Recombinant Human Blood group Rh (D) polypeptide (RHD)
Description
Definition and Biological Role
The RHD gene encodes the RhD protein, a 417-amino-acid transmembrane glycoprotein expressed on erythrocytes. It determines Rh(D) antigen positivity, distinguishing Rh-positive (+) from Rh-negative (−) individuals . The RhD protein is part of a heterotrimeric complex with RhAG and RhCE, forming a channel thought to transport ammonia (NH₃) or carbon dioxide (CO₂) . While its precise physiological role remains debated, it is essential for maintaining proper erythrocyte membrane integrity and function .
Key Features of RHD
Transfusion Medicine
RHD’s presence or absence determines blood compatibility. Rh-negative individuals lack RHD, making Rh-positive blood incompatible for transfusion . Recent advances include:
-
Gene Editing: TALEN-mediated RHD knockout in erythroid progenitor cells converts Rh D-positive cells to D-negative, enabling universal donor blood production .
-
HDFN Prevention: Anti-D immunoglobulin is administered to Rh-negative mothers to prevent alloimmunization .
Molecular Insights
-
Transport Function: Structural homology to ammonium transporters (AmtB) suggests a role in NH₃ transport, though erythrocyte-specific evidence is limited .
-
RhAG Dependency: RhAG stabilizes RhD on the cell membrane; mutations in RHAG lead to Rh(null) phenotypes with absent Rh antigens .
-
D Antigen Variants: Partial D and weak D alleles alter epitope expression, affecting clinical outcomes. For example, weak D type 160 retains partial D antigenicity .
Functional Studies
-
Gas Transport: In vitro models propose RhD as part of an ammonia transporter complex, but in vivo evidence in erythrocytes is inconclusive .
-
Toxoplasmosis Interaction: RhD-positive individuals show reduced cognitive deficits post-Toxoplasma infection compared to RhD-negative individuals .
Challenges in Production
Product Specs
Note: If you have specific requirements regarding glycerol content, please specify them when placing the order.
For lyophilized powder delivery forms, the buffer used before lyophilization is a Tris/PBS-based buffer containing 6% Trehalose.
Note: We will prioritize shipping the format we currently have in stock. However, if you have a specific requirement for the format, please indicate it when placing your order. We will then prepare the product according to your request.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Note: The complete sequence including tag sequence, target protein sequence and linker sequence could be provided upon request.
Target Background
- Four novel RHD alleles, each distinguished by a single nucleotide substitution, were identified. RHD*67T, RHD*173T, and RHD*579C result in weak D phenotypic expression. The corresponding amino acid changes are predicted to be located within the membrane-spanning or intracellular domains of the RhD protein. RHD*482G represents the fourth substitution. PMID: 29052223
- Extensive research indicates that RHD*1227A is the most prevalent DEL allele in East Asian populations, potentially confounding initial molecular studies. PMID: 29214630
- The most frequent DEL allele, RHD*DEL1 (c.1227G>A), has been confirmed to be immunogenic. A high frequency of RHD*Psi was observed in donors with non-deleted RHD alleles (31%), surpassing the frequency of RHD variant alleles (15.5%). PMID: 29193119
- The complete absence of the RHD gene is common among RhD negative blood donors from the Qingdao region, with notable genetic polymorphisms at this locus. PMID: 29188626
- The RHD 1227G>A mutation contributes to the molecular basis of the Del phenotype in the Taiwanese population. This point mutation leads to aberrant frameshift or exon deletion transcripts, resulting in a D protein with weak antigen-presenting function. PMID: 26774048
- Within this mixed Brazilian population, the most frequent weak D types were 1, 4, 3, and 2 (frequencies of 4.35%, 2.32%, 1.46%, and 0.29%, respectively; total of 8.41%). Partial D was detected in 2.90% of samples carrying the RHD gene. For samples with inconclusive RhD typing, 53.33% exhibited weak and partial RHD, and 43.75% presented more than one RHD variant concurrently. PMID: 27184292
- Sequence comparisons revealed high sequence similarity between Patr_RHbeta and Hosa_RHCE, while the chimpanzee Rh gene most closely related to Hosa_RHD was not Patr_RHa but rather Patr_RHy. PMID: 26872772
- Six weak D types were identified in the Russian Federation: the most prevalent being type 3 (49.2%) and type 1 (28.6%), followed by type 2 (14.3), type 15 (4.8%), type 4.2 (DAR) (1.6%), and type 6 (1.6%). PMID: 27459619
- The frequency of RhD negative homozygosity in the Cypriot population was estimated to be 7.2%, while the frequencies of RHD hemizygosity and RhD positive homozygosity were calculated to be 39.2% and 53.6%, respectively. PMID: 27036548
- The occurrence of partial RhD alleles in the Tunisian population was reported. PMID: 26482434
- Reduced expression of the D antigen is attributed not only to missense mutations within the RHD gene but also to silent mutations that may affect splicing. PMID: 26340140
- Loss of heterozygosity of the RhD gene on chromosome 1p has been observed in acute myeloid leukemia. PMID: 25495174
- Data suggests that partial DEL women may be at increased risk of alloimmunization to the D antigen. PMID: 26033335
- Weak D type 4.0 appears to be the most prevalent weak D in the studied population. However, all samples must undergo sequencing to precisely determine the subtype of weak D type 4, as weak D type 4.2 holds significant clinical importance. PMID: 25369614
- Paternal RHD zygosity determination in Tunisians was evaluated using three molecular tests. PMID: 24960665
- Serologic findings of RhD alleles in Egyptians and their clinical implications were reported. PMID: 25219636
- Despite the vast diversity of RHD alleles, first-line weak D genotyping proved to be remarkably informative, enabling rapid classification of most samples with distinct RhD phenotypes in Flanders, Belgium. PMID: 25413499
- Splicing is altered in the RHD*weak D Type 2 allele, a rare variant most commonly found in Caucasians. RHD, including the full-length Exon 9, is transcribed in the presence of the c.1227G>A substitution frequently observed in Asians with DEL phenotypes. PMID: 25808592
- Among all donors, 89.00% were D-positive and 10.86% were D-negative, while 0.14% (n=55) were found to be weak D-positive. PMID: 24960662
- The frequency of D variants detected by IAT allele RHD(M295I) was 1:272 in D negative donors. Evidently, the DEL phenotype is more common in certain segments of the European population than initially believed. PMID: 24556127
- New RHD variant alleles were identified. PMID: 25179760
- Currently, the identification of novel RHD alleles in the Han Chinese population seems challenging. D prediction in this population is relatively straightforward as common alleles are dominant, accounting for approximately 99.80% of alleles in D-negative individuals. PMID: 24333088
- In Han Chinese individuals with weak D serotyping, 8 weak D and 4 partial D alleles were detected. Three novel weak D alleles (RHD weak D 95A, 779G, and 670G) and one new partial D allele (RHD130-132 del TCT) were identified. PMID: 25070883
- DEL/weak D-associated RHD alleles were found in 2.17% of Australian D-, C+ and/or E+ blood donors. PMID: 24894016
- RHD alleles and D antigen density were examined among serologically D- C+ Brazilian blood donors. PMID: 24267268
- In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was exceptionally low, with very few antigenic molecules per cell, but the D antigen epitopes appeared largely complete. PMID: 24333082
- The prevalence of D-/RHD+ samples is higher than that observed in Europeans. Over 50% of the RHD alleles identified were represented by RHDpsi and RHD-CE-D(s), highlighting the African contribution to the genetic pool of the admixed population analyzed. PMID: 24819281
- A genotyping method was developed in the laboratory. Genotyping results from 200 pregnant women were compared with RH1 phenotype at birth. PMID: 24559796
- Non-invasive fetal RHD genotyping from maternal blood provides accurate results, indicating its feasibility as a clinical tool for managing RhD-negative pregnant women in an admixed population. PMID: 24615044
- Two molecular polymorphisms were identified for detecting the (C)ce(s) type 1 haplotype. PMID: 24333080
- This study analyzes the phenotype and frequency of RhD and tetanus toxoid-specific memory B cells in limiting dilution culture. PMID: 24965774
- Data suggests that non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can accurately predict fetal RhD type in D-negative pregnant women. PMID: 24204719
- DIV alleles arose from at least two independent evolutionary events. DIV Type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV Type 2 to Type 5 with DIVb phenotype emerged from more recent gene conversions. PMID: 23461862
- RHD*DARA and RHD*DAR2 represent the same allele. Additionally, RHD*DAR1.2 and RHD*DAR1.3 both exist; however, the silent mutation 957G>A (V319) did not demonstrate any influence on the RhD phenotype. PMID: 23902153
- All novel weak D types expressed all tested D epitopes. PMID: 23550956
- Only 0.2% of D- Polish donors carried fragments of the RHD gene, all of whom were C or E+. Nearly 60% of the detected RHD alleles could potentially be immunogenic when transfused into a D- recipient. PMID: 23634715
- This study is the first to describe weak D types caused by intronic variations near the splice sites in the RHD gene. This finding is supported by the genotyping results combined with serologic profiles and bioinformatics analysis. PMID: 23216299
- RHD variants were identified in 91.6% of the 430 samples studied. Two of the nine previously unreported variants, c.335G>T and c.939G>A, were found to cause aberrant mRNA splicing through a splicing minigene assay. PMID: 23228153
- Hemizygous RHD subjects demonstrated significantly higher platelet increases and peak platelet counts compared to homozygous RHD subjects. PMID: 23712954
- The RHD*weak 4.3 allele, exhibiting markedly reduced D antigen expression, was shown to be associated with an altered RHCE gene formation leading to the expression of C(X) and VS. PMID: 22288371
- This factor modulates the influence not only of latent toxoplasmosis but also of at least two other potentially detrimental factors, age and smoking, on human behavior and physiology. PMID: 23209579
- RHD*DIVa and RHCE*ceTI are almost always, but not invariably, linked. This haplotype is found in individuals of African ancestry, and their red blood cells can exhibit aberrant reactivity with anti-C. PMID: 22804620
- RHD*DOL2, like RHD*DOL1, encodes a partial D antigen and the low-prevalence antigen DAK. PMID: 22738288
- The utilization of cell-free fetal DNA in non-invasive prenatal early detection of fetal RhD status and gender by real-time PCR demonstrates high sensitivity and accuracy as early as the 11th week of gestation for RhD status and the 7th week of gestation for fetal sex. PMID: 21488716
- This deletion appears to represent not only the first large deletion associated with weak D but also the weakest of weak D alleles reported to date. PMID: 22420867
- Novel RHD alleles were characterized. PMID: 22320258
- RHD genotyping has proven to be an essential tool for characterizing RHD alleles in donors phenotyped as D- or weak D, enhancing transfusion safety in highly racially mixed populations. PMID: 22211984
- RHD homozygotes exhibited almost twice as many D antigen sites as hemizygotes. Expression of c or E antigens was associated with increased RBC D antigen expression, while the presence of C or e antigens reduced expression. PMID: 22121029
- Anti-D investigations were conducted in individuals expressing weak D Type 1 or weak D Type 2. PMID: 21658048
- The distribution of weak D types in the Croatian population was reported. PMID: 21269342
Q&A
What is the Recombinant Human Blood group Rh(D) polypeptide?
Recombinant Human RhD protein is a fragment protein typically expressed in systems such as Escherichia coli, with high purity levels exceeding 90%. The protein belongs to the ammonium transporter family (specifically the Rh subfamily) and is believed to function as part of an oligomeric complex with transport or channel functions in the erythrocyte membrane . The commercially available recombinant versions typically cover specific amino acid ranges, such as positions 108-165, and undergo post-translational modifications including palmitoylation . For research applications, these proteins are suitable for multiple experimental methods including SDS-PAGE, ELISA, and Western blotting.
How does recombinant RhD protein differ from native RhD protein?
The recombinant RhD protein, while maintaining the essential antigenic epitopes of the native protein, is produced in controlled expression systems rather than being isolated from human sources. This creates important functional distinctions researchers must consider. When using recombinant RhD for immunological studies, it's essential to note that quantification methods differ fundamentally between recombinant and plasma-derived products. The European Pharmacopoeia "AutoAnalyzer" assay measures agglutinating activity in plasma-derived products, while recombinant versions are quantified by biochemical protein determination . Research indicates that recombinant anti-RhD (such as MonoRho) is underestimated by a factor of 4-5 in the AutoAnalyzer assay , necessitating different dosing considerations in experimental designs.
What experimental approaches can be used to study RhD protein function?
Methodologically, researchers can employ several techniques to investigate RhD protein function:
-
Flow cytometry analysis: Using fluorescently labeled antibodies to detect RhD expression and binding interactions on red blood cells. This method has been validated for measuring both the clearance of RhD-positive RBCs and the saturation of RhD antigen sites with anti-RhD IgG .
-
Half-life determination: Calculating elimination half-lives of RhD-positive RBCs following administration of anti-RhD antibodies through log-linear regression of concentration-time curves, using the formula t₁/₂ = ln 2/λz (where λz is the disposition rate constant) .
-
Saturation binding assays: Determining the percentage of RhD-positive antigen sites occupied by anti-RhD IgG, calculated as the ratio of median fluorescence obtained from samples with and without anti-RhD treatment during staining .
How do Fcγ receptor polymorphisms influence RhD clearance mechanisms?
The clearance of RhD-positive red blood cells demonstrates significant associations with Fcγ receptor polymorphisms, particularly FcγRIIA and FcγRIIIA, but not with FcγRIIIB variants . Research methodologies for investigating these associations include genotyping subjects for Fcγ receptor polymorphisms and correlating these with clearance rates.
Recent findings show that RhD-positive RBC clearance rates are strongly influenced by specific allotypes: subjects homozygous for FcγRIIA-131H or FcγRIIIA-158V demonstrate faster clearance compared to both heterozygotes and alternative homozygote allotypes . This effect appears more pronounced with recombinant anti-RhD than with polyclonal preparations. To properly investigate this phenomenon, researchers should:
-
Conduct comprehensive Fcγ receptor genotyping of study subjects
-
Measure clearance rates using flow cytometry at multiple time points
-
Apply statistical analyses to correlate specific polymorphisms with clearance parameters
-
Compare effects between recombinant and polyclonal anti-RhD preparations
What are the comparative efficacies of single recombinant versus polyclonal anti-RhD antibodies?
For researchers investigating this comparison, the following methodological approach is recommended:
-
Challenge study design: Administer a controlled volume of RhD-positive RBCs (e.g., 15 mL represents a worst-case scenario) followed by anti-RhD treatment 24 hours later .
-
Clearance measurement: Monitor RhD-positive RBC concentration over time using flow cytometry, calculating elimination half-lives.
-
Immunization prevention assessment: Test for anti-RhD alloantibodies at 3 and 6 months post-challenge .
-
Dose determination: Establish comparative dosing by escalation studies, considering that recombinant antibodies may be underestimated by standard assays used for polyclonal products .
What mechanisms explain the prevention of RhD immunization by recombinant anti-RhD antibodies?
The successful prevention of RhD immunization by a single recombinant IgG1 anti-RhD antibody represents a significant finding, as demonstrated in phase 1 clinical trials where no evidence of immunization was detected at 6 months post-challenge with 15 mL RhD-positive RBCs . This contradicts previous assumptions that multiple epitope recognition and diverse IgG subclasses (particularly IgG3) might be necessary for effective prophylaxis.
To investigate the underlying mechanisms, researchers should consider:
-
Epitope mapping: Determine which specific RhD epitopes are recognized by the recombinant antibody and compare with the epitope spectrum recognized by polyclonal preparations.
-
Fc-mediated effector functions: Examine antibody-dependent cellular cytotoxicity (ADCC), complement activation, and phagocytosis initiated by the recombinant antibody.
-
RBC clearance patterns: Compare intravascular versus extravascular clearance patterns between recombinant and polyclonal antibodies.
-
Immune modulation assessment: Investigate whether the recombinant antibody induces immune tolerance mechanisms beyond simple antigen clearance.
How should researchers design dose-finding studies for recombinant anti-RhD products?
When designing dose-finding studies for recombinant anti-RhD products, researchers must account for the fundamental differences in quantification between plasma-derived and recombinant antibodies. The European Pharmacopoeia "AutoAnalyzer" assay measures agglutinating activity for plasma-derived products, while recombinant antibodies are quantified by biochemical protein determination .
A methodological approach should include:
-
Establish a dose-equivalence framework: Since recombinant anti-RhD may be underestimated by a factor of 4-5 in the AutoAnalyzer assay , an escalating dose range should be evaluated in vivo.
-
Define clearance efficacy endpoints: Set predefined clearance thresholds (e.g., >92.5% clearance by day 3 or >50% clearance by day 7) as markers of adequate dosing .
-
Implement safety monitoring: Include rescue protocols with established products if clearance thresholds aren't met by specific timepoints.
-
Consider pharmacokinetic parameters: Measure serum anti-RhD IgG concentration over time using sensitive assays with appropriate detection limits (e.g., 0.39 ng anti-RhD/mL) .
What controls and standards are essential for researching recombinant RhD proteins?
For rigorous research with recombinant RhD proteins, the following controls and standards are methodologically essential:
-
RhD-negative RBC controls: Include RhD-negative RBCs as negative controls in all assays involving RBC binding or clearance to establish background levels.
-
Spike samples: Incorporate low, intermediate, and high anti-RhD content samples to validate assay performance, ensuring measurements fall within 25% of theoretical concentrations .
-
Reference standards: Use established polyclonal anti-RhD products as comparative references when evaluating recombinant antibody functions.
-
Purity verification: Confirm recombinant protein purity (>90% recommended) using SDS-PAGE and other analytical methods prior to experimental use .
-
Functional validation: Verify that the recombinant protein maintains expected biochemical properties and domain functions, especially if studying specific protein regions like the 108-165 amino acid fragment .
How should researchers interpret variable clearance rates observed with recombinant anti-RhD antibodies?
Clinical studies with recombinant anti-RhD antibodies have revealed considerable variation in RhD-positive RBC clearance rates among subjects receiving identical doses, with no clear dose-response relationship . When analyzing such data, researchers should:
What parameters should be measured to comprehensively evaluate recombinant anti-RhD efficacy?
A comprehensive evaluation of recombinant anti-RhD efficacy requires measurement of multiple parameters:
| Parameter | Methodology | Significance |
|---|---|---|
| RhD-positive RBC clearance | Flow cytometry quantification | Primary marker of antibody functionality |
| Elimination half-life | Log-linear regression of concentration-time curves | Indicator of clearance kinetics |
| RhD antigen site saturation | Flow cytometry with/without anti-RhD staining | Measure of binding efficiency |
| Serum anti-RhD concentration | Modified European Pharmacopoeia FACS assay | Pharmacokinetic profile |
| Anti-RhD alloantibody development | Antibody screening at 3 and 6 months post-challenge | Ultimate measure of immunization prevention |
| FcγR genotype correlation | Genotyping for FcγRIIA, FcγRIIIA, and FcγRIIIB polymorphisms | Explanation for inter-subject variation |
This multifaceted approach provides a complete picture of recombinant anti-RhD performance, allowing researchers to fully characterize its efficacy relative to plasma-derived products .
