assay using freshly prepared blocking reagent and ensure reagent reservoirs are clean or replaced. Confirm that all wash steps are performed using the correct buffer and that full plate coverage is achieved during the blocking step. Use new pipette tips for all reagent transfers to prevent cross-contamination.
Problem
All Wells Show Low Signal
Solution
The issue may be due to inactive streptavidin-HRP or TMB substrate, incorrect incubation temperature, or systematic pipetting errors. To address this, verify reagent activity by testing streptavidin-HRP and TMB substrate using a known positive control. Additionally, confirm that all incubation steps were performed at room temperature or in accordance with SOP requirements. Inspect the performance of the plate reader to ensure proper function. If necessary, repeat the assay using a new high-binding ELISA plate and freshly prepared signal amplification reagents from different lots or new aliquots.
Problem
High Variability Between Duplicates
Solution
The issue may be caused by inconsistent pipetting, incomplete mixing of reagents, or non-uniform loading of samples and reagents. To address this, use calibrated pipettes and maintain a consistent pipetting technique; the use of a multichannel pipette is recommended for uniform application. Additionally, ensure that all samples and reagents are thoroughly mixed before use.
Before start
Note on Concentration Units: All antigen and antibody dilutions in this protocol are prepared using molar concentrations (e.g., nM) to ensure accurate comparison across immunoglobulin types with different molecular weights. When applicable, molecular weight values are used to convert between ng/mL and nM to reflect molar equivalency (e.g., IgG and monomeric IgA ~150 kDa, dimeric IgA ~300 kDa, IgM ~950 kDa).
Concentrations are lot-dependent; always make sure to check the lot even if the catalog number is the same.

Example conversion: To convert 1 mg/mL of monomeric IgA (~150 kDa) to molarity (mM), ensure that the units are consistent: mass in mg/mL and molecular weight in kDa. If using µg/mL, your result will be in µM instead of mM.

(1 mg/mL)/(150 kDa) = 0.00667 mM = 6.67 µM

Alternatively, [(1 mg/mL)/(150 kDa)] x 1000 = 6.67 µM
Prepare all the calculations beforehand.
Definitions and Acronyms
Definitions

Analyte / Antigen (recombinant rhesus IgA, monomeric and dimeric)
Recombinant rhesus IgA, present in either monomeric or dimeric form, is coated onto the ELISA plate and serves as the target analyte. Anti-rhesus IgA antibodies in the sample bind specifically to the immobilized IgA. The assay signal generated reflects the extent of antibody binding to the coated antigen.

Test Articles (TAs), TA-pAb, and TA-mAb
Test Articles (TAs): Anti-rhesus IgA antibody reagents that are being evaluated for identity and specificity.
TA-pAb: Goat anti-rhesus IgA polyclonal antibody Test Article.
TA-mAb: Mouse anti-rhesus IgA monoclonal antibody Test Article. These reagents are tested in parallel on plates coated with monomeric and dimeric IgA to assess binding profiles and confirm identity.

Reference Control (Positive Control) Antibody
A previously qualified anti-rhesus IgA antibody lot which is included on each plate (typically as a serial dilution/titration curve) to confirm that the assay produces the expected binding behavior (signal range and curve shape) and to support run-to-run trending and lot-to-lot comparability of reagents. (Abcam, 2024). In this ELISA, the positive control is an anti-rhesus IgA antibody sample that is known to specifically bind both monomeric and dimeric recombinant rhesus IgA. Its binding curve serves as a benchmark for the expected signal and plate validity.
Note
If the positive control fails to generate the expected signal, the assay may be invalid, and the run should be repeated (BosterBio, n.d.).

Negative Control Antibody
An antibody of the same species, isotype, and format as the Test Articles (TAs), but known not to bind rhesus IgA. The negative control antibody should produce only baseline signal. It is used to verify assay specificity and to help identify non-specific binding or background issues.

Blank
A buffer-only well that is used to establish the background signal and verify the absence of non-specific binding. Unexpected signal indicates contamination or assay error.
Note
If a blank well exhibits an unexpected signal, this may indicate contamination or non-specific binding, and the assay should be considered invalid (Jackson ImmunoResearch, n.d.).

Monoclonal Antibody (mAb) An antibody preparation derived from a single B-cell clone, recognizing one specific epitope on an antigen and therefore having a single binding specificity. In this SOP, the monoclonal antibody Test Article (TA-mAb) is a mouse anti-rhesus IgA antibody evaluated for its binding to recombinant monomeric and dimeric rhesus IgA.

Polyclonal Antibody (pAb)
An antibody preparation containing a mixture of immunoglobulin molecules produced by multiple B-cell clones, recognizing multiple epitopes on the same antigen. In this SOP, the polyclonal antibody Test Article (TA-pAb) is a goat anti-rhesus IgA antibody evaluated for its binding profile to recombinant monomeric and dimeric rhesus IgA.

Monomeric IgA
A single IgA immunoglobulin unit composed of two heavy and two light chains (one “Y-shaped” IgA molecule). In this SOP, recombinant monomeric rhesus IgA is used as a coated antigen on ELISA plates to assess antibody binding to the monomeric form of IgA.

Dimeric IgA
A form of IgA composed of two IgA monomers covalently linked, typically via a joining (J) chain, representing the main configuration of secretory IgA at mucosal surfaces. In this SOP, recombinant dimeric rhesus IgA is used as a coated antigen on ELISA plates to assess antibody binding to the dimeric form of IgA.

Direct ELISA A plate-based immunoassay format in which the antigen is immobilized on the plate and detected using a single enzyme-labeled (e.g. HRP-conjugated) primary antibody that binds directly to the antigen. Signal is generated when the enzyme acts on a chromogenic substrate (e.g. TMB), and the measured absorbance is proportional to the amount of antigen–antibody complex formed. Direct ELISA uses fewer steps and reagents than indirect ELISA but offers less signal amplification because only one labeled antibody binds per antigen site. (ELISA Guide; Part 1: Introduction to ELISA, Formats and Signal Amplification, 2023; NCBI, 2020)

Indirect ELISA A plate-based immunoassay format in which the antigen is immobilized on the plate, followed by incubation with an unlabeled primary antibody specific for the antigen, and then a labeled secondary antibody (e.g. HRP-conjugated anti-species antibody) that recognizes the primary antibody. The enzyme on the secondary antibody converts a chromogenic substrate (e.g. TMB) to a colored product measured by absorbance. Indirect ELISA provides signal amplification because multiple secondary antibodies can bind each primary antibody, increasing assay sensitivity compared with direct ELISA. (ELISA Guide; Part 1: Introduction to ELISA, Formats and Signal Amplification, 2023; NCBI, 2020)

Dilution Plate A low-binding microplate used to prepare antibody dilutions and perform vertical serial dilutions of TAs, controls, and HRP-conjugated antibodies before transfer to the working plate. The dilution plate mirrors the layout of the working plate but is not read in the plate reader.

Working Plate The high-binding ELISA plate on which antigen coating, blocking, antibody incubation, washing, substrate addition, and OD reading at 450 nm are performed. The working plate contains the bound analyte and is the plate that generates the analytical signal. 

Blocking Buffer
In this SOP, the blocking buffer is 5% non-fat dry milk in PBS with 0.05% Tween-20. It is applied after the coating step to occupy unbound sites on the plate surface and is also used as a diluent for antibodies. Blocking buffer minimizes non-specific binding and reduces background signal. (Excedr, 2023)

Washing Buffer
PBS containing 0.05% Tween-20 (1× DPBS or PBS base, as prepared under “Wash Buffer Preparation”). It is used between assay steps to remove unbound reagents and reduce non-specific background.

Detection Reagent (HRP-conjugated antibody)
For the indirect ELISA, the detection reagents are HRP-conjugated secondary antibodies (mouse anti-goat HRP and goat anti-mouse HRP) that bind the species-specific primary anti-IgA antibodies. For the direct ELISA, the detection reagents are HRP-conjugated anti-rhesus IgA antibodies themselves. In both formats, the detection reagent binds to the antibody–antigen complex and enables signal generation via HRP-mediated TMB conversion. (ELISA Guide; Part 1: Introduction to ELISA, Formats and Signal Amplification, 2023; NCBI, 2020)

TMB Substrate and Stop Solution
TMB (3,3′,5,5′-Tetramethylbenzidine) is a chromogenic substrate that is oxidized by HRP, producing a blue color proportional to bound HRP. The Stop solution (acid) halts the HRP reaction and converts the color to yellow, allowing stable measurement at 450 nm. 

Optical Density at 450 nm (OD₄₅₀)
The absorbance measured at 450 nm using an ELISA plate reader after TMB development and stopping. OD₄₅₀ values reflect the amount of HRP-mediated color change and are used to construct binding curves for each antibody.

Acronyms:

CV – Coefficient of Variation.
DPBS – Dulbecco’s Phosphate-Buffered Saline.
EC₅₀ – Half-maximal effective concentration.
ELISA – Enzyme-Linked Immunosorbent Assay.
HRP – Horseradish Peroxidase.
IgA – Immunoglobulin A.
kDa – KiloDalton (molecular mass unit).
LoD – Limit of Detection.
NHP – Nonhuman Primate.
NHPRR – Nonhuman Primate Reagent Resource.
OD – Optical Density (absorbance).
O/N – Overnight (incubation at 4 °C unless otherwise stated).
PBS – Phosphate-Buffered Saline.
RT – Room Temperature.
TA – Test Article.
TA-pAb – Test Article polyclonal anti-rhesus IgA antibody (goat).
TA-mAb – Test Article monoclonal anti-rhesus IgA antibody (mouse).
TMB – 3,3′,5,5′-Tetramethylbenzidine.
Principle
Analyte/Antigen: recombinant rhesus IgA, monomeric and dimeric forms (two plates processed in parallel).

Test Articles (TA):
TA-pAb: goat anti-rhesus IgA (polyclonal)
TA-mAb: mouse anti-rhesus IgA (monoclonal)

Controls:
Negative Control Ab: same species/format as TA, known non-binder to rhesus IgA
Reference Positive Control Ab: previously qualified anti-IgA (optional but recommended for plate validity)

Identity is confirmed by a specific binding signature to both monomeric and dimeric IgA with acceptable curve fit and specificity, and by matching expected relative response with defined limits.
See Acceptance Criteria Section below.


Figure 1: Workflow for Identification of Anti-Rhesus IgA by ELISA
Publication citation: Created in BioRender. de Vasconcellos Castro, J. (2026) https://BioRender.com/m2svlm2
Concentration & Dilution Notes
Please read the guidelines on concentration units under Guidelines & Warnings.

Use the dilution formula:
C₁V₁= C₂V₂

Where:
  • C₁ = stock concentration
  • V₁ = volume of stock needed
  • C₂ = desired final concentration
  • V₂ = final total volume

Example: To prepare 1 mL of antigen at 1 µM from a 1 mM stock:
  • C₁ = 1 mM = 1000 µM
  • C₂ = 1 µM
  • V₂ = 1 mL = 1000 µL

  • V₁=(C₂ × V₂)/ C₁= 1 μM × 1000 μL/1000 μM = 1 μL

So: Add 1 µL of stock + 999 µL of DPBS to reach 1 µM in 1 mL total volume.
Wash Buffer Preparation (1x DPBS + 0.05% Tween-20)
Add Amount18 L of deionized water in a container.

Add Amount2 L of 10x DPBS and mix gently.

Add Amount50 mL of Tween-20 and mix gently.
Note
Scale up or down according to the need.

Day 1 - Plate Coating (~30 min preparation + O/N incubation)
1d
Prepare Concentration10 nanomolar (nM) (C2) of IgA in 1x DPBS.

Two working plates in parallel:
  • Plate 1: Monomeric IgA
  • Plate 2: Dimeric IgA

Plate Layout (See Figure 2):
  • Columns 1-9: Coating with IgA (as per plate type);
  • A10-A11: Coat only (IgA);
  • B10-F11: 1x DPBS only controls;
  • G10-G11: Blank (1x DPBS)

Figure 2: Coating Plate Layout
Publication citation: Created in BioRender. de Vasconcellos Castro, J. (2026) https://BioRender.com/rh7lu1d

Calculation Volumes (in Table 1)
V2 = 74 wells/plate x 100 µL = 7400 µL; prepare 7800 µL/plate to allow excess.
ReagentReference rhesus monomeric IgA [CC6.29]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00377
ReagentReference rhesus dimeric IgA [CC6.29]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00378
AntibodyInitial Concentration (C1)Final Concentration (C2)Final Volume (V2)Initial Volume (V1)
Reference rhesus monomeric IgA2.69 mg/mL, 146.6 kDa, 18.3 µM10 nM7800 µL4.3 µL
Reference rhesus dimeric IgA0.56 mg/mL, 308.7 kDa, 1.8 µM10 nM7800 µL43.3 µL
Table 1: Calculation for the coating solution.
Note
For accuracy, remove the equivalent DPBS volume and add the same volume of stock.




Add Amount100 µL /well.

Seal the plate/s and incubate DurationOvernight at Temperature4 °C .

Day 2 - Blocking (~15 min preparation + 1 h incubation)
1h 15m
Wash the working plates twice with wash buffer.
2m
Prepare the Blocking buffer with Concentration5 % volume milk in wash buffer:

For Amount150 mL : add Amount7.5 g milk to Amount150 mL PBS + Concentration0.05 % volume Tween-20 .

9m
Add Amount200 µL /well.

3m
Seal the plate/s.
1m
Incubate Duration01:00:00 at TemperatureRoom temperature
Note
Potential STOP point: store up to 1-2 days at Temperature4 °C in blocking buffer



1h
OPTION A - Indirect ELISA: Primary antibody preparation and incubation (~30 min preparation + 1 hour incubation)
1h 30m
Prepare Concentration40 nanomolar (nM) (C₂) working stocks of TA-pAb, TA-mAb, and Negative Control Ab in blocking buffer.

Plate Layout (Figure 3):

  • Columns 1-3: TA-pAb (goat anti-rhesus IgA, pAb);
  • Columns 4-6: TA-mAb (mouse anti-rhesus IgA, mAb);
  • Columns 7-9: Negative control Ab (non-binder);
  • Row A10-11: Blocking buffer (coated wells only);
  • Row B10-11: TA-pAb controls;
  • Row C10-11: TA-mAb controls;
  • Row D10-11: Negative control Ab controls;
  • Row E10-F11: Reserved for secondary-only controls;
  • Row G10-11: Blank.


Figure 3: Primary Antibodies Plate Layout
Publication citation: Created in BioRender. de Vasconcellos Castro, J. (2026) https://BioRender.com/kvj3gee

Volume Calculations V2 (in Table 2):
5 wells/plate/each antibody (3 wells for the triplicate + 2 wells for the control)
2 plates total
200 µL of primary antibody solution/well
V₂ = 5 x 2 x 200 = 2000 µL - Make it in excess
V₂ = 2500 µL

ReagentGoat Anti-Rhesus IgA [GARI]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00366
ReagentAnti-IgA [NHPMab001]Nonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00423
AntibodyInitial Concentration (C1)Final Concentration (C2)Final Volume (V2)Initial Volume (V1)
Goat anti-rhesus IgA [GARI]6.64 mg/mL, 150 kDa, 44 µM40 nM2500 µL2.272 µL
Mouse anti-rhesus IgA [NHPMab001]3.78 mg/mL, 150 kDa, 25 µM40 nM2500 µL4 µL
Table 2: Calculation for the working stock solution.

Note
For accuracy, remove the equivalent blocking buffer volume and add the same volume of stock.





In a dilution plate, dispense Amount200 µL of working stock to row A of designated wells (Figure 3). Control wells will also receive the working stock.
Note
  • The dilution plate allows for preparing the dilutions ahead of time, while the working plate is for blocking. Also, in case of mistakes, you can toss the dilution plate without having to repeat the whole experiment.
  • The dilution plate is an exact same copy of the working plate.

Add Amount150 µL of blocking buffer to the remaining wells.

Perform 1:4 vertical serial dilutions(Amount50 µL transfers, mix 5x, discard the very last Amount50 µL ). Ensure equal volumes visually.

See Figure 4 for reference.
Figure 4. Schematic representation of 1:4 vertical serial dilution.
Publication Citation: Created in BioRender. de Vasconcellos Castro, J. (2026) https://BioRender.com/vc899id
Wash the working plate twice.
Transfer Amount100 µL /well from the dilution plate to the working plate.

Seal the plate and incubate for Duration01:00:00 at TemperatureRoom temperature .

1h
OPTION B - Direct ELISA: Primary antibody preparation and incubation (~30 min preparation + 1 hour incubation)
1h 30m
Use the same coated and blocked plates as in Section 6.
Prepare HRP-conjugated TA-pAb and TA-mAb at Concentration40 nanomolar (nM) working stocks in blocking buffer.

Note
The layout of the working plate is identical to that in Section 7.

Volume Calculations V2 (in Table 2):
5 wells/plate/each antibody (3 wells for the triplicate + 2 wells for the control)
2 plates total
200 µL of primary antibody solution/well
V₂ = 5 x 2 x 200 = 2000 µL - Make it in excess
V₂ = 2500 µL

ReagentGoat Anti-Rhesus IgA [GARI]-HRPNonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00365
ReagentAnti-IgA [NHPMab001]-HRPNonhuman Primate Reagent Resource (NHPRR)Catalog #CAT-00392
AntibodyInitial Concentration (C1)Final Concentration (C2)Final Volume (V2)Initial Volume (V1)
HRP Goat anti-rhesus IgA [GARI]0.5 mg/mL, 150 kDa, 3.3 µM40 nM2500 µL30 µL
HRPMouse anti-rhesus IgA [NHPMab001]0.5 mg/mL, 150 kDa, 3.3 µM40 nM2500 µL30 µL
Table 2: Calculation for the working stock solution.

Note
For accuracy, remove the equivalent blocking buffer volume and add the same volume of stock.



Set up the dilution plate as in sections 7.1, 7.2, and 7.3.
Wash the working plate twice.
Transfer Amount100 µL /well from the dilution plate to the working plate.
Seal the plate and incubate for Duration01:00:00 at TemperatureRoom temperature .
Note
For Direct ELISA, skip the secondary antibody preparation and incubation in Section 9, and go directly to Detection & Reading (Section 10).

Secondary antibody preparation and incubation (~20 min preparation + 1 hour incubation)
1h 20m
Prepare each HRP secondary antibody at Concentration1 nanomolar (nM) in blocking buffer:
  • mouse anti-goat HRP (detects TA-pAb)
  • goat anti-mouse HRP (detects TA-mAb)
  • Assign secondary for the Negative control Ab species as applicable.

Plate layout (Figure 5):

  • Columns 1-3: HRP anti-TA-pAb, mouse anti-goat HRP;
  • Columns 4-6: HRP anti-TA-mAb, goat anti-mouse HRP;
  • Columns 7-9: HRP anti-negative control Ab, depending on the control antibody of your choice;
  • Row A10-11: Blocking buffer (coat only);
  • Row B10-11: Blocking buffer (TA-pAb controls);
  • Row C10-11: Blocking Buffer (TA-mAb controls);
  • Row D10-11: Blocking buffer (Negative control Ab controls);
  • Row E10-F11: HRP secondary antibodies controls;
  • RowG10-11: Blocking buffer, blank.



Figure 5: Secondary Antibody Plate Layout
Publication Citation: Created in BioRender. de Vasconcellos Castro, J. (2026) https://BioRender.com/n5v2prn

Volume calculations V2 (in Table 3):
26 well/plate will receive the secondary antibody
2 plates total
100 µL of coating solution/well
V₂ = 26 x 2 x 100 = 5200 µL - Make it in excess
V₂ = 5600 µL
ReagentMouse Anti-Goat IgG Fc-HRP (SB115d)Southern BiotechCatalog #6158-05
ReagentPeroxidase AffiniPure® F(ab)₂ Fragment Goat Anti-Mouse IgG, Fcγ fragment specificJackson ImmunoResearch Laboratories, Inc.Catalog #115-036-071
AntibodyInitial Concentration (C1)Final Concentration (C2)Final Volume (V2)Initial Volume (V1)
Mouse anti-goat IgG Fc-HRP (SB115d)0.75 mg/mL, 150 kDa 5 µM1 µM5600 µL1.12 µL
HRP F(ab)2 Fragment Goat Anti-Mouse IgG0.6 mg/mL, 100 kDa (it’s a F(ab')2 ) = 6 µM1 µM5600 µL0.933 µL
Table 3: Calculation for the secondary antibody solution.
Note
Depending on the Control Antibody of your choice, you will need to adjust the volume of secondary antibody (V₂).

Note
For accuracy, remove the equivalent blocking buffer volume and add the same volume of stock.

Wash working plates twice in wash buffer.

Add Amount100 µL /well.

Seal the plate and incubate for Duration01:00:00 at TemperatureRoom temperature .

1h
Detection & Reading (~15 min)
15m
Wash the working plates twice with wash buffer.
Add Amount100 µL /well of TMB substrate.

Incubate for 5-10 min.
Note
Monitor the color development as this can vary depending on the antibodies.

Stop the reaction with Amount100 µL /well ELISA Stop solution.

Read the plates at 450 nm.

Data Extraction and Organization
Export OD450 readings from the plate reader as a ".csv" or ".xlsx" file.

Option A - Excel
  • Organize the data with columns for concentration (nM) and raws for the TAs (raw OD450 values);
  • Average the triplicates and calculate %CV;
Note
%CV = Coefficient of Variation, measures the variability among replicates. Lower values indicate precision between replicates. For ELISA assays ≤15% is generally acceptable.

Calculate it as follows:
%CV = (Standard Deviation, SD / Mean) x 100
  • Save the table and import into GraphPad Prism or other analysis software;
  • Go to Section 12.
Option B - GraphPad Prism
  • Open GraphPad Prism and create a new XY data table (X = Concentration, Y = OD450 readings);
  • Under X, enter the antibody concentrations and label the column "Concentration (nM)";
  • Under Y, paste the corresponding raw OD450 values in three replicate subcolumns for each sample;
  • Prism will automatically generate a graph, under the "Graphs" tab;
  • Adjust the graph layout as desired (color, font, size, legends, etc...);
  • Since concentrations are expressed in linear values, set the X-axis to logaritmic scale:
Double-click the X-axis -> under Scale, select "Log 10".
Now you are ready to analyze the samples.
Data Analysis & Interpretation
In Prism, choose Analyze -> Nonlinear regression (curve fit)
Select "Dose-response - sigmoidal, 4PL, X is the concentration"
Under Settings -> Constraints, leave unconstrained unless you have historical limits already set.
Outputs per curve:
  • R2
  • EC50
  • Top (maximum OD)
  • Bottom (baseline OD)
  • Hill slope
Acceptance Criteria
The acceptance criteria will be the following:

ParameterAcceptance CriterionRationale
Goodness of fit (R2)≥0.98Indicates strong fit of the 4PL model to the experimental data
Top (maximum OD)within 1.0 and 4.0 OD units (depends on the LoD of the reader)Ensures the signal is within the reader's linear range and avoids saturation
Bottom (baseline)≤0.10 ODConfirms minimal backgroud and proper washing/blocking
Hill SlopeBetween 0.8 and 2.0Reflects a good dose-response curve steepness. Values outside may indicate hook effect or non specific binding
EC50 (half-maximal concentration)within 0.5 to 2 x of an established range or a reference lot if availableEnsures the antibody affinity is consistent with its identity
Replicate precision (%CV)≤15 %Confirms intra-assay repeatability and accurate pipetting
Specificity check (Negative control Ab)≤0.10 ODVerifies that binding is specific to IgA and not background or nonspecific interactions
Hook effect checkNo drop in OD at highest concentrationsEnsures no antigen excess. You can exclude the point and refit the curve
Summary of acceptance criteria

The test articles (antibodies) identity is confirmed when the 4PL regression meets the above criteria on both monomeric and dimeric IgA plates.
Protocol references
Abcam. (2024). Required controls for ELISA. Abcam.com. https://www.abcam.com/en-us/technical-resources/guides/elisa-guide/controls-for-elisa

BosterBio. (n.d.). How to design positive and negative controls for ELISA. https://www.bosterbio.com/how-to-design-positive-negative-controls-ihc-western-blot-elisa

de Vasconcellos Castro, J. (2026) https://BioRender.com/m2svlm2

de Vasconcellos Castro, J. (2026) https://BioRender.com/rh7lu1d

de Vasconcellos Castro, J. (2026) https://BioRender.com/kvj3gee

de Vasconcellos Castro, J. (2026) https://BioRender.com/vc899id

de Vasconcellos Castro, J. (2026) https://BioRender.com/n5v2prn

ELISA Guide; Part 1: Introduction to ELISA, Formats and Signal Amplification. (2023, November 30). https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/elisa-guide-part-1/

Jackson ImmunoResearch. (n.d.). ELISA Guide Part 3: ELISA Optimization.https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/elisa-guide-part-3-elisa-optimization/
Acknowledgements
This protocol and reagents were developed by the Nonhuman Primate Reagent Resource (NHPRR; RRID:SCR_012986; nhpreagents.org)