The author would like to thank Miss Lora Matthews for performing the technique and Mr Paul Shepherd for cinematography. This project was funded by the University of South Australia, Laboratory Medicine program.

Preparation of 5x Veronal Buffered Saline (VBS)
<ol>
 <li>To prepare the Veronal Buffered Saline (VBS), three separate solutions need to be prepared.</li>
 <li>Prepare solution 1 by dissolving 21.25gm of NaCl and 0.94gm of Sodium Barbitone in 350ml of distilled water. The final concentrations of the NaCl and Sodium Barbitone are 1.02M and 13mM respectively.</li>
 <li>Prepare solution 2 by dissolving 1.44gm of Barbitone in 125ml of Hot distilled water. The final concentration of Barbitone is 62.5mM. </li>
 <li>Prepare solution 3 by dissolving 20.33gm of MgCl2 and 4.41gm of CaCl2 in 100ml of distilled water. The final concentration of MgCl2 and CaCl2 is 2.18M and 440mM respectively.</li>
 <li>Mix solutions 1 and 2 and cool to room temperature.</li>
 <li>Once the combined solution has cooled, add 1.25 ml of solution 3 and adjust the pH to 7.3-7.5 using 1M HCl.</li>
 <li>Adjust the final volume to 500ml with distilled water to prepare a 5x stock solution.</li>
 <li>To prepare a 1x working solution, dilute the stock 1:5 with distilled water.</li>
</ol>
Dilution of Revercells
<ol>
 <li>Gently resuspend the stock solution of Revercells by inversion and then remove 1ml of cells and centrifuge for 5 min at 600g at 4&deg;C.</li>
 <li>After centrifugation, carefully remove the supernatant and resuspend the cells in 3ml of 1x Veronal Buffered Saline.</li>
 <li>The cells can now be stored at 4&deg;C until use.</li>
</ol>
Procedure
<ol>
 <li>Transfer 50ml of 1x VBS into rows A1-A12 of a 96 well U bottom plate.</li>
 <li>To Well A1, add 50&mu;l of anti-A serum and mix thoroughly using a pipette.</li>
 <li>Using a fresh pipette tip, remove 50&mu;l of liquid from well A1 and transfer it to well A2. Mix thoroughly and repeat this process until you reach well A11 and then discard the last 50&mu;l from this well.</li>
 <li>The serum sample has now been serially diluted 2-fold from 1:2 to 1:2048. No serum is added to Well A12 as this is the VBS negative control.</li>
 <li>If more than one serum sample is to be assessed, repeat steps 4.1-4.4 for row B etc.</li>
 <li>Once all the dilutions have been made, add 50&mu;l of 1% Revercells to all wells. Note: The Revercells will settle over time and should be gently suspended prior to being dispensed into the wells.</li>
 <li>Mix the samples by gently tapping on the side of the microtitre tray, cover and leave the haemagglutination reaction to proceed for 1h at 37&deg;C. An incubator or appropriately heated room can be used.</li>
 <li>Following incubation, carefully remove the tray from the incubator and examine the plate for haemagglutination. The VBS control well should be the first well examined. If the reaction is negative, then the results are valid. If the VBS sample shows a positive haemagglutination result, then the results cannot be used and the process should be repeated. A positive haemagglutination reaction will appear as a broad sheet on the base of the well, while a negative reaction will appear as a small concentrated pellet of cells in the centre of the well. An intermediate result will have a halo of positive cells with a central core of pelleted cells. This result occurs as the serum has some Ab that can react, but insufficient amounts of Ab to cause a full reaction.</li>
 <li>The representative results section has two diagrammatic representations of the expected results. An example of an invalid and valid set of results is provided. The titre for the anti-serum will also be determined.</li>
</ol>
Representative Results:
The video includes an example of representative results. Below (Fig1 and Fig 2) are two examples shown in a diagrammatic manner. The first example is of a reaction that cannot be interpreted as the VBS control is positive, while the second is a valid assay that allows for the titre to be determined.
<img src="/files/ftp_upload/1752/1752fig1.jpg" alt="Figure 1" /> Figure 1. An example of a haemagglutination where the positive result has failed. In the diagrammatic representation above, the heamagglutination reaction cannot be accepted. The VBS serum negative control displays a positive haemagglutination reaction as evidenced by the sheet formation at the base of the well. This indicates that an error has occurred and the results cannot be accepted. In this case, the process should be repeated.
<img src="/files/ftp_upload/1752/1752fig2.jpg" alt="Figure 2" /> Figure 2. An example of a haemagglutination where the positive result has worked correctly. In the diagrammatic representation, the heamagglutination reaction is valid. The VBS negative control displays a negative haemagglutination reaction as evidenced by the pellet that has formed at the base of the well. The last positive well is row 7, which indicates that the titre for this serum sample is 128 (i.e the reciprocal of 1/128). This would be an example of an antibody that is present in moderate amounts in the serum.

The haemagglutination assay is a very simple technique that allows for a large number of serum samples to be assessed within a short period of time. It also has the advantage of being read without the need for specialised equipment, with the eye being suitable. This technique can also be modified for the detection of human chorionic gonadotrophin hormone present in urine, as part of a haemagglutination-inhibition test. In relation to a standard haemagglutination test, there are a few areas where care needs to be taken in order for the assay to be performed correctly. Firstly, this technique is based on the settling of cells onto a concave surface. As such, it is essential that a U- or round bottom microtitre plate is used. The use of a flat bottom plate as is commonly used for ELISA is not suitable, as negative and positive reactions will appear identical. It should also be appreciated that when different serum samples are used, reactive antibodies are likely to be present at differing concentrations. At high antibody concentration, the antigen (Revercells) may be limiting and a prozone effect may be observed. However this will not affect the determination of the titre value, since this is determined as being the last well where a positive reaction was last seen. Lastly, this technique is not quantitative, the absolute level of reactive antibody in the serum is not known. However, in most cases this is not necessary, particularly when the determination of reactivity to a known antigen is all the information that is required.

<table><tbody><tr><td>Revercells</td><td>CSL</td><td>02490201</td><td>Use at 1% final</td></tr><tr><td>Anti-A serum</td><td>CSL</td><td>02611305</td><td></td></tr><tr><td>96 well U bottom plate</td><td>TRP</td><td>92097</td><td>Must be U-bottom</td></tr><tr><td>Veronal buffered saline</td><td></td><td></td><td>Use at 1x final</td></tr><tr><td>37&amp;#x00B0;C room/incubator</td><td></td><td></td><td></td></tr><tr><td>Disposable gloves</td><td></td><td></td><td></td></tr></tbody></table>

determining the reactivity and titre of serum using a haemagglutination assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Watch this Scientific Journal Video about Determining the Reactivity and Titre of Serum using a Haemagglutination Assay at JoVE.com

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

determining-reactivity-titre-serum-using-haemagglutination

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.9K Views.  University of South Australia. Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Video: Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Medicine

Streamlined Preparation of 4&#8212HAU Antigen for Accurate NDV Antibody Detection

Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article ...

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

Source: Gross, F. L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56448">Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing &#945;2,6-linked sialic acid in human sera. This assay is often used for influenza human serology

Test mikroneutralizacji do pomiaru miana przeciwciał neutralizujących w surowicy ludzkiej

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Begin by labeling 96-well microtiter plates with the appropriate experimental information. Then, turn the plate in the vertical orientation and use a multichannel pipette to add 25 microliters of PBS to every well except the first well of the bottom back titration row. Add 50 microliters of the freshly prepared antigen solution of interest to the first well of the back titration row, followed by the addition of 25 microliters of RDE-treated serum samples to the first wells of the top 10 rows.
Add 25 microliters of the appropriate anti-serum to the first well of the 11th row as a positive control. Then, transfer 25 microliters from the first well of each row to their successive wells to perform serial two-fold dilutions. Pipette up and down 10 to 15 times for each dilution step, discarding the final 25 microliters from the last wells.
Next, add 25 microliters of the antigen solution to each well of rows 1 through 11, and 25 microliters of PBS only to each well of the back titration row. Tap the plate carefully 10 times on all four sides to mix, and cover the plate for 30 minutes at room temperature. At the end of the incubation, add 50 microliters of red blood cell solution to each well, and mix the plate with more tapping. Then, cover the plate again and incubate the red blood cells according to the species used, as outlined in the table.
After adding the red blood cells to the plate, adhere to the appropriate incubation time for the type of blood used in the assay. A too-short or too-long incubation will lead to an incorrect interpretation of the results.
At the end of the incubation, assess the hemagglutination by tilting the plate 90 degrees for 25 seconds, and mark the results for each well while the plate is still tilted on a printed scheme of the 96-well plate.

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Measuring the 50% Haemolytic Complement (CH₅₀) Activity of Serum

Determining the Phagocytic Activity of Clinical Antibody Samples

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Test mikroneutralizacji do pomiaru miana przeciwciał neutralizujących w surowicy ludzkiej

Test zahamowania hemaglutynacji w celu wykrycia przeciwciał w surowicy przeciwko antygenowi docelowemu